THE AEROBIOLOGY OF THE AECIAL STATE OF THE COMANDRA BLISTER RUST, CRONARTIUM COMANDRAE PECK, I N ALBERTA by JOHN MARTIN POWELL B . S c , U n i v e r s i t y o f London, 1956 M . S c , M c G i l l U n i v e r s i t y , 1959 A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f BOTANY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , I969 0 J o h n M a r t i n P o w e l l 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r ee t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y I f u r t h e r a g r ee t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n Depar tment o f Z> 0>"T A NY The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8 , Canada i ' ABSTRACT Cronartium comandrae Peck is a heteroecious native rust which is damaging to several Pinus species in North America. It grows peren-n i a l l y in the li v i n g bark of hard pines producing pycniospores and aeciospores, and develops annually on species of Comandra and Geocaulon producing urediospores, teliospores and basidiospores. Studies were carried out to determine the effect of various meteorological and biological environmental factors on the aeciospore aerobiological phase of the rust. The main aspects considered included the factors affecting aeciospore production and release; and the factors affecting aeciospore transport, dispersion, deposition, germination and v i a b i l i t y . Aeciospore production occurred from mid May to late August, with the peak period between late May and mid June. An average aecium produced spores for 35-50 days, and individual cankers produced spores for up to 95 days. Much variation occurred from year to year and between trees. Aecial production was interfered with by the activity of rodents, insects and other fungi on the canker and through resinosis, which pro-bably accounted for a 50-55$ reduction of the potential aeciospore pro-duction in any year. Fresh rodent damage was recorded on U0-52$ of the cankers at about 20 locations in the years I966 to -I968, insect damage on 39-^6$, Tuberculina and Cladosporium infection on YJ-33?o, and resinosis on 67-71$. Rodent damage was mainly caused by squirrels. Some 6k micro-f l o r a l organisms were isolated from the cankers and spores. Tuberculina maxima was mainly responsible for k i l l i n g the infected canker bark and i i g r e a t l y r e d u c e d t h e p r o d u c t i o n o f a e c i o s p o r e s . P e n i c i l l i u m spp. and an u n d e s c r i b e d C l a d o s p o r i u m were a l s o p r o m i n e n t on t h e c a n k e r s b u t p l a y e d a l e s s e r r o l e i n r e d u c i n g p r o d u c t i o n . One hundred and s e v e n t e e n s p e c i e s o f i n s e c t s , m i t e s and s p i d e r s were a s s o c i a t e d w i t h t h e r u s t c a n k e r . E p u r a e a o b l i q u u s , an u n i d e n t i f i e d c e c i d o m y i i d a e and P a r a c a c o x e n u s g u t t a t u s were t r u e m y c e t o b i o n t s . V a r i o u s o t h e r s p e c i e s caused damage t o t h e ca n k e r , i n c l u d i n g D i o r y c t r i a , P i s s o d e s s c h w a r z i and C y l i n d r o c o p t u r u s d e l e o n i . D a i l y a e c i o s p o r e p e r i o d i c i t y showed s p o r e s t o be n o r m a l l y d i s -p e r s e d between 0800 and 1900 h o u r s , w i t h some e v i d e n c e o f a d o u b l e peak between 1000 and 1600 h o u r s , and l i t t l e d i s p e r s a l between 2000 and 0700 h o u r s . T u r b u l e n t a t m o s p h e r i c c o n d i t i o n s were a s s o c i a t e d w i t h a l l peak a e c i o s p o r e c o n c e n t r a t i o n s . Heavy r a i n s i n i t i a l l y i n c r e a s e d s p o r e c oncen-t r a t i o n s , b u t no d i s p e r s a l o c c u r r e d d u r i n g l o n g humid c o o l p e r i o d s . S p o r e d e p o s i t i o n c o n c e n t r a t i o n s were v e r y s t e e p c l o s e t o s o u r c e , and showed a t y p i c a l h o l l o w c u r v e d e p o s i t i o n g r a d i e n t . Spore c o n c e n t r a t i o n s f r o m a r t i f i c i a l r e l e a s e p o i n t s were s i m i l a r l y r e d u c e d w i t h d i s t a n c e f r o m s o u r c e , l a r g e l y b y d i f f u s i o n . A e c i o s p o r e s had an average v e l o c i t y o f 3.23 cm/sec i n c a lm a i r . R a p i d d e p l e t i o n o f spor e c o n c e n t r a t i o n o c c u r r e d under t h e f o r e s t canopy, m a i n l y b y s e d i m e n t a t i o n , a l t h o u g h a e c i o s p o r e s had good im-p a c t i o n e f f i c i e n c y . A e c i o s p o r e s g e r m i n a t e d on w a t e r agar o v e r t h e t e m p e r a t u r e r a n g e 1-30°C, w i t h optimum f o r g e r m i n a t i o n and germ t u b e g r o w t h c l o s e t o 15°C. Most a e c i o s p o r e s g e r m i n a t e d w i t h i n k-5 h o u r s , w i t h a r e d u c t i o n i n r a t e o f germ t u b e e l o n g a t i o n a f t e r 8 h o u r s and l i t t l e i n c r e a s e a f t e r 2k h o u r s . F r e e w a t e r was n e c e s s a r y f o r g e r m i n a t i o n ; a l l s p o r e s s w e l l e d p r i o r t o g e r m i n a t i o n . A e c i o s p o r e s g e r m i n a t e d e q u a l l y w e l l i n t h e d a r k and l i g h t , i i i and g e r m i n a t e d o v e r t h e pH ra n g e k.5-8. G e r m i n a t i o n r e s p o n s e on sugar media was b e t t e r t h a n on some o t h e r media, b u t a d d i t i o n o f a l t e r n a t e h o s t m a t e r i a l t o me d i a d i d not improve g e r m i n a t i o n . D a i l y a e c i o s p o r e c o l l e c t i o n s gave h i g h g e r m i n a t i o n p e r c e n t a g e s f o r 2-k weeks, b u t much l o w e r p e r c e n t a g e s d u r i n g l a t e r s p o r u l a t i o n p e r i o d . A e c i o s p o r e g e r m i n a t i o n was l o w e r f r o m exposed t h a n p r o t e c t e d a e c i a , and wet s p o r e s g e r m i n a t e d p o o r l y . V i a b i l i t y was r e d u c e d b y c o n t a m i n a t i o n f r o m a s s o c i a t e d f u n g i . A e c i o s p o r e s l o s t v i a b i l i t y v e r y r a p i d l y when ex-pos e d t o t e m p e r a t u r e s above 25°C; t e m p e r a t u r e s c l o s e t o 0°C were most f a v o u r a b l e . H i g h h u m i d i t y a f f e c t e d v i a b i l i t y , b u t u l t r a d r y c o n d i t i o n s were a l s o a d v e r s e . D i r e c t s u n l i g h t r e d u c e d v i a b i l i t y r a p i d l y . G e n e r a l l y , d a i l y c o n d i t i o n s f a v o u r i n g d i s p e r s a l were l e a s t f a v o u r a b l e f o r g e r m i n a t i o n and v i a b i l i t y r e t e n t i o n . i v TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i v LIST OF TABLES i x LIST OF FIGURES x i i i ACKNOWLEDGMENTS x x i v INTRODUCTION 1 THE ORGANISM 5 HISTORY OF THE FUNGUS 5 DISTRIBUTION AND HOSTS 10 D i s t r i b u t i o n o f t h e r u s t on P i n u s 11 D i s t r i b u t i o n o f t h e r u s t on S a n t a l a c e a e Ik L I F E CYCLE 2k SYMPTOMS 37 DAMAGE kl AREA OF STUDY 1+8 LOCATION, PHYSIOGRAPHY AND GEOLOGY 48 SOILS 50 CLIMATE 50 VEGETATION 6 l STUDY LOCATIONS 62 V AECIOSPORE PRODUCTION 65 PERIODS OF AECIOSPORE PRODUCTION AND ENVIRONMEfflTAL FACTORS AFFECTING SPORULATION 65 Methods 65 R e s u l t s 66 D i s c u s s i o n 75 BIOLOGICAL FACTORS AFFECTING AECIOSPORE PRODUCTION 78 Methods 78 R e s u l t s 82 M i c r o f l o r a 82 M i c r o f a u n a 91 Rodents 102 Response o f t h e t r e e 107 D i s c u s s i o n 109 AECIOSPORE DISPERSAL 128 METEOROLOGICAL FACTORS AFFECTING DISPERSAL 128 Methods and M a t e r i a l s 128 E x p e r i m e n t a l s i t e s 128 Spore c o l l e c t o r s 131 M e t e o r o l o g i c a l i n s t r u m e n t s 136 R e s u l t s 139 D i u r n a l p e r i o d i c i t y o f m e t e o r o l o g i c a l f a c t o r s , and t h e m i c r o c l i m a t e o f s t u d y l o c a t i o n 1 139 D i u r n a l s p o r e p e r i o d i c i t y l 4 l E f f e c t o f r e l a t i v e h u m i d i t y and t e m p e r a t u r e 146 E f f e c t o f r a i n f a l l 150 v i E f f e c t o f dew 15I4. E f f e c t o f w i n d 155 A v a i l a b i l i t y o f s p o r e s 155 S e a s o n a l s p o r e p e r i o d i c i t y 156 AECIOSPORE DISPERSAL FROM A NATURAL POINT SOURCE 159 Methods and M a t e r i a l 159 R e s u l t s 162 OTHER DATA ON DISTANCE OF AECIOSPORE DISPERSAL 171 AECIOSPORE DISPERSAL EXPERIMENTS FROM POINT SOURCES 173 M a t e r i a l s and Methods 173 E x p e r i m e n t a l arrangement 175 E x p e r i m e n t a l p r o c e d u r e 179 A n a l y s i s o f d a t a l 8 l R e s u l t s 18U C o n c e n t r a t i o n p a t t e r n s l8k Change o f c o n c e n t r a t i o n w i t h d i s t a n c e l8h RATE OF FALL OF AECIOSPORES I N CALM AIR 197 M a t e r i a l s and Methods 197 R e s u l t s 200 AECIOSPORE DISPERSAL DISCUSSION 205 M e t e o r o l o g i c a l f a c t o r s and s p o r e p e r i o d i c i t y 205 S p o r e d i s p e r s a l 213 AECIOSPORE GERMINATION 228 FACTORS AFFECTING AECIOSPORE GERMINATION 230 Methods and M a t e r i a l s 230 v i i Spore m a t e r i a l and methods of handling 230 Methods f o r e x p l o r a t o r y experiments 231 Methods f o r subsequent experiments . 23^ R e s u l t s 2k0 E f f e c t of temperature on germination 2k0 E f f e c t of humidity on germination 2k7 S w e l l i n g of spores on ... l i q u i d media 2^9 E f f e c t of h y d r a t i o n of spores on germination 251 E f f e c t of l i g h t on germination 252 E f f e c t of hydrogen i o n concentration on germination 255 E f f e c t of substrate on germination 257 a. Various agar media 257 b. A d d i t i o n of sucrose to media 259 c. Presence of host leaves on, or l e a f e x t r a c t s i n the media 2.6o D i s c u s s i o n 262 DAILY AECIOSPORE GERMINATION 270 Methods and M a t e r i a l s 270 R e s u l t s and D i s c u s s i o n 272 AECIOSPORE VIABILITY 283 Methods and M a t e r i a l 283 R e s u l t s 287 E f f e c t of temperature on spore v i a b i l i t y 287 E f f e c t o f humidity on spore v i a b i l i t y 292 • E f f e c t of l i g h t on spore v i a b i l i t y 29U v i i i D i s c u s s i o n 297 SUMMARY AM) CONCLUSION 302 LITERATURE CITED 321 APPENDIX I 353 APPENDIX I I 356 i x LIST OF TABLES TABLE Page I Monthly and annual c l i m a t i c summaries f o r Kananaskis, 53 l a t . 51°02'W, long. 115°03'W; e l e v . k,560 f t MSL, f o r the p e r i o d o f record (1939-1968). I I Monthly and annual c l i m a t i c summaries f o r s e l e c t e d 54 s t a t i o n s i n the Marmot Creek Research Basin, l a t . 50°57'N, long. 115° 1 0 % elev. 5,300-^8,000 f t MSL (1962-1967). I I I Monthly temperature and p r e c i p i t a t i o n averages at Kan- 55 anaskis Boundary Ranger S t a t i o n , l a t . 50°55'N, long. 115°08'W, elev. 4,800 f t MSL (1962-1968)., and Pigeon Mountain Lookout, l a t . 51°03'N, long. 115°04% el e v . 6,000 f t MSL (1960-1968), compared w i t h those at Kananaskis f o r a s i m i l a r p e r i o d (1963J-I968). IV Date of beginning and end of Cronartium comandrae 67 aeciospore production, and main s p o r u l a t i o n p e r i o d at a number of l o c a t i o n s i n the years 1964 - 1968' i n c l u s i v e . V Date of beginning and end of spore production, and 68 : t o t a l number of days of production from i n d i v i d u a l cankers at l o c a t i o n 1 from 1965 to 1968 i n c l u s i v e . Main spore production p e r i o d i s i n c l u d e d f o r 3 to 6 cankers f o r the years 1965 to I967. VI Average number of days and range o f days of ae c i o - 74 spore production f o r i n d i v i d u a l p u s t u l e s on s e v e r a l cankers i n 1966 and I967 at l o c a t i o n 1. V I I Summary of sequence of s p o r u l a t i o n f o r 30 and 20 74 p u s t u l e s on two cankers at l o c a t i o n 1 i n 1967. V I I I The recorded incidence of Tuber c u l i n a maxima on 83 Cronartium comandrae cankers on lodgepole pine and the t o t a l number of a c t i v e and i n a c t i v e cankers ob-served at var i o u s l o c a t i o n s i n southwest A l b e r t a during the years 1964 to 1968. IX The incidence of Tuber c u l i n a maxima on observed 87 a c t i v e and i n a c t i v e Cronartium comandrae cankers at 7 l o c a t i o n s during the years 1964 to 1968. X X The number of Cronartium comandrae a e c i a l cankers ( l ) producing aeciospores, (2) s p o r u l a t i n g but i n f e c t e d w i t h Tuberculina, (3) w i t h i n a c t i v e a e c i a l zones i n -fecte d w i t h Tuberculina, (k) w i t h i n a c t i v e or dead cankers, at two l o c a t i o n s during the years 1966 to 1968. XI The recorded incidence of Cladosporium tax. sp. 1 on Cronartium comandrae cankers observed at v a r i o u s l o c a t i o n s i n southwest A l b e r t a during the years I965 to 1968. X I I The incidence of i n s e c t damage on Cronartium comandrae cankers on lodgepole pine and the num-ber o f cankers observed at v a r i o u s l o c a t i o n s i n southwest A l b e r t a during the years 1964 to 1968. 92 X I I I The incidence of new rodent damage on Cronartium 10k comandrae cankers, and the number of cankers observed at va r i o u s l o c a t i o n s i n southwest A l b e r t a during the years 1966 to I968. XIV The percentage incidence of f r e s h rodent chewing 106 of Cronartium comandrae cankers at s e l e c t e d l o c a t i o n s d u r ing the years 1966 to 1968. XV XVI The incidence of f r e s h r e s i n o s i s on Cronartium comandrae cankers on lodgepole pine and the number of cankers observed at a number of l o c a -t i o n s i n southwest A l b e r t a during the years 1966 to 1968. The h o u r l y mean temperature, r e l a t i v e humidity and wind speed at two canker s t a t i o n s , compared w i t h records from the Bay s t a t i o n f o r temperature and humidity, and from Kananaskis f o r wind speed, f o r the p e r i o d May 17 to J u l y 11, 1966. 109 iko XVII Number of aeciospores deposited on n a t u r a l l e a f surfaces c o l l e c t e d along the four c a r d i n a l r a d i i at v a r i o u s d i s t a n c e s from a s p o r u l a t i n g Cronartium comandrae canker on J u l y 7, 1968, at l o c a t i o n 3-170 X V I I I Average number of aeciospores deposited per square centimeter on spore c o l l e c t o r coated s l i d e s at d i s -tances of 5, 10 and 15 feet along ei g h t r a d i i a-round a s p o r u l a t i n g Cronartium comandrae canker at l o c a t i o n 33 on 9 days between June 2k and J u l y 15, 1968. 170 x i XIX P a r t i c u l a r s of experiments on d i s p e r s i o n of Cron- l80 artium comandrae aeciospores from a p o i n t source. XX T o t a l number of spores trapped at one foot above 185 ground at each distance on a l l r a d i i . XXI T o t a l number of spores trapped at 5 and 10 f e e t a- 185 bove ground at 20, 50, 100 and 150 f e e t on f i v e r a d i i . XXII R e s u l t s of Experiment X, showing number of spores 190 trapped on an area of 13-5 sq cm at the one foot l e -v e l at each sampling p o i n t . X X I I I Comparison of the observed and expected concentra- 195 t i o n of spores at four distances from the 5 f o o t r e l e a s e p o i n t during ei g h t t e s t s . XXIV Percentages of i n d i v i d u a l t e s t s of aeciospores 201 of Cronartium comandrae deposited 3 meters from p o i n t of l i b e r a t i o n on gl a s s s l i d e s exposed suc-c e s s i v e l y f o r 15 second periods i n a closed c y l i n -der. XXV Average r a t e of f a l l i n s t i l l a i r of ten aeci o - 202 spore t e s t s . XXVI Number of aeciospore clumps deposited 3 meters from 203 p o i n t of l i b e r a t i o n on glass s l i d e s exposed s u c c e s s i v e l y f o r 15 second periods i n a closed c y l i n d e r . XXVII Average r a t e of f a l l i n s t i l l a i r of dry and wet 20k aeciospores during two t e s t s . XXYIII Average l e n g t h and width (|a) of aeciospores 205 deposited on s l i d e s during the dry and wet-spore t e s t s o f r a t e o f f a l l . XXIX Average number of germ tubes per spore, and the 2^3 range of the average f o r four t e s t s e r i e s at var i o u s temperatures. XXX Average l e n g t h and width ( i n microns) of 25 a e c i o - 250 spores from 5 f r e s h or stored samples. Measure-ments were made on dry spores and when spores were placed on a Czapek-Dox agar medium. XXXI Average germination percentages f o r spores 252 stored f o r v a r i o u s periods of time at -k°C, and then germinated, dry or a f t e r 2k hours hy-d r a t i o n i n a saturated humidity, on Czapek-Dox agar at 15°'C. X l l E f f e c t of l i g h t and dark c o n d i t i o n s on percent germination and germ tube growth of three s e r i e s of aeciospore samples, germinated on Czapek-Dox agar at 15-C or under f l u c t u a t i n g outside tem-peratures f o r 2k hours. E f f e c t of dark and three colored l i g h t wave bands on percent germination and germ tube growth of three s e r i e s of aeciospore samples, germinated at 15°C on Czapek-Dox agar f o r 2k hours. Average percentage and range of germination of two s e r i e s of aeciospores a f t e r 2k hours on d i f f e r e n t media at 15°C. Average number of germ tubes and lengths of germ tubes per spore f o r three samples on seven d i f f e r e n t media a f t e r 2k hours at 15°C. Average germination percentages o f f i v e a e c i o -spore samples stored at s i x temperatures and germinated on water agar at 15°C a f t e r v a r i o u s i n t e r v a l s of storage. Average germination percentages o f aeciospore samples stored f o r four to s i x days at v a r i o u s temperatures and germinated on water agar at 15°C Average percentage germination of a number of spore samples given two methods of r a p i d c o o l i n g and stored at -20°C f o r periods between 7 and 18 days, and then germinated on water agar at 15°C f o r 2k hours. A p o r t i o n of the l i q u i d n i t r o g e n cooled spores was r a p i d l y thawed f o r one minute at 38°C p r i o r t o seeding on the agar. Average number of germ tubes per spore and average l e n g t h of longest germ tube i n three dry-humid treatments a f t e r v a r i o u s i n t e r v a l s of storage. x i i i LIST OF FIGURES Figure Page 1 D i s t r i b u t i o n of Cronartium comandrae on 15 Pinus species i n western Canada. 2 D i s t r i b u t i o n of Cronartium comandrae on 16 Pinus species i n eastern Canada. 3 A e r i a l stems of Geocaulon l i v i d u m , w i t h 17 a x i l l a r y greenish f l o w e r s . h A e r i a l stems of Comandra umbellata ssp. 17 p a l l i d a , w i t h t e r m i n a l white flowers about to break open. 5. S e r i e s of a e r i a l stems of Comandra umbellata 17 ssp. p a l l i d a , which branch j u s t below the s o i l , and are attached to an underground rhizome. Some a e r i a l stems dead from pre-vious year. 6 Large haustorium of Comandra umbellata ssp. 17 p a l l i d a attached to Populus tremuloides Michx. r o o t , w i t h smaller haustorium on smaller r o o t . 7 D i s t r i b u t i o n of Cronartium comandrae on the 22 S a n t a l a c e a e , Comandra umbellata and Geocaulon ' l i v i d u m , i n western Canada. 8 D i s t r i b u t i o n o f Cronartium comandrae on the 23 Santalaceae, Comandra umbellata and Geocaulon l i v i d u m , i n eastern Canada. 9 Transverse s e c t i o n s taken from the c e n t r a l 26 p o r t i o n of four Cronartium comandrae cankers on lodgepole pine showing e c c e n t r i c growth caused by the r u s t . Note the r e s i n impreg-n a t i o n of the outer sapwood. 10 Transverse s e c t i o n s taken from the c e n t r a l 26 p o r t i o n of f i v e Cronartium comandrae cankers on lodgepole pine showing e c c e n t r i c growth caused by the r u s t . x i v Transverse s e c t i o n taken through the a e c i a l zone of a Cronartium comandrae canker showing the a e c i a s i t u a t e d i n the bark t i s s u e s . Note the r e s i n impregnation of the bark between aeci a , and the s w e l l i n g of the outer r i n g - o f sapwood caused by the fungus. P y c n i a l drops on the p y c n i a l zone of a Cronar-tium comandrae canker on the stem and branches of a lodgepole pine. Note the s w e l l i n g of the stem a s s o c i a t e d w i t h the canker. Group of p y r i f o r m pycniospores of Cronartium comandrae. X 2500. P y r i f o r m aeciospores of Cronartium comandrae showing s i z e and shape v a r i a t i o n of the spore t a i l . X 700. P y r i f o r m aeciospores of Cronartium comandrae showing the spore w a l l ornamentation. X 700. Canker.of Cronartium comandrae on a young branch of Pinus contorta, w i t h the p e r i d i a of the a e c i a s t i l l unruptured and forming t y p i c a l b l i s t e r s pushing through the bark. Canker of Cronartium comandrae w i t h abundant ruptured a e c i a covering the a e c i a l zone. B r i g h t orange-colored aeciospores of Cronartium comandrae covering the exposed a e c i a of the canker. Young canker of Cronartium comandrae on Pinus c o n t o r t a w i t h rough bark covering the o l d a e c i a l zone around the branch stub t h a t acted as entry p o i n t to the stem. Abundant a e c i a were produced around the o l d a e c i a l zone. S l i g h t hypertrophy of the young Pinus c o n t o r t a stem caused by Cronartium comandrae, w i t h c h a r a c t e r i s t i c rough, cracked bark, i n the o l d e r canker area caused by a e c i a r u p t u r i n g . T y p i c a l b a s a l stem canker of Cronartium coman-drae showing o l d e r rough bark zone and current year a e c i a l zone w i t h many dispersed aeciospores caught i n the c r e v i c e s of the bark. XV 22 Aeciospores of Cronartium comandrae, stained' 33 w i t h HC1 - Giemsa, each w i t h two n u c l e i . X 700. 23 Aeciospore of Cronartium comandrae, st a i n e d w i t h 33 HC1 - Giemsa, w i t h two n u c l e i . X 1200, 2k I n i t i a t i o n o f aeciospore germination, w i t h four 33 germ tubes developing through the spore w a l l , a f t e r 1 hour. X 1200. 25 Cronartium comandrae aeciospore w i t h s e v e r a l 33 germ tubes, but only one germ tube w e l l developed a f t e r 6 hours. Note the short-m u l t i p l e branches on the non-septate germ tube. X 380. 26 Group of germinated aeciospores w i t h t y p i c a l 33 branching of germ tubes a f t e r 2k hours. X 125. 27 Aeciospore of Cronartium comandrae w i t h one 3^ developed germ tube, a f t e r 3 hours. Note the non-septate c o n d i t i o n of the germ tube and t h a t the two n u c l e i have migrated towards the t i p of the germ tube. X 830. 28 Germ tubes of two Cronartium comandrae aeci o - - 3^+ spores a f t e r 3 hours, showing development of a globose and club-shaped appressorium on the developed germ tube, i n t o which the two n u c l e i have migrated. X 700. 29 Germ tube development of a Cronartium comandrae 3^+ aeciospore a f t e r 3 hours, showing a globose ap-pressorium w i t h two n u c l e i and an i n f e c t i o n peg. x 1080. 30 Germ tube development of two Cronartium coman- 3^ drae aeciospores a f t e r 3 hours, showing two types of a p p r e s s o r i a each w i t h two n u c l e i and an i n f e c t i o n peg. X 6l0. 31 U r e d i a of Cronartium comandrae on c e n t r a l p o r t i o n 35 of a Comandra umbellata ssp. p a l l i d a l e a f . Dark s t r u c t u r e s on bottom are young t e l i a . 32 T e l i a of Cronartium comandrae on both surfaces 35 of a p o r t i o n - o f a Comandra umbellata ssp. p a l l i d a l e a f . x v i Branch i n f e c t i o n o f Cronartium comandrae on Pinus contorta, w i t h mycelium causing s w e l l i n g i n the main stem around the branch stub. Tree w i t h dead spike-top above a canker of Cronartium comandrae g i r d l i n g the t r e e ; lower branches are p r o g r e s s i v e l y k i l l e d by downward growth of r u s t . Heavy r e s i n o s i s forming a d r i e d c r u s t over a p o r t i o n of a Cronartium comandrae stem canker. Note rough bark of o l d branch canker (upper l e f t ) which served as entry f o r the r u s t to the stem. Annual rodent damage on a l a r g e Cronartium comandrae stem canker on Pinus contorta. Note the s t r i p of d r i e d dead bark not removed on each annual v i s i t , and the abundant exudation of r e s i n . Rodentcdamage on a Cronartium comandrae stem canker around a branch which acted as an entry p o i n t f o r the r u s t i n t o the stem. Sporu-l a t i o n of the a e c i a l zone can be seen outside the chewed area. Map of the lower Kananaskis R i v e r V a l l e y , A l b e r t a , showing the l o c a t i o n s o f the main study areas. Study l o c a t i o n 1, on the Kananaskis Forest Experiment S t a t i o n , near the northeast shore of B a r r i e r Lake, showing the l o c a t i o n of i n f e c t e d lodgepole pine, Comandra p l a n t p l o t s , and i n -struments used during the study. Summary of the d a i l y maximum and minimum temperatures and r e l a t i v e humidity taken from hygrothermograph records, the d a i l y r a i n f a l l , and the phenology of the a e c i a l , p y c n i a l , u r e d i a l and t e l i a l s t a t e s of Cronartium comandrae, May to August i n I965 and 1966. Summary of the d a i l y maximum and minimum tem-peratures and r e l a t i v e humidity taken from hy-grothermograph records, the d a i l y r a i n f a l l , and the phenology of the a e c i a l , p y c n i a l , u r e d i a l and t e l i a l s t a t e s of Cronartium comandrae, May to August i n I967 and 1968. XV11 A p l a s t i c screening c y l i n d r i c a l - s l e e v e cage used f o r c o l l e c t i n g i n s e c t s from Cronartium comandrae cankers on stems of Pinus contorta. Rough, cracked bark of the a e c i a l zone of a Cronartium comandrae canker i n f e c t e d w i t h the purple mold, Tuberculina maxima, which i s conspicuous as a darker area where the surface bark has been removed, or cracked. B a s a l canker of Cronartium comandrae w i t h t y p i c a l rough bark i n a e c i a l zone and showing evidence of i n s e c t damage. Note e x i t holes and Lepidoptera f r a s s at top of canker, and f u r t h e r f r a s s i n lower rough zone. Pupal chambers of Pissodes schwarzi scored i n t o the sapwood throughout the Cronartium comandrae canker area on s m a l l Pinus c o n t o r t a stem. View of s i t e no. 2 at study l o c a t i o n 1, showing a 24-hour impaction spore c o l l e c t o r by Cronartium comandrae canker no. 2721 on a s m a l l Pinus contorta, and instruments f o r r e c o r d i n g the weather. Instruments are, from l e f t to r i g h t , b l a c k porous d i s c atmometer, instrument s h e l t e r c o n t a i n i n g hygrothermograph, Wallin-Polhemus dew d u r a t i o n recorder on ground, mast w i t h anemometer cups f o r wind speed recorder and spore c o l l e c t o r . (Rain gauge i s out of the p i c t u r e ) Standard Stevenson screen i n an opening, con-t a i n i n g hygrothermograph and thermometers used as reference weather s t a t i o n at study area 1, w i t h a b i - m e t a l actinograph f o r r e c o r d i n g i n -coming r a d i a t i o n seen at the back. A r e c o r d -i n g r a i n gauge and a wind d i r e c t i o n recorder (out of the p i c t u r e ) were maintained at t h i s open s i t e . A H i r s t spore trap w i t h sampling o r i f i c e one foot above ground, close to a Comandra um-b e l l a t a p l o t . x v i i i 49 Seven-day p o l l e n sampler of the Sarvas type, 130 used to c o l l e c t aeciospores at set distances from s p o r u l a t i n g Cronartium comandrae cankers. Spores pass through the sampler o r i f i c e and are deposited on a vaseline-coated'sampling band placed around a c l o c k - d r i v e n drum housed w i t h i n the i n t a k e c y l i n d e r . 50 A 24-hour impaction spore c o l l e c t o r , w i t h the 133 sealed l i d removed to show the a c r y l i c p l a s t i c d i s c h o l d i n g 24 microscope s l i d e s upon which spores are deposited, and the fan f o r drawing a i r through the box at a c o n t r o l l e d r a t e . 51 A 24-hour impaction spore c o l l e c t o r i n opera- 133 t i o n , w i t h i t s sampling o r i f i c e placed close to the sporulating surface of a Cronartium comandrae canker. 52 C e n t r a l instrument power and r e c o r d i n g box, 133 w i t h Thorntbmte f o u r - u n i t wind speed r e g i s t e r recorder ( l e f t ) , wind d i r e c t i o n recorder, and d i g i t a l p r i n t o u t recorder w i t h p o l a r o i d camera f o r wind speed recorder system ( r i g h t ) . Behind are the transformers and r e c t i f i e r s f o r reducing the 110 v o l t power supply and c o n t r o l l i n g an output of 12 v o l t s f o r operation of wind i n -struments, dew instruments and 24-hour impaction spore c o l l e c t o r s . A standby 12 v o l t b a t t e r y i s a l s o present. 53 Set of anemometer cups of the Thornthwaite 133 wind speed r e g i s t e r recorder system operated at canker height near a s p o r u l a t i n g Cronartium comandrae canker. 54 Mean d i u r n a l aeciospore p e r i o d i c i t y curves 143 f o r three cankers over v a r y i n g periods of t r a p p i n g i n the years 1964 to I967, expressed as a percentage of the peak geometric mean hou r l y concentration. 55 Frequency of h o u r l y maximum aeciospore concen- l45 t r a t i o n s from three Cronartium comandrae can-kers on 91 dry and 40 r a i n y days i n 1966 and 95 dry and 29 r a i n y days i n 1967, during the main spore production p e r i o d s . 56 Average hou r l y aeciospore concentrations 147 c o l l e c t e d from three Cronartium comandrae cankers i n June 1966. x i x Average h o u r l y aeciospore concentrations c o l l e c t e d from three Cronartium comandrae cankers from June 6 to 25, I967. Average hou r l y aeciospore concentration c o l -l e c t e d from Cronartium comandrae canker no. 2713 during periods without r a i n (average 19 to 2k days) i n June 1966, compared w i t h the aver-age h o u r l y a i r temperature, r e l a t i v e humidity and wind speed. Hourly number of aeciospores c o l l e c t e d from Cronartium comandrae canker no. 25l6 during the p e r i o d June 7 to J u l y 3, I965, r e l a t e d to hourl y a i r temperature, r e l a t i v e humidity, r a i n f a l l , incoming r a d i a t i o n and wind speed. Hourly number of aeciospores c o l l e c t e d from Cronartium comandrae canker no. 2721 during the p e r i o d May 29 to June 95 1966, r e l a t e d to a i r temperature, r e l a t i v e humidity, r a i n -f a l l , incoming r a d i a t i o n and wind speed. D a i l y number of aeciospores c o l l e c t e d from two Cronartium comandrae cankers during the 1966 spore production p e r i o d , p l o t t e d on semi-log s c a l e . D a i l y number of aeciospores c o l l e c t e d from Cronartium comandrae canker no. 2713 during I965, I966 and 1967, p l o t t e d on semi-log s c a l e . P a t t e r n of aeciospore d e p o s i t i o n around Cronartium comandrae canker no. 2713 during a ICH5- hour p e r i o d on June 23, 1967-The average percentage change of aeciospore d e p o s i t i o n concentration w i t h distance on eight r a d i i around Cronartium comandrae canker no. 2713 on June 23, 1967, and the percentage change on the southeast r a d i i , p l o t t e d on semi-log s c a l e . P a t t e r n of aeciospore d e p o s i t i o n around Cronartium comandrae canker no. 2689, on three days (0800 to IcOO'J-hours) i n June 1968. XX The average percentage change o f aecio -spore d e p o s i t i o n w i t h d i s t a n c e around Cronartium comandrae canker no. 2689, on three days (0800 to l600 hours) i n June 1968, p l o t t e d on semi-log s c a l e . Spore-ejector used f o r l i b e r a t i n g a e c i o -spores i n d i s p e r s a l experiments from p o i n t source. Compressor u n i t v w i t h connecting rubber tubing, f o r r e g u l a t i n g a i r flow f o r r e -lease of aeciospores i n d i s p e r s a l experiments. W i n d - d i r e c t i o n a l p l a s t i c s l i d e holder spore c o l l e c t o r s (without microscope s l i d e s ) used f o r c o l l e c t i n g aeciospores i n d i s p e r s a l experiments and from n a t u r a l sources. P o r t i o n of spore c o l l e c t o r network, w i t h c o l l e c t o r s on d i f f e r e n t r a d i i at var i o u s distances from r e l e a s e p o i n t . Wind d i r e c t i o n vane and wind speed anemometer can be seen to the l e f t o f the stand f o r the spore-ejector. Note spore c o l l e c t o r s at 5 and 10 f e e t on the masts. P o r t i o n of spore c o l l e c t o r network w i t h c o l l e c t o r s on d i f f e r e n t r a d i i at var i o u s distances from spore-ejector at r e l e a s e p o i n t . Wind speed anemometer at l e f t of s p o r e - e j e c t o r . Diagram of p o i n t source aeciospore d i s p e r s a l experiment sampling g r i d , w i t h 113 sampling c o l l e c t o r s l a i d out f o r southwest winds. Spore c o l l e c t o r s were a l s o l o c a t e d at the 300 and kOO foot distances along the m i d - l i n e . Aeciospore concentration p a t t e r n s at three sampling heights f o r a spore r e l e a s e one foot above ground (Experiment V I I ) . Aeciospore concentration patterns at three sampling heights f o r a spore r e l e a s e f i v e • f e e t above ground (Experiment X ) . xx i Composite aeciospore concentration p a t t e r n s at three sampling heights f o r a l l e i g h t spore r e l e a s e s f i v e f e e t above ground (Experiments I I - VI, V I I I - X ) . V e r t i c a l p r o f i l e s of aeciospore concen-t r a t i o n p atterns along the m i d - l i n e of the sampling network f o r two spore r e -leases at one foot (Experiments I and V I I ) , and two spore r e l e a s e s f i v e f e e t above ground (Experiments V and X ) . V e r t i c a l p r o f i l e s of aeciospore concen-t r a t i o n p atterns along the m i d - l i n e and f o r k5° and 90° sectors of the sampling network, f o r one spore r e l e a s e at f i v e f e e t above ground (Experiment V I I I ) . Composite v e r t i c a l p r o f i l e s of aeciospore concentration p a t t e r n s along the m i d - l i n e and f o r ^5° and 90° sectors of the sam-p l i n g network, f o r a l l e ight spore r e l e a s e s f i v e f e e t above ground (Experiments I I -VI, V I I I - X ) . E f f e c t of temperature on per cent germina-t i o n of three Cronartium comandrae aecio-spore samples a f t e r 2k hours on water agar (pH 6 .8 ) . E f f e c t of temperature on Cronartium com-andrae aeciospore germination. Average percent germination of f i v e s e r i e s and v a r i o u s numbers of r e p l i c a s w i t h three to f i v e samples each, a f t e r 2k hours on water agar. Average l e n g t h of. germ tubes of three Cronartium comandrae aeciospore samples germinated oh water agar (pH 6.8). a f t er 2k hours at v a r i o u s temperatures. (a) Length of longest germ tube per spore, (b) Length of a l l germ tubes per spore. Inf l u e n c e of v a r i o u s temperatures on r a t e of Cronartium comandrae aeciospore germina-t i o n a f t e r v a r i o u s time i n t e r v a l s on water agar. x x i i 83 Average r a t e of germination of three Conartium 246 comandrae aeciospore samples at d i f f e r e n t tem-peratures and a f t e r v a r i o u s time i n t e r v a l s on water agar. 8 4 Average l e n g t h of the longest germ tube from 2 4 8 three to f i v e Cronartium comandrae aeciospore samples germinated on..water agar at d i f f e r e n t temperatures and a f t e r v a r i o u s time i n t e r v a l s . 85 E f f e c t o f hydrogen i o n concentration on germ- 256 i n a t i o n of Cronartium comandrae aeciospores a f t e r 24 hours at 15 and 20°C. 86 E f f e c t of hydrogen i o n concentration on 256 germination of Cronartium comandrae aec i o -spores a f t e r 2 4 hours at 5, 15 and 25°C. Spore samples were stored f o r 4 and 6 weeks at 0°C p r i o r to use. 87 Average d a i l y per cent germination of f i v e 273 to seven Cronartium comandrae aeciospore samples a f t e r 24 hours on water agar at 15°C, f o r the years 1965, 1966 and 1967. 88 D a i l y per cent germination of aeciospores 275 from two Cronartium comandrae cankers f o r the years 1965, 1966 and 1967. 89 V a r i a t i o n i n the d a i l y per cent germination 276 of aeciospores from seven Cronartium com-drae cankers i n I965. 90 D a i l y per cent germination of aeciospores 277 from f i v e i n d i v i d u a l a e c i a of a Cronartium comandrae canker during the 1966 s p o r u l a t i o n p e r i o d . 91 D a i l y per cent germination of aeciospores from 278 f i v e i n d i v i d u a l a e c i a of a Cronartium comandrae canker during the I967 s p o r u l a t i o n p e r i o d . 92 Average per cent germination o f three Cronartium 288 comandrae aeciospore samples stored at three temperatures, and germinated on water agar at 15°C a f t e r v a r i o u s hours o f storage. XX 111 Average per cent germination of f i v e Cronartium comandrae aeciospore samples stored i n a wet, dry or u l t r a dry atmos-phere, and germinated on water agar at 15°C a f t e r v a r i o u s i n t e r v a l s of storage. E f f e c t of exposure of Cronartium comandrae aeciospores to d i r e c t l i g h t f o r v a r y i n g lengths of time on c l e a r sunny days (June 16, and J u l y 5, 1966; June 23, 1967), and on a cloudy day (June 16, 1967). x x i v ACKNOWLEDGMENTS I wish to express my sin c e r e g r a t i t u d e to the l a t e Dr. J . E. Bi e r f o r h i s enlightened guidance and communicative enthusiasm throughout-much of t h i s i n v e s t i g a t i o n . I wish a l s o to thank Dr. R. J . Bandoni, Dr. A. L. F a r l e y , Dr. P. G. Haddock, Dr. T. M. C. Taylor, Dr. G. H. N. Towers, and Dr. B. J . van der Kamp, f o r t h e i r counseling and as s i s t a n c e i n the pr e p a r a t i o n o f the t h e s i s . Thanks are extended to the Canada Department of F i s h e r i e s and F o r e s t r y f o r permission to use, f o r t h i s t h e s i s , data gathered i n the course o f work on a Department p r o j e c t . A p p r e c i a t i o n i s expressed to Mr. W. Morf and Mr. L. S. Skaley, t e c h n i c i a n s of the Forest Research Laboratory, Calgary, A l b e r t a , f o r a s s i s t a n c e i n the c o l l e c t i o n of data. Thanks are due to Dr. B. C. Sutton, F o r e s t Research Laboratory, Canada Department of F i s h e r i e s and F o r e s t r y , Winnipeg, Manitoba, f o r the i d e n t i f i c a t i o n o f the m a j o r i t y of the f u n g i . Other s p e c i a l i s t s who con-t r i b u t e d to the i d e n t i f i c a t i o n of va r i o u s f l o r a l groups are: Dr. A. M. Adams, H o r t i c u l t u r a l Experiment S t a t i o n , Ontario Department of A g r i c u l t u r e , Vineland S t a t i o n , Ontario; Dr. F. D. Cook, Department o f S o i l Science, U n i v e r s i t y of A l b e r t a , Edmonton, A l b e r t a ; Dr. Y. H i r a t s u k a , Forest Research Laboratory, Canada Department of F i s h e r i e s & F o r e s t r y , Calgary, A l b e r t a ; and Dr. D. B. P r e s t , Department of B a c t e r i o l o g y , U n i v e r s i t y of Wisconsin, Madison, Wisconsin. Many i n s t i t u t i o n s provided Cronartium comandrae ma-t e r i a l f o r the d i s t r i b u t i o n a l aspects o f t h i s study, and I would l i k e to e s p e c i a l l y thank the curators of the f o l l o w i n g h e r b a r i a f o r t h e i r a s s i s -tance: Arthur Herbarium, Purdue U n i v e r s i t y , L a f a y e t t e , Indiana; The xxv N a t i o n a l Fungus C o l l e c t i o n s , B e l t s v i l l e , Maryland; The New York B o t a n i c a l Garden, New York; Mac Donald College, Ste. Anne-de-Bellevue, Quebec; The W. P. Fraser Memorial Herbarium, U n i v e r s i t y of Saskatchewan, Saskatoon, Saskatchewan; U n i v e r s i t y of A l b e r t a , Edmonton, A l b e r t a ; M y c o l o g i c a l Herbarium, Canada Department of A g r i c u l t u r e , Ottawa; Ontario; and the M y c o l o g i c a l Herbaria, Canada Department of F i s h e r i e s and F o r e s t r y , at Calgary, Quebec, V i c t o r i a and Winnipeg. I am indebted to the f o l l o w i n g taxonomic s p e c i a l i s t s of the Entomology Research I n s t i t u t e , Canada Department of A g r i c u l t u r e , Ottawa, Ontario, who made a u t h o r i t a t i v e i d e n t i f i c a t i o n s o f f a u n a l m a t e r i a l : Dr. E. C. Becker, Dr. D. Brown, Mr. W. J . Brown, Dr. J . M. Campbell, Dr. T. N. Freeman, Dr. M. Ivanochko, Mr. G. Lewis, Dr. E. E. L i n d q u i s t , Miss M. R. MacKay, Mr. J . E. H. M a r t i n , Dr. W. R. M. Mason, Dr. J . F. McAlpine, Mr. C. D. F. M i l l e r , Dr. A. Mutuura, Dr. 0. Peck, Dr. B. V. Peterson, Dr. W. R. Richards, Mr. R. de Ruette, Dr. L. K..Smith, Dr. J . R. Vockeroth and Mr. G. S. Walley. Other s p e c i a l i s t s who c o n t r i b u t e d to the i d e n t i f i c a t i o n of v a r i o u s f a u n a l groups are: Dr. G. A. Bradley, Forest Research Laboratory, Canada Department of F i s h e r i e s & F o r e s t r y , Winnipeg, Manitoba; Dr. C. W. O'Brien, Department of Entomology, Univer-s i t y of C a l i f o r n i a , Berkeley, C a l i f o r n i a ; Dr. C. V. G. Morgan, Entomology Laboratory, Canada Department of A g r i c u l t u r e , Summerland, B r i t i s h Colum-b i a ; Dr. C. T. Parsons, Manchester Depot, Vermont; Mr. B. A. Sugden, Forest Entomology Laboratory, Canada Department of F i s h e r i e s & F o r e s t r y , Vernon, B r i t i s h Columbia; Dr. A. L. T u r n b u l l , Department of B i o l o g i c a l Science, Simon Fraser U n i v e r s i t y , Burnaby, B r i t i s h Columbia; and Dr. T. A. Woolley, Department of Zoology, Colorado State U n i v e r s i t y , F o r t C o l l i n s , xxv i Colorado. Thanks are extended to my w i f e , Margaret, who proof-read the manuscript during i t s p r e p a r a t i o n , and to Miss Barbara Brown who typed the f i n a l manuscript. - 1 -INTRODUCTION A knowledge of the e f f e c t of environment on a pathogen during that p a r t of i t s l i f e c y c l e when i t i s outside the host i s of prime im-portance to an understanding of the f a c t o r s i n f l u e n c i n g the spread and i n t e n s i f i c a t i o n of an airborne disease. For a pathogen to be able to spread and i n t e n s i f y e f f e c t i v e d i s p e r s a l must be achieved. This i m p l i e s the p r o d u c t i o n of spores i n p o s i t i o n s from which they can be tran s p o r t e d and deposited a l i v e on host p l a n t s under c o n d i t i o n s and i n q u a n t i t i e s t h a t w i l l lead to i n f e c t i o n . The environmental c o n d i t i o n s under which a pathogen can i n t e n s i f y have been f a i r l y a c c u r a t e l y defined f o r a few diseases, but estimates of the distance of spread or l o s s through non-v i a b i l i t y under d i f f e r e n t environmental c o n d i t i o n s have been l a r g e l y guesses. Besides a f f e c t i n g d i s p e r s a l , environment a l s o a f f e c t s the pro-d u c t i o n of inoculum on the host, spore germination and subsequent i n f e c -t i o n of the host, a l l o f which should t h e r e f o r e be considered i n t h i s phase of disease epidemiology. Factors o f the environment are both l i -m i t i n g and opt i m a l f o r spore s u r v i v a l and germination and must be known before i n f e c t i o n can be p r e d i c t e d . The phase i n the l i f e c y c l e of a pathogen, when a spore s t a t e proceeds independently of i t s hosts, con-cerns the f i e l d of aerobiology. Gregory (1961) considered aerobiology as "the study of p a s s i v e l y airborne-.macro-organisms t h e i r i d e n t i t y , behaviour, movements and s u r v i v a l " . This phase has been stud i e d i n a few diseases a f f e c t i n g a g r i c u l t u r a l crops ( C a r t e r 1963; H i r s t and Stedman 1961, 1962; J a r v i s 1962; Meredith 1961a, 1961b; Shanmuganathan and - 2 -Arulpragasam 1966; TSireeramulu 1 9 6 2 ; Wilson and Baker 1 9 4 6 ) , but few-studies have i n v o l v e d aspects of the aerobiology of f o r e s t pathogens (Bega i 9 6 0 ; R i s b e t h 1959; i^reeramulu 1 9 6 3 ; Van A r s d e l et a l . 1 9 6 1 ) . The comandra b l i s t e r r u s t , Cronartium comandrae Peck, an im-por t a n t n a t i v e disease damaging s e v e r a l species of hard pine i n North America, was s e l e c t e d f o r t h i s study, because of i t s d i s t i n c t i v e a e c i o -spore, a unique p y r i f o r m shape, which i s e a s i l y i d e n t i f i e d against the background a i r spora. Except f o r an o u t l i n e of the C_. comandrae r u s t — i t s h i s t o r y , d i s t r i b u t i o n , l i f e c y c l e , symptoms and damage, t h i s study i s l i m i t e d to the a e r o b i o l o g i c a l aspects of the a e c i a l s t a t e of the r u s t . The r u s t has been known on p i n e f o r n e a r l y 90 years and was f i r s t recorded i n western Canada from A l b e r t a i n 1907 on lodgepole pine, Pinus c o n t o r t a Dougl., and from B r i t i s h Columbia i n 1913 on ponderosa pine P. ponderosa Laws. (Arthur and Kern 1 9 1 4 ). Widespread damage by C_. comandrae i n western North America was not recorded u n t i l the mid -1950's (Mielke 1957), but during the l a s t decade there have been s e v e r a l r e -ports (Andrews and H a r r i s o n 1959; K r e b i l l 1 9 6 5 ; Peterson 1 9 6 2 a ) , and it -has r e c e n t l y become a p l a n t a t i o n problem i n other p a r t s o f North America ( C o r d e l l et a l . 1 9 6 7 ; Dance and Lynn 1 9 6 5 ; Wolfe et a l . 1 9 6 8 ) . The genus Cronartium i n c l u d e s the world's most damaging t r e e r u s t f u n g i (Peterson 1 9 6 2 b ) ; C. comandrae has been reported the most d e s t r u c t i v e stem r u s t on hard pines of western North Americai•(Peterson 1 9 6 2 a ; Hawksworth 1 9 6 4 ) . Hawksworth ( 1 9 6 4 ) suggested t h a t C. comandrae was one of the three major diseases of P. contorta. The l i f e - c y c l e of C. comandrae has been summarized by Hedgcock - 3 -and Long ( 1 9 1 5 a ) , Mielke ( 1 9 6 1 ) , and Mielke et a l . ( 1 9 6 8 ) , but the e n v i r -onmental c o n d i t i o n s under which each spore s t a t e develops or survives are not w e l l known. C. comandrae i s a heteroecious r u s t which grows p e r e n n i a l l y i n the l i v i n g bark of hard pines, and develops annually on the a l t e r n a t e hosts of the genera Comandra and Geocaulon. The pine i s i n f e c t e d during the l a t e summer and f a l l ; the p y c n i a appear i n the summer 2 or 3 years a f t e r i n i t i a l i n f e c t i o n , and a e c i a are produced the f o l l o w i n g year. The a e c i o -spores are d i s p e r s e d and i n f e c t the a l t e r n a t e hosts, where s e v e r a l genera-t i o n s of u r e d i a may be produced. T e l i a f o l l o w the u r e d i a l s t a t e , and these germinate t o produce ba s i d i o s p o r e s which are able to i n f e c t pine, com-p l e t i n g the l i f e c y c l e of the r u s t . Heavy pine i n f e c t i o n occurs at i n f r e -quent i n t e r v a l s ( K r e b i l l 1 9 6 5 ; Mielke 1957; Peterson 1 9 6 2 a ; Wagener i 9 6 0 ) g i v i n g r i s e to the use of the term "wave" years of i n f e c t i o n . This sug-gests that s a t i s f a c t o r y environmental c o n d i t i o n s f o r i n f e c t i o n do not occur every year, or even every decade. C h a r a c t e r i s t i c a l l y a n a t i v e pathogen i s i n s t a b l e balance w i t h i t s hosts and n a t u r a l environment, but i f t h i s balance i s upset then l o c a l or widespread epidemics w i l l r e s u l t . For an outbreak — an i n t e n s i f i c a t i o n or spread of the comandra b l i s t e r r u s t to occur, environmental f a c t o r s would have to be n o n - l i m i t i n g f o r three se-parate a e r o b i o l o g i c a l phases during the l i f e c y c l e of the r u s t i n any one year. Although the present study concentrates on the f i r s t phase of a e c i o -spore s p o r u l a t i o n on pine to d i s p e r s a l and germination on the a l t e r n a t e host, s i m i l a r f a c t o r s would a l s o a f f e c t the r e p e t i t i v e u r e d i a l phase on the a l t e r n a t e host, and the phase between b a s i d i o s p o r e production on the a l t e r n a t e host and i n f e c t i o n on s u s c e p t i b l e p i n e . The b a s i d i o s p o r e phase, - k -i s probably the most c r i t i c a l f o r determining outbreaks or wave years of pine i n f e c t i o n . The b a s i d i o s p o r e phase was not s e l e c t e d f o r study here, as i t s spore s t a t e was not very s u i t a b l e f o r d i s t a n c e d i s p e r s a l s t u d i e s . The i n f o r m a t i o n gained i n t h i s study can be r e l a t e d to the probable aero-b i o l o g y of the other r u s t spore s t a t e s , and to t h a t of other important Cronartium r u s t s , and f o r e s t pathogens i n general. The a e r o b i o l o g i c a l aspects which p l a y a r o l e i n the sequence of development of each spore s t a t e of the r u s t and which must be considered i n any aerobiology phase, i n c l u d e both m e t e o r o l o g i c a l and b i o l o g i c a l en-vironmental f a c t o r s . The main aspects which are considered i n the present study i n c l u d e , a) the character of the aeciospores, t h e i r p r o d u c t i o n and r e l e a s e , b) the boundary l a y e r and t u r b u l e n t f a c t o r s o f the atmosphere as they, a f f e c t escape, t r a n s p o r t , d i s p e r s i o n and d e p o s i t i o n of aeciospores, c) the environmental f a c t o r s a f f e c t i n g aeciospore germination, d) the environmental e f f e c t s on aeciospore v i a b i l i t y during these events, and e) b i o l o g i c a l f a c t o r s a f f e c t i n g aeciospore production, d i s p e r s a l , germina-t i o n and v i a b i l i t y . - 5 -THE ORGANISM HISTORY OF THE FUNGUS In the 1870*s C. H. Peck described two new r u s t f u n g i . T h i r t y -f i v e years l a t e r they were connected as the a e c i a l and t e l i a l s t a t e s of the same fungus. I n l875.Peck described as a new species, under the name Periderm!um pyriforme, a c a u l i c o l o u s or stem-inhabiting Peridermium w i t h a e c i a having obovate, p y r i f o r m , or oblong-pyriform spores, from a s p e c i - . men c o l l e c t e d by J . B. E l l i s on pine branches at Newfield, New Jerse y . Peck was informed by E l l i s t h a t the c o l l e c t i o n may have been made i n Georgia, and placed by accident among h i s New Jersey specimens. Arthur and Kern (l9lk), a f t e r examining the type specimen, b e l i e v e d the i n -s c r i p t i o n on the o r i g i n a l specimen voucher was c o r r e c t . There are s t i l l no records of any c o l l e c t i o n of t h i s r u s t i n Georgia. Hedgcock and Long (1915b) suggested Pinus r i g i d a M i l l , was the host of the type s p e c i -men, as t h i s was the only n a t i v e species of pine i n the type l o c a l i t y which was found i n f e c t e d elsewhere (Vermont). I n 1879 Peck described the second fungus, the t e l i a l s t a t e on the stems of Comandra p a l l i d a A.DC. from Colorado, as a new species, Cronartium comandrae. He st a t e d that t h i s species appeared to be a p a l e r form of the same species which occurred on leaves of Comandra umbellata (L.) Nutt. A specimen of C. comandrae was c o l l e c t e d by E l l i s on _C. umbellata ( E l l i s and Everhart, North American Fungi, No. 1082) near New-f i e l d , New Jersey, i n 1879, which was used by Hedgcock and Long (1915a) as more evidence t h a t the type l o c a t i o n f o r P. pyriforme was c o r r e c t . An - 6 -e a r l y c o l l e c t i o n by T. G-. Lea from Ohio On Thesium umbellatum (C. umbel-l a t a ) was given the name Cronartium asclepiadeum Kze. var. T h e s i i by Berkeley ( l 8 4 5 ) , to d i s t i n g u i s h the r u s t from the European C. a s c l e p i a -deum. Berkeley s t a t e d then that t h i s v a r i e t y might be a d i s t i n c t species. I n 1895 Lagerheim, elev a t e d i t to species status and termed i t Cronar-tium t h e s i i . In the meantime Peck (1879) had c o r r e c t l y given the t e l i a l s t a t e of the r u s t f u l l species status as C. comandrae, i t s present v a l i d name. Fol l o w i n g Peck's d e s c r i p t i o n of Peridermium pyriforme, con-s i d e r a b l e confusion developed i n the use of t h i s b i n o m i a l , which produced many m i s - i d e n t i f i c a t i o n s of specimens and erroneous r e p o r t s of the occur-rence of the comandra r u s t . The:specimens which probably i n i t i a t e d t h i s confusion were c o l l e c t i o n s by E l l i s of Peridermium r u s t s from near the type l o c a t i o n of P. pyriforme at Newfield. One was c o l l e c t e d on Pinus inops A i t (P. v i r g i n i a n a M i l l s ) i n May 1882 ( i s s u e d as P. pyriforme, Wo. 1021, E l l i s , North American Fungi), and the others were c o l l e c t e d on P. v i r g i n i a n a i n A p r i l 1890, and on P. r i g i d a i n May 1890 (both i n the Herbarium, New York B o t a n i c a l Garden). The l a t t e r two were o r i g i n a l l y l a b e l l e d Peridermium p i n i W a l l r . , a European Peridermium, but were l a t e r r e f e r r e d to P. pyriforme. However, these have now been r e f e r r e d to P. comptoniae (Arth.) Orton and Adams. These l a t e r c o l l e c t i o n s of E l l i s lead Arthur and Kern (1906) and Arthur (1907a) to describe as P. p y r i -forme what i s now known as P. comptoniae, completely i g n o r i n g Peck's d e s c r i p t i o n s t a t i n g that the aeciospores were p y r i f o r m . C l i n t o n (1908) l a t e r connected t h i s so c a l l e d P. pyriforme (P. comptoniae) w i t h - 7 -Cronartium comptoniae A r t h . , a t e l i a l s t a t e o c c u r r i n g on Comptonia p e r e g r i n a (L.) Coult. and Myr i c a spp. P r i o r to t h i s , Underwood and E a r l e (1896) had cast doubt on the status of P. pyriforme and had sug-gested that i t was p o s s i b l y a synonym of Peridermium cerebrum Peck, a species described i n 1873. They suggested t h i s , because they found the s t r i k i n g p y r i f o r m spores of P. pyriforme occurred " i n both forms of P. cerebrum as they occur i n the South". This i n d i c a t e s f u r t h e r con-f u s i o n of the i d e n t i t y of Peridermium forms on pine, as only the t r u e P. pyriforme has p y r i f o r m spores. Hedgcock and Long (1913) described a c a u l i c o l o u s species of Peridermium w i t h ovoid to p y r i f o r m aeciospores on Pinus c o n t o r t a c o l -l e c t e d by E. B e t h e l from Colorado, as a new species, Peridermium b e t h e l i . Unable to compare the specimen of B e t h e l w i t h type m a t e r i a l of P. p y r i -forme , they r e l i e d (Hedgcock and Long 1915a) on the published statement of Arthur and Kern (1906) which concluded that Peck's d e s c r i p t i o n was inaccurate concerning the p y r i f o r m shape of the aeciospores. Arthur and Kern (1913 5 1914), however, discarded t h e i r o r i g i n a l i n t e r p r e t a t i o n of P. pyriforme a f t e r seeing a specimen submitted i n 1913 by W. P. Frase r , c o l l e c t e d on Pinus ponderosa at Vernon, B r i t i s h Columbia. This specimen had the t y p i c a l p y r i f o r m spores described o r i g i n a l l y by Peck. Arthur and Kern (1914) speculated t h a t the a l t e r n a t e s t a t e s o f t h i s Peridermium would be found on species of Comandra, as t h i s genus had the only unattached species of Cronartium then known. A I907 c o l -l e c t i o n of P. pyriforme on Pinus c o n t o r t a from D e v i l ' s Lake (now Lake Minnewanka), Banff, A l b e r t a , by E. W. D. Holway, gave support to t h i s - 8 -s p e c u l a t i o n , as Holway wrote on the packet t h a t the Peridermium on pine was undoubtedly a s s o c i a t e d w i t h a Cronartium on Comandra. Orton and Adams (igik) found true P. pyriforme on Pinus pungens Lamb., and Cronartium comandrae on Comandra umbellata w i t h i n ho f e e t of the i n -f e c t e d pine. They concluded t h a t t h i s Cronartium was the a l t e r n a t e s t a t e of P. pyriforme. They a l s o suggested t h a t P. b e t h e l i was a synonym of P. pyriforme. Hedgcock and Long (lyik) s u c c e s s f u l l y i n f e c t e d Comandra umbellata w i t h aeciospores of P. pyriforme, confirming the l i f e c y c l e of the r u s t and gave i t the new combination Cronartium pyriforme. In the f o l l o w i n g year (1915a) they gave the morphology, l i f e h i s t o r y , and d i s t r i -b u t i o n of t h i s r u s t as then known under the new combination C. pyriforme. Kirkwood (1915) i n o c u l a t e d Pinus ponderosa w i t h spores from Comandra ( p a l l i d a ? ) through i n c i s i o n s i n the bark, which r e s u l t e d i n the develop-ment of r u s t hyphae w i t h i n the bark and wood of-.--the pine. This was the f i r s t r e cord of the s u c c e s s f u l t r a n s f e r of C. comandrae from Comandra t o pine. He als o s u c c e s s f u l l y i n o c u l a t e d Comandra p l a n t s w i t h aeciospores from pine. More r e c e n t l y , Andrews e t a l . (1963) reported s u c c e s s f u l i n -f e c t i o n of Pinus c o n t o r t a w i t h b a s i d i o s p o r e s of C. comandrae. T e l i a l horns were placed among and above the needles, w i t h p y c n i a being observed a year l a t e r . Boyce (1916) f i r s t described the p y c n i a and pycniospores of C. comandrae from a c o l l e c t i o n on Pinus ponderosa made i n J u l y 1916 i n C a l i f o r n i a . I n the same year, Weir and Hubert (1917) observed p y c n i a and pycniospores o c c u r r i n g on P. ponderosa and P. contorta, confirming the f i n d i n g s of Boyce. Meinecke (1929) t r i e d u n s u c c e s s f u l l y f o r 6 years to i n o c u l a t e d i r e c t l y from pine to pine w i t h aeciospores. This i n d i c a t e s _ 9 -t h a t C. comandrae was only heteroecious and can not be autoecious as i s Peridermium h a r k n e s s i i J.P. Moore (Meinecke 19 l6 , 1920, I929; Hedgcock and Hunt 1920). Many e a r l y herbarium specimens of C. comandrae are s t i l l l a -b e l l e d w i t h synonyms or combinations o f them. Some e a r l y c o l l e c t i o n s were placed under the form-genus Aecidium (Farlow 1905) i n s t e a d of P e r i -dermium, some of which were even n o n - a e c i a l c o l l e c t i o n s (eg. Aecidium asclepiadeum var. t h e s i i on Comandra p a l l i d a from Kamloops, B r i t i s h Columbia). Hedgcock and Long (1915a) gave Caeoma comandrae Peck as a synonym of Cronartium pyriforme. Caeoma comandrae was described by Peck (1884) from a specimen c o l l e c t e d by M. E. Jones on Comandra p a l l i d a i n Utah., However, a recent check of the New York B o t a n i c a l Garden Herbarium, where Hedgcock and Long reported the specimen deposited, r e -vealed no specimen he l d as Caeoma comandrae, but one held as Uredo comandrae by the same c o l l e c t o r . This specimen proved to be P u c c i n i a comandrae Peck, a species a l s o described by Peck i n 1884 but from a c o l -l e c t i o n by T. S. Brandeges i n Washington T e r r i t o r y . This leaves the question of the v a l i d i t y of Caeoma comandrae as a synonym i n doubt, as i t has not been l i s t e d by others. - 10 -DISTRIBUTION AND HOSTS Comandra b l i s t e r r u s t has been reported on one introduced and ten n a t i v e hard pine species i n North America, ranging from New Brunswick to the Yukon and southward to Tennessee, northern Alabama, M i s s i s s i p p i , New Mexico and C a l i f o r n i a . I t has not been reported from A l a s k a (Cash 1953; Laurent, p e r s o n a l communication 1966) or Mexico although suscept-i b l e pines do occur. I t has not been introduced outside North America but i s a p o t e n t i a l l y dangerous r u s t to na t i v e hard pines of Europe and A s i a . The u r e d i a l and t e l i a l s t a t e s of the r u s t have been recorded over a s i m i l a r range on the a l t e r n a t e hosts, Comandra and Geocaulon of the f a m i l y Santalaceae. The e a r l i e s t l i s t s of d i s t r i b u t i o n and hosts were given by Hedgcock and Long (1915a) and by Arthur (1907b, 1925, 1927, 1934). More recent c o l l e c t i o n s have extended the known d i s t r i -b u t i o n of the r u s t and r e - i d e n t i f i c a t i o n has e l i m i n a t e d some of the e a r l i e r r e p o r t s . A l l the Cronartium comandrae m a t e r i a l has been seen i n the f o l l o w i n g h e r b a r i a (Herbarium codes are those used i n the "index Herb-ariorum", Lanjouw and S t a f l e u (196U): CFB, DAOM, DAVFP, PUR, UBC, WIN, and c r i t i c a l . . ' m a t e r i a l has been seen from the f o l l o w i n g h e r b a r i a : BPI, MISSA, NY. L i s t s or in f o r m a t i o n have been obtained from the f o l l o w i n g h e r b a r i a not included above: ALTA, FFB, MFB, MONT, QFB, QMP, SASK, WINF, WSP, MacDonald College, Ste. Anne-de-Bellevue, and many of the r e g i o n a l l a b o r a t o r i e s of the United States F o r e s t S e r v i c e . - 11 -D i s t r i b u t i o n o f the Rust on Pinus Pinus attenuata Lemm. This host was not recorded by Arthur (1934) or more r e c e n t l y , by Peterson (1962b), but there are specimens from Grant Pass, Oregon, c o l l e c t e d by J . R. Weir, Sept. 6, 1916, i n the PUR and BPI h e r b a r i a . A recent p u b l i c a t i o n by Peterson (1967) does i n c l u d e t h i s pine as a host. Pinus banksiana Lamb. Arthur (1934) reported C. comandrae occurred on t h i s host i n Michigan, Wisconsin, A l b e r t a and Saskatchewan. Hedgcock and Long (1915b) reported s e v e r a l c o l l e c t i o n s from Michigan and. Minnesota on P. d i v a r i c a t a (=_P. banksiana). I t was a l s o reported from New Brunswick (Davidson and Newall 1957), Quebec (Pomerleau 1942), Ontario (Conners 1934) and Mani-toba ( R i l e y et a l . 1952). Pinus c o n t o r t a Dougl. Recorded by Arthur (1934) from Colorado, Idaho, Wyoming and A l b e r t a . Hedgcock and Long (1915b) reported the occurrence i n Michigan, and Peterson (1962b) i n C a l i f o r n i a , Montana, Oregon, Utah and Washington. Al s o recorded i n B r i t i s h Columbia ( Z i l l e r 1953) and the Yukon (Molnar i960). In I968 two specimens were c o l l e c t e d from the Cypress H i l l s , Saskatchewan (CFB 8406, 8407). Pinus e c h i n a t a M i l l . Four recent r e p o r t s of the r u s t on t h i s host are from M i s s o u r i (Berry et a l . 1961), Arkansas (U.S. Forest Service I962; P o o l i n g et a l . 1964), and Tennessee (Powers et a l . I967). - 12 -Pinus j e f f r e y i Grev. & B a l f . Peterson (1962b) reported t h i s species as a host i n C a l i f o r n i a and Oregon, and I have seen specimens c o l l e c t e d by Lewis, from near the S i s k i y o u N a t i o n a l F o r e s t , Oregon (BPI, PUR). Pinus ponderosa Laws. Arthur (193^) reported i t from C a l i f o r n i a , Colorado, Montana, Oregon, South Dakota, Utah, Washington and B r i t i s h Columbia. Peterson (1962b) added the st a t e s of Ar i z o n a , Idaho, Nevada and Wyoming. C o l l e c -t i o n s on P. ponderosa var. a r i z o n i c a (Engelm.) Shaw are included i n t h i s d i s t r i b u t i o n . P l a n t a t i o n s of ponderosa pine have r e c e n t l y been reported i n f e c t e d i n Tennessee (Powers et. a l . 1967). Pinus pungens Lamb. Orton and Adams (l^lh) reported the r u s t from Pennsylvania. Pinus r i g i d a M i l l . There i s a n o n - v e r i f i a b l e specimen from t h i s host, c o l l e c t e d at Mt. Calvary, New Je r s e y i n 1917 (PUR), but Hedgcock and Long (1915b) reported a c o l l e c t i o n from Vermont. Pinus s y l v e s t r i s L. The r u s t has been reported on t h i s introduced pine, from Wash-ington (Shaw 1958) and Connecticut (Spaulding 1958). In Canada i t has been reported from Manitoba (Thomas 1953), Saskatchewan (Ives et a l . I968), and A l b e r t a (Powell and Morf 1965). Specimens from Roscommon, Michigan are deposited i n the BPI and PUR h e r b a r i a , n e i t h e r i s v e r i f i a b l e , - 13 -and i s probably C. comptoniae. A specimen c o l l e c t e d i n 1887 i n the Mis-s o u r i B o t a n i c a l Garden, St. L o u i s , M i s s o u r i (Arthur and Kern I906) and now i n the BPI herbarium, was l a b e l l e d Peridermium pyriforme, but i s ' probably Cronartium comptoniae as i t does not have the p y r i f o r m spores. Pinus taeda L. M i l e s (1934) f i r s t reported the r u s t on t h i s host from M i s s i s -s i p p i , and more r e c e n t l y i t has been, reported from p l a n t a t i o n s i n Tennessee and the northeastern corner of Alabama (Powers et a l . 1967). Pinus v i r g i n i a n a M i l l . There i s a lQlk c o l l e c t i o n by Hedgcock from Peterburg, Penn-s y l v a n i a , i n the PUR herbarium a t t r i b u t e d to t h i s host, but the c o l l e c t i o n i s not v e r i f i a b l e , and was not l i s t e d by Hedgcock and Long (1915a, 1915b) or Arthur (193*+). Peterson (1967) reported a Pinus sp. specimen as . l i k e l y from t h i s p ine. C o r d e l l et a l . (1967) noted t h a t p l a n t e d and n a t u r a l P. v i r g i n i a n a were not i n f e c t e d i n Tennessee, although at s e v e r a l l o c a t i o n s they were intermixed w i t h i n f e c t e d P. taeda. Other r e p o r t s The introduced species P. n i g r a A r n o l d and P. p i n a s t e r A i t . have a l s o been reported as hosts of C. comandrae (Boyce 19^3; Spaulding 1958; Mielke 196I; V e r r a l l 1964), but Peterson (1962b) p o i n t s out th a t these were based on a re p o r t by C l i n t o n (1912) at the time when P e r i -dermium pyriforme was o f t e n l i n k e d w i t h C. comptoniae (Arthur and Kern I906; C l i n t o n 1908). There i s al s o a I918 n o n - v e r i f i a b l e specimen l a -b e l l e d as C. comandrae from Roscommon, Michigan on P. n i g r a i n the BPI - Ik -herbarium. Peterson. (1966b) i n d i c a t e d the erroneous r e p o r t (Gremmen 1964). of P. r e s i n o s a as a host f o r C. comandrae i n Vermont. The c o l l e c t -i o n upon which t h i s r e p o r t was based was again C. comptoniae. The d i s t r i b u t i o n of C. comandrae on Pinus spp. i n western and eastern Canada i s shown i n F i g s . 1 and 2. D i s t r i b u t i o n of the r u s t on Santalaceae Two genera of the Santalaceae serve as a l t e r n a t e hosts f o r C. comandrae. The d i s t r i b u t i o n f o r the genus Comandra has r e c e n t l y been modified as P i e h l (1965) rearranged the genus, and placed what were f i v e species (C. umbellata (L.) Nutt., C. c a l i f o r n i c a Eastw., C. r i c h a r d s i a n a Fernald, C_. p a l l i d a A. DC., C. elegans (Rochel) Reichenb.) as four sub-species of one species (C. umbellata). This i s not too s a t i s f a c t o r y , e s p e c i a l l y i n Canada, as h i s d i s t r i b u t i o n map i n d i c a t e s that there are large zones which have intermediates between two subspecies. However, h i s arrangement has been followed, as i t does e l i m i n a t e the previous problem of d i s t i n g u i s h i n g between C_. umbellata and C. r i c h a r d s i a n a , "since they are now one subspecies (ssp. umbellata). E a r l i e r , F e r n a l d (1928) had separated the species C. l i v i d a Richardson from the genus Comandra, and formed a new genus, Geocaulon w i t h G. l i v i d u m (Richardson) Fernald as the type. P i e h l (1965) agreed w i t h t h i s separation, as Geo-caulon has s e v e r a l features that separate i t from Comandra. Both Comandra and Geocaulon are hemiparasites ( F i g s . 3, k, 5 and 6 ) . S e v e r a l papers have l i s t e d the p l a n t species upon which Comandra i s p a r a s i t i c (Brooks '.v 1937; F i s h e r 1922; Harrington 19^5; Hedgcock 1915; Moss 1926; P i e h l . F i g . 1. D i s t r i b u t i o n of Cronartium comandrae on Pinus species i n western Canada. F i g . 2. D i s t r i b u t i o n of Cronartium comandrae on Pinus species i n eastern Canada. F i g . 3- A e r i a l stems of Geocaulon l i v i d u m , w i t h a x i l l a r y greenish f l o w e r s . F i g . k. A e r i a l stems of Comandra umbellata ssp. p a l l i d a , w i t h t e r -m inal white flowers about to break open. F i g . 5. S e r i e s of a e r i a l stems of Comandra umbellata ssp. p a l l i d a , which branch j u s t below the s o i l and are attached to an . underground-rhizome. Some a e r i a l stems dead from previous year. F i g . 6. Large haustorium of Comandra umbellata ssp. p a l l i d a a t t a -ched to Populus tremuloides Michx. r o o t , w i t h smaller haustorium on smaller r o o t . 17 -- 18 -1 9 6 5 ) 5 but I am aware of only one published r e p o r t f o r Geocaulon (Moss 1926, as C. l i v i d a ) . Geocaulon i s a genus w i t h a d i s t i n c t l y northern d i s t r i b u t i o n i n North America, extending from Newfoundland to Alaska, and only as f a r south as northern New England, Wisconsin, Michigan, Minnesota, Idaho and Washington (Fernald 1928; H a r r i s 1965; Hitchcock et a l . 1964; Raup 1947 p i . X X I I ) . Comandra umbellata (L.) Nutt. ssp. umbellata P i e h l (=C. umbellata; CJ. r i c h a r d s i a n a ) . Arthur (1934) reported C. comandrae occurred on t h i s host i n C a l i f o r n i a , Delaware, I l l i n o i s , Indiana, Iowa, Massachusetts, Michigan, M i s s i s s i p p i , New Hampshire, New Jersey, New York, Ohio, Oregon, Pennsyl-van i a , Washington, Wisconsin, Ontario and Quebec. P i e h l ' s (1965') d i s -t r i b u t i o n map suggests that the records f o r C a l i f o r n i a , Oregon, and Washington were on the ssp. c a l i f o r n i c a and not umbellata. Hedgcock and Long (1915a) reported a. c o l l e c t i o n from Vermont, and Powers et a l . (1967) record i t from Alabama and Tennessee. Specimens from the D i s -t r i c t of Columbia and Minnesota are deposited i n the BPI herbarium. C o l -l e c t i o n s l i s t e d as C. umbellata or C. r i c h a r d s i a n a from Manitoba and Saskatchewan are i n the UBC and WINF h e r b a r i a , and are probably t r u e C. umbellata spp. umbellata, or an intermediate between t h i s and ssp. p a l -l i d a . Comandra umbellata (L.) Nutt. ssp. p a l l i d a (A.DC.) P i e h l ( =C. p a l l i d a ) Arthur (1934) reported i t from A r i z o n a , Colorado, Idaho, Iowa, Minnesota, M i s s o u r i , Montana, New Mexico, North Dakota, Oregon, South - 19 -Dakota, Utah, Washington, Wyoming, A l b e r t a , B r i t i s h Columbia, Northwest T e r r i t o r i e s and Saskatchewan. Hedgcock and Long ( 1915a) reported c o l -l e c t i o n s from Nebraska, and a c o l l e c t i o n from Michigan i s deposited i n the PUR herbarium. There i s a specimen of t h i s host from Texas i n the BPI herbarium, and one reported as Comandra sp. (NY herbarium) which should probably be r e f e r r e d to ssp. p a l l i d a . Bisby ( 1 9 3 8 ) reported i t s occurrence i n Manitoba, and Conners ( 1 9 3 4 ) i n Ontario. There i s some doubt about the specimen upon which Arthur ( 1 9 3 4 ) based h i s Northwest T e r r i t o r i e s r e c o r d . J . A. Parmelee (personal communication 1966) be-l i e v e d t h i s r ecord was based on the DAOM specimen, No. l 8 6 l , from near M a r t i n Cabin, Slave Lake A l b e r t a , c o l l e c t e d i n 1929, and that A rthur i n t e r p r e t e d 'Slave Lake' as Great Slave Lake, Northwest T e r r i t o r i e s , i n s t e a d of Lesser Slave Lake, A l b e r t a . However, Arthur ( 1 9 2 7 ) i n c l u d e d the Northwest T e r r i t o r i e s i n h i s d i s t r i b u t i o n l i s t p r i o r to the c o l l e c t i o n of the above DAOM specimen. There i s no record of Comandra from the Northwest T e r r i t o r i e s or the Yukon i n the DAO or CAN h e r b a r i a , or more r e g i o n a l h e r b a r i a .(ALTA, CAFB, UAC, UBC), and i t has not been c o l l e c t e d north of about 59°N. l a t i t u d e i n A l b e r t a . P o r s i l d and Cody ( 1 9 6 8 ) , i n t h e i r c h e c k l i s t of v a s c u l a r p l a n t s i n the c o n t i n e n t a l Northwest T e r r i -t o r i e s , i n d i c a t e d t h a t C_. p a l l i d a was expected to occur, but at present there i s no rec o r d from the. area. Comandra umbellata (L.) Nutt, ssp. c a l i f o r n i c a (Eastw.) P i e h l . I am aware of no herbarium specimens under t h i s subspecies name, but according to P i e h l ' s ( 1 9 6 5 ) d i s t r i b u t i o n map, the specimens recorded by Hedgcock and Long ( 1 9 1 5 a ) and Arthur ( 1 9 3 4 ) under C. - 20 -umbellata from C a l i f o r n i a , Oregon, and Washington, should be ssp. c a l i f o r -n i c a . Geocaulon l i v i d u m (Richardson) F e r n a l d Arthur (1934) gave the f o l l o w i n g d i s t r i b u t i o n of the r u s t on t h i s host (as Comandra l i v i d a ) , Wisconsin, Manitoba and Quebec. Z i l l e r and Molnar (1953) f i r s t r eported the occurrence i n B r i t i s h Columbia, and Molnar (i960) i n the Yukon. Baranyay et a l . (1962) f i r s t reported the i n f e c t e d host i n A l b e r t a , and a year l a t e r Baranyay and Bourchier (1963) reported i t i n the Northwest T e r r i t o r i e s , though i n a c c u r a t e l y reported to be on Comandra. A c o l l e c t i o n made i n 1937 by D. V. Baxter (DAOM 5558 and PUR 48514) from Great Slave Lake, Northwest T e r r i t o r i e s , and l a b e l l e d Comandra sp. proved to be G. l i v i d u m . Other unreported herbarium specimens, some l a b e l l e d as C. l i v i d a , extend the known d i s -t r i b u t i o n to Idaho (NY), Washington (DAOM) and Ontario (DAOM). Other r e p o r t s Arthur (1934) r e f e r r e d to Buckleya d i s t i o c h o p h y l l a (Nutt.) Torr. as a host of C_. comandrae i n Tennessee. This species i s no longer regarded as a host of C. comandrae, as Hepting (1957) made the connection o f the Cronartium o c c u r r i n g on Buckleya w i t h Peridermium appalachianum Hepting and Cummins, g i v i n g the combination Cronartium appalachianum Hepting, a r u s t which occurs i n North C a r o l i n a , Tennessee and V i r g i n i a . The d i s t r i b u t i o n of C_. comandrae on Comandra and Geocaulon i n western and eastern Canada i s shown i n F i g s . 7 and 8. Very r e c e n t l y a - 21 -d i s t r i b u t i o n map f o r C. comandrae on Pinus spp. and Comandra spp.has been p u b l i s h e d by K r e b i l l (1968b). G e n e r a l l y h i s northern d i s t r i b u t i o n l i n e f o r Canada does not extend f a r enough north, e s p e c i a l l y i n the Yukon and Northwest T e r r i t o r i e s , and i n Manitoba and Quebec. The c o l -l e c t i o n from Great Whale R i v e r , has been misplaced i n Ontario i n s t e a d of Quebec. F i g . 7. D i s t r i b u t i o n o f Cronartium comandrae on the Santalaceae, Comandra umbellata and Geocaulon l i v i d u m , i n western Canada. F i g . 8. D i s t r i b u t i o n of Cronartium comandrae on the Santalaceae, Comandra umbellata and Geocaulon l i v i d u m , i n eastern Canada. - 2k -LIFE CYCLE Pine i n f e c t i o n by Cronartium comandrae u s u a l l y takes place during summer and autumn through the needles on branches or main stems (Andrews et a l . I963). I t i s p o s s i b l e that the fungus i n f e c t s young t r e e s d i r e c t l y through the bark or through wounds (Kirkwood 1915)5 but t h i s mode of entry i s of minor importance. P e n e t r a t i o n of the needle by the germ tube of the b a s i d i o s p o r e or sporidium may be through the stomatal pore or d i r e c t l y through the epidermal c e l l s . Following;pene-t r a t i o n a mass of hyphae develops w i t h i n the needle t i s s u e near the p o i n t of i n f e c t i o n , then hyphal growth penetrates downwards through the v a s c u l a r bundle of the needle and i n t o the bark which becomes d i s c o l o r e d The length of the p e r i o d between needle i n f e c t i o n and bark d i s c o l o r a t i o n the i n c u b a t i o n p e r i o d , v a r i e s according to the length o f growing season, the seasonal weather, and the distance the hyphae must grow between p o i n t of i n f e c t i o n and the bark. The mycelium of the fungus, which branches i r r e g u l a r l y , r a m i f i e s i n t e r c e l l u l a r l y through a l l l i v e pine bark t i s s u e s , and extends i n t o the outer sapwood t i s s u e s , mainly i n the medullary rays (Adams 1919; K r e b i l l 1968a). K r e b i l l found the advancing hyphae i n the phloem, never more than 2.cm.beyond the v i s i b l e s w e l l i n g of the i n f e c t e d stem. The i n f e c t e d bark increased i n th i c k n e s s due to the i n t e r c e l l u l a r hyphae and expanding parenchyma c e l l s . With the i n -crease i n th i c k n e s s the conducting sieve c e l l s i n the phloem c o l l a p s e . One or more u n i c e l l u l a r h a u s t o r i a may penetrate i n t o a s i n g l e parenchyma c e l l from the i n t e r c e l l u l a r hyphae. K r e b i l l a l s o r e p o r t e d h a u s t o r i a sometimes present i n sieve elements and xylem ray t r a c h e i d s , and noted - 25 -t h a t the h a u s t o r i a v a r y i n s i z e and morphology depending on t h e i r p o s i -t i o n . Peterson (1966a) reported the i n t e r e s t i n g occurrence o f C. comandrae hyphae i n t i s s u e s of the dwarf m i s t l e t o e , Arceuthobium ameri-canum Nutt. ex Engelm., on Pinus contorta, w i t h h a u s t o r i a p e n e t r a t i n g i n t o Arceuthobium parenchyma c e l l s . The r e a c t i o n o f the sapwood to i n -f e c t i o n i s a decrease or c e s s a t i o n of growth i n the canker area ( F i g s . 9 and 10). Resinosus a l s o occurs and the r e s i n may thoroughly impregnate the bark and outer sapwood ( F i g s . 9? 10 a n ( i l l ) • The p y c n i a l or spermogonium s t a t e appears as a d i s t i n c t c r u s t -l i k e l a y e r of p a r a l l e l pycniophores or spermatiophores pushing up between the outer l a y e r of c o r t i c a l parenchyma c e l l s and the o v e r l y i n g t h i n w a l l e d c e l l s of the phellogen (Adams 1919). The pycniophores a r i s e from a zone of i n t e r t w i n e d u n i n u c l e a t e hyphae which make up the base of each pycnium. The pycniophores c o n t a i n a s i n g l e nucleus which d i v i d e s , one nucleus m i g r a t i n g i n t o the immature pycniospore or spermatium which are abstr a c t e d from the f r e e ends o f the pycniophores at ma t u r i t y . The pycn-iospores appear to escape through the i r r e g u l a r c r a c k i n g of the over-l y i n g pheHum t i s s u e , and are exuded w i t h a t h i n g e l a t i n o u s f l u i d to form orange-colored d r o p l e t s ( F i g . 12) , which e v e n t u a l l y dry on the sur-face o f the bark to form dark-colored spots c a l l e d " p y c n i a l s c a r s " . Insects are a t t r a c t e d to the d r o p l e t s and help d i s t r i b u t e the pycniospores. The pycniospores are 3 to k by 3 to 7M-, p y r i f o r m i n shape ( F i g . 13) , and probably have a sexual f u n c t i o n s i m i l a r to th a t shown by P i e r s o n (1933) f o r pycniospores of Cronartium r i b i c o l a F i s c h e r . The p y c n i a appear one to three years a f t e r the i n i t i a l i n f e c t i o n and precede the a e c i a l s t a t e F i g . 9- Transverse sec t i o n s taken from the c e n t r a l p o r t i o n of ' four Cronartium comandrae cankers on lodgepole pine showing e c c e n t r i c growth caused by the r u s t . Note the r e s i n impregnation of the outer sapwood. F i g . 10. Transverse sec t i o n s taken from the c e n t r a l p o r t i o n of f i v e Cronartium comandrae cankers on lodgepole pine • showing e c c e n t r i c growth caused by the r u s t . F i g . 11. Transverse s e c t i o n taken through the a e c i a l zone of a Cronartium comandrae canker showing the a e c i a s i t u a t e d i n the bark t i s s u e s . Note the r e s i n impregnation of the bark between a e c i a , and the s w e l l i n g of the outer r i n g of sapwood caused by the fungus. - 26 -F i g . 12. P y c n i a l drops on the p y c n i a l zone of a Cronartium coman-drae canker on the stem and branches of a lodgepole pine. Note the s w e l l i n g of the stem a s s o c i a t e d w i t h the canker. F i g . 13. Group of p y r i f o r m pycniospores of Cronartium comandrae. x 2500. F i g . Ik. P y r i f o r m aeciospores of Cronartium comandrae showing s i z e and shape v a r i a t i o n o f the-spore t a i l . X 700. F i g . 15. P y r i f o r m aeciospores of Cronartium comandrae showing the spore w a l l ornamentation. X 700. - 28 -on the i d e n t i c a l area by at l e a s t one season. The p y c n i a occur i n t i s -sues j u s t beneath the periderm (Adams 19195 K r e b i l l 1968a) and o v e r l i e the a e c i a which develop s e v e r a l c e l l s deeper i n the cortex ( F i g . l l ) . H i r a t s u k a and Cummins (1963) found that the Cronartium species were the on l y r u s t s to develop an i n t r a c o r t i c a l pycnia, and one of the few r u s t genera i n which p y c n i a l growth was indeterminate. Pycniospore product-io n u s u a l l y begins i n June, or s h o r t l y a f t e r the main a e c i a l p r o d u c t i o n p e r i o d , and i n some seasons they may continue to be produced i n t o October. The p r i m o r d i a of the a e c i a appear as a mass of uninucleate hyphal c e l l s , o r i g i n a t i n g i n the c o r t i c a l parenchyma of the host some 3 to 8 c e l l s below the phellogen (Adams 1919)- The c e l l s o f the c e n t r a l p o r t i o n of the matured primorddfa become d i f f e r e n t i a t e d f i r s t to form b i -nucleate b a s a l c e l l s . Each of the f i r s t b a s a l c e l l s formed cuts o f f an i n t e r c a l a r y c e l l and a c e l l which becomes metamorphosed to form a p e r i -d i a l c e l l of the c e n t r a l arch. The peridium so formed c o n s i s t s o f a continuous membrane one to three c e l l s t h i c k , and the aecium i s d e l i m i t e d by the p e r i p h e r a l p e r i d i a l chains. The b i n u c l e a t e b a s a l c e l l s cut o f f chains of c e l l s which f u r t h e r d i v i d e i n t o an i n t e r c a l a r y c e l l and an aeciospore. The c e l l w a l l s of the aeciospores begin to t h i c k e n when they are about the t h i r d or f o u r t h spore from the b a s a l c e l l . The aeci o -spores then become acuminate above (Adams 1919? Arthur 193^) or below (Arthur and Kern l^lk; Hedgcock and Long 1915a) to give a t y p i c a l p y r i -form shape, are quite v a r i a b l e i n s i z e , 17 to 37 by 38 to 77u, w i t h c o l o r l e s s 2 to 3u t h i c k w a l l s , which are f i n e l y verrucose w i t h s m a l l i r -r e g u l a r t u b e r c l e s ( F i g s . Ik and 15) . As the a e c i a continue to mature the F i g , 16. Canker of Cronartium comandrae on a young "branch of Pinus contorta, w i t h the p e r i d i a of the a e c i a s t i l l unruptured and forming t y p i c a l b l i s t e r s pushing through the bark. F i g . 17. Canker of Cronartium comandrae w i t h abundant ruptured a e c i a covering the a e c i a l zone. F i g . 18. B r i g h t orange-colored aeciospores of Cronartium comandrae covering the exposed a e c i a of the canker. - 29 -- 30 -o v e r l y i n g host t i s s u e becomes r a i s e d and g r a d u a l l y ruptures exposing the white b l i s t e r s c o n s i s t i n g of the peridium ( F i g . 16) e n c l o s i n g a powdery mass of p y r i f o r m orange-yellow aeciospores ( F i g . 1 8 ) . The a e c i a ( F i g s . 17 and 18) are produced over the area which p r e v i o u s l y produced pycnia, and occur over or between p y c n i a l scars, u s u a l l y i n the year f o l l o w i n g p y c n i a l production, although a e c i a may not occur u n t i l the second year or even l a t e r . Aeciospore p r o d u c t i o n v a r i e s w i d e l y w i t h seasonal con-d i t i o n s , l o c a l i t y , and bark t h i c k n e s s . I n the study area the main p e r i o d of production began i n l a t e May and continued i n t o J u l y and up to the end of August at a reduced r a t e . In some years the cankers may produce a second crop of aeciospores l a t e i n the season, but i n the current year p y c n i a l zone. There may be a great v a r i a t i o n between i n d i v i d u a l a e c i a from one canker which can prolong the production p e r i o d , but u s u a l l y production from an i n d i v i d u a l aecium only l a s t s f o r two to f i v e weeks. Frequently the i n d i v i d u a l a e c i a are so close together that s e v e r a l ap-pear to coalesce to form one l a r g e i r r e g u l a r aecium. A f t e r the aeci o -spores are r e l e a s e d , the peridium d i s i n t e g r a t e s , and bark n e c r o s i s oc-curs around the a e c i a l ruptures. Adams (1919) reported that a new cork l a y e r i s formed below the a e c i a , and the dead t i s s u e i s sloughed o f f or remains as a rough, deeply-cracked bark ( F i g s . 19, 20 and 2 l ) . K r e b i l l (1968a) found no evidence of the development of a secondary cork l a y e r beneath a e c i a , but a l a y e r occurred at times beneath p y c n i a . The mature aeciospores, which are g e n e r a l l y dispersed by the wind to s u s c e p t i b l e Comandra or Geocaulon p l a n t s , are b i n u c l e a t e ( F i g s . 22 and 2 3 ) . Upon germinating one to s i x germ tubes may emerge through F i g . 19. Young canker of Cronartium comandrae on Pinus c o n t o r t a w i t h rough bark covering the o l d a e c i a l zone around the branch stub that acted as entry p o i n t to the stem. A-bundant a e c i a were produced around the o l d a e c i a l zone. F i g . 20. S l i g h t hypertrophy of the young Pinus c o n t o r t a stem caused by Cronartium comandrae, w i t h c h a r a c t e r i s t i c rough, cracked bark, i n the o l d e r canker area caused by a e c i a r u p t u r i n g . F i g . 21. T y p i c a l b a s a l stem canker of Cronartium comandrae showing older rough bark zone and current year a e c i a l zone w i t h many dispersed aeciospores caught i n the c r e v i c e s of the bark. - 32 -the spore w a l l ( F i g . 2h), but u s u a l l y the two n u c l e i migrate i n t o one of the developing germ tubes ( F i g . 25) and only t h i s germ tube continues to elongate to any extent. The germ tubes have m u l t i p l e short branches which may occur i r r e g u l a r l y throughout the length of the germ tube ( F i g s . 26 and 27). The germ tubes are non-septate, w i t h the two n u c l e i continu-i n g to migrate toward the t i p of the tube during e l o n g a t i o n . I f condi-t i o n s are s u i t a b l e an appressorium forms ( F i g . 28) from which an i n -f e c t i o n peg may develop ( F i g s . 29 and 30) and penetrate the host through the stomata to i n f e c t and develop a b i n u c l e a t e mycelium. This type of aeciospore germination i s t y p i c a l of the heteroecious r u s t s , and i s i n marked contrast to that of the autoecious r u s t s ( H i r a t s u k a 1968; H i r a t -suka et a l . 1966; P o w e l l and Morf 1966). The stem, l e a f p e t i o l e s , leaves, f l o r a l b r a c t s and f r u i t s of the a l t e r n a t e host may a l s o be i n f e c t e d . One to three weeks a f t e r aeciospore i n f e c t i o n , the u r e d i a appear on the sur-face of the leaves as s m a l l y e l l o w p u s t u l e s ( F i g . 31)• The u r e d i a de-velop from a hyphal plexus o f t e n beneath a stoma of the host l e a f (Moss I 9 2 8 ) . The v e r t i c a l hyphae a r i s e i n a p a l i s a d e f a s h i o n and become d i -v i d e d i n t o three c e l l s ; the p e r i d i a l , i n t e r c a l a r y and sporogenous c e l l s . At a l a t e r stage the i n t e r c a l a r y c e l l s disorganize, the b a s a l sporogenous c e l l s give r i s e to s p o r e - i n i t i a l s , and the p e r i d i a l c e l l s give r i s e to the peridium of the u r e d i a l sorus. The peridium of the u r e d i a l sorus q u i c k l y ruptures exposing a, mass of e l l i p t i c a l to g l o b o i d , orange-yellow urediospores, 16 to 23 by 19~28u, w i t h n e a r l y c o l o r l e s s 1.5 to 2\x t h i c k w a l l s , which are s p a r s e l y e c h i n u l a t e . The urediospores are e a s i l y disseminated by wind or i n s e c t s and are able to germinate and r e - i n f e c t F i g . 22. Aeciospores o f Cronartium comandrae, st a i n e d w i t h HC1-Giemsa, each w i t h two n u c l e i . X 700. F i g . 23. Aeciospore of Cronartium comandrae, s t a i n e d w i t h HC1-C-iemsa, w i t h two n u c l e i . X 1200. F i g . 2k. I n i t i a t i o n of aeciospore germination, w i t h four germ tubes developing through the spore w a l l , a f t e r 1 hour. X 1200. F i g . 25. Cronartium comandrae aeciospore w i t h s e v e r a l germ tubes, but only one germ tube w e l l developed a f t e r 6 hours. Note the short m u l t i p l e branches on the non-septate germ tube. X 380. F i g . 26. Group of germinated aeciospores w i t h t y p i c a l branching of germ tubes a f t e r 2k hours. X 125. - 33 -F i g . 27. Aeciospore of Cronartium comandrae w i t h one developed germ tube, a f t e r 3 hours. Note the non-septate c o n d i t i o n of the germ tube and th a t the two n u c l e i have migrated towards the t i p o f the germ tube. X 830. F i g . 28. Germ tubes of two Cronartium comandrae aeciospores a f t e r 3 hours, showing development of a globose and club-shaped appressorium on the developed germ tube, i n t o which the two n u c l e i have migrated. X 700. F i g . 29. Germ tube development o f a Cronartium comandrae a e c i o -spore a f t e r 3 hours, showing a globose appressorium w i t h two n u c l e i and an i n f e c t i o n peg. X 1080. F i g . 30. Germ tube development of two Cronartium comandrae a e c i o -spores a f t e r 3 hours, showing two types of a p p r e s s o r i a each w i t h two n u c l e i and an i n f e c t i o n peg. X 6l0. F i g . 31. U r e d i a of Cronartium comandrae on c e n t r a l p o r t i o n of a Comandra umbellata ssp. p a l l i d a , l e a f . Dark s t r u c t u r e s on bottom are young t e l i a . F i g . 32. T e l i a of Cronartium comandrae on both surfaces of a por-t i o n of Comandra umbellata ssp. p a l l i d a l e a f . - 35 -- 36 -the same a l t e r n a t e host, thus spreading and i n t e n s i f y i n g the i n f e c t i o n during the summer, o f t e n over a distance of a hundred miles or more. In the present study u r e d i a l i n f e c t i o n s were found hundreds of miles out on the P r a i r i e s away from any pine source. A l l p a r t s of Comandra p l a n t s were h i g h l y s u s c e p t i b l e t o i n f e c t i o n at any stage i n t h e i r develop-ment. Four to seven generations of the u r e d i a l s t a t e may be produced i n a s i n g l e season, i f weather co n d i t i o n s permit, which g r e a t l y increases the p o s s i b i l i t i e s of pine i n f e c t i o n . T e l i a may form about two to three weeks a f t e r the u r e d i a ap-pear on the p l a n t s and may occur on l e s i o n s i n i t i a t e d by u r e d i a or on new l e s i o n s l a t e i n the season. The t e l i a are reddish-brown, slender, o f t e n curved, h a i r - l i k e s t r u c t u r e s , 80 to 120u t h i c k and about 1 mm i n length, which stand up from the host t i s s u e ( F i g . 32). They are com-posed of columnar rows of t e l i o s p o r e s , 12 to 16 by 28 to kk\i, w i t h n e a r l y c o l o r l e s s , 2 to 3M- t h i c k , smooth w a l l s , h e l d together i n a g e l a -t i n o u s matrix. I n time, u s u a l l y under moist c o n d i t i o n s , each t e l i o s p o r e germinates i n s i t u by means of a f i v e - c e l l e d promycelium, each of the four upper c e l l s bearing one s m a l l t h i n - w a l l e d globose b a s i d i o s p o r e , 2 t o 6 by 2 to 6\i, on the t i p of a stout sterigma. The b a s i d i o s p o r e s are disseminated by the wind, and i f deposited on s u s c e p t i b l e pine needles w h i l e s t i l l v i a b l e , can germinate under favourable c o n d i t i o n s and i n -f e c t the pine, thus completing the l i f e c y c l e of the fungus. - 37 -SYMPTOMS The f i r s t v i s i b l e s i g n of comandra b l i s t e r r u s t i n f e c t i o n on a pine i s a yellow-brown spot on a needle. This i s at the p o i n t of i n -f e c t i o n , but i t i s d i f f i c u l t to d i s t i n g u i s h m a c r o s c o p i c a l l y from s i m i l a r spots caused by other agencies. In the f i r s t , second or t h i r d season f o l l o w i n g i n f e c t i o n an area of the bark a t the base of the needle f a s c -i c l e becomes d i s c o l o r e d , and a s m a l l spindle-shaped s w e l l i n g may develop. This i s the beginning of a canker, which mostly begins on needle-bearing branches and stems. I n f e c t i o n s on main stems u s u a l l y enter v i a s m a l l branches ( F i g . 3 3 ) . M y c e l i a l growth i n the host t i s s u e i s more r a p i d l o n g i t u d i n a l l y than l a t e r a l l y , and more r a p i d towards the base than the t i p of the branch or stem. Reddish-orange p y c n i a l drops appear on the bark two or more years a f t e r i n f e c t i o n . In subsequent years the p y c n i a form i n a zone behind the p e r i p h e r y o f the i n f e c t e d t i s s u e and outside the a e c i a l zone. P y c n i a l drops are not v i s i b l e long; they may be eaten by l a r v a l and adu l t i n s e c t s , dry up, or be washed by r a i n . " P y c n i a l s c a r s " may s t i l l be seen i f the bark i s r e l a t i v e l y smooth. A e c i a appear as white b l i s t e r s pushing through the bark over p y c n i a l zones of the previous years. The a e c i a l p e r i d i a rupture r e l e a s -i n g masses of orange aeciospores. A e c i a are only present i n one area f o r one season as the p y c n i a l and a e c i a l zones c o n t i n u a l l y form around the pe r i p h e r y o f the canker. Not a l l a e c i a appear as b l i s t e r s through the bark. In thick-barked trees the a e c i a are o f t e n b u r i e d and the aeciospores F i g . 33- Branch i n f e c t i o n of Cronartium comandrae on Pinus contorta, w i t h mycelium causing s w e l l i n g i n the main stem around the branch stub. Fig,. 35-. Heavy r e s i n o s i s forming a d r i e d c r u s t over a p o r t i o n of a Cronartium comandrae stem canker. Note rough bark of o l d branch canker (upper l e f t ) which served as entry f o r the r u s t to the stem. F i g . 3^. Tree w i t h dead spike-top 'above a canker of Cronartium comandrae g i r d l i n g the t r e e ; lower branches are progres-s i v e l y k i l l e d by downward growth of r u s t . - 39 -appear through cracks i n the bark. On removing the bark large areas of a e c i a w i t h aeciospores are exposed. Under n a t u r a l c o n d i t i o n s only a sm a l l p r o p o r t i o n of these aeciospores would be dispersed. Some t r e e s , e s p e c i a l l y those w i t h marked r e s i n o s i s , seldom produce aeciospores, and i t i s d i f f i c u l t to e s t a b l i s h the l i m i t s of the canker m i c r o s c o p i c a l l y . Where a e c i a have been produced, the bark cracks and the cambium and u n d e r l y i n g wood d i e . As the fungus grows the branch or stem even-t u a l l y i s g i r d l e d and p a r t s beyond the canker d i e . Spike-topped t r e e s ( F i g . 3*0 and flagged branches are conspicuous symptoms of the disease. When cankers develop on the main stem, the canker zone i s o c c a s i o n a l l y c o n s t r i c t e d i n s t e a d of swollen, as increased diameter growth of the healthy stem continues above and below the canker during canker g i r d -l i n g . Resin exudation i s a conspicuous symptom of disease, e s p e c i a l l y on Pinus c o n t o r t a ( F i g . 35). Much of the r e s i n o s i s i s caused by the fungus but some i s caused by rodents chewing the succulent i n f e c t e d bark of the canker. Rodent damage i s usua l l y a good i n d i c a t i o n of the pre-sence of the disease. ( F i g s . 36 and 37). The f i r s t symptom of the r u s t on the a l t e r n a t e host i s the y e l -low s w e l l i n g s of the developing u r e d i a l s o r i , which rupture to r e l e a s e the urediospores. A few weeks l a t e r the t e l i a l "horns" are e a s i l y v i s i -b l e developing on the same area as the u r e d i a l s o r i or from new l e s i o n s . U r e d i a and t e l i a may be present over the whole p l a n t . There i s some c h l o r o s i s of the leaves i n h e a v i l y i n f e c t e d p l a n t s , s l i g h t evidence of reduced l e a f growth and d e f o l i a t i o n i s u s u a l l y premature. F i g . 36. Annual rodent damage on a la r g e Cronartium comandrae stem canker on Pinus contorta. Note the s t r i p of d r i e d dead bark not removed on each annual v i s i t , and the abundant exudation of r e s i n . F i g . 37. Rodent damage on a Cronartium comandrae stem canker a-round a branch which acted as an entry p o i n t f o r the r u s t i n t o the stem. S p o r u l a t i o n of the a e c i a l zone can be seen outside the chewed area. - ko -- 1+1 -DAMAGE Comandra b l i s t e r r u s t damage i n n a t u r a l pine stands i s not u s u a l l y s p e c t acular, i n f e c t i o n being l i m i t e d to some extent by the d i s -t r i b u t i o n of i t s a l t e r n a t e hosts, which, although ranging over much of North America, are r e s t r i c t e d l o c a l l y to s m a l l areas. Comandra umbel-l a t a i s r a r e l y found w i t h i n pine stands as i t favours dry open areas, but Geocaulon l i v i d u m occurs on wetter areas w i t h i n spruce-pine stands. Heavy pine i n f e c t i o n i s u s u a l l y found adjacent to areas of i n f e c t e d a l -ternate hosts. The r u s t a t tacks t r e e s of a l l ages and s i z e s and causes m o r t a l i t y by b a s a l or stem g i r d l i n g . The p e r i o d between i n i t i a l i n f e c -t i o n and death o f the t r e e may be as much.as t h i r t y years, or more, and then only a few tre e s d i e each year. Seedlings may be k i l l e d w i t h i n a few years f o l l o w i n g i n f e c t i o n , because the r u s t can enter through needles on the main stem and the fungus soon g i r d l e s the s m a l l stem. The fungus c h a r a c t e r i s t i c a l l y enters the main stem of the o l d e r t r e e v i a branch i n -f e c t i o n s . The fungus only grows one to. three inches down the branch each year so that the rust.may be present i n the branch many years be-fore i t reaches the main stem. I f the branch i s k i l l e d before the r u s t reaches the main stem, the main stem w i l l not u s u a l l y become i n f e c t e d . Dead tops or spike-tops were common damage features i n mature stands where the fungus had g i r d l e d the stem thereby k i l l i n g the upper stem and branches. C h i l d s (1968) showed t h a t downward growth on the stem by the r u s t i s l e s s than 6 inches per year, thus the lower p o r t i o n of the t r e e may l i v e f o r many years i f vigorous lower branches remain. I n f e c t i o n w i t h i n a stand may have occurred i n only a few years, or even i n one year, when c o n d i t i o n s - h2 -f o r i n f e c t i o n were favourable and a supply of basi d i o s p o r e s was a v a i l -able. S e v e r a l r e p o r t s show that pine i n f e c t i o n by:the r u s t i s i n -frequent. Observations were made f o r over 50 years i n an i n f e c t e d Pinus ponderosa stand i n S i s k i y o u County, C a l i f o r n i a , and i t appeared t h a t current damage was from i n f e c t i o n kO years ago w i t h l i t t l e or no recent i n f e c t i o n ( C a l i f o r n i a Forest Pest C o n t r o l A c t i o n C o u n c i l 1958; U. S. Forest S e r v i c e 1955; Wagener i 9 6 0 ) . I n f e c t i o n of P. ponderosa i n south-ern Nevada was reported as sporadic by Wagener ( i 9 6 0 ) w i t h a wave of i n f e c t i o n by the r u s t i n .1932 or 1 9 3 3 " and almost no new i n -f e c t i o n on pine since Wagener ( 1 9 5 0 ) reported the r u s t on P. ponderosa i n a southeastern Idaho p l a n t a t i o n " . . . . c h i e f l y on the 19^0 and 19^1 growth of branches and no evidence was found i n I 9 6 2 to i n d i c a t e any more recent i n f e c t i o n ( K r e b i l l , p e r s o n a l communication 1 9 6 2 ) . L i t t l e or no recent i n f e c t i o n was found on P. ponderosa i n the Black H i l l s , South Dakota, and areas of Colorado i n stands where damage was reported 2 5 - 3 5 years p r e v i o u s l y (Peterson 1 9 6 2 a ) . Peterson ( 1 9 6 2 a ) pre-sented evidence that i n f e c t i o n occurred two to s e v e r a l decades ago on P. co n t o r t a i n the Bighorn and Shoshone N a t i o n a l F o r e s t s , Wyoming, w i t h almost no recent i n f e c t i o n . One of the f i r s t r e p o r t s of damage i n the United States was made by Boyce (Hedgcock and Long 1915a) i n Klamath N a t i o n a l F o r e s t , C a l i -f o r n i a , where he found 34% of the young P. ponderosa dead and a f u r t h e r 17% i n f e c t e d i n a stand r e p r e s e n t i n g average r a t h e r than heavy i n f e c t i o n . Meinecke ( 1 9 2 8 ) reported 35% of P. ponderosa dead or doomed i n an area - k3 -near Mt. Lassen, C a l i f o r n i a , and c a l c u l a t e d t h a t t h i s r e s u l t e d i n a 21$ r e d u c t i o n of f o r e s t cover. Other r e p o r t s o f damage to P. ponderosa have been noted from other p a r t s of C a l i f o r n i a (U.S. For e s t S e r v i c e 195'5, 1956), a 30-year o l d p l a n t a t i o n i n Caribou N a t i o n a l F o r e s t , Idaho (Wegener 1950), Charleston Mountains, southern Nevada (Wegener i960), the Black H i l l s of South Dakota (Hedgcock and Long 1915a; L u c k i n b i l l 1935)> and the east side of the Cascade Range i n Oregon and Washington ( C h i l d s 1968). Damage to young P. pungens i n Pennsylvania was noted by Adams and Orton (191^) and Hedgcock and Long (1915a); the l a t t e r reported t h a t 58$ of the pine which were producing a e c i a at the end of June were dead by autumn from the g i r d l i n g e f f e c t of the fungus. U n t i l r e c e n t l y , P. co n t o r t a was considered only an o c c a s i o n a l host f o r t h i s pathogen (Mielke 1957) although Peterson (1962a) quotes an unpublished I925 r e p o r t by Be t h e l of damage to t h i s host i n Colorado. Mielke (1957) reported mature stands covering s e v e r a l square miles where 50 - 98$ of the tre e s were i n f e c t e d i n areas of Idaho, Utah and Wyoming. Andrews and H a r r i s o n (1959) a l s o reported s e v e r e l y i n f e c t e d P. c o n t o r t a stands i n Wyoming adjacent to sagebrush areas supporting the a l t e r n a t e host. Peterson (1962a) studied damage on 2k p l o t s i n i n f e c t e d stands i n northern Wyoming and found 21$ of the t r e e s i n f e c t e d . The stands were l a r g e l y 50 - 100 years o l d w i t h i n f e c t i o n centered i n t i s s u e u s u a l l y over 20 years o l d , w i t h the cankers at an average height o f 12 f e e t . K r e b i l l (1965) s t u d i e d i n f e c t e d stands i n 12 N a t i o n a l Forests l o c a t e d from c e n t r a l Montana to northern Utah and found t h a t some cankers were 100 years o l d , and that there had been a b u i l d up between 1910 to 19^5, - 44 -w i t h only sparse recent i n f e c t i o n . I n most cases over 90% of- the sampled cankers were i n trunks and from one-fourth to one-half of these had caused spiketops. The centers of cankers ranged from 1.5 to 69 f e e t above ground w i t h the mean height around 26 f e e t . About 90% of the cankers had been scarred by rodent chewing. I n one sample p l o t 26% of a l l l i v e lodgepole pines were i n f e c t e d , and there was considerable e v i -dence of recent m o r t a l i t y by r u s t g i r d l i n g . R e cently C. comandrae has become a problem i n young p l a n t a t i o n s of P. ponderosa and P. taeda i n the Cumberland P l a t e a u area of eastern Tennessee and northeastern Alabama (Powers et a l . 1967). Powers e t a l . r e p o r t p l a n t a t i o n s w i t h over 9°% of the l e s s than 10 y e a r - o l d t r e e s i n -f e c t e d , and one case, w i t h 57% of the 2 y e a r - o l d P. taeda t r e e s i n f e c t e d . More r e c e n t l y , C o r d e l l et a l . (1967) reported t h a t 40% of the P. taeda p l a n t a t i o n s l e s s than 10 years o l d were i n f e c t e d on the Cumberland Pla t e a u , and on these p l a n t a t i o n s 6% of the t r e e s were i n f e c t e d . They als o detected severe i n f e c t i o n and m o r t a l i t y i n a P. ec h i n a t a p l a n t a t i o n i n the same area. C. comandrae i s a l s o proving a p l a n t a t i o n problem on P. ec h i n a t a i n the C l a r k N a t i o n a l Forest, M i s s o u r i (Berry et a l . 1961), and Ozark N a t i o n a l Forest, Arkansas (Dooling et a l . 1964). I n the l a t t e r area 44% of the t r e e s on three p l o t s planted i n 1958 i n the B u f f a l o D i s -t r i c t were dead by 1967 (Dooling 1967). Wolfe et a l . (1968) r e c e n t l y found t h a t the r u s t was now present on a l l 8 d i s t r i c t s of the Ozark N a t i o n a l Forest, being observed i n 19% of the P. echi n a t a stands examined. In Canada there have been few recorded r e p o r t s o f extensive damage, although the r u s t has been reported as f a i r l y common throughout - 45 -the P r a i r i e Provinces. I n 1959 two area r e p o r t s of m o r t a l i t y were r e -corded i n P. con t o r t a stands: one i n young t r e e s near Robb, A l b e r t a (Thomas et a l . i960) , and the other i n the Teslin-Whitehorse area o f the Yukon w i t h 5 - 10$ of the s a p l i n g s i z e t r e e s i n f e c t e d (Molnar i960) . L a t e r , Baranyay and Stevenson (1964) reported 3$ of the l i v i n g t r e e s on seven 0.05-acre p l o t s i n 20 y e a r - o l d regeneration near Robb were i n f e c t e d by C. comandrae, the range between p l o t s v a r y i n g from 1.4 to 10.8$. I n eastern Canada, 22$ of the t r e e s i n a P. banksiana p l a n t a t i o n at Thunder Bay, Ontario, were i n f e c t e d (Dance and Lynn 1965), and more than 10$ of young P. banksiana i n a sm a l l area at Sa i n t Urbain, Quebec, were i n f e c t e d (Martineau and O u e l l e t t e 1966). Meinecke (1928), Horton (1955), and Mielke et a l . (1968) men-t i o n the value o f the r u s t as a b i o t i c t h i n n i n g agent causing m o r t a l i t y i n overstocked young pine stands. Peterson (1962a), however, s t a t e s that m o r t a l i t y caused by the r u s t "could seldom or never be w r i t t e n o f f as harmless or b e n e f i c i a l n a t u r a l t h i n n i n g " . He found t h a t the i n f e c t e d t r e e s were l a r g e r , and that suppressed t r e e s d i d not respond to r e l e a s e by the r u s t . He a l s o found t h a t over h a l f the i n f e c t e d l i v e lodgepole pine t r e e s had a lower v i g o r r a t i n g than they would have i f r u s t - f r e e . C h i l d s (1968) found t h a t t o p - k i l l i n g of P. ponderosa by the r u s t was not i n d i c a t i v e of h i g h - r i s k t r e e s , unless the remaining crown was s m a l l and of poor v i g o r . Nordin (195*0 reported the r u s t was more pr e v a l e n t i n pure than i n mixed stands i n A l b e r t a . Damage a p p r a i s a l surveys c a r r i e d out i n a s s o c i a t i o n w i t h the present s t u d i e s , i n d i c a t e d that 44$ o f t t h e 725 t r e e s on a 0.1 acre p l o t - k6 -i n a 20 y e a r - o l d P. banksiana stand south of Rae, N.W.T., were i n f e c t e d i n I965 w i t h an average of 1.6 i n f e c t i o n s per tree;.. By 1966, 7.9% of the i n f e c t e d t r e e s had died and many others had dead tops ( J . A. Baranyay, unpublished data I965, I966). Nearly a t h i r d o f the cankers noted i n I965 were i n a c t i v e i n 1966, but many new a c t i v e cankers were recorded. A 1966 survey on a 0.1 acre p l o t i n a 25 y e a r - o l d P. con t o r t a stand near Saskatchewan R i v e r Crossing, Banff N a t i o n a l Park, A l b e r t a , i n d i c a t e d 32.7% of the tre e s i n f e c t e d by the r u s t , Peridermium s t a l a c t i f o r m e A r t h . . & Kern, and 10% by C. comandrae ( J . A. Baranyay, unpublished data 1966). Over h a l f the C_. comandrae i n f e c t e d t r e e s were dead by 1968. A I92U p l a n t a t i o n of Pinus s y l v e s t r i s near Beaver Mines, A l b e r t a , was reported to have extensive rodent damage, some w i t h f r e s h chewing which l e a d me to suspect a r u s t i n f e c t i o n . A f t e r considerable search, one s t r i p of a c t i v e canker w i t h spores was found at the edge of a chewed area ( P o w e l l and Morf 1965). Of 50 t r e e s t a l l i e d a l l had rodent damage, suggesting r u s t i n f e c t i o n was once widespread throughout the p l a n t a t i o n . H e a v i l y i n -f e c t e d C. umbellata ssp. p a l l i d a grew w i t h i n a few f e e t i n an open meadow. About 500 cankers were measured and observed on trees v a r y i n g i n age from 3 to 125 years from 23 areas i n the Rocky Mountains and Foot-h i l l s regions of A l b e r t a . The average height of the center of the can-kers was l.k f e e t , w i t h the highest centered at 15 f e e t . The average length of the cankers was 1.2 f e e t , w i t h the longest 15 f e e t . In 1966 and 1967, kkO l i v e cankers were tagged (103 i n 1967), but by 1968 101 of of these were dead, of which only 13 were branch cankers. Canker m o r t a l i t y at one l o c a t i o n amounted to 82%, and at another 55%5 both occurred i n 10 - kl -to 20 y e a r - o l d stands. I n a regeneration area where few tre e s were over 5 years o l d , kk% of the t r e e s w i t h cankers died i n one year. T o t a l t r e e i n f e c t i o n i n t h i s area amounted to 23% on a 0.22 acre p l o t i n 19°75 but i t was u n l i k e l y t h a t any of the i n f e c t e d t r e e s would s u r v i v e f o r more than a few years at the current m o r t a l i t y r a t e . The reg e n e r a t i o n oc-curred i n an area of Comandra and chances f o r f u r t h e r i n f e c t i o n i n these young age tre e s were high. - hd -AREA OF STUDY LOCATION, PHYSIOGRAPHY AND GEOLOGY The main area of study was r e s t r i c t e d to a p o r t i o n of the Kananaskis R i v e r v a l l e y centered on the Kananaskis Forest Experiment S t a t i o n some 5 miles south of Seebe, A l b e r t a ( F i g . 38), where the f o o t -h i l l s merge w i t h the f r o n t range of the Rocky Mountains. The Kananaskis Forest Experiment S t a t i o n , an area of about 2k square m i l e s , i s bounded to the north by the Stoney In d i a n Reservation, and on a l l other sides borders on the Bow R i v e r F o r e s t , of the A l b e r t a Rocky Mountains Forest Reserve. The Kananaskis R i v e r , which flows north to j o i n the Bow R i v e r at Seebe, l i e s i n a north-south v a l l e y s i t u a t e d between two of the east-ern s e r i e s of ranges of the Rocky Mountains. In the northern p a r t of the v a l l e y the Kananaskis Range l i e s to the west and the F i s h e r Range to the east. The a l t i t u d e of the area v a r i e s between U,200 and 95 500 f e e t , w i t h timber l i n e between 6,500 and 7 , 0 0 0 f e e t . B a r r i e r Lake i s a man-made water body over four miles long, f i l l i n g a p o r t i o n o f the f l a t -bottomed U-shaped v a l l e y between the northern end of the Kananaskis Forest Experiment S t a t i o n and a p o i n t about a mile south o f the Evans -Thomas Creek. The v a l l e y bottom, which at some p o i n t s i s !§• miles wide, i s f l a n k e d at many places by te r r a c e s of g l a c i a l t i l l and morainic ma-t e r i a l , and l a c u s t r i n e and a l l u v i a l d e p o s i t s ; evidence of previous g l a c i a t i o n s and r e t r e a t s to which the area was subjected. From approxi-mately the 5,000 f o o t l e v e l the slopes r i s e g r a d u a l l y or p r e c i p i t o u s l y to masses of upthrust and f o l d e d sedimentary rocks deposited from F i g . 38- Map o f t h e l o w e r K a n a n a s k i s R i v e r V a l l e y , A l b e r t a , show-i n g t h e l o c a t i o n s o f t h e main s t u d y a r e a s . - k9 -- 50 -Cambrian to Cretaceous times, which form the mountains and ri d g e tops of the ranges. The g e o l o g i c a l formations, of the area were stud i e d by Bawling (1905) and Beach (19U3). SOILS A p r e l i m i n a r y survey of the s o i l s of the area was made by Crossl e y (1951), who found w e l l drained a l l u v i a l s o i l s and eroded s o i l and rock areas dominant i n the f l o o d p l a i n s of the Kananaskis R i v e r and i t s major east bank t r i b u t a r i e s . Shallow stomy/ l i t h o s o l s o i l s are pro-minent on moderate to steep slopes where e r o s i o n i s q u i t e r a p i d . The dominant s o i l s on the lower slopes of the v a l l e y are grey podzols of calcareous or non-calcareous o r i g i n , w i t h brown podzols and podzols gen-e r a l l y at higher e l e v a t i o n s . L o c a l i z e d areas support hangmoor peat, h a l f -bog and sod s o i l s , which r e s u l t from excessive moisture, poor drainage, or s o i l s at high a l t i t u d e s under grass or open f o r e s t . Brown f o r e s t s o i l s , and l o c a l i z e d chernozem and re n d z i n a s o i l s are found around the northern end of B a r r i e r Lake and to the north, where the F o o t h i l l s abut against the Rocky Mountains. A f o r e s t land c l a s s i f i c a t i o n map, based on s u r f i c i a l m a t e r i a l , was r e c e n t l y prepared f o r some of the Kananaskis Research Forest (Duffy and England I967), and a ge n e r a l i z e d s o i l s map was prepared f o r the Marmot Creek b a s i n ( J e f f r e y 1965). CLIMATE The main c l i m a t i c c h a r a c t e r i s t i c of the Kananaskis V a l l e y i s - 51 -i t s v a r i a b i l i t y , t y p i c a l of most mountain regions i n c o n t i n e n t a l l o c a t i o n s . This v a r i a b i l i t y i s only now becoming more a c c u r a t e l y determined through the increase i n the number of c l i m a t o l o g i c a l s t a t i o n s i n the v a l l e y , and e s p e c i a l l y through the i n t e n s i v e instrumentation a s s o c i a t e d w i t h the A l b e r t a Watershed Research Program i n the b a s i n of Marmot Creek (Munn and S t o r r 1967), and other s p e c i a l i z e d p r o j e c t s (MacHattie 1966, 1967). Kananaskis ( e l e v a t i o n k,560 f e e t MSL, l a t i t u d e 51°02'N, longitude 1 1 5 ° 0 3 ' W) i s the only s t a t i o n w i t h a r e l a t i v e l y long c l i m a t o l o g i c a l record upon which acceptable average conditions can be based, but even some of the parameters recorded at t h i s s t a t i o n are based on short term, and some-times i r r e g u l a r p e r i o d s . This s t a t i o n i s l o c a t e d 300 f e e t to the east of study l o c a t i o n 1 ( F i g s . 38 and 39)j on a k n o l l on the edge of a large grassed c l e a r i n g , open to the south and east, w i t h k-0 f o o t trees im-mediately to the north, thus the s i t e i s not p a r t i c u l a r l y t y p i c a l of t h i s f o r e s t e d r e g i o n . A l l other c l i m a t o l o g i c a l s t a t i o n s i n the lower Kananaskis v a l l e y have been e s t a b l i s h e d during the l a s t decade, and again records f o r some parameters a r e • i n t e r m i t t e n t , and i n the case of Pigeon Lookout ( e l e v . 6,000 f t MSL, l a t . 51°03'N, long. 1 1 5 ° 0 V w ) depend on the d u r a t i o n of the summer f o r e s t f i r e season. Tables I, I I and I I I , give the c l i m a t i c summaries f o r the m e t e o r o l o g i c a l parameters recorded at Kananaskis, Kananaskis Boundary Ranger S t a t i o n ( e l e v . k,800 f t MSL, l a t . 50°55'W, long. 115°08'W), Pigeon Lookout, and f o r a few s t a t i o n s i n the Marmot Creek Basin ( e l e v . 5,200 to 9,200 f t MSL, l a t . 5 0 ° 5 7 % long. 115°10'W). Data f o r Kananaskis, Kananaskis Boundary and Pigeon Lookout s t a t i o n s were e x t r a c t e d from the "Monthly Record", Canada Department of F i g . 39- Study l o c a t i o n 1, on the Kananaskis Forest Experiment S t a t i o n , near the northeast shore of B a r r i e r Lake, showing the l o c a t i o n of i n f e c t e d lodgepole pine, Comandra p l a n t p l o t s , and instruments used during the study. Table I. Monthly and annual climatic summaries for Kananaskis, lat. 51°02'N, long. 115 03'W, elev. 4,560 ft MSL, for the period of record (1939 - 1968). TEMPERATURE "F 1 JAN FEB MAR. APR MAY JUN JUL AUG SEP OCT NOV DEC YEAI Daily mean 15.2 2 0 . 4 2 4 . 8 34.3 45.0 51.4 57-6 55.7 48.7 4 0 . 9 27.2 21 .8 36. c Extreme maximum 59 61 64 75 82 88 93 92 86 80 66 64 93 Extreme minimum -50 -42 -41 -24 - 7 23 23 29 15 - 8 -32 -44 -50 1 PRECIPITATION inches Total 1.05 1.41 i . 4 o 2.49 3.06 4 . 0 8 2.49 2.78 2.28 1.44 1.15 1.17 24.7S Snowfall 10.24 14.07 12.89 20.18 7.48 1.53 0.00 0.16 0.34 8 .88 10.52 11.24 9 7 . 5 : SUNSHINE DURATION hours 2 Average 69 138 150 246 214 236 308 254 163 121 71 61 2031 Per cent of possible 26 41 59 45 48 62 57 43 37 27 25 43 Years of data 1 2 2 l 2 4 5 5 5 2 1 1 WIND SPEED mph3 Mean 6.8 6 . 8 5.9 5-9 5-3 5-4 5.0 4 . 8 5-1 7.2 7-2 7.7 6 .1 WIND DIRECTION FREQUENCY *3 C A co F i g . hO. Summary of the d a i l y maximum and minimum temperatures : and r e l a t i v e humidity taken from hygrothermograph records, the d a i l y r a i n f a l l , and the phenology, of the a e c i a l , py-c n i a l , u r e d i a l and t e l i a l s t a t e s of Cronartium comandrae, " May to August i n 1965 and 1966. - 69 -10 15 20 15 30 10 15 20 25 30 10 15 20 15 30 10 15 10 MAXIMUM 20 AND MINIMUM T E M P E R A T U R E °C -10 RELATIVE 2 4 HUMIDITY 16 NO.OF H O U R S 80 AND 100% * 0 2.0 1.5 RAINFALL INCHES 1.0 0.5 0 A E C I O S P O R E S P Y C N I O S P O R E S U R E D I O S P O R E S TEL IOSPORES K> 15 20 25 30 MAY 10 15 20 25 30 JUNE 10 15 20 25 30 JULY 5 10 15 20 A U G U S T 10 15 10 25 30 10 15 20 25 30 10 15 20 25 30 MAXIMUM j o AND MINIMUM T E M P E R A T U R E "> °C RELATIVE HUMIDITY 16 NO. OF H O U R S 80 AND 100% RAINFALL 1.0 INCHES AEC IOSPORES PYCNIOSPORES UREDIOSPORES TEL IOSPORES 10 13 20 25 30 MAY 10 15 20 25 30 JUNE 15 20 25 30 JULY 5 10 15 20 A U G U S T 80n HUMIDITY 100| % Light Moderate Heavy F i g . 1+1. Summary of the d a i l y maximum and minimum temperatures and r e l a t i v e humidity taken from hygrothermograph records, the d a i l y r a i n f a l l , and the phenology of the a e c i a l , py-c n i a l , u r e d i a l and t e l i a l s t a t e s of Cronartium comandrae, May to August i n 1967 and 1968. - 70 -10 15 20 25 30 10 IS 20 25 30 10 15 20 25 30 10 15 20 MAXIMUM 20 AND MINIMUM T E M P E R A T U R E 1 0 °C -10 24 r RELATIVE HUMIDITY 1 6 NO. OF H O U R S 8 80 AND 100% o 2.0 1.5 1.0 0.5 RAINFALL INCHES A E C I O S P O R E S P Y C N I O S P O R E S UREDIOSPORES TEL IOSPORES l_ MAXIMUM AND MINIMUM T E M P E R A T U R E °C RELATIVE HUMIDITY NO. OF H O U R S 8 80 AND 100% o RAINFALL INCHES A E C I O S P O R E S PYCNIOSPORES UREDIOSPORES TEL IOSPORES L_ 5 10 15 20 25 30 MAY 10 15 20 25 30 10 15 20 25 30 5 JUNE 10 15 20 25 30 10 15 20 25 30 JULY 10 15 20 25 30 1 1 5 10 15 20 A U G U S T 10 15 20 25 30 MAY 80n HUMIDITY , 100i % U g h , Moderate Heavy 10 15 20 25 30 JUNE 10 15 20 25 30 JULY NR = No Record 5 10 15 20 A U G U S T - 71 -f i r s t from cankers on the smaller branches, seedlings and young t r e e s , a l l having t h i n smooth bark, somewhat l a t e r on large branches w i t h t h i c k e r bark, and l a t e r s t i l l on stems of o l d e r t r e e s w i t h a t h i c k e r rough bark. This was probably because the developing a e c i a were deeper seated i n the t h i c k e r roughened bark than i n t h i n smoother bark, and because of the mechanical problems of r u p t u r i n g t h i c k bark. U s u a l l y the a e c i a l b l i s t e r s of t h i c k barked cankers erupted i n the bark f i s s u r e s , but removal of bark o f t e n exposed a large a e c i a l area underneath where spores remain unexposed f o r much of the season. Cankers on trees i n the open or on a south or east aspect of a t r e e a l s o began s p o r u l a t i o n s l i g h t l y e a r l i e r than those in a dense stand or l o c a t e d w i t h a dominantly west or north aspect. Even on trees which were n e a r l y g i r d l e d by the fungus, s p o r u l a t i o n began from pustules on the southeast or south aspect and was l a s t from those on the north. Cankers a few f e e t up the stem als o sporulated before b a s a l cankers s i t u a t e d i n the t h i c k e r bark zone of a t r e e . I n i t i a t i o n of s p o r u l a t i o n was about two to three weeks a f t e r evidence of shoot bud break on the lodgepole pine which u s u a l l y commenced i n l a t e A p r i l or e a r l y May. S i m i l a r l y , growth of Comandra p l a n t s began before aeciospore s p o r u l a t i o n . Growth amounted .to an i n c h or more of a e r i a l p l a n t development by May 12 i n 1965 and May 10 i n I966, and the Comandra p l a n t s were f l o w e r i n g by the l a s t week i n May i n favourable s i t e s . Development was l a t e r i n 1967 and 1968. I n 1963 and I96U Comandra p l a n t s were f i r s t noted as producing urediospores at l o c a t i o n 1 on June 18 and 28 r e s p e c t i v e l y . I n l a t e r years the incidence of i n f e c t i o n and u r e d i a l and t e l i a l development was recorded on Comandra p l a n t s on the four p l o t s at .-.location 1 ( F i g . 39) . Each p l o t having an average of 53 - 72 -a e r i a l shoots each year. These p l o t s were l a i d out w i t h i n 100 f e e t of i n f e c t e d t r e e s , and i n most cases were w i t h i n 1+0 f e e t . I n 1966 no i n -f e c t i o n s were.recorded on the p l a n t s u n t i l 39 days (on June 28) a f t e r aeciospore r e l e a s e from the adjacent t r e e s ( F i g . 1+0). A s i m i l a r long i n t e r v a l occurred i n 1965 ( F i g . 1+0), but i n I968 ( F i g . l+l) u r e d i a were present 12 days (on June 17) a f t e r the f i r s t aeciospores were recorded. E a r l y springs may advance and l a t e springs r e t a r d somewhat the advent of a e c i a l production. S p o r u l a t i o n was three weeks e a r l i e r i n 1966 than i n 1968. E a r l y May (May 1-15) was u n u s u a l l y warm i n 1966 there being twice the number of degree days above 0°C than i n the same p e r i o d i n 1967 and 1968, and one and a h a l f times the number i n 1965. During May 1966 the r e g i o n was i n f l u e n c e d by a prevalence of mP a i r , but i n 1967 and 1968, May was dominated by mA and cA a i r masses. Wo cA a i r was experienced i n May 1966", but t h i s a i r mass was present f o r a few days i n the f i r s t h a l f of May 1967 and 1968, b r i n g i n g w e l l below normal tem-peratures to the r e g i o n . May 1965 d i d not experience the c o l d cA a i r , as occurred i n 1967 and 1968, but was dominated by mA a i r during the month. The presence of warmer a i r masses, w i t h t h e i r corresponding warmer surface temperatures, could w e l l account f o r s p o r u l a t i o n being e a r l i e r i n 1966 than the other years, and~.'why s p o r u l a t i o n was delayed to a l e s s e r extent i n 1965 compared w i t h 1967 and 1968. A p r i l had a l s o been warmer i n I965 and 1966 than i n the l a t t e r two years. I n i t i a t i o n of s p o r u l a t i o n i n most years seemed to s t a r t on a day of, or a day f o l l o w i n g , r a i n , suggesting that moisture was r e q u i r e d to rupture the p e r i d i a . The end of the main spore production p e r i o d o f t e n coincided w i t h a heavy r a i n f a l l which washed the spores from the a e c i a . This was most n o t i c e a b l e i n 1965 and - 73 -I966. The warm dry weather of J u l y 1966 and 1967 tended to extend the spore production p e r i o d . There was a great range i n the d u r a t i o n of spore production from i n d i v i d u a l cankers. (Table V) and pustules (Table V i ) . P e r i o d of spore production on some tre e s was c o n s i s t e n t l y short, e.g. #2711, and on others long, e.g. #2713 and 2719. G e n e r a l l y production tended to be longest on the l a r g e s t t r e e s , s h o r t e s t on the sma l l e s t , w i t h the i n t e r -mediates i n between. As w e l l as v a r i a t i o n i n the spore d u r a t i o n from i n -d i v i d u a l cankers there was considerable range between i n d i v i d u a l p u s t u l e s on the same canker (Table V i ) . I n both years i n d i v i d u a l p u s t u l e s pro-duced spores f o r a shorter p e r i o d on #2715, & younger, t h i n suppressed t r e e , than on the o l d e r dominant t r e e , #2719- Tree #2717 was s i m i l a r to #2719, but #2710 was again a smaller diameter t r e e , and on average, sporulated f o r a shorter p e r i o d although the pustu l e s p o r u l a t i o n range was s i m i l a r . P u s t u l e s tended to sporulate f i r s t towards the center of a canker and l a s t towards the periphery. G e n e r a l l y pustules on one canker sporulated at about the same time, although on occasion there could be two waves of pustul e s p o r u l a t i o n , as occurred on t r e e #25l6 i n 1965 (Table V) which accounted f o r the two main periods of spore p r o d u c t i o n . Table V I I summarizes the sequence of development of i n d i v i d u a l pustules on two cankers i n I967, and shows how a large percentage of the pustules rupture w i t h i n a few days of each other and produce spores f o r very s i m i -l a r periods of time. - 7h -Table VI. Average number of days and range o f days of aeciospore production f o r i n d i v i d u a l p u s t u l e s on s e v e r a l cankers i n 1966 and 1967 at l o c a t i o n 1. Year Canker No. No. of pustules Spore Production followed Aver. no Range of days 1966 2715 8 21 17-38 2719 6 k6 21-64 1967 2715 20 3k 25-58 2719 9 60 43-75 2710 30 52 37-87 2717 3 60 53-71 Table V I I . Summary of sequence of s p o r u l a t i o n f o r 30 and 20 pu; t u l e s on . two cankers at l o c a t i o n 1 i n 1967. Canker Date State o f Aeciospore pustules No. Closed Ruptured S p o r u l a t i o n F i n i i Main Diminished 2710 May 31 13 2 15 0 0 June 1 6 4 20 0 0 June 3 4 1 25 -0 0; June 6 ,'0 5 25 0 0 June 8 0 1 29 0 0 June 9 0 0 30 0 0 June 23 0 0 Ik 16: 0 June 28 0 0 :o 30 0 J u l y 25 0 0 0 . 11 19 Aug. 9 0 0 0 6 24 2715 June 7 19 1 "0 0 0 June 8 12 7 1 0 0 June 9 9 10 1 . 0 0 June 12 4 14 2 0 0 June 16 2 6 12 0 0 June 17 0 3 17 0 0 June 22 0 0 20 0 0 J u l y 4 0 0 0 20 0 J u l y 25 0 0 0 k 16 - 75 -"V ' ' D i s c u s s i o n Annual production of the a e c i a may be short l i v e d on s m a l l seedlings, young trees and s m a l l diameter branches. Production may only l a s t f o r two or at the most, three years on t h i s s m a l l diameter stem or branch m a t e r i a l before the fungus e f f e c t i v e l y g i r d l e s and k i l l s the stem or branch. I f branch i n f e c t i o n s are s u f f i c i e n t l y close to the main stem the fungus w i l l grow i n t o the main stem and continue to pro-duce spores annually, although there i s o f t e n a year without a e c i a l production at time of t r a n s f e r from a k i l l e d branch to the stem. On large branches and stems w i t h t h i c k bark, a e c i a may be produced annually u n t i l t hat p o r t i o n beyond the canker i s k i l l e d . a s a r e s u l t of the g i r d -l i n g a c t i o n of the fungus. This may r e q u i r e many years and depends on the s i z e and s t a t e of the i n f e c t e d t r e e . K r e b i l l (1965) found t h a t the m a j o r i t y o f cankers of h i s study o r i g i n a t e d from 20 to 50 years ago, and t h a t a few were more than 100 years o l d . I n the present study some tree s under observation succumbed every year, but the p e r i o d of study was not long enough to f o l l o w f r e s h stem cankers from i n i t i a l stem i n -f e c t i o n to stem g i r d l i n g , although one branch to stem i n f e c t i o n had a l -most g i r d l e d a 1.4 i n c h d.b.h. stem i n three years. Evidence of annual rodent chewing on one 10.0 i n c h d.b.h. t r e e i n d i c a t e d t h a t the i n f e c t i o n had been present f o r at l e a s t 13 years and probably f o r 20 years. Dur-in g the p e r i o d of i n f e c t i o n there i s an annual increase i n canker s i z e , thus t h e o r e t i c a l l y , a greater volume o f aeciospores i s produced each succeeding season u n t i l death of the a f f e c t e d p a r t occurs. I n most cases, however, a e c i a l . p r oduction i s i n t e r f e r e d w i t h by the a c t i v i t y o f other f u n g i , i n s e c t s or rodents, at l e a s t i n some p o r t i o n of the canker, - 76 -and there i s o f t e n a r e d u c t i o n i n annual a e c i a l p r o d u c t i o n d e s p i t e the increased canker s i z e . The e f f e c t of these b i o l o g i c a l agents on produc-t i o n i s discussed l a t e r . Mielke (19^3) noted the c o r r e l a t i o n between s i z e of canker, age of host t i s s u e s and bark t h i c k n e s s , and aeciospore production f o r C. r i b i c o l a . Death o f stem or branch cankers i n any one year i n one l o c a l i t y w i l l g r e a t l y reduce the volume of aeciospores pro-duced the f o l l o w i n g year i n the same area. I n 1968, a dying t r e e pro-duced only two a e c i a l pustules whereas i n the three previous y e a r s " i t had produced abundant spores (#2713, Table V). I n 1968 i n f e c t i o n on the nearby Comandra p l a n t s was low and l a t e o c c u r r i n g , whereas i n other years i t appeared about 10 days a f t e r the f i r s t aeciospores were r e l e a s e d and was abundant. Widespread canker death may t h e r e f o r e be a f a c t o r con-t r i b u t i n g to the wavelike 'character of spread and i n t e n s i f i c a t i o n of the r u s t . K r e b i l l (1968c) has r e c e n t l y p u b l i s h e d i n f o r m a t i o n on the phenology o f C!. . comandrae i n the Rocky Mountain S t a t e s . Data from a number of p l o t s showed great v a r i a t i o n i n d u r a t i o n of aeciospore pro-duction, varying, from 3 to 17 weeks, but ae c i a were only abundant through June and J u l y . Development of a e c i a was e a r l i e s t at the low-est e l e v a t i o n s , and the d u r a t i o n of a e c i a was s h o r t e s t at the higher e l e v a t i o n s . Boyce (1916) reported t h a t CJ. comandrae a e c i a u s u a l l y commence s p o r u l a t i o n i n l a t e A p r i l or e a r l y May, and f i n i s h by l a t e June or e a r l y J u l y . Spaulding (1922) gives the d u r a t i o n of C_. r i b i c o l a aeciospore production i n d i f f e r e n t years f o r a number of areas i n east-ern North America. Duration i n most years l a s t e r f o r k8 to 90 days, although he gives extremes of 12 and lk-0 days. The 12 day p e r i o d appears - 77 -to be very short e s p e c i a l l y as he records closed b l i s t e r s appearing kl days e a r l i e r I The long aeciospore p e r i o d r e s u l t e d from l a t e a e c i a being noted i n mid-September at Amery, Wisconsin, p o s s i b l y a second p e r i o d of a e c i a l production as occurred at l o c a t i o n 2 of the present study i n I966. Pennington ( c i t e d i n Spaulding 1922) noted that more a e c i a were produced per canker i n 1919 than i n 1918 i n the Adirondacks, a l s o that there were more new s p o r u l a t i n g cankers. However, he b e l i e v e d that fewer aeciospores were set f r e e i n 1919 than i n 1918, from r e s u l t s of spore-trapping and observations on f i r s t generation u r e d i a . York ( c i t e d i n Spaulding 1922), working i n the White Mountains, found t h a t a e c i a matured f o r about 30 days on s i n g l e cankers, but may take longer on l a r g e r branch or stem cankers. Rhoads (1920) followed p r o d u c t i o n of 300 a e c i a on v a r i o u s t r e e s i n Maine from the time they f i r s t broke on May 3- He found t h a t by May 20, only 63 a e c i a were s t i l l s p o r u l a t i n g , by May 29 only 19 and none by June k. Others have reported periods of 20 to 30 days, and more than Ik days ( c i t e d i n Spaulding 1922) f o r aeciospores to be produced by an i n d i v i d u a l aecium. I n western North America, Lachmund (1933) reported t h a t C_. r i b i c o l a aeciospore p r o d u c t i o n at low e l e v a t i o n s may begin as e a r l y as l a t e February although more u s u a l dates occur i n l a t e March or e a r l y A p r i l . A t the higher e l e v a t i o n s s p o r u l a t i o n u s u a l l y begins by June. The main aeciospore production p e r i o d occurs between mid May and mid J u l y , and heavy spore production may extend i n t o August. Lachmund a l s o noted t h a t , r a t h e r f r e q u e n t l y at lower e l e v a t i o n s , a sporadic and l i g h t aeciospore production may occur i n October or November. The a e c i a were c h a r a c t e r i s t i c a l l y s m a l l , and t h i s second wave of aeciospore p r o d u c t i o n - 78 -occurred under the i n f l u e n c e of warm f a l l weather i n years having an e a r l y s p r i n g . He a l s o noted i t s occurrence on C. comptoniae and C_. filamentosum Pk. cankers (Lachmund 1929). Dates o f aeciospore produc-t i o n given by Mielke and Kimmey (1935) i n d i c a t e d t h a t the main aeciospore p e r i o d occurs from m i d - A p r i l to mid-June, but may be a month l a t e r at higher e l e v a t i o n s . Mielke (19*1-3) s t a t e d t h a t during warm and dry springs the main a e c i a l s p o r u l a t i o n may only l a s t two or three weeks, but during c o o l , wet springs may l a s t two or more months. Lachmund (1933) noted that t h i c k bark tended to r e t a r d a e c i a l production, and Mielke (19*1-3) f u r t h e r discusses t h i s f a c t o r i n r e l a t i o n to the d i f f e r e n t s o f t pine species t h a t C. r i b i c o l a i n f e c t s . Lachmund (1933) found t h a t f o r cankers t h a t produce p y c n i a f o r the f i r s t time i n a given year only 57$ produce a e c i a the f o l l o w i n g s p r i n g , the r e s t produce a e c i a i n the second year, although a few do not produce a e c i a u n t i l subsequent years. . Cankers a f f e c t e d by secondary organisms may not produce a e c i a at a l l . BIOLOGICAL FACTORS AFFECTING AECIOSPORE PRODUCTION ;[••.:) Methods Observations were made on the frequency and type of as s o c i a t e d f u n g i seen on the a e c i a l zone of cankers at d i f f e r e n t l o c a t i o n s . These f u n g i were i s o l a t e d from the canker, c u l t u r e d and i d e n t i f i e d . On one occasion pieces of a e c i a l canker were shaken i n d i s t i l l e d water f o r sev-e r a l hours, and the r e s u l t i n g s o l u t i o n streaked on agar p l a t e s . Micro-organisms which developed on the streaks were i s o l a t e d and c u l t u r e d . A large number of f u n g i , yeasts and b a c t e r i a were f r e q u e n t l y found as-s o c i a t e d w i t h f r e s h and stored spore c o l l e c t i o n s . These microorganisms - 79 -were allowed to e s t a b l i s h c o l o n i e s on t e s t agar media f o r a few days before being i s o l a t e d and grown i n pure c u l t u r e s f o r i d e n t i f i c a t i o n . Records were kept of the frequency of these a s s o c i a t e d microorganisms during the d a i l y germination t e s t s at l o c a t i o n 1 from 1965 to I967 i n an e f f o r t to e s t a b l i s h the importance o f the va r i o u s microorganisms. Tentative i d e n t i f i c a t i o n s were made ,;6f many of the i s o l a t e s of organisms f r e q u e n t l y encountered. Many!isolates, i n c l u d i n g the l e s s f r e q u e n t l y o c c u r r i n g , were i d e n t i f i e d or v e r i f i e d by taxonomic s p e c i a l i s t s . I n s e c t s were observed to be i n f e s t i n g the r u s t cankers i n most areas v i s i t e d , and i n many of these areas the frequency and amount of i n s e c t damage was noted. On a few occasions i n s e c t s were c o l l e c t e d d i r e c t l y from the a e c i a l canker surface and placed i n p r e s e r v a t i o n v i a l s . • To gain a b e t t e r i d e a of the number of i n s e c t s which used the r u s t can-ker as a h a b i t a t niche f o r a l l or p a r t of t h e i r l i f e c y c l e a c y l i n d r i c a l -sleeve i n s e c t cage was developed which could be pla c e d around the r u s t canker on the i n f e c t e d t r e e stem or branch ( E l l i o t t and P o w e l l 1966). The cage was constructed of p l a s t i c screening and cotton f a b r i c closed by an open-ended z i p p e r , the cage being attached to the t r e e by nylon draw-s t r i n g s at each end ( F i g . k-2). The drawstrings held the cage i n p o s i t i o n when the zip p e r was opened f o r removal of i n s e c t s , but to ensure t h a t a l l i n s e c t s were c o l l e c t e d , the cage was removed and a l l f o l d s of the cage m a t e r i a l examined. Ten to 12 cages were operated at l o c a t i o n 2 from 1965 to 1968, 8 to 10 cages at l o c a t i o n 3 from 1966 to 1968, and 1 or 2 cages near l o c a t i o n 1 f o r the same p e r i o d . The cages were v i s i t e d f o r i n s e c t c o l l e c t i o n at weekly i n t e r v a l s from mid May to October. Many of the cages were l e f t on the tre e s overwinter and were p r o t e c t e d by l / 2 - i n c h F i g . k2. A p l a s t i c s c r e e n i n g c y l i n d r i c a l - s l e e v e cage u s e d f o r c o l -l e c t i n g i n s e c t s f r o m C r o n a r t i u m comandrae c a n k e r s on stems o f P i n u s c o n t o r t a . - 8o -- 81 -wire mesh to prevent damage from bears or other mammals. I n many cases a cage was l e f t around a canker f o r two or more seasons, thus any i n -sects w i t h a long l i f e c y c l e would have been obtained. I n each year some new cankers were caged i n each area. In a d d i t i o n to the above c o l l e c t i o n methods, i n s e c t s were reared from i n f e c t e d cankers which were placed i n cardboard or metal r e a r i n g containers i n the i n s e c t a r y . These containers were u s u a l l y placed i n a c o o l area (about 2-5°C) f o r a p e r i o d of 3 to k months i n an e f f o r t to s a t i s f y any i n s e c t requirements o f a diapause p e r i o d . Each c o l l e c t i o n f o r r e a r i n g came from a s i n g l e canker, or as many as 20 can-kers. The c o l l e c t i o n s came from many l o c a t i o n s i n A l b e r t a , a few from the Northwest T e r r i t o r i e s , and in c l u d e d some C_. comandrae cankers on Pinus banksiana. G e n e r a l l y the l a r g e r c o l l e c t i o n s f o r r e a r i n g were made i n September or October, thus any i n s e c t s which overwintered i n the s o i l had u s u a l l y departed the environs of the canker by t h i s time. I t was o f t e n noted l a t e i n the season, t h a t a number of l a t e i n s t a r l a r v a e or pupae had c o l l e c t e d at the bottom of the cage, thwarted i n t h e i r e f f o r t s to crawl or drop to the s o i l surface. C o l l e c t e d or reared i n s e c t s were pinned, mounted or placed i n an appropriate amount of a l c o h o l f o r pre-s e r v a t i o n . Aeciospores t h a t had passed through ad u l t or immature i n s e c t s were checked f o r v i a b i l i t y by attempting to germinate spores obtained from f a e c a l p e l l e t s . Estimates were a l s o made of the number of spores i n the f a e c a l p e l l e t s from some species, and whether the f a e c a l p e l l e t s were composed e n t i r e l y of spores or p a r t l y of bark and other m a t e r i a l from the canker area. - 82 -O b s e r v a t i o n s were made on t h e f r e q u e n c y o f o l d and f r e s h r o d e n t chewing a s s o c i a t e d w i t h t h e r u s t c a n k e r a t about 20 l o c a t i o n s i n t h e y e a r s 1966 t o 1968 and a t a few l o c a t i o n s i n 196k and 1965. A t most l o c a t i o n s i n I967 and I968 t h e amount o f chewing and i t s e f f e c t on f u t u r e a e c i a l p r o d u c t i o n was n o t e d . The e f f e c t o f r e s i n f l o w on a e c i o -s p o r e p r o d u c t i o n was n o t e d . R e s u l t s M i c r o f l o r a A l l t h e i d e n t i f i e d m i c r o f l o r a l o r g a n i s m s t h a t were i s o l a t e d f r o m a e c i o s p o r e c o l l e c t i o n s o r d i r e c t l y f r o m t h e a e c i a l zone o f t h e ca n k e r a r e g i v e n i n A p p e n d i x I . The l i s t g i v e s a t o t a l o f 6k o r g a n i s m s , a l t h o u g h t h e number might be r e d u c e d i f or g a n i s m s o n l y i d e n t i f i e d t o genus p r o v e d t o be i d e n t i c a l w i t h o t h e r s i d e n t i f i e d t o s p e c i e s . The l i s t i n c l u d e s 8 b a c t e r i a and 56 f u n g i , i n c l u d i n g 5 y e a s t s o r y e a s t - l i k e f u n g i . The f u n g i and y e a s t s l a r g e l y b e l o n g t o t h e c l a s s D e u t e r o m y c e t e s (43) w i t h t h e l a r g e s t number b e l o n g i n g t o t h e f a m i l y M o n i l i a c e a e ( 2 6 ) . W i t h i n t h e M o n i l i a c e a e , 18 b e l o n g t o P e n i c i l l i u m and i t s c l o s e l y r e -l a t e d g e n e r a P a e c i l o m y c e s and S p i c a r i a . Many o f t h e s e o r g a n i s m s a r e p r o b a b l y o n l y a i r s p o r a c o n t a m i n a n t s a l t h o u g h , as such, t h e y may w e l l a f f e c t t h e v i a b i l i t y o f t h e a e c i o s p o r e s w i t h w h i c h t h e y come i n t o con-t a c t . S e v e r a l o f t h e s e o r g a n i s m s a r e common s o i l i n h a b i t i n g f u n g i , e.g. Mucor, B o t r y t i s , A s p e r g i l l u s , P e n i c i l l i u m and E p i c o c c u m . P o l y p o r u s a d u s t u s W i l l d . ex F r . i s known t o be a p r o m i n e n t p a t h o g e n on s p e c i e s o f P o p u l u s , b u t was i s o l a t e d f r o m a v e r y l a r g e number o f spo r e c o l l e c t i o n s a t l o c a t i o n 1 i n 1966. An u n i d e n t i f i e d s t e r i l e m y c e l i u m w i t h clamp con-n e c t i o n s was a l s o q u i t e common i n t h e same y e a r . - 83 -Two of the organisms were commonly found s p o r u l a t i n g on the a e c i a l zone o f the canker and played a l a r g e r o l e i n reducing aeciospore production and aeciospore v i a b i l i t y . They were the purp l e mold, Tu b e r c u l i n a maxima Rost. ( F i g . 4 3 ) , and an undescribed dark green species, Cladosporium tax. sp. 1. The l a t t e r fungus does not f i t any of the species described from r u s t s and has been given a taxonomic niche, number X L I I , i n the Commonwealth M y c o l o g i c a l I n s t i t u t e , Kew, England, by Dr. M. B. E l l i s (B. C. Sutton,personal communication 1966). Table V I I I summarizes the incidence of T. maxima and the num-ber of cankers observed during the years 1964 to 1968 at 3 to 23 l o c a t i o n s . Table V I I I . The recorded incidence of Tu b e r c u l i n a maxima on Cronartium comandrae cankers on lodgepole pine and the t o t a l number of a c t i v e and i n a c t i v e cankers observed at va r i o u s l o c a t i o n s i n southwest A l b e r t a during the years 1964 to 1968. Year No. of No. of T o t a l no. of Tu b e r c u l i n a Per cent l o c a t i o n s l o c a t i o n s a c t i v e and i n f e c t e d T u b e r c u l i n a observed w i t h no i n a c t i v e can- cankers i n f e c t e d T u b e r c u l i n a kers observed 1964 5 2 38 7 18.5 1965 3 0 26 5 19.2 I966 20 6 31+1 82 24 .0 1967 23 10 424 55 13.2 1968 22 9 313 42 13-4 The number of cankers f o r which observations were recorded v a r i e d each year depending on the number of l o c a t i o n s v i s i t e d and on canker m o r t a l i t y . The number of cankers observed at any one l o c a t i o n v a r i e d from 1 to 51 w i t h most l o c a t i o n s having 15 to 20 a c t i v e cankers i n the i n i t i a l year F i g . U-3. Rough, cracked bark of the a e c i a l zone of a Cronartium comandrae canker i n f e c t e d w i t h the purple mold, Tuber-c u l i n a maxima, which i s conspicuous as a darker area where the surface bark has been removed, or cracked. - 8k -- 85 -observations were recorded. On subsequent v i s i t s the same cankers were observed, but some had become i n a c t i v e through the a c t i o n of T u b e r c u l i n a or other agents.. T^ maxima was not observed at s i x of the l o c a t i o n s i n any year, and the numbers of cankers i n f e c t e d at other l o c a t i o n s v a r i e d from year to year. Examples of the v a r i a t i o n . i n d i f f e r e n t years i s given f o r 7 l o c a t i o n s i n Table IX. At A l t r u d e , V i c a r y and Marmot Creeks, and the area near Saskatchewan R i v e r Crossing, there was l e s s t r e e m o r t a l i t y r e s u l t i n g from canker i n f e c t i o n s , but considerable m o r t a l i t y was ob-served at the other l o c a t i o n s , e s p e c i a l l y at C l i n e R i v e r , where the stands were g e n e r a l l y s l i g h t l y younger. At C l i n e R i v e r the s i t e was p a r t i c u l a r l y dry and 25 trees succumbed during a two year p e r i o d . The number of T u b e r c u l i n a i n f e c t e d trees at A l t r u d e Creek and at the Wedge has remained r e l a t i v e l y constant, but the number has f l u c t u a t e d at the other l o c a t i o n s , w i t h a peak year i n 1966 at most l o c a t i o n s . On some cankers the T u b e r c u l i n a completely covered the a e c i a l zone and prevented any aeciospore production. On other cankers only s m a l l areas were covered and aeciospore production continued from the u n i n f e c t e d areas. Tuberculina d i d not appear to sporulate to any degree on the p y c n i a l zone, but was most abundant on the a e c i a l zone. T u b e r c u l i n a was pre-sent from the beginning of aeciospore production, although there was a marked increase i n the production of i t s purple spores as the season advanced. I n i t i a l l y i t appeared as a purple, v e l v e t y l a y e r , but soon a mass of spores were produced which continued w e l l past the normal a e c i o -spore s p o r u a l t i o n p e r i o d . No evidence could be found t h a t T u b e r c u l i n a was a hyperparasite of the r u s t , but i t was c e r t a i n l y a p a r a s i t e on the r u s t canker, not being found outside the r u s t zone on the t r e e . I t was - 86 -u s u a l l y r e s t r i c t e d to the current year's a e c i a l zone, or what would normally have been the a e c i a l zone i f a e c i a l p r oduction had not been a f f e c t e d , although on occasions i t spread i n t o o l d a e c i a l zones or i n t o the current p y c n i a l zone. Only current season a e c i a l or p y c n i a l cankers appeared to be s u s c e p t i b l e to T u b e r c u l i n a as no cankers-which had been i n a c t i v e the previous season were recorded w i t h Tuberculina. In 1966 and 1967 a general view was obtained t h a t the amount of a e c i a l zone covered by T u b e r c u l i n a increased as the season progressed. In I968 e i g h t cankers w i t h Tuberculina, i n i t i a l l y covering between 5 and 40% of the a e c i a l zone, were checked at weekly i n t e r v a l s and there appeared to be no appreciable increase i n the amount of the a e c i a l zone covered by Tuberculina. Although there may be o n l y a s l i g h t increase i n the Tuber- . c u l i n a covered area during the aeciospore production p e r i o d , there was g e n e r a l l y an increase i n the f o l l o w i n g season, and i n many cases no aeciospores were produced. When the p y c n i a l zone became i n f e c t e d there was no aeciospore production the f o l l o w i n g year, thus T u b e r c u l i n a was a favourable b i o l o g i c a l c o n t r o l of C. comandrae. Tub e r c u l i n a appeared to hasten the death of the r u s t i n f e c t e d bark. A t r e e p r a c t i c a l l y g i r d l e d s o l e l y by C. comandrae tended to l i v e and continue producing aeciospores f o r at l e a s t a year, but one g i r d l e d by C. comandrae and i n f e c t e d by T u b e r c u l i n a was soon k i l l e d . At the two l o c a t i o n s w i t h T u b e r c u l i n a ob-servations f o r f i v e years no t r e e was i n f e c t e d by T u b e r c u l i n a f o r more than three years, and at the l o c a t i o n s w i t h three years of data no t r e e was recorded w i t h T u b e r c u l i n a f o r more than two years. Generally, Tu-b e r c u l i n a i n f e c t e d the whole of the canker and the canker became i n a c t i v e , but o c c a s i o n a l l y one area of the canker escaped i n f e c t i o n and aeciospore Table IX. The incidence of Tu b e r c u l i n a maxima on observed a c t i v e and i n a c t i v e Cronartium comandrae cankers at 7 l o c a t i o n s during the years 196k to 1968. Year A l t r u d e Creek V i c a r y Creek M i s t Creek No. of cankers (A) No. i n f e c t e d (B) (A) (B) (A) (B 1964 5* 3 10 1 - -1965 2* 2 - - - -1966 7 5 23 9 20 k 1967 8 3 22 3 15 6 1968 5 0 21 0 11 l * Incomplete t a l l y Marmot Creek The Wedge C l i n e R i v e r Saskatchewan RiVer Crossing (A) (B) (A) (B) (A) (B) (A) (B -• - 12* 3 - - - -- - 10* 2 - - - -25 13 16 3 33 9 17 k 25 6 20 2 18 2 16 6 2k 5 15 ' 5 7 0 16 2 - 88 -production continued from t h i s area f o r a year or two. Table X shows some of the e f f e c t of Tuberculina and other f a c t o r s on continued ae c i o -spore production at two l o c a t i o n s . At both l o c a t i o n s there was a t r e n d f o r the number of cankers producing aeciospores to decrease and the num-ber of cankers becoming i n a c t i v e to in c r e a s e . Most of these cankers were i n a c t i v e as a r e s u l t of Tuberculina, although i n both areas a few cankers were i n a c t i v a t e d through rodent chewing. When the cankers w i t h aeciospore producing zones or zones i n f e c t e d w i t h T u b e r c u l i n a were tagged at Marmot Creek i n 1966,'.a number of other i n a c t i v e cankers were observed i n the stand but not tagged. None of these cankers became a c t i v e a f t e r I966 and evidence i n d i c a t e d that many were probably i n -a c t i v a t e d by Tuberculina. From the evidence of the present study the Table X. The number of Cronartium comandrae a e c i a l cankers ( l ) producing aeciospores, (2) s p o r u l a t i n g but i n f e c t e d w i t h Tuberculina, (3) w i t h i n a c t i v e a e c i a l zones i n f e c t e d w i t h Tuberculina, (4) w i t h i n a c t i v e or dead cankers, at two l o c a t i o n s during the years I966 to 1968. Year Cankers producing aeciospores.; 1966 19 1967 14 10 1966 14 1967 12 1968 4 A e c i a l zone A e c i a l zone a c t i v e w i t h i n a c t i v e w i t h Tu b e r c u l i n a T u b e r c u l i n a Marmot Creek 7 6 3 3 l 4 Saskatchewan R i v e r Crossing 2 1 3 4 2 2 0 Other i n a c t i v e cankers 0 Dead cankers 10 0 3 12 0 0 1 0 0 1 - 8 9 ' -b u i l d up and spread of T u b e r c u l i n a i n an i n f e c t e d stand i s slow, but once e s t a b l i s h e d i t i s e f f e c t i v e i n i n a c t i v a t i n g the canker and markedly reducing the aeciospore production. Germination t e s t s o f T u b e r c u l i n a spores gave average per cent germination of about 80$. V i a b i l i t y was maintained f o r a p e r i o d of at l e a s t a year. The r o l e of Cladosporium tax sp. 1. i n reducing aeciospore production i s not as c l e a r . G e n e r a l l y i t became obvious only on the a e c i a l zone towards the end of the aeciospore production p e r i o d , although evidence of Cladosporium i n f e c t i o n could be observed at the beginning of the aeciospore s p o r u l a t i o n p e r i o d on the o l d a e c i a l zones of the canker. The fungus was t y p i c a l l y shades of dark green although at times, i t appeared black-green. I t sporulated abundantly i n the l a t t e r h a l f of the summer and covered both the a e c i a l and p y c n i a l zones of the canker. Some evidence was obtained t h a t i t p a r a s i t i z e d the r u s t a e c i o -spores, and i t was an extremely common i s o l a t e from spore c o l l e c t i o n s where i t s : mycelium was found throughout many c o l l e c t i o n s . When present i n stored spore c o l l e c t i o n s , the v i a b i l i t y of the aeciospores was u s u a l l y reduced to zero. The incidence of Cladosporium i s shown i n Table XI f o r the years 1965 to 1968, and was g e n e r a l l y lower than f o r Tu b e r c u l i n a at most l o c a t i o n s . The percentage f o r 1965 was obtained from a very s m a l l l o c a t i o n sample, a n d ' i f percentages from the same three l o c a t i o n s were only considered i n the other years percentages of 19 .0 , 23 .4 and 15.8 were obtained, values much higher than those f o r the l a r g e r l o c a t i o n sample. Nearly h a l f (46$) of the Cladosporium i n f e c t i o n s occurred on the same cankers as T u b e r c u l i n a was observed, but due to the very low incidence i n 1968, Cladosporium was only recorded from two - 90 -Table X I . The recorded incidence of Cladosporium tax. sp. 1 on Cron-artium comandrae cankers observed at va r i o u s l o c a t i o n s i n southwest A l b e r t a during the years I965 to 1968. Year 1965 1966 1967 1968 No. of l o c a t i o n s observed 3 21 23 21 No. of l o c a t i o n s w i t h no Cladosporium 1 7 10 16 T o t a l no. of cankers observed 26 341 424 313 Cladosporium i n f e c t e d cankers 47 40 .14 Per cent Cladosporium i n f e c t e d 3 0 . 8 13-8 9-5 4 . 5 l o c a t i o n s i n a l l three years. The low incidence of 1968 may have been due to most of the observations being made by another observer and a l s o be-cause many l o c a t i o n s were v i s i t e d at a s l i g h t l y e a r l i e r date than i n other years. G e n e r a l l y a higher percentage of the Cladosporium i n f e c t i o n s was a s s o c i a t e d w i t h a c t i v e s p o r u l a t i n g a e c i a l zones than occurred w i t h T u b e r c u l i n a and only a few occurred on non-sporulating cankers which didn' t a l s o have Tuberculina. Cladosporium was recorded on a few tre e s f o r four years and on many, aeciospores were produced f o l l o w i n g two or even three years of Cladosporium i n f e c t i o n , thus Cladosporium d i d not appear to act as a b i o l o g i c a l c o n t r o l agent of the r u s t i n the same way as Tuberculina. Despite the low annual incidence of Cladosporium, 80$ of the cankers at Marmot Creek were i n f e c t e d by Cladosporium during the three yecar:. r e c o r d i n g p e r i o d , and 68$ of the cankers at The Wedge during a four year p e r i o d , Cladosporium was not observed at f i v e l o c a t i o n s i n any year, and at a f u r t h e r f i v e l o c a t i o n s o n ly one canker was observed i n f e c t e d i n three years of observing. Other organisms observed i n f e c t i n g the a c t i v e zone of the - 91 -a e c i a l canker were white m y c e l i a l growths, which were l a t e r i d e n t i f i e d as Fusarium sp., MonociIlium sp., Coniothyrium olivaceum Bonard., and Sclerophoma p i t y o p h i l a (Cda.) Htihn. Their incidence i n any year was very low, l e s s than 1% i n the years I966 to 1968. At one l o c a t i o n they were present on 9% of the cankers i n 1967, DUT-- were not observed at t h i s l o c a t i o n i n 1968. P e n i c i l l i u m spp., notably P. tardum Thorn, P. god l e w s k i i Z a l e s k i , P. rugulosum Thorn, P. funiculosum Thorn s e r i e s , P. brevi-compactum Dierckx. and P. nr. r o l f s i i Thorn, were a l l i s o l a t e d on one or s e v e r a l occasions from the a e c i a l zone, but a l l were probably i n c i d e n t a l and not p a r a s i t i c . A l t e r n a r i a t e n u i s auct. and Epicoccum nigrum Link were al s o o c c a s i o n a l l y i s o l a t e d from the current year a e c i a l zone, along w i t h Sporobolomyces sp., Rhodotorula spp., and b a c t e r i a belonging to Ar t h r o b a c t e r sp., Pseudomonas and a number of u n i d e n t i f i e d species. A number of secondary organisms, not p a r a s i t i c on the r u s t , were found to f r u i t on non-current year a e c i a l zones of o l d cankers, such as L a c h n e l l u l a a r i d a ( P h i l l . ) Dennis, but they were only observed i n f r e q u e n t l y on C. comandrae, being more p l e n t i f u l on P. s t a l a c t i f o r m e can-kers . Microfauna Insect damage was widespread on C. comandrae cankers. Table X I I records the incidence of obvious i n s e c t damage on the cankers ob-served during the years 1964 to 1968 at 3 to 22 l o c a t i o n s . Data f o r 1964 and 1965 are based on only a s m a l l sample of observed cankers and are t h e r e f o r e l e s s r e l i a b l e , although the percentages are close to the range of the other years. Insect damage was recorded at each l o c a t i o n on at l e a s t one occasion. At four l o c a t i o n s more than 60% of the cankers - 92 -Table X I I . The incidence of i n s e c t damage on Cronartium comandrae cankers on lodgepole pine and the number of cankers ob-served at various l o c a t i o n s i n southwest A l b e r t a during the years 1964 to 1968. Year Wo. of l o c a t i o n s observed No. of l o c a t i o n s w i t h no damage T o t a l no. of cankers observed Cankers w i t h i n s e c t damage Per cent • i n s e c t damage 1964 4 2 47 15 31.9 1965 3 1 22 13 59-1 1966 20 4 299 137 4 5 . 8 1967 22 2 4o4 154 3 8 . 8 I968 22 5 317 132 ' 41 .6 had some evidence of damage i n each of the years I966 to 1968, and at one l o c a t i o n over 88% were damaged each year. Much of the f l u c t u a t i o n i n incidence was r e l a t e d to as s o c i a t e d rodent damage, as w i t h the r e -moval of i n f e c t e d bark there was l i t t l e s u i t a b l e h a b i t a t remaining f o r the i n s e c t s . Table X I I records only the obvious i n s e c t damage, o f t e n recorded on only one annual v i s i t to a canker, and must be considered an underestimate of the incidence of damage. Often signs cf i n s e c t damage were not v i s i b l e on the canker surface f o r aeciospores may cover the b o r i n g holes, o r i n s e c t s may mine t h e i r way i n t o the canker through bark c r e v i c e s . Insects may al s o have been present i n the a e c i a and were not v i s i b l e without i n v e s t i g a t i o n , t h i s was e s p e c i a l l y so of young l a r v a l i n s t a r s which only became v i s i b l e at a l a t e r stage. Some lar v a e a l s o took on the c o l o r of the spores when feeding on them and even a t r a i n e d eye could e a s i l y miss them i n a mass of aeciospores. Insect-damage took s e v e r a l forms, o f t e n the r u s t i n f e c t e d bark was h e a v i l y chewed and there was much b o r i n g w i t h i t s a s s o c i a t e d f r a s s ( F i g . 4 4 ) . F i g . kk. B a s a l canker of Cronartium comandrae w i t h t y p i c a l rough bark i n a e c i a l zone and showing evidence of i n s e c t damage. Note e x i t holes and Lepidoptera f r a s s at top of canker, and f u r t h e r f r a s s i n lower rough zone. F i g . 45. Pupal chambers of Pissodes schwarzi scored i n t o the sap-wood throughout the Cronartium comandrae canker area on sm a l l Pinus c o n t o r t a stem. - 93 -- 9*+ -At the time of a e c i a l s p o r u l a t i o n the i n s e c t s , both larvae and adu l t forms, fed e x t e n s i v e l y on the aeciospores, reducing the number a v a i l a b l e f o r d i s p e r s a l . They s i m i l a r l y fed on the pycniospores and the p y c n i a l drops which have a high concentration of sugars. Often the aeciospores became matted together and gave a mealy, bleached appearance, having l o s t t h e i r v i a b i l i t y ' . Much of the canker became a mass of i n s e c t f a e c a l p e l l e t s composed almost e x c l u s i v e l y of aeciospores, or w i t h cer-t a i n species of i n s e c t s , a mixture of i n f e c t e d bark and spores. The phloem i n f e c t e d t i s s u e o f t e n became a mass of feeding g a l l e r i e s and pupal chambers ( F i g . 45). The i n s e c t f r a s s o f t e n hung together on the surface of the canker as large aggregates h e l d together by i n s e c t s i l k . The d i f f e r e n t forms of i n s e c t damage were l a r g e l y d i c t a t e d by the species of i n s e c t i n h a b i t i n g the canker. In the s p r i n g and e a r l y summer the i n s e c t s occurred mainly i n the current a e c i a l zone and past a e c i a l zones towards the center of the canker. As the season advanced the l a r v a l forms and v i s i t i n g a d u lts were a t t r a c t e d more to the s p o r u l a t i n g p y c n i a l zone. By l a t e summer and e a r l y f a l l the whole p y c n i a l zone was h e a v i l y damaged. Many, i n s e c t s overwintered i n the canker, thus i n s e c t s were o f t e n present i n an a e c i a l p u s t u l e before the peridium of the pustu l e broke open. On some occasions cankers were so h e a v i l y i n f e s t e d w i t h larvae that very few aeciospores were dispersed from the canker d e s p i t e being produced i n great abundance. Many of these aeciospores were eaten by l a r v a e , but many others were aggregated together and held by f i n e s i l k threads. A l l the i d e n t i f i e d microfauna — i n s e c t s , mites and s p i d e r s , t h a t were c o l l e c t e d from the exposed surface of the canker or were reared from the i n f e c t e d cankers are given i n Appendix I I . The l i s t contains a - 95 -t o t a l of 117 species. A few of the organisms o n l y i d e n t i f i e d to genus, many being immatures or of the nondiagnostic sex, may be i d e n t i c a l w i t h others i d e n t i f i e d to species. The l i s t i n c l u d e s 98 i n s e c t s , 17 mites and 2 s p i d e r s . The most important insect:, orders represented were the Coleoptera w i t h 25 species belonging to 12 f a m i l e s , the D i p t e r a w i t h 17 species i n 9 f a m i l i e s , the Hymenoptera w i t h 2k species i n 7 f a m i l i e s , the Lepidoptera w i t h 10 species i n k f a m i l i e s , and the Collembola w i t h 8 species i n 2 f a m i l i e s . The number of specimens of each species c o l l e c t e d or reared from the cankers i s recorded i n Appendix I I . In many cases not a l l specimens of a species were removed from a canker, a l s o on numerous occasions the more common or f r e q u e n t l y o c c u r r i n g species were not c o l l e c t e d from a canker. This group of common species are u s u a l l y i n d i c a t e d i n the l i s t by a p l u s (+) f o l l o w i n g the number, which i n d i c a t e s t h a t they were f a r more common than the number of specimens c o l l e c t e d would i n d i c a t e . Mycetocoles, or animals a s s o c i a t e d w i t h f u n g i , have.been c l a s s i f i e d i n three main categories: mycetobionts, mycetophiles and mycetoxenes (Benick 1952). Animals which cannot complete t h e i r develop-ment without u t i l i z i n g the fungus as food are known as mycetobionts. Mycetophiles are those organisms t h a t are not a b s o l u t e l y dependent upon f u n g i f o r development, w h i l e organisms that are simply chance v i s i t o r s or users of the fungus are termed mycetoxenes. Many of the species c o l l e c t e d from the canker i n the present study are probably mycetoxenes — u s i n g the canker as s h e l t e r or food, or seeking out species upon which they feed which happen to be u s i n g the canker. Many of the Hymenoptera probably f a l l i n t o t h i s l a s t category as they are l a r g e l y - 96 -p a r a s i t i c on other i n s e c t s . A few of the species c o l l e c t e d appear to be true mycetobionts — the Coleoptera, Epuraea obliquus Hatch, and two d i p t e r a , an u n i d e n t i f i e d Cecidomyiidae and Paracacoxenus guttatus Hardy and Wheeler. These three were very important i n reducing the production of aeciospores and w i l l be discussed i n some d e t a i l . Among the others c o l l e c t e d there appeared to be a number of mycetophiles which were u t i l i -z i n g the fungus but known more commonly to develop i n other h a b i t a t s . The most commonly observed species on the cankers were the o r i b a t i d mite, Ceratozetes sp., the f l a t brown n i t i d u l i d b e e t l e , Epuraea obliquus i n the l a r v a l and adu l t stages, v a r i o u s d i p t e r a l a r v a e , i n c l u d -i n g Paracacoxenus guttatus and an u n i d e n t i f i e d Cecidomyiidae, and larvae of a number of Lepidoptera. Epuraea obliquus was prominent on the canker throughout the a e c i a l and p y c n i a l s p o r u l a t i o n p e r i o d . A d u l t b e e t l e s were observed i n unruptured p u s t u l e s , and on two occasions a b e e t l e was observed chewing through the peridium and e f f e c t i v e l y r u p t u r i n g the p u s t u l e . Evidence from r e a r i n g s and from f i n d i n g specimens at the bottom of cages i n d i -cated that the larvae probably pupate and overwinter i n the s o i l or d u f f l a y e r , from which the adu l t seeks out the canker on emerging i n the s p r i n g . Some may pupate i n the canker zone, but b e e t l e s were only reared once d i r e c t l y from l o g s . A d u l t b e e t l e s were f a i r l y common from' mid May u n t i l August, and a few were present on the cankers through to September" or l a t e r ( l a t e s t c o l l e c t e d October 17, 1966). Ge n e r a l l y from one to six'were observed on an i n d i v i d u a l canker. Epuraea were observed on 80$ of the cankers at l o c a t i o n 2 i n 1965, and at l e a s t 50$ of the cankers i n 1966 and 1968. A s i m i l a r high incidence was recorded at - 97 -l o c a t i o n 3 i n the years 1966 to 1968. The f i r s t l a r v a e were found from mid June onwards, and some were c o l l e c t e d as l a t e as October 9 i n 1968, although i n other years few were observed a f t e r August. The adults and l a r v a e became covered w i t h aeciospores; the average number of spores on the body of 8 b e e t l e s was 580, and on 3 l a r v a e was 1198. A t the time of aeciospore production the Epuraea e x i s t e d e n t i r e l y on spores. A number of f a e c a l p e l l e t s was examined from b o t h adults and la r v a e , and only spores and p e r i d i a l c e l l s were present. The number of spores i n l 6 p e l l e t s was counted from adult b e e t l e s and gave an average of 586 spores, very few of which r e t a i n e d t h e i r form. These spores were checked f o r v i a b i l i t y but none germinated. The number of a e c i o -spores made i n v i a b l e through being eaten by the Epuraea b e e t l e s and l a r v a e •was enormous. Counts of adu l t Epuraea p e l l e t s from C_. comptoniae, which has a smaller aeciospore, gave lk^8 spores per p e l l e t . The l a r v a e fed e x t e n s i v e l y on the p y c n i a l drops c o n t a i n i n g the pycniospores, and were observed crawling from one drop to another. G e n e r a l l y the la r v a e oc-curred i n l a r g e r numbers on an i n d i v i d u a l canker than the a d u l t s , but r a r e l y more than 12 were counted. Epuraea a l s o fed e x t e n s i v e l y on Tuber-c u l i n a and to a l e s s e r extent on Cladosporium. The la r v a e and b e e t l e s o f t e n c a r r i e d a l a r g e number of spores on t h e i r bodies, which made them appear purple, and probably a s s i s t e d i n d i s p e r s i n g the spores over the canker zone to areas not i n f e c t e d by Tuberculina. I n a l l thar- t r a v e l s , the l a r v a e tended to leave slime t r a i l s to which aeciospores became a t -tached. Other spores became attached to f a e c a l p e l l e t s , and these attached spores were e f f e c t i v e l y prevented from being dispersed. On one occasion three b e e t l e s were allowed to crawl over a malt agar p l a t e to a s c e r t a i n what spores they might be c a r r y i n g . On t h i s occasion c o l o n i e s of - 98 -Cladosporium herbarum and Cladosporium tax. sp. 1 developed. Epuraea and other i n s e c t s no doubt act as c a r r i e r s o f many f u n g i and b a c t e r i a , and may w e l l be r e s p o n s i b l e f o r a number of s o i l organisms being present among the aeciospores. Epuraea obliquus were c o l l e c t e d or reared from s i x d i f f e r e n t l o c a t i o n s (#1, 2, 3, 5, 7 and 9) i n the study area on C. comandrae, and were reared from two canker c o l l e c t i o n s on P. banksiana, one from a p o i n t 33 miles southwest of Rae, N.W.T., and the other from Twatinaw i n northern A l b e r t a . I t was probably much more widespread than the c o l l e c t i o n s would i n d i c a t e , f o r i t was c o l l e c t e d on a number of other stem r u s t s i n the r e g i o n . A d d i t i o n a l c o l l e c t i o n data i s given f o r some of my E. obliquus c o l l e c t i o n s on pine stem r u s t s i n the paper by Parsons (1967). D i p t e r a larvae were commonly observed e a t i n g among the a e c i o -spores and often-caused the aeciospores t o become-aggregated together i n a mass o f f i n e s i l k and gave a mealy bleached appearance to the spores. These la r v a e were o f t e n present i n very large numbers on an i n d i v i d u a l canker and as many as f i f t y were counted. Between 25 and 70$ of the cankers at l o c a t i o n s 2 and 3 were observed w i t h dipterous larvae i n the various years. An average of 253 aeciospores were counted attached to the bodies of two young l a r v a e . The larvae took on the c h a r a c t e r i s t i c orange-yellow c o l o r of the spores. I n f f a c t , s i m i l a r dipterous larvae feeding on the white form of P. s t a l a c t i f o r m e (Powell 1966), were a l l white i n c o l o r . These dipterous larvae belong to at l e a s t two species and probably more, although the larvae of only two species were con-s i s t e n t l y c o l l e c t e d . One i s an u n i d e n t i f i e d Cecidomyiidae which u s u a l l y occurred i n l a r g e r numbers. Cecidomyiidae larvae on C. comandrae cankers - 99 -were c o l l e c t e d from l o c a t i o n s 1, 2, 3, and A l t r u d e Creek, B a r i l Creek and Saskatchewan R i v e r Crossing. Dr. J . R. Vockeroth (personal com-munication 1966) considered the larvae c o l l e c t e d i n large numbers from the u r e d i a l and t e l i a l s t ates of C. comandrae on Comandra p l a n t s , i n widel y s c a t t e r e d l o c a l i t i e s , and from a l t e r n a t e host p l a n t s of P. s t a l a c t i f o r m e to be the same species. U n f o r t u n a t e l y , only one Cecido-myiidae a d u l t f l y was c o l l e c t e d from the cankers during the study p e r i o d and could only be i d e n t i f i e d to genus, belonging to the t r i b e L e s t r e m i i n i . This was obtained from a canker cage i n June. Larvae of one Cecidomyiidae c o l l e c t i o n were i d e n t i f i e d as belonging to sub-family Lestremiinae. Cecidomyiidae larvae were f i r s t c o l l e c t e d i n the l a t t e r h a l f of June on C. comandrae cankers, and an odd one could s t i l l be found i n September and October. The other common dipterous l a r v a e were t e n t a t i v e l y i d e n t i -f i e d i n I965 as Dros o p h i l i d a e or Lauxaniidae, but were l a t e r reared to adult and i d e n t i f i e d as the d r o s o p h i l i d Paracacoxenus guttatus (McAlpine I968), a d u l t s of which had been reared or c o l l e c t e d e a r l i e r . This was the f i r s t i n f o r m a t i o n on the h a b i t a t of t h i s species and on the l a r v a l and pupal stages. Paracacoxenus had only been reported once p r e v i o u s l y from sweepings over muddy ground along streams or lakes i n a f o r e s t e d area. P. guttatus were c o l l e c t e d from cankers at l o c a t i o n s 1, 2, 3 and 5 i n the study area, and adults were c o l l e c t e d when v i s i t i n g the p y c n i a l drops of exposed cankers. A d d i t i o n a l data i s given f o r some of my Paracacoxenus c o l l e c t i o n s i n the paper by McAlpine (1968). The Paracaco-xenus l a r v a e , l i k e the Cecidomyiidae l a r v a e , fed e x t e n s i v e l y on the aeci o -spores and p y c n i a l drops. Adults were taken i n d i f f e r e n t years between June lk and October 17, but larvae were not observed u n t i l mid J u l y i n - 100 -most years. Specimens of another important d i p t e r a genus, which l a r g e l y occurred i n overwinter r e a r i n g s , belonged to one or more species of Bradysia. These emerged over a long p e r i o d of time i n the i n s e c t a r y and the larvae presumably fed e x t e n s i v e l y on the i n f e c t e d bark, although they were not observed doing so i n the f i e l d . S e v e r a l adult S c i a r i d a e , a l l of which were i d e n t i f i e d as Bradysia. were c o l l e c t e d i n the f i e l d . A S t a p h y l i n i d a e b e e t l e , of the genus Atheta was al s o very prominent i n r e a r i n g s , but was not observed i n the f i e l d . Coleoptera that were observed i n the f i e l d , other than the Epuraea, and were ap-p a r e n t l y feeding i n the i n f e c t e d canker t i s s u e s and p o s s i b l y on the spores, belonged to some of the f o l l o w i n g genera: Fissodes, C y l i n d r o -copturus, C o r t i c a r i a , Melanopthalma and Ernobius. The Cu r c u l i o n i d a e , Pissodes schwarzi Hopkins and Cylindrocopturus d e l e o n i Buchanan were observed feeding on t h e ' a e c i a l zone o f the canker, and t h e i r feeding g a l l e r i e s and pupal chambers ( F i g . h^) could be found i n the i n f e c t e d r u s t t i s s u e s . These weevils probably hasten the death of the t r e e as t h e i r l a r v a l g a l l e r i e s u s u a l l y scored the outer sapwood. P. schwarzi and C. d e l e o n i were c o l l e c t e d or reared from m a t e r i a l from l o c a t i o n s 1, 3, 5, and from C l i n e R i v e r , Saskatchewan R i v e r Crossing, and an area 3 mies northeast of Robb, w h i l e P. schwarzi was a d d i t i o n a l l y c o l l e c t e d from the Yukon, and C. d e l e o n i from l o c a t i o n 2. The a d u l t C o r t i c a r i a , -Melanopthalma and Ernobius observed i n the f i e l d , were a l l c o l l e c t e d between l a t e May and the end of J u l y at l o c a t i o n s 1, 2 and 3. Three genera of Lepidoptera were f r e q u e n t l y encountered i n the canker, t h e i r damage was c h a r a c t e r i z e d by aggregates of f r a s s at the entrances to t h e i r feeding g a l l e r i e s and over t h e i r pupal chambers - 101 -which were o f t e n found i n the f i s s u r e s of the rough c e n t r a l p o r t i o n of the canker. The great e s t damage was done by the l a r g e r larvae of D i o r y c t r i a zimmermani Grt., or specimens i d e n t i f i e d o n ly as D i o r y c t r i a sp., which could o f t e n be seen momentarily on the surface of the canker, and which pupated i n a s i l k - l i n e d chamber i n the i n f e c t e d t i s s u e . These larvae mine e x t e n s i v e l y i n t o the phloem t i s s u e and destroy l a r g e areas of the a e c i a l and p y c n i a l zone o f the canker. D i o r y c t r i a were c o l l e c t e d or reared from l o c a t i o n s 1, 2, 3 and 55 Saskatchewan R i v e r Crossing and C l i n e R i v e r , as w e l l as a number of other p o i n t s i n A l b e r t a , the North-west T e r r i t o r i e s , and the Cypress H i l l s , Saskatchewan. The larvae of the L a s p r e y r e s i a and Coleotechnites (Recurvaria) were much smaller, but s t i l l caused some damage i n the current or o l d a e c i a l zone. On occasion an empty cocoon could, be found s t i c k i n g out from the canker surface, or fromtthe a s s o c i a t e d r e s i n c r u s t . -The o r b a t i d mite, Ceratozetes sp., was present at s e v e r a l l o -cations i n the study area -and could be found on most i n f e c t e d t r e e s . However, i t was not r e s t r i c t e d to the canker area but could be found on many areas of the stem and apparently l i v e d i n the s o i l or d u f f l a y e r . Large numbers were o f t e n found i n the a e c i a l zone of the canker from the beginning of spore production, but were encountered l e s s o f t e n as aeci o -spore s p o r u l a t i o n ended. A few of the other mites could have been phyto-phagous, but most are probably 'classed as mycetoxenes. The Eupodidae and Tydeid mites taken from aeciospore c o l l e c t i o n s were approximately the same c o l o r as the spores, and were probably, feeding on them. Aphids of the genus Cin a r a were observed covering the whole a e c i a l and p y c n i a l zone of a few cankers, and were attended by species - 102 -of ant belonging to Lasius and Camponatus. The aphids f ed e x t e n s i v e l y on the aeciospores and l a t e r on the p y c n i a l drops, they probably a l s o ob t a i n n u t r i e n t s from the i n f e c t e d bark t i s s u e s . I n one case the ants associated w i t h the Cih a r a had b u i l t up e a r t h to enclose the canker and aphids, e f f e c t i v e l y preventing aeciospore d i s p e r s a l . The aphids were of t e n found sucking the a e c i a l area o f the canker and had worked i n un-der the bark. Of the other orders of insects, some of the Collembola and Thysanoptera probably scavenged on the a e c i a l zone of the canker, and ate spores. The Hymenoptera, excluding the Formicidae, were l a r g e l y p a r a s i t i c on Coleoptera, Lepidoptera or D i p t e r a species i n h a b i t i n g the canker and t h e r e f o r e were i n c i d e n t a l . A l l others given i n the l i s t may a l s o f a l l i n t o t h i s i n c i d e n t a l or mycetoxenes category, and were c e r t a i n l y not mycetobionts and probably not mycetophiles. Rodents Rodent damage was widespread at most l o c a t i o n s . The Richardson red s q u i r r e l , Tamiasciurus hudsonicus r i c h a r d s o n i (Bachman), B r i t i s h Columbia v a r y i n g hare, Lepus americanus columbiensis Rhoads, and the dusky porcupine, E r e t h i z o n dorsat um nigrescens A l l e n , were a l l observed chewing on the a e c i a bearing bark at one time or another, and judging from t e e t h marks, chipmunks and mice were a l s o r e s p o n s i b l e f o r removing some bark. Rodents g e n e r a l l y removed a l l the bark down to the sapwood, but u s u a l l y r e s t r i c t e d t h e i r a c t i v i t y , except i n the case of porcupines, to the i n f e c t e d bark. They showed a preference f o r the p y c n i a l and a e c i a l zones, but o f t e n ate the i n f e c t e d bark outside the p y c n i a l zone. The gnawing r a r e l y extended i n t o non-infected healthy bark. I n most - 103 -cases the rodents f a i l e d to eat the e n t i r e diseased area, enabling the r u s t to produce a e c i a i n l i m i t e d bark areas, or to continue to grow beyond the chewed area where p y c n i a l and a e c i a l s p o r u l a t i o n would again take place i n fu t u r e years. I n some cases the rodents hastened the death o f the t r e e by completely g i r d l i n g the stem or by removing the one remaining l i v e i n f e c t e d s t r i p of bark. Such trees would g e n e r a l l y succumb to the attack of the r u s t i n a year or two. I n many cases the rodent-chewing helped to suppress or destroy the canker f o r around the edge of the chewed areas c a l l u s t i s s u e developed. I n s e v e r a l 20 to 30 year o l d stands a f a i r p r o p o r t i o n of the tre e s showed o l d chewed areas where presumably a r u s t canker was .once a c t i v e , but had been i n a c t i v a t e d - b y rodent chewing. In these cases the whole canker area was chewed out and presumably no i n f e c t e d bark remained from which the r u s t could continue to i n f e c t the t r e e and produce a e c i a . One of the most s t r i k i n g cases of t h i s form of b i o l o g i c a l c o n t r o l encountered i n the present study, was the case of the P. s y l v e s t r i s p l a n t a t i o n near Beaver Mines, already r e f e r r e d to i n the s e c t i o n on damage. Rodent damage was c h a r a c t e r i s t i c of i n f e c t e d t r e e s , as i n i n -f e c t e d stands only r u s t i n f e c t e d t r e e s showed evidence of rodent damage. The one exception to t h i s was an area of porcupine damage where the por-cupine had been l e s s s e l e c t i v e although s t i l l showing a preference f o r r u s t i n f e c t e d t r e e s . Rodents appeared to only a t t a c k t r e e s which had a c t i v e l y producing pycnial and a e c i a l zones, and p r e f e r e d the p y c n i a l zones. Most of the damage probably occurred during the winter or e a r l y s p r i n g , but.some damage was recorded s p o r a d i c a l l y throughout the summer and e a r l y f a l l . I n many cases, cankers showed evidence of annual rodent v i s i t s . - 10k Each year the rodents removed the succulent p y c n i a l zone near the l i m i t s of the canker o f t e n l e a v i n g a t h i n s t r i p of c a l l u s t i s s u e adjacent to the previous year's chewed area. An example of such annual chewing i s shown i n F i g . 36, which had probably been v i s i t e d f o r at l e a s t 10 conse-c u t i v e years. Often the rodents removed a l l the p y c n i a l zone and some of the a e c i a l zone, completely r i n g i n g the canker, but l e f t untouched the center of the canker composed o f dead bark t i s s u e s . At l o c a t i o n s 2 and 3, where many of the cankers were p r o t e c t e d by i n s e c t cages, i t was not uncommon to di s c o v e r , i n the sp r i n g , t h a t the rodents had chewed the newly i n f e c t e d bark beyond the top and bottom of the cage. There were als o a few cases where the rodents damaged the i n s e c t cage and were able to chew the canker beneath. During the p e r i o d 1966 to 1968, kkY cankers were observed f o r rodent damage, and of these 320 or 71.7$ r e c e i v e d some damage i n one of the years or i n many cases each year. This percentage was s l i g h t l y lower than would have occurred normally as some 20 of the cankers were pr o t e c t e d by i n s e c t cages during two or three years of the p e r i o d . Fresh chewing was only recorded on 6 of these "protected" cankers, f a r lower than the average rodent damage incidence at these l o c a t i o n s . Table X I I I Table X I I I . The incidence of new rodent damage on Cronartium comandrae cankers, and the number of cankers observed at v a r i o u s l o -cations i n southwest A l b e r t a during the years 1966 to 1968. Year Wo. of l o c a t i o n s T o t a l no. of No. of can- Per cent observed cankers kens w i t h new new rodent observed rodent damage damage 1966 19 333 133 " 39-9 1967 21 385 166 4 3 . 1 1968 21 307 158 51.5 - 105 -shows the incidence of f r e s h rodent damage recorded each year and the number of cankers observed, excluding the cankers w h o l l y or p a r t l y pro-t e c t e d by i n s e c t cages. At two l o c a t i o n s no rodent damage was recorded i n any of the years. These, were areas of young open grown i n f e c t e d t r e e s . G e n e r a l l y the g r e a t e s t incidence of damage occurred i n the o l d e r stands where damage by s q u i r r e l s was predominant. At some l o c a t i o n s the rodent p o p u l a t i o n must remain high, but at others considerable f l u c t u a t i o n must occur. Table XIV shows the percentage incidence of f r e s h canker chewing at a number of selected' l o c a t i o n s . At M i s t Creek the stand was very open which may have accounted f o r the lower incidence, a l s o l e s s cankers were a c t i v e i n I968 which perhaps reduced t h e i r n a t u r a l a t t r a c t i v e n e s s to the rodents. The l a t t e r f a c t o r may a l s o be r e s p o n s i b l e f o r the lower i n c i -dence of rodent damage at Honeymoon and Ribbon Creeks i n 1968, as much of the a c t i v e p o r t i o n s of cankers had been removed i n the two previous years. The l a s t three l o c a i o n s given i n Table XIV were about h a l f a mile apart, but Saskatchewan R i v e r Crossing 2 was an o l d e r stand and probably more s u i t a b l e as permanent s q u i r r e l t e r r i t o r y . I n the o l d e r stands i t was not uncommon to f i n d s q u i r r e l nests i n r u s t i n f e c t e d t r e e s . At l o -c a t i o n 1 a nest occurred at the l e v e l of a canker, but a e c i a were never observed during the 5 years, although f r e s h chewing occurred each year. The main r e s u l t of the rodent-feeding was the-removal of the p y c n i a l zone, and the f u t u r e a e c i a l zone, which brought about an enormous r e d u c t i o n i n the volume of p o t e n t i a l aeciospores f o r the f o l l o w i n g season. Often cankers were prevented from producing any a e c i a f o r a number of years through the rodent a c t i v i t y . A secondary f a c t o r was t h a t the death of a diseased t r e e was o f t e n hastened, which f u r t h e r reduced the r T a b l e XIV. The p e r c e n t a g e i n c i d e n c e o f f r e s h r o d e n t chewing o f C r o n a r t i u m comandrae c a n k e r s a t s e l e c t e d l o c a t i o n s d u r i n g t h e y e a r s 1966 t o 1968. L o c a t i o n V i c a r y Honeymoon B a r i l M i s t R i b b o n Marmot Robb Saskatchewan R i v e r C r o s s i n g Creek Creek Creek Creek Creek Creek Road b u r n #1 #2 #3 1 H Year £ 1966 78 81 25 25 31 83 67 0 77 0 1967 . 55 90 26 13 68 74 32 17 80 9 1968 77 53 82 0 33 63 2 6 ' 83 100 1+6 - 107 -p o t e n t i a l of a canker to produce aeciospores. A l s o , a s s o c i a t e d w i t h the rodent chewing damage there was g e n e r a l l y copious r e s i n flow, which o f t e n contaminated the a e c i a l zone preventing aeciospore d i s p e r s a l . Dur-ing the w i n t e r - s p r i n g season of 1966-67 at k l o c a t i o n s , an average of 25$ (range f o r i n d i v i d u a l l o c a t i o n s 10 to 3*+$) of the canker bark was removed from a l l cankers showing any rodent damage, and i n many cases the bark not removed was no longer productive. S i m i l a r values f o r the 1967-68 w i n t e r - s p r i n g season, from cankers chewed at 17 l o c a t i o n s , showed th a t 30$ (range f o r i n d i v i d u a l l o c a t i o n s 7 to 73$) of the canker bark was removed, most being from the p o t e n t i a l aecia-bearing zone o f the canker. There were numerous cases where 70 to 100$ of the p o t e n t i a l a e cia-bearing bark on a canker was removed. Rodents tended to remove the i n f e c t e d bark nearest the center of the canker, the p o r t i o n on which the f i r s t a e c i a would develop, thus even i f some i n f e c t e d bark remained, a e c i a l s p o r u l a -t i o n was s t i l l delayed on these cankers. Often the few remaining a e c i a would not sporulate u n t i l two weeks a f t e r the main aeciospore s p o r u l a -t i o n p e r i o d . I t was common to f i n d rodent gnawing a l l round a branch stub which had served as the mode of entry of the r u s t i n t o the main stem: Response of the t r e e The main response of the t r e e to the canker was the pro d u c t i o n of r e s i n . The most n o t i c e a b l e r e s i n production occurred i n the p y c n i a l zone, although considerable amounts were a l s o produced i n the t i s s u e s of the a e c i a l zone. C h a r a c t e r i s t i c a l l y the r e s i n flowed down over the canker and the stem area below, causing most damage when o r i g i n a t i n g i n the upper p y c n i a l zone. The r e s i n o f t e n prevented f u r t h e r d i s p e r s a l of aeciospores during the season, and formed a cr u s t over the bark which may have - 108 -e f f e c t i v e l y prevented, a e c i a from pushing through the c r u s t i n f u t u r e years to disperse aeciospores. I t was not uncommon f o r the r e s i n flow to take on an orange-yellow c o l o r where p y c n i a l drops or aeciospores were i n c o r -porated i n t o the r e s i n . Most r e s i n flow occurred towards the end o f the main aeciospore s p o r u l a t i o n p e r i o d , c o n t i n u i n g u n t i l at l e a s t l a t e Septem-ber. Resin was probably produced because the r e s i n canal c e l l s were rup-tured by the a e c i a l zone cracking and d r y i n g out, and through pressure produced on the r e s i n canal c e l l s by the increased amount of i n t e r c e l l u l a r r u s t mycelium. Further r e s i n flow occurred through the i n s e c t and rodent-damage, e s p e c i a l l y that of the l a t t e r , and helped s u b s t a n t i a l l y to pro-duce a crust- of d r i e d r e s i n over much of the canker. Often r e s i n flow extended f o r s e v e r a l f e e t below cankers o c c u r r i n g higher upon t r e e s . R e s in production o f t e n occurred annually on a c t i v e cankers. TableXV r e -cords the incidence of f r e s h r e s i n o s i s on cankers i n the years I966 to 1968 at v a r i o u s l o c a t i o n s . Most of those cankers not r e c o r d i n g f r e s h r e s i n o s i s were i n a c t i v e cankers, although some cankers showing no a c t i v e p y c n i a l or a e c i a l s p o r u l a t i o n zones or the presence of T u b e r c u l i n a or other f u n g i , s t i l l produced some surface r e s i n . f l o w presumably i n r e s -ponse to the extension of the rust- mycelium around the p e r i p h e r y of the canker,.- Resin production was more extensive i n a s s o c i a t i o n w i t h Tuber-c u l i n a than w i t h the r u s t alone. I n the three main years of observation only 37 cankers (7.9$) were observed that d i d not show evidence of some r e s i n o s i s during t h e i r h i s t o r y . The m a j o r i t y of these occurred on very young trees ( l l ) , or on t r e e s that were observed f o r only one or two years before dying (20). P r a c t i c a l l y every t r e e w i t h an i n a c t i v e canker showed evidence of some p e r i o d of r e s i n o s i s , and o f t e n the v a s t m a j o r i t y of the - 109 -canker and area below was covered i n d r i e d r e s i n . The r e s i n o f t e n im-pregnated l a r g e areas of the bark and outer sapwood. Table XV. The incidence- of f r e s h r e s i n o s i s on Cronartium comandrae cankers on lodgepole pine and the number of cankers obser-ved at a number of l o c a t i o n s i n southwest A l b e r t a during the years 1966 to 1968. Year Wo. of l o -cations -" observed No. of cankers observed No. of cankers w i t h f r e s h r e s i n Per cent of cankers w i t h f r e s h r e s i n 1966 20 356 237 . 66 .6 1967 22 398 282 71.1 1968 22 320 212 66.2 D i s c u s s i o n T u b e r c u l i n a maxima i s reported to be a hyperparasite o f r u s t s , i n c l u d i n g those of the Cronartium (Peridermium) group (Hedgcock 1935; Hubert 1935a; Mielke 1933; Tubeuf 1901, 19lk; Weir and Hubert 1917). However, present observations, and the study of Wicker on C_. r i b i c o l a i n Idaho (Leaphart and Wicker 1968) i n d i c a t e that T. maxima i s not a tru e hyperparasite o f the r u s t , but i s p a r a s i t i c on the r u s t canker area where the mycelium invades the bark t i s s u e . This i s i n agreement w i t h the e a r l y work of Lechmere (1914) who found t h a t T. maxima d i d not att a c k or destroy the r u s t mycelium. A l l e a r l i e r r e p o r t s are i n agreement t h a t the cankered bark dies a f t e r i n v a s i o n by T. maxima and tha t i t g r e a t l y reduces or-• e n t i r e l y ' i n h i b i t s the production of aeciospores where i t occurs. T. maxima was described by Rostrup (1890) as a t t a c k i n g C. r i b i c o l a ( P u c c i n i a klebahni) on Pinus strobus i n Europe, and was f i r s t r eported on C. comandrae - 110 -on P. ponderosa and P. c o n t o r t a "by Weir and Hubert (1917) from Montana. Tuberculina was not found on t h i s group of r u s t s i n A l b e r t a u n t i l 1964 (Powell and Morf 1965), but has been known since I926 on C.' r i b i c o l a i n B r i t i s h Columbia (Mielke 1933). I n 1964, T u b e r c u l i n a was found at 11 l o c a t i o n s on C_. comandrae on P. c o n t o r t a and one on P. s y l v e s t r i s between Robb and Beaver Mines, A l b e r t a . Since that time T u b e r c u l i n a has been found at a f u r t h e r 11 l o c a t i o n s i n A l b e r t a , and one l o c a t i o n i n Kootenay N a t i o n a l Park, B r i t i s h Columbia, on P. contorta, the l a t t e r • r e c o r d being the f i r s t on C. comandrae i n that Province. S e v e r a l T u b e r c u l i n a c o l l e c -t i o n s were a l s o made on C. comandrae on P. banksiana from one l o c a t i o n on the Mackenzie Highway, 110 miles south of Rae, Northwest T e r r i t o r i e s (Baranyay 1968), but Tu b e r c u l i n a has not been recorded on t h i s pine host i n A l b e r t a , Saskatchewan or Manitoba. P o w e l l and Morf (1965) reported the occurrence, of Tu b e r c u l i n a on Peridermium s t a l a c t i f o r m e i n A l b e r t a , but not on C. r i b i c o l a or P. h a r k n e s s i i . However i n 1965 T u b e r c u l i n a was found on P. h a r k n e s s i i on P. co n t o r t a (CFB 6895) i n Kootenay N a t i o n a l Park, B r i t i s h Columbia, the f i r s t r e c o r d on t h i s r u s t i n western Canada. In 1966 T u b e r c u l i n a was c o l l e c t e d on C_. comptoniae on P. c o n t o r t a x P. banksiana h y b r i d (CFB 7736), 40 miles north of Nahanni Butte, Northwest T e r r i t o r i e s , which was r e c e n t l y reported by Baranyay (1968). Tu b e r c u l i n a was p r e v i o u s l y only known on t h i s r u s t host from B r i t i s h Columbia (Mielke 1933). I t s occurrence on C. comandrae and other Cronartium (Peridermium) spp. i n western North American i s probably more widespread than the c o l -l e c t i o n s i n d i c a t e . On C. r i b i c o l a and C. comptoniae, T. maxima i s r e -ported to sporulate most abundantly on the p y c n i a l zones o f the canker (Hubert 1935a,b; Mielke 1933; Wicker, p e r s o n a l communication 1968), but on C_. comandrae i t r a r e l y sporulates on the p y c n i a l zone but abundantly - I l l -on the a e c i a l zone. I t was found i n the present study t h a t only current season p y c n i a l and a e c i a l zones appear to be s u s c e p t i b l e to T. maxima:, no f r e s h i n f e c t i o n occurred on i n a c t i v e cankers. I n o c u l a t i o n t e s t s , r e -ported by Wicker and Kimmey (1967) s i m i l a r l y showed th a t n o n f r u i t i n g cankers were not s u s c e p t i b l e to i n f e c t i o n , p roving that i n f e c t i o n e n t r y was only gained through s p o r u l a t i n g p y c n i a or a e c i a . Wicker and Wells (1968) reported t h a t T. maxima was able to overwinter as spores, sporo-dochia, or i n the m y c e l i a l stage w i t h i n the pine cortex. Some spore v i a b i l i t y was r e t a i n e d a f t e r 19 months storage at -31°C. T. maxima was always found i n d i r e c t a s s o c i a t i o n w i t h the r u s t , and from the evidence th a t s m a l l areas remained u n i n f e c t e d and were able to s p o r u l a t e , i t -appeared that T. maxima d i d not extend to or beyond the l i m i t of the r u s t mycelium i n the i n f e c t e d bark and t h e r e f o r e d i d not give complete b i o l o g i -c a l c o n t r o l of the r u s t . A number of people i n Europe and America have used T. maxima as a c o n t r o l of C. r i b i c o l a w i t h l i m i t e d or no success (Hubert 1935a,b; Mielke 1933, Quick and Lamoureaux 1967, Tubeuf 1914, 1917, 1930). Mielke (1933) and Hubert (1935a,b) were of the o p i n i o n that i t s p o s s i b i l i t i e s as a c o n t r o l agent were remote, but the more r e -cent work of Quick and Lamoureaux (1967), Wicker and Wells (1968) and Wicker (personal communication 1968) i n d i c a t e d greater p o t e n t i a l . T. maxima was c e r t a i n l y able to cause marked r e d u c t i o n i n aeciospore pro-d u c t i o n (Lechmere 191k; Spaulding 1929, Tubeuf 1 9 l 4 ; and the present study). Leaphart and Wicker (1968) reported a marked increase i n the percentage of C. r i b i c o l a cankers p a r a s i t i z e d by T. maxima. Ranges of incidence at the s t a r t were 37 to 80%, but by 1966 were 83 to 100%, a l -though some may not have been i n o c u l a t e d n a t u r a l l y . I n the present study, - 112 -average annual incidence of 13 to 24$vas recorded f o r a l l l o c a t i o n s . The high l e v e l o f incidence, plus the f a c t that i t was r a r e l y present on the same canker f o r more than one or two years and th a t i t u s u a l l y i n a c t i v a t e d the canker, i n d i c a t e s that Tuberculina was p l a y i n g an important r o l e i n c o n t r o l l i n g C. comandrae and presumably other Cronartium r u s t s . A Cladosporium sp. has not been reported as an ass o c i a t e d fungus of Cronartium stem, r u s t s , although Keener (1964) found Cladosporium spp. on the a e c i a l s o r i of the cone r u s t Cronartium conigenum Hedge. & Hunt.. He als o s t a t e d that Cladosporium a e c i d i i c o l a Thum was the most f r e q u e n t l y encountered fungus on a l l types of r u s t s o r i , and reported s e v e r a l cases on r u s t s i n Worth America (Keener 1954, 1956, 1964). Cladosporium exoasci Lindau, C. e x o b a s i d i i Jaap. (C. cl a d o s p o r i o i d e s Thum), and C. hemileiae Steyaert were a l s o reported to be hyperparasites on other p a r a s i t i c f u n g i (de V r i e s 1952). According to Dr. E l l i s (see above, page 83) Cladosporium tax. sp. 1. does not f i t any of the d e s c r i p t i o n s f o r Cladosporium spp. asso c i a t e d w i t h r u s t s . Cladosporium tax. sp. 1 was f a i r l y widespread as i n a d d i t i o n to the l o c a t i o n s reported above, i t was found on C. comandrae on P. banksiana (CFB 7447), 60 m i l e s northeast of F o r t Providence, W.W.T., and i s qui t e common (9 CFB c o l l e c t i o n s ) on P. h a r k n e s s i i g a l l s , on P. con t o r t a i n A l b e r t a and the Wational Parks i n B r i t i s h Columbia. Dr. Sutton (personal communication 1966) a l s o reported i t on C. comandrae and P. h a r k n e s s i i on P. banksiana i n the Saskatchewan and Manitoba area. Some evidence was gained that Cladosporium tax. sp. 1 might be p a r a s i t i c on C. comandrae aeciospores. de V r i e s (1952) doubted whether a l l of the described r u s t a s s o c i a t e d Cladosporium species were a c t u a l l y "hyperpara-s i t e s " as he was unable to detect connections between Cladosporium hyphae - 113 -and uredospores of Melampsora l a r i c i - e p i t e a Kleb. on S a l i x sp., the s o r i of which were covered by C. c l a d o s p o r i o i d e s , but the r e p o r t o f Steyaert (1930) f o r C_. hemileiae on Hemil e i a v a s t a t r i x Berk. & Br. i s convincing. Keener (1964) was of the o p i n i o n that species such as Cladosporium ap-p a r e n t l y lacked the c a p a c i t y to destroy r u s t spores and probably only impeded spore dissemination. Smith (1905) reported t h a t a species of Cladosporium on Asparagus r u s t i n C a l i f o r n i a , which he b e l i e v e d to be C. herbarum Link, was able to destroy r u s t spores and i l l u s t r a t e d a Clados-porium germinating from a dead r u s t spore. C. herbarum was observed l e s s f r e q u e n t l y i n the present study from spore c o l l e c t i o n s and may be p l a y i n g a r o l e i n reducing the v i a b i l i t y of aeciospores, along w i t h C_. tax. sp. 1. C. herbarum was r e c e n t l y i s o l a t e d from d e t e r i o r a t i n g t e l i a l g a l l s of of Gymnosporangium juvenescens Kern ( E s l y n i 9 6 0 ) . Cladosporium spp. are common among the primary f u n g a l c o l o n i z e r s on p l a n t m a t e r i a l (Greene 1952; Leben .1965). A number of people have i n d i c a t e d t h a t there i s a succession of f u n g a l c o l o n i z e r s on p l a n t m a t e r i a l s (Hudson 1962; Last 1955), and tha t Cladosporium o f t e n overruns the other c o l o n i z e r s , e s p e c i a l l y under h i g b humidity c o n d i t i o n s . When i n s e c t cages were maintained on cankers i n the present study, o f t e n causing a higher humidity l e v e l , Cladosporium, when present, r a p i d l y . c o v e r e d the whole canker. Of the other m i c r o f l o r a l species i s o l a t e d i n the present study a few, or species of the same genus, have been reported as p l a y i n g a pos-s i b l e r o l e i n reducing the e f f e c t of other p l a n t pathogens. Wollenweber (1934) described Fusarium b a c t r i d i o i d e s which was found to p a r a s i t i z e the cone b l i s t e r r u s t , Cronartium conigenum. Goodding was al s o able to demon-s t r a t e the a b i l i t y of F. b a c t r i d i o i d e s to at t a c k C. r i b i c o l a , P. (C.) - 114 -h a r k n e s s i i and P. (C.) filamentosum from s u c c e s s f u l i n o c u l a t i o n s i n Oregon and Idaho (Goodding, i n Wollenweber 1934). Goodding (1932) a l s o b r i e f l y discussed another Fusarium sp. which occurred on C. r i b i c o l a cankers i n -dependent o f the a e c i a and was as s o c i a t e d w i t h a H e c t r i a sp. There are a number of r e p o r t s of the secondary f u n g i a s s o c i a t e d w i t h C. r i b i c o l a can-kers (Bingham 1942; Goodding 1932; Rhoads 1920; S n e l l 1929a,b; Spaulding 1922, 1929; S t o u f f e r 1932) which are not p a r a s i t i c on the r u s t . Rhoads (1920), Spaulding (1922) and Hubert (1935b), b e l i e v e d t h a t the secondary f u n g i a c c e l e r a t e d the g i r d l i n g of the r u s t and hastened the death of the canker t i s s u e s . Posey and Gravatt ( c i t e d i n Spaulding 1929) reported t h a t 15$ of the r u s t i n f e c t e d t r e e s recovered through the a c t i o n of the secondary f u n g i i n k i l l i n g the i n f e c t e d pine bark, and through suppres-s i o n of lower branches before the r u s t spread to the stem. Bingham (1942) and Bingham and E h r l i c h (1943) b e l i e v e d t h a t the secondary f u n g i reduce the a e c i a l s p o r u l a t i o n of the r u s t . A l s o t h a t one of these, a Dasyscypha sp., once e s t a b l i s h e d k i l l e d the bark much more r a p i d l y than C. r i b i c o l a (Bingham and E h r l i c h 1943). Among the secondary f u n g i reported by others . on C. r i b i c o l a and i d e n t i f i e d i n the present study were L a c h n e l l u l a (Dasyscypha) spp. (Bingham 1942; Bingham and E h r l i c h : . l 9 4 3 ; Goodding 1932; S n e l l 1929a,b; S t i l l i n g e r 1929), Tympanis spp. (Bingham 1942; Hubert 1931), Phoma sp. ( S n e l l 1929a) and Phomopsis sp. (Goodding 1932). Keener (1964) considered t h a t V e r t i c i l l i u m along w i t h D a r l u c a and Tuberculina were species capable of de s t r o y i n g r u s t spores. C a s t e l l a n i and G r a n i t i (1949) reported a V e r t i c i l l i u m t h a t was p a r a s i t i c on Cronartium asclepiadeum aeciospores causing them to become h y a l i n e . Sukapure and Thirumalachar (1966) have reported a Cephalosporium p a r a s i t i c on the u r e d i a - 115 -of three r u s t s i n I n d i a . Greene (1952) r e p o r t s a Coniothyrium sp. on the t e l i a of P u c c i n i a anemones-virginianae Schw. i n Wisconsin, and a Phoma sp. on Taphrina m i r a b i l i s (Atk.) Giesenhag. Myren (1964) i s o l a t e d various imperfect f u n g i from a l l of the Cronartium fusiforme cankers c o l -l e c t e d from three l o c a t i o n s . T h i r t y per cent of the 132 cankers a l s o y i e l d e d b l u e - s t a i n f u n g i , and 13%-basidiomycetes. He thought t h a t the associated i n s e c t g a l l e r i e s served as i n f e c t i o n courts f o r v a r i o u s wood-i n h a b i t i n g f u n g i . Microorganisms have al s o been reported a s s o c i a t e d w i t h Hypoxylon pruinatum (Klotzsch) Cke. cankers. B i e r and Rowat (1962a) r e -ported P u l l u l a r i a sp. and Epicoccum nigrum to be common, and Wood and French (1965) added species of A l t e r n a r i a , Chaetomium, Cytospora and b a c t e r i a . S e v e r a l of these f u n g i i n h i b i t e d growth of Hypoxylon i n dual c u l t u r e , and prevented canker formation ( B i e r and Rowat 1962a, b, 1963). B i e r (1963) l a t e r reported that healthy bark-water suspensions c o n t a i n i n g f u n g i and b a c t e r i a c o n t r o l l e d a l e a f r u s t and a number of canker and decay diseases. Wood and French (1965) found that the b a c t e r i a were capable of reducing or preventing ascospore germination. P e n i c i l l i u m spp. are very common on a number of surfaces. P. brevi-compactum Dierckx. has been i s o l a t e d from decaying f l e s h y f u n g i , P. funiculosum has been reported from lumber and i s one of the world's com-mon s o i l f u n g i , and P. cyclopium causes a bulb r o t (Raper and Thorn 19^9). I n a s s o c i a t e d studies v a r i o u s P e n i c i l l i u m spp. were i s o l a t e d from P e r i -dermium h a r k n e s s i i g a l l s , where they appeared to be f a r more common than on C. comandrae cankers. Paecilomyces f a r i n o s u s ( D i c k s , ex Fr.) Brown & Smith i s a common i n s e c t p a r a s i t e (Brown & Smith 1957). Seimatosporium d i s c o s i o i d e s ( E l l . & Ev.) Shoemaker has been found on stems, and leaves of - n 6 -Rosa (Shoemaker 1964). The Monoc i l l i u m sp. i n the present study may be an undescribed species as i t d i f f e r s from the type species of the genus, M. indicum Saksena, i s o l a t e d from s o i l (Saksena 1955), i n having much smaller c o n i d i a and d i f f e r e n t c u l t u r a l features (B. C. Sutton, p e r s o n a l communication 1966). Barron (1961) r e c e n t l y added the species M. humicola, i s o l a t e d from v a r i o u s f o r e s t s o i l s i n Ontario. The c o n i d i a of M. humicola are s i m i l a r i n s i z e to those found i n the present study. The bacterium Pseudomonas fluorescens (Fliigge) M i g u l a was found, to i n h i b i t a number of b a c t e r i a l pathogens ( T e l i z - O r t i z and Burkholder i 9 6 0 ) . A number of other e p i p h y t i c b a c t e r i a are reported to reduce or c o n t r o l other diseases (Leben 1965). P u l l u l a r i a p u l l u l a n s and Sporobolo-myces sp. are widespread e p i p h y t i c yeasts or y e a s t - l i k e f u n g i (Leben 1965). Voznyakovskaya (1963) discusses the widespread occurrence of 19 e p i p h y t i c yeasts on p l a n t m a t e r i a l s and reported that they sometimes c o n s t i t u t e k-0 to 100$ of the t o t a l number of e p i p h y t i c microorganisms, and that the m y c e l i a l y e a s t - l i k e forms o f t e n predominate on the leaves of t r e e s , es-p e c i a l l y P u l l u l a r i a p u l l u l a n s . Of the non-mycelial forms, species of Rhodotorula ( i n c l u d i n g R. a u r a n t i c a ) , Sporobolomyces, and Cryptococcus were most common. K a i s (1963) found Sporobolomyces on the t e l i a l columns of Cronartium f us i f or me. Levine e_t a l . (1936) described a B a c i l l u s which i n h i b i t e d c e r e a l r u s t development i n the f i e l d , and Poh et a l . (195*0 described a Xanthomonas p a r a s i t i c on u r e d i a of c e r e a l r u s t s . S i m i l a r l y , Morgan.(1963) and French et a l . (1964) discussed the e f f e c t s of Pseudomonas fluorescens and B a c i l l u s spp. on c e r e a l r u s t spore germination and i n f e c -t i o n . Further study may i n d i c a t e that b a c t e r i a and 'yeasts' p l a y a r o l e . i n reducing the production and v i a b i l i t y of C. comandrae aeciospores. - 117 -Leben (1965) comments that the e p i p h y t i c b a c t e r i a , which seem to c o l o n i z e a c t i v e l y growing t i s s u e f i r s t , should be i n v e s t i g a t e d i n t e n s i v e l y , as many have been shown to be i n h i b i t o r y to t e s t b a c t e r i a or f u n g i i n d u a l c u l t u r e . I n s e c t s have been reported from Cronartium stem r u s t s , but there i s l i t t l e reference to the damage caused by them. Peterson ( i 9 6 0 ) r e f e r s to i n s e c t s e a t i n g aeciospores of P. h a r k n e s s i i , and Gravatt and Posey (1918), and S n e l l (1929a) noted i n s e c t s feeding on aeciospores and p y c n i a l drops of C. r i b i c o l a . A number of i n s e c t s are reported to c a r r y Cronartium aeciospores e x t e r n a l l y on t h e i r bodies which aids i n spore d i s s e m i n a t i o n (Gravatt and M a r s h a l l 1917; Gravatt and Posey 1918; Peterson i 9 6 0 ; S n e l l 19193 1929a). Myren (1964) reported from surveys on s l a s h and l o b l o l l y pine p l a n t a t i o n s , that i n s e c t a t t a c k occurred i n kk to 100% of the C. fusiforme cankers on 6 to 15 year o l d pine, and t h a t the percent-age was higher i n o l d e r p l a n t a t i o n s . The most common invaders were D i o r y c t r i a amatella ( H u l s t ) , a p i t c h moth, Eurytoma s c i r o m a t i s Bugbee, a c h a l c i d wasp, and Pissodes nemorensis Germar., the deodar w e e v i l . A few other w r i t e r s have mentioned i n s e c t s a s s o c i a t e d w i t h Cronartium cankers. These cankers o b v i o u s l y provide a microhabitat i n which many mi c r o f a u n a l organisms spend a p a r t or a l l of t h e i r l i v e s . S n e l l (1919) l i s t e d Ik species of i n s e c t s , as w e l l as a crustacean and s p i d e r , which he c o l l e c t e d on or near cankers of C. r i b i c o l a and which were l a t e r found to be c a r r y -in g aeciospores on t h e i r bodies. Eleven of these species were b e e t l e s , and i n c l u d e d three genera represented i n the present study, Melanophthalriia gibbosa Hbst., Pissodes s t r o b i Peck, and Dendroctonus valens Lec. He reported specimens w i t h as many as 16,500 spores on t h e i r bodies, a l -though few c a r r i e d more than 1 ,000. The only Lepidoptera c o l l e c t e d was - 118 -a L i p a r i d a e , P o r t h e t r i a d i s p a r L., and Gravatt and Posey (1918) reported that the lar v a e of t h i s moth r a p i d l y destroyed the a e c i a and the under-l a y i n g C. r i b i c o l a i n f e c t e d t i s s u e s . They noted that the larvae o f t e n destroyed a la r g e percentage of the next year's s p o r u l a t i n g zone and t h a t aeciospore production was prematurely a r r e s t e d i n 25 'to 100% of the pus t u l e s . They observed an average of 18,100 spores on the surface of each s m a l l l a r v a , and counted a f u r t h e r 26,000 spores i n the alimentary t r a c t . During a 13 hour period.2 0 l a r v a e feeding on aeciospores produced k23 f a e c a l p e l l e t s , • e a c h of which contained an average of 8 , l 6 o spores, but from t e s t s they found t h a t many spores were s t i l l v i a b l e . From t h i s they estimated t h a t each P o r t h e t r i a larvae was capable of e a t i n g 3 1 8 , 6 l 6 spores a day. The D i o r y c t r i a spp., found i n the present study, may w e l l cause s i m i l a r damage to that caused by the P o r t h e t r i a l a r v a e . H e i n r i c h (1956), i n h i s monograph on the moths of the subfamily P h y c i t i n a e , mentioned t h a t D. zimmermani was the most economically important D i o r y c t r i a and th a t the larvae of t h i s species bore i n t o the cambium causing considerable damage. The D i o r y c t r i a as a group, may w e l l be im p o r t a n t . i n reducing the q u a n t i t y of Cronartium aeciospores produced. As already mentioned, Myren (1964) found D. amatella was one of the three most important i n s e c t invaders. E b e l (1965b) a l s o found t h i s species was common i n C. fusiforme cankers, t h a t D. a b i e t e l l a (Denis & S c h i f f e r m u l l e r ) occurred s p o r a d i c a l l y , and D. c l a r i o r a l i s (Walker) r a r e l y . E b e l noted t h a t the larvae of D. amatella, fed f i r s t among the spore masses upon the canker from January to March, and then entered the cankered t i s s u e to complete development. Heikkenen (1964) found t h a t 35% of the C. fusiforme cankers i n p l a n t a t i o n s of - 119 -l o b l o l l y pine were i n f e c t e d by D. amatella, and a s i m i l a r percentage oc-curred i n cankers on s l a s h p i n e . D. amatella were a l s o prevalent i n f i r s t - y e a r cones i n f e c t e d by cone r u s t , Cronartium s t r o b i l i n u m (Arth.) Hedge. & Hahn, on s l a s h pine ( E b e l 1965b; Merkel 1958). E b e l (1965b) reared s e v e r a l hymenopterous p a r a s i t e s from D i o r y c t r i a , i n c l u d i n g species of Agathis and Apanteles, which were both reared from cankers c o n t a i n i n g D i o r y c t r i a i n the present study. I n B r i t i s h Columbia, Ross and Evans (1957) reared D. a b i e t e l l a and D. zimmermani from Cronartium g a l l s on Pinus contorta, and D. sp. nr. zimmermani from the area surrounding a patch of rodent-damaged bark. Anderson and French (1964) reported that D. zimmermani was a common i n h a b i t a n t of C. comptoniae cankers, causing considerable r e s i n flow. Specimens o f D. zimmermani were a l s o reared from P. h a r k n e s s i i and P. s t a l a c t i f o r m e cankers i n A l b e r t a . The Las-p r e y r e s i a spp. are o f t e n seed and cone feeders, and the R e c u r v a r i a are l a r g e l y needle miners ( P r e n t i c e et a l . 1965), thus the occurrence of both i n the cankers was probably due to the larvae seeking s h e l t e r f o r pupation. A L a s p r e y r e s i a sp. gp. 2 was a l s o obtained from a P. s t a l a c t i -forme canker. This species does not f i t the d e s c r i p t i o n of the others placed i n L a s p r e y r e s i a Htm., group 2 (MacKay 1959> and p e r s o n a l communi-c a t i o n 1968). The P u l i c a l v a r i a sp. represents new host and geographical records, according to T. N. Freeman (pe r s o n a l communication 1968). Lar-vae of the f a m i l y Blastobasidae are o f t e n scavangers, although one species (Holcocera immaculella McD.) has been found b o r i n g i n g a l l s on pines ( P r e n t i c e et a l . I965). Nothing was known about the h a b i t a t of Epuraea obliquus u n t i l the present study, as i t was only r e c e n t l y described (Hatch 1962) from two Oregon specimens. Parsons (1967) gives f u r t h e r d e t a i l e d d e s c r i p t i o n s - 120 -based l a r g e l y on my m a t e r i a l . I n a d d i t i o n to the ei g h t r e p o r t s of i n c i -dence on C. comandrae cankers, i t was reared or c o l l e c t e d from one P. s t a l a c t i f o r m e canker,.and k P. h a r k n e s s i i g a l l s i n A l b e r t a , and from one P. h a r k n e s s i i g a l l from B r i t i s h Columbia and one from Quebec. Parsons (1967) reported seeing a specimen c o l l e c t e d from near O l i v e r , B r i t i s h Columbia, and one reared from a C. quercuum g a l l from New Brunswick. There are many r e p o r t s o f other members of the genus c o l l e c t e d from f u n g i . Hubert (1935b) mentions E. o r a t a (probably E. ovata Horn) causing damage on C. r i b i c o l a cankers. BoVing and Rozen (1962) mention la r v a e of E. avara (Randall) from a fungus on Pinus taeda, which could have been C. fusiforme. Hubbard (1892) and Leech (1947) reported E. monogama Cr. found covered w i t h fungus spores. Benick (1952), Hatch (1962) and others, men-tio n e d s e v e r a l other Epuraea species found i n f u n g i . Parsons (1967) r e -ported that E. avara, E. c o r t i c i n a E r., and E. t e r m i n a l i s Mann, were reared from oak w i l t fungus mats, and Yount et a l . (1955) reported the i s o -l a t i o n of v i a b l e Endoconidiophora fagacearum B r e t z . c o n i d i a from f a e c a l m a t e r i a l of Epuraea spp. The aeciospores from E. obliquus f a e c a l m a t e r i a l were not v i a b l e . Of the other Coleoptera genera represented i n the present study, both larvae and ad u l t s of the t r i b e C o r t i c a r i i n i ( L a t h r i d i i d a e ) feed ex-t e n s i v e l y on f u n g i (Beaver 1966; Hatch 1962). Members of the genera Atheta are one of the most common i n f u n g i (Benick 1952; Donisthorpe 1935; Graves i 9 6 0 ) , and were termed mycetocoles by Benick (1952). The Tenebri-onidae, except f o r a few species, have l i t t l e connection w i t h f u n g i (Graves i 9 6 0 ) . Benick (1952) s t a t e d that Aphodinus f i m e t a r i u s (L.) was r a r e l y found i n f u n g i , but t h a t c e r t a i n members of the f a m i l y Anobiidae, to which - 121 -Ernobius belongs, were s t r o n g l y mycetophilous, almost mycetobionts, l i v i n g and breeding on f u n g i . The Curculionidae and the S c o l y t i d a e were probably secondary species, a t t r a c t e d to the canker zone of the r u s t as they do not normally i n f e s t healthy t r e e s . Other species of the same genera are primary species. I n the d e s c r i p t i o n of the species Cylindrocopturus d e l e o n i Buchanan, Buchanan (Vjko) mentioned that some specimens from Idaho were c o l l e c t e d from a "fungus g a l l i n f e s t e d w i t h coleopterous larvae on y e l l o w pine". This could have been a g a l l of P. h a r k n e s s i i which i s common on Pinus ponderosa. Wood (1964) reported l a r v a e , pupae or t e n e r a l adults of Pissodes schwarzi and P. c u r r i e i Hopk. i n the root c o l l a r s of dead or dying Pinus monticola s a p l i n g s i n B r i t i s h Columbia, which had been i n f e c t e d w i t h C. r i b i c o l a or root r o t . Wood's d i s t r i b u t i o n map f o r P. schwarzi showed that i t ranged throughout'..the i n t e r i o r from the United States border and north i n t o the Yukon T e r r i t o r y . As mentioned above, P. nemorensis was a common invader, of C. fusiforme cankers (Myren 1964). S n e l l (1919) found P. s t r o b i a s s o c i a t e d w i t h C. r i b i c o l a . S n e l l a l s o mentioned Dendroctonus valens Lec. at the base of an i n f e c t e d C. r i b i c o l a t r e e . The specimen of D. murrayanae Hopkins of the present study, was found burrowing i n t o a b a s a l canker. Rhoads (1920) reported t h a t the b e e t l e Pityogenes h o p k i n s i Swaine attacked t r e e s weakened by C. r i b i c o l a , and a c c e l e r a t e d " t h e i r death. Many of these were t h r i f t y young trees which would not have died f o r at l e a s t one to three years from the a c t i v i t y of the r u s t . A s i m i l a r r e l a t i o n s h i p was noted i n the pre-sent study, trees from which C_. d e l e o n i and P. schwarzi were c o l l e c t e d were o f t e n dying or dead by the f o l l o w i n g year. K a i s (1963) reported t h a t larvae of the. d i p t e r a species Mycophila f u n g i c o l a F e l t fed on the spores of 0. fusiforme, and S n e l l (1919) found - 122 -a species Rhagio ( L e p t i s ) mystaceus (Macquart), c o l l e c t e d from the base of an i n f e c t e d C_. r i b i c o l a t r e e , w i t h 500 aeciospores on i t s body. . At present no other type of h a b i t a t i s known f o r Paracacoxenus guttatus and i t seems that t h i s species may be a true mycetobiont. A d u l t f l i e s were reared from cankers, and ad u l t s were c o l l e c t e d when v i s i t n g the a e c i a l and e s p e c i a l l y the p y c n i a l zone. The larvae of t h i s d i p t e r a and other larvae on the cankers may p l a y a r o l e i n the exchange o f (+) and (-) pycniospores between the h a p l o i d pustules of the r u s t ( C r a i g i e 1931) 5 as they move from one p y c n i a l drop to another, thus h e l p i n g the r u s t to com-p l e t e i t s l i f e c y c l e . E r a d y s i a were reported i n moist places wherever:; f u n g i grew (Stone et a l . I965). A few of the Cecidomyiidae cause primary damage to f u n g i (Stone et a l . 1965), and P i e l o u (1966) reported many speimens c o l l e c t e d from Polyporus b e t u l i n u s ( B u l l i a r d ) F r i e s , which i n -cluded the most abundant species found. Many Phoridae have been reared from f l e s h y and woody f u n g i , and the P i o p h i l i d a e are scavengers i n f u n g i (Stone et a l . 1965). Members of the genus Medetera are common predators on immature stages o f bark b e e t l e s (Beaver 1966). Jackson and Parker (1958), as w e l l as Myren (196U) have noted the occurrence of c h a l c i d wasp larvae i n C_. fusiforme cankers. They r e -ported that 2 to 30 larvae could be found i n the canker and t e n t a t i v e l y assigned the i n s e c t to the genus Bephratoides. They were of the o p i n i o n t h a t the canker a f f o r d e d an i d e a l h a b i t a t f o r breeding. Most of the hymenoptera reared i n the present study were probably p a r a s i t i c on other species and were a t t r a c t e d by the numerous larvae t h a t developed i n the cankers. A few may be phytophagous l i k e Eurytoma s c i r o m a t i s on C. fusiforme.(Krombein and Burks 1967, Myren 1964). Coelichneumon - 123 -b r u n n e r i Rohw. has been reported as a p a r a s i t e of D i o r y c t r i a aurantice 1 1 a Grt. (Krombein and Burks 1967), and was reared from a c o l l e c t i o n o f D. zimmermani i n the present study. Agathis binominata M.&W., Apanteles, Phaeogenes, Glypta, Microchelonus, and Copidosoma are probably p a r a s i t e s of Lepidoptera l a r v a e (Krombein and Burks 1967; Muesbeck et_ a l . 1951). Phygadeunon are u s u a l l y p a r a s i t e s of muscoid D i p t e r a (Muesbeck et a l . 1951) and were reared from a canker•that produced P^iacacoxenus g u t t a t u s . T r i a s p i s are p a r a s i t i c on Coleoptera larvae (Muesbeck et a l . 1 9 5 l ) , and Dolichomitus terebrans nublipennis has been reported from Pissodes (Krom-b e i n and Burks 1967). Pissodes and Cylindrocopturus were both reared from the same c o l l e c t i o n s i n t h i s study. Many collembolan species are herbivorous but have o f t e n been found a s s o c i a t e d w i t h f u n g i , and i t i s not unreasonable to assume that the c o r t i c a l species l i v e on fun g a l hyphae and spores (Graves i 9 6 0 ) . P i e l o u and Matthewman (1966) recorded s e v e r a l Collembola species from bracket f u n g i i n Quebec. The C o r r e n t i a were mostly from r e a r i n g s and form an important group of mycetophages, as Graves ( i 9 6 0 ) found i n fun g a l conks.. Gnophothrips fuscus (Morgan) has been reported damaging pine species i n a number o f areas ( E b e l 1961,- 1965a; L i n d q u i s t and Harn-den 1957; O ' N e i l l I965). G. fuscus were a l s o reared from a P. s t a l a c t i -forme canker i n a companion study of i n s e c t s a s s o c i a t e d w i t h other Cronartium cankers. Stannard (1957) pointed out th a t t h r i p s of the fam-i l y P h l o e o t h r i p i d a e , to which G. fuscus belongs, feed e x t e n s i v e l y on spores of f u n g i , thus cankers of Cronartium are a l i k e l y h a b i t a t f o r t h i s species. The L a e l a p t i d a e mites are u s u a l l y p a r a s i t e s on v e r t e b r a t e s and - 12k -i n v e r t e b r a t e s , and the B d e l l i d a e are predaceous on mites and s m a l l i n -sects such as Collembola (Graves i960) . The Eupopidae and Tydeidae ap-peared to be fungus feeders. The Anystidae are predaceous on mites and small i n s e c t s , and the Erythraeidae are p a r a s i t e s of i n s e c t s , although the a d u l t s are f r e e - l i v i n g predators (Baker and Wharton 1952). The Acaridae l i v e on a l l kinds;-of organic m a t e r i a l , some f a i r l y exclusively on f u n g i (Baker and Wharton 1952). The o r i b a t i d mites are l a r g e l y found i n s o i l d e bris where they feed upon organic matter, i n c l u d i n g f u n g i (Graves i960), although l i t t l e i s known of the i n d i v i d u a l species h a b i t a t s . A number of d i f f e r e n t species of rodents are b e l i e v e d to eat the i n f e c t e d l i v i n g bark from Cronartium r u s t cankers. They-have not „ a l l been observed, as many rodents are s t r i c t l y n o c t u r n a l , and most ob-servat i o n s are made during the day. S q u i r r e l s are regarded as the most important i n f e c t e d bark removers, and have been reported by a la r g e num-ber of people (Hedgcock and Hunt 1920; Hubert 1935b, Mielke 1935, 1956; Peterson i960; Rhoads 1920; S n e l l 1929a; Spaulding 1918, 1922, 1929; S t i l l i n g e r l^kk). Mielke (1935) l i s t e d many examples of s q u i r r e l damage that occurred on C. r i b i c o l a cankers, and were l a r g e l y reported i n "The B l i s t e r Rust News" from 1928 to 1933- Damage by porcupines was mentioned by Pennington ( c i t e d i n Spaulding 1922), Hedgcock and Hunt (l<920\ Hubert (1935b) and Mielke (1935, 1957), and s e v e r a l of these authors r e f e r r e d to damage r e s u l t i n g from mice and r a b b i t s . Mielke (1935) mentioned chip-munks and the p o s s i b i l i t y o f a p i k a . Rodent feeding on cankers g e n e r a l l y occurred during winter and e a r l y s p r i n g and was u s u a l l y r e s t r i c t e d to the l i v i n g i n f e c t e d t i s s u e s of the canker. There are s e v e r a l r e p o r t s of the incidence of rodent damage on C. r i b i c o l a cankers from both eastern and - 125 -western North America. Gravatt and Posey ( c i t e d i n Mielke 1935) reported that 17% of the cankers had been p a r t i a l l y eaten o f f at K i t t e r y P o i n t , Maine, and 75% of these had kO to 100% of the bark removed. Pennington ( c i t e d i n Spaulding 1922) estimated that the production of aeciospores i n the Adirondacks was reduced about 15% by the e a t i n g of i n f e c t e d bark. S n e l l (1929a) i n New York S t a t e , found that 1+1% of 11,000 cankers had been gnawed by rodents and that the e n t i r e crop of p y c n i a was consumed i n some l o c a t i o n s . P e r r y ( c i t e d i n Mielke 1935) estimated t h a t the f r u i t -i n g area of cankers was reduced by 95% at Pembroke, Massachusetts i n 1928. At Cheekye, B r i t i s h Columbia, Lachmund ( c i t e d i n Mielke 1935) found 1+5.2% of the cankers were gnawed to some degree i n 192k, and tha t 25% of the aecia-bearing bark was removed. He a l s o noted that o l d e r cankers, which had sporulated f o r more than one year, had a greater incidence of chewing. I n a l i g h t i n f e c t e d stand near Revelstoke the number of cankers gnawed i n -creased from 13.5% i n 1931, to 25% i n 1933- Mielke (1935) estimated that rodents removed 10 to 35% of the aecia-bearing bark i n the ol d e r i n f e c -t i o n areas of the west. S t i l l i n g e r (l9kk) reported that the Richardson red s q u i r r e l chewed 28% of 1575 cankers and 38% of 10,360 C. r i b i c o l a can-kers observed at two l o c a t i o n s i n Idaho. Mielke (1956) found only a few small cankers of P. s t a l a c t i f o r m e , i n many thousands observed, that had not had zones o f i n f e c t e d bark removed annually by rodents. He reported a margin or ri d g e of dry and hardened bark on the P. s t a l a c t i f o r m e can-kers which was not u s u a l l y fed upon, as observed on many C. comandrae can-ke r s . Anderson et a l . (1967) reported incidences where 37 to 50% of the P. s t a l a c t i f o r m e cankers had rodent feeding, and i n many instances the feeding appeared to have prevented f u r t h e r development of the cankers. - 126 -Mielke (1957) reported rodent damage on C. comandrae cankers on lodgepole pine, and some r e p o r t s of severe porcupine damage a s s o c i a t e d w i t h t h i s r u s t . He f u r t h e r observed t h a t the v a r i o u s rodents causing the damage tended to congregate more or l e s s w i t h i n the diseased stands. C h i l d s (1968) r e f e r r e d to o v a l gnawed areas centered at the branch where C. comandrae i n f e c t i o n s t a r t e d on Pinus ponderosa. K r e b i l l (1965), i n a study i n 12 N a t i o n a l Forests i n the Rocky Mountain States area, found over 90$ °f the sampled C. comandrae cankers had been scarred by r o -dent chewing. C o r d e l l et a l . (1967) found that over 75$ of the f r u i t i n g _C. comandrae cankers i n a P. taeda p l a n t a t i o n had been chewed, and i n many cases the cankers were completely gnawed o f f . Peterson ( i 9 6 0 ) r e -ported the gnawing of P. h a r k n e s s i i g a l l s , and t h e i r removal from s m a l l branches by s q u i r r e l s , chipmunks and porcupines. I n the study area, and e s p e c i a l l y near l o c a t i o n s 2 and 5, large numbers of P. s t a l a c t i f o r m e can-kers were e x t e n s i v e l y gnawed by rodents each year, and l i g h t e r damage was observed on P. h a r k n e s s i i cankers. A number of references mention r e s i n exuding abundantly from cankers of C. comandrae (Mielke 1957, I 9 6 I ; Mielke et a l . 1968; Peterson and K r e b i l l 1967), e s p e c i a l l y on l a r g e r stem i n f e c t i o n s . Some r e f e r to the a d d i t i o n a l r e s i n flow associated w i t h rodent damage on the canker, but there i s no quantitative data on the incidence. K r e b i l l (1968a) found some s m a l l patches of r e s i n o s i s i n the bark and wood i n f e c t e d by C_. com-andrae but d i d not b e l i e v e t h i s was s u f f i c i e n t to impair the conducting a b i l i t y o f the xylem. Mielke (1961) noted that r e s i n o s i s was l e s s marked on C. comandrae cankers on Pinus ponderosa than on P. contorta. Mielke (I956) mentioned t h a t impregnation of the wood by r e s i n u s u a l l y occurred i n P. s t a l a c t i f o r m e cankers. Spaulding (1929) reported t h a t abundant - 127 -r e s i n p r oduction i s a good symptom of the disease caused by C_. r i b i c o l a . C o l l e y (1918) described the manner i n which r e s i n canals were broken w i t h the consequent exudation of r e s i n i n large q u a n t i t i e s . He i n d i -cated that the r e s i n impregnated the whole cortex and phloem of the cracked C. r i b i c o l a canker area, r e s u l t i n g i n stoppage of the conducting elements i n the phloem hastening the g i r d l i n g of the t r e e . H i r t (1964) suggested th a t the increase i n number and s i z e of the e p i t h e l i a l c e l l s adjacent to vegelative hyphae i n i n f e c t e d bark, which tend to f i l l the r e s i n canals, may p a r t i a l l y account f o r the abundance of r e s i n a s s o c i a t e d w i t h C_. r i b i -c o l a cankers. The impregnation of the bark and wood around the canker prevents d e s i c c a t i o n of the sapwood, and hinders the growth of the fungus, so that complete g i r d l i n g of the t r e e i s g r e a t l y delayed. - 1 2 8 -AECIOSPORE DISPERSAL This s e c t i o n of the study aims: ( l ) to e s t a b l i s h the d a i l y and seasonal p e r i o d i c i t y and concentration of aeciospore rele a s e , from i n d i -v i d u a l cankers, and to r e l a t e t h i s to v a r i o u s m e t e o r o l o g i c a l parameters; (2) to e s t a b l i s h the n a t u r a l p a t t e r n and gradient of aeciospore d i s p e r s a l and d e p o s i t i o n around i n d i v i d u a l cankers, and the distance of d i s p e r s a l i n the f o r e s t ; (3) to e s t a b l i s h from experimental t e s t s the aeciospore concentration p a t t e r n and distance o f d i s p e r s a l i n the open from two source r e l e a s e heights under d i f f e r e n t wind v e l o c i t i e s and to r e l a t e t h i s to d i f f u s i o n theory; and (k) to e s t a b l i s h the r a t e of f a l l of aeciospores i n s t i l l a i r , a f a c t o r which i s important i n understanding d i s p e r s a l d i s -tance and spore d e p o s i t i o n . METEOROLOGICAL FACTORS AFFECTING DISPERSAL Methods and M a t e r i a l s Two areas were instrumented w i t h a number of m e t e o r o l o g i c a l instruments to re c o r d the various m e t e o r o l o g i c a l parameters close to the ground w i t h i n the f o r e s t stands. A v a r i e t y of spore c o l l e c t o r s were set up i n the same areas to re c o r d the numbers, periods and distance o f spore d i s p e r s a l . Experimental S i t e s I n I96U an area was instrumented at 5,000 f e e t i n a dense 25 year o l d lodgepole pine stand on the north slope of The Wedge and on the south side of Evans-Thomas Creek, 50°53' N, U5 ° 0 9 ' W, ( l o c a t i o n 2, F i g . - 129 -3 8 ) . This s i t e was r e l a t i v e l y l e v e l as i t formed p a r t of an o l d r i v e r t e r r a c e , but on the west and north the t e r r a c e sloped s t e e p l y towards the Kananaskis R i v e r , and Evans-Thomas Creek. The s i t e was l a r g e l y abandoned i n I965 f o r t h i s aspect of the study, except f o r the mainten-ance of a few weekly r e c o r d i n g m e t e o r o l o g i c a l instruments and weekly ob-ser v a t i o n s on the development of the r u s t cankers, which was continued through to I968. Operation of spore c o l l e c t o r s on t h i s s i t e i n 1964 i n -volved the use of s i x and 1 2 . v o l t b a t t e r i e s , which f o r s a t i s f a c t o r y op-e r a t i o n were changed d a i l y , and i n the case of the H i r s t spore trap changed twice d a i l y f o r continuous o p e r a t i o n . I n 1964 a few instruments were run near the shore of B a r r i e r Lake, 51°02' W, 115°Q2' W, ( l o c a t i o n 1, F i g . 38) , i n an area of minor un-d u l a t i n g r e l i e f at about 4,550 f e e t , w i t h f a i r l y open grown lodgepole pine of uneven age. I n the p e r i o d 1965 to 1968 t h i s s i t e became the main e x p e r i -mental area and was f u l l y instrumented ( F i g s . 46 to 53) . Many of the spore c o l l e c t o r s , and the wind and some dew r e c o r d i n g instruments, were opera-ted from a temporary 1 1 0-volt power l i n e from the Kananaskis•Forest Ex-periment S t a t i o n . A sketch showing the area and the p o s i t i o n s o f the instruments and i n f e c t e d t r e e s used i n the study i s shown i n F i g . 39- In a d d i t i o n to the t r e e s shown i n F i g . 39? "two other t r e e s (#2710 and 271 l ) were observed near l o c a t i o n 1. These were on a k n o l l , 1600 f e e t to the east of t r e e #2720, where a hygrothermograph and rain-gauge were maintained i n a l l the summers, and a spore c o l l e c t o r was operated next to #2710 i n 1964, I965 and I967. The number of i n d i v i d u a l instruments and c o l l e c t o r s used v a r i e d from year to year depending on the number of cankers used to observe d a i l y F i g . 46. View of s i t e no. 2 at study l o c a t i o n 1, showing a 24-hour impaction spore c o l l e c t o r by Cronartium comandrae canker no. 2721 on a s m a l l Pinus contorta, and i n s t r u -ments f o r r e c o r d i n g the weather. Instruments are, from l e f t to r i g h t , b l ack porous d i s c atmometer, instrument s h e l t e r c o n t a i n i n g hygrothermograph, Wallin-Polhemus dew d u r a t i o n recorder on ground, mast w i t h anemometer cups f o r wind speed recorder and spore c o l l e c t o r . (Rain gauge i s out of the p i c t u r e ) F i g . 47. Standard Stevenson screen i n an opening, c o n t a i n i n g hy-grothermograph and thermometers used as reference weather . s t a t i o n at study l o c a t i o n 1, w i t h a b i - m e t a l actinograph f o r r e c o r d i n g incoming r a d i a t i o n seen at the back. A r e c o r d i n g r a i n gauge and a wind d i r e c t i o n recorder (out of the p i c t u r e ) were maintained at t h i s open s i t e . F i g . 48. A H i r s t spore trap w i t h sampling o r i f i c e one foot above ground, close to a Comandra umbellata p l o t . F i g . 49. Seven-day p o l l e n sampler of the Sarvas type, used to c o l l e c t aeciospores at set distances from s p o r u l a t i n g Cronartium comandrae cankers. Spores, pass through the sampler o r i f i c e and are deposited on a v a s e l i n e - c o a t e d sampling band placed around a c l o c k - d r i v e n drum housed w i t h i n the intak e c y l i n d e r . - 131 -spore d i s p e r s a l , and the requirements f o r s a t i s f a c t i o n of the v a r i o u s aspects of the study. Spore c o l l e c t o r s F i v e types of spore c o l l e c t o r s were used over a p e r i o d of f i v e seasons. The e f f i c i e n c y and usefulness o f each c o l l e c t o r type v a r i e d considerably. One " C a s e l l a " model of the H i r s t spore trap ( H i r s t 1952) and f i v e weekly p o l l e n c o l l e c t o r s of a design s i m i l a r to Sarvas (1952) proved un-s a t i s f a c t o r y f o r o b t a i n i n g d e t a i l e d i n f o r m a t i o n on the d i u r n a l d i s p e r s a l of aeciospores. This type of wind d i r e c t i o n a l recorder cannot be placed close to a s p o r u l a t i n g . r u s t canker, consequently they were only used to gain i n f o r m a t i o n on di s t a n c e of spore d i s p e r s a l . I n 1964 the H i r s t spore trap was operated i n a s m a l l c l e a r i n g at l o c a t i o n 2 approximately k2 f e e t from the nearest s p o r u l a t i n g cankers. From 1965 to 1967 the trap was operated at l o c a t i o n 1 throughout the s p o r u l a t i o n p e r i o d near a patch of Comandra p l a n t s and 3**- f e e t from the nearest i n f e c t e d pine t r e e ( F i g . k8). The tr a p sampled a i r at the r a t e of 10 l i t e r s / m i n u t e through the operation o f a sm a l l separate e l e c t r i c a i r s u c t i o n pump. The o r i f i c e was one foot above ground and was c o n t i n -uously d i r e c t e d i n t o the wind. The microscope s l i d e s were prepared f o r exposure according to the method described by H i r s t (1953), and were changed d a i l y between 0845 and 0900 M.S.T. The s l i d e was moved past an o r i f i c e , 2 mm wide, at a r a t e of 2 mm per hour, thus h o u r l y deposits of spores throughout a 2k hour period, were obtained. The s l i d e s were scanned under a stereomicroscope w i t h a 50x power, and the number o f spores de-p o s i t e d on each 2 mm i n t e r v a l or band were counted. - 132 -The c l o c k - d r i v e n p o l l e n c o l l e c t o r s ( F i g . 1+9) were i d e n t i c a l t o those used by E b e l l and Schmidt (1964, p. 4-5) i n t h e i r study of p o l l e n d i s p e r s a l on Vancouver I s l a n d . The spores were deposited on a v a s e l i n e -coated c e l l u l o i d sampling band which was placed round the drum of an ;8-day clock enclosed i n a removable c y l i n d e r which allowed f o r the passage of a i r through the o r i f i c e around the drum and out of a hole i n the r e a r of the c y l i n d e r . The sampler i s wind d i r e c t i o n a l and i n the study was run one f o o t above ground. The c e l l u l o i d band had v e r t i c a l l i n e s p r i n t e d on i t at 2 hour i n t e r v a l s . Approximately 2 hours of the band were ex-posed at one time. These p o l l e n samplers were operated at v a r i o u s i n t e r v a l s during the s p o r u l a t i o n p e r i o d . o f I965, 1966 and I967, and at v a r i o u s p o i n t s removed from s p o r u l a t i n g cankers, i n an e f f o r t to o b t a i n distance of spore d i s p e r s a l data. Impaction spore c o l l e c t o r s of a design s i m i l a r to Panzer e_t a l . (1957) were used i n 1964. They could be placed close to a s p o r u l a t i n g canker, but were not wind d i r e c t i o n a l . These c o l l e c t o r s had some d i s a d -vantages; a low spore c o l l e c t i o n e f f i c i e n c y , a non-continuous r e c o r d of c o l l e c t i o n f o r a 24 hour p e r i o d , and were time consuming i n changing 24 s l i d e s d a i l y , e s p e i c a l l y during inclement weather when the chances of contamination were increased. Some features of the b a s i c design of the Panzer e_t a l . model were modified i n the winters of 1964 and 1965, and a model was developed which proved e n t i r e l y s a t i s f a c t o r y f o r . t h e d a i l y c o l l e c t i o n of dispersed spores (Powell and Morf 1967? and F i g s . 50 and 5 l ). The fan of the impaction spore c o l l e c t o r was operated at a c o n t r o l -l e d 12 v o l t s i n 1965, 1966 and 1967, through a transformer r e c t i f i e r u n i t powered by a 110 v o l t power l i n e . To improve on the c o l l e c t i o n and F i g . 50. A 24-hour impaction spore c o l l e c t o r , w i t h the sealed l i d removed to show the a c r y l i c p l a s t i c d i s c h o l d i n g 24 mi-croscope s l i d e s upon which spores are deposited, and the fan f o r drawing a i r through the box at a c o n t r o l l e d r a t e F i g . 51- A 24-hour impaction spore c o l l e c t o r i n operation, w i t h i t s sampling o r i f i c e placed close to the s p o r u l a t i n g surface of a Cronartium comandrae canker. F i g . 52. C e n t r a l instrument power and r e c o r d i n g box, w i t h Thorn-thwaite f o u r - u n i t wind speed r e g i s t e r recorder ( l e f t ) , wind d i r e c t i o n recorder, and d i g i t a l p r i n t o u t recorder w i t h p o l a r o i d camera f o r wind speed recorder system ( r i g h t ) . . Behind are the transformers and r e c t i f i e r s f o r reducing the 110 v o l t power supply and f o r c o n t r o l l i n g an output of 12 v o l t s f o r operation of wind instruments, dew instruments and 24-hour impaction spore c o l l e c t o r s . A standby 12 v o l t b a t t e r y i s a l s o present. F i g . 53. Set of anemometer cups of.the Thornthwaite wind speed r e g i s t e r recorder system operated at canker height near a s p o r u l a t i n g Cronartium comandrae canker. - 133 -- 13h -r e t e n t i o n e f f i c i e n c i e s of the rPanzer et a l . c o l l e c t o r our model was designed to increase the volume of a i r sampled to 13 tl l i t e r per minute (O.78 m /hr.) through the use of a l a r g e r diameter sampling o r i f i c e ( 0 . 8 cm diameter, compared to a O.k cm diameter). A l s o the gaps between the 2k microscope s l i d e s of the Panzer et a l . model were e l i m i n a t e d by a r -ranging the s l i d e s side by sid e , held together along the bottom by a s t r i p of masking tape w i t h an overlap of tape at each end f o r a t t a c h i n g to a p l a s t i c holder. The s l i d e s were prepared i n the l a b o r a t o r y and' attached to spare holders which f a c i l i t a t e d the d a i l y change at approxi-mately 0900 hours; holders were changed i n the same order each day. The prepared s l i d e s and holders were c a r r i e d to the f i e l d i n dust proof boxes which f u r t h e r minimized the chance of contamination. I n 196I+ two c o l -l e c t o r s of t h i s type were operated at l o c a t i o n 1 and four at l o c a t i o n 2 . From 1965 to 1967, three to s i x c o l l e c t o r s . w e r e used each year at l o c a -t i o n 1 f o r the d u r a t i o n of aeciospore s p o r u l a t i o n . The same four cankers at l o c a t i o n 1 were used i n three of the four years, although they were not the same years. The same trees could not be used each year because the t r e e had died or because of la c k of s p o r u l a t i o n i n some seasons. Counts were made of the t o t a l number of spores deposited on 5 . ^ cm of each s l i d e , and no c o r r e c t i o n was a p p l i e d f o r the e f f i c i e n c y of the trap which depended on wind speed. The e f f i c i e n c y of the t r a p was un-• known, although i t was apparently good. I t was i m p r a c t i c a l to scan a l l s l i d e s d a i l y but during i n i t i a l and c e s s a t i o n spore periods s l i d e s were examined soon after- exposure. A f t e r long storage spores tended to lose c o l o r and counts at these times were probably underestimated, although the o v e r a l l p i c t u r e was probably not changed. Spore t r a p p i n g u s u a l l y - 135 -s t a r t e d j u s t before rupture of the a e c i a which was e s t a b l i s h e d through frequent v i s i t s to the i n f e c t e d t r e e , and continued f o r two to three months u n t i l no spores were found on s l i d e s exposed on three consecutive days and when macroscopic examination of the canker revealed no a c t i v e s p o r u l a t i n g a e c i a . O c c a s i o n a l l y spore t r a p p i n g was stopped because of mechanical f a i l u r e , or f o r placement of the tr a p on a d i f f e r e n t s p o r u l a -t i n g canker. Another simple wind d i r e c t i o n a l p l a s t i c c o l l e c t o r was designed i n 1966 f o r the d i s p e r s a l experiments i n v o l v i n g the r e l e a s e of spores from a f i x e d p o i n t (see page 175), and were al s o used i n 1966, 1967 and I968 to catch spores at set distances (5 to 100 f e e t ) from s p o r u l a t i n g cankers. These c o l l e c t o r s were mounted at one foot above ground, or at 1, 5 and 10 f e e t above ground on a supporting stand. S l i d e s were ex-posed f o r 2k hour periods being changed between 0800 and 0830 hours. One or two ( i n I967 and 1968) r o t o r o d samplers of a modified type, s i m i l a r to that described by Per k i n s (1957) were used to catch spores on a d a i l y b a s i s at set distances from s p o r u l a t i n g cankers and during the d i s p e r s a l experiments. Rotorod samplers c o l l e c t e d spores by impaction on a p a i r of small c l e a r p l a s t i c c o l l e c t o r rods which were hel d i n a s p e c i a l l y designed U-shaped holder and r o t a t e d at a constant speed of 2k00 r.p.m. by means o f a'miniature battery-operated e l e c t r i c motor. The l i g h t l y v a s e l i n e coated surfaces of the c o l l e c t o r rods sam-p l e d at an average r a t e of 60 l i t e r s of a i r per.iminute. This impaction c o l l e c t o r had a high r e t e n t i o n and e f f i c i e n c y l e v e l , i n excess o f 90% (Metronics A s s o c i a t e s , 1966). The ro t o r o d samplers were operated on a sma l l t r i p o d at approximately 3 f e e t above the ground. I n most cases the c o l l e c t o r rods were exposed f o r 2k hours being changed between 0800 - 136 -and 0830 hours. M e t e o r o l o g i c a l instruments Temperature and r e l a t i v e humidity were recorded at both s i t e s by weekly r e c o r d i n g Fuess hygrothermographs operated i n standard Steven-son screens or modified instrument s h e l t e r s ( F i g s , 46 and 4 7 ) . I n both areas one was operated at standard height (4-1/2 f e e t ) and up to f i v e others were operated close to and at the height o f the cankers (between 6 inches and 1 foot above ground). The instruments at l o c a t i o n 1 were checked d a i l y , and standard c e r t i f i e d maximum and minimum thermometers . .r were maintained at some s t a t i o n s as a check against the performance of the temperature sensor of the hygrothermograph. The r e l a t i v e humidity sen-sor of the recorder was checked at r e g u l a r i n t e r v a l s by readings from a s l i n g psychrometer. A l l instruments, i n c l u d i n g c l o c k s , were c a l i b r a t e d at the beginning and end of the f i e l d season over a range of temperature and moisture c o n d i t i o n s as a f u r t h e r check on the accuracy of t h e i r op-e r a t i o n . P r e c i p i t a t i o n i n t e n s i t y and d u r a t i o n were recorded by two C a s e l l a n a t u r a l siphon weekly r e c o r d i n g rain-gauges, one at each l o c a t i o n . A s e r i e s of p l a s t i c wedge-shaped and MSC ( M e t e o r o l o g i c a l S e r v i c e of Canada) standard non-recording rain-gauges were a l s o placed throughout the study area at l o c a t i o n 1 and attended on a d a i l y b a s i s . Huff (1955) showed that the p l a s t i c wedge-shaped gauge, w i t h a 2.5 by 2.3 i n c h r e c t a n g u l a r o r i f i c e , was s a t i s f a c t o r y i n i t s performance when compared w i t h an 8 i n c h U.S. Weather Bureau standard gauge. Four types of dew d u r a t i o n or leaf-wetness recorders were used at l o c a t i o n 1 f o r v a r y i n g p e r i o d s , none of which were 100$ s a t i s f a c t o r y , - 137 -but y i e l d e d s u f f i c i e n t dew and l e a f wetness i n f o r m a t i o n f o r short p e r i o d a n a l y s i s . One Wallin-Polhemus dew d u r a t i o n recorder ( W a l l i n and Polhemus 195*1-) employing a lambs-gut or cellophane s t r i p sensor was operated on the ground at one canker s i t e (#2721 - F i g . k6) over the p e r i o d 1965 to I967. Two types of ground glass dew d u r a t i o n recorders were used i n 1965, one, a 7-day recorder of a type s i m i l a r to th a t described by Theis and Calpouzos (1957), and the other, a d a i l y recorder modified from Taylor (1956) by Dr. A. K. Parker, Department of F i s h e r i e s and F o r e s t r y , V i c t o r i a , B r i t i s h Columbia, and used i n h i s s t u d i e s of the Rhabdocline needle cast on Douglas f i r . An e l e c t r i c a l g r i d wetness recorder system w i t h four sensors was developed, employing two p a r a l l e l s t r i p s of metal set i n a p l a s t i c d i s c , and attached to a Rustrak four-channel on-off recorder. The sensors were placed close to s p o r u l a t i n g cankers and up to 500 f e e t from the recorder. When moisture was deposited on the d i s c the c i r c u i t between the two metal s t r i p s was completed and the recorder a c t i v a t e d to the "on" p o s i t i o n . The p e r i o d of contact denoted the length of the wet p e r i o d . A grass minimum thermometer was operated i n the open at 6 inches above the ground which gave a d d i t i o n a l i n f o r m a t i o n on the d a i l y occurrence of dew and minimum temperatures at t h i s height. F i v e atmometer assemblies were run i n 1965, 1966 and 1967 at location' ;.l, to o b t a i n estimates of the d a i l y amounts of evaporation. Two were the " s h i e l d e d p l a s t i c mount" type, which employed the b l a c k B e l l a n i p l a t e as an evaporating surface, and three were the "black porous d i s c " type, an assembly which f u n c t i o n s on the p r i n c i p l e of the Piche atmometer. Carder ( i 9 6 0 ) compared both, f i n d i n g that the "black porous d i s c " had s e v e r a l advantages over the other. Two "black porous d i s c " atmometers were operated i n the open, and the others w i t h i n the - 138 -stand, near i n f e c t e d t r e e s . The amount .of water evaporated by each a t -memeter ( i n c . c ) , was recorded d a i l y between 0815 and 08^5 hours. A C a s e l l a model of the improved R o b i t z s c h b i - m e t a l actinograph ( F i g . 1+7) was run d a i l y i n 19&5, 1966 and 1967 i n an open c l e a r i n g at l o c a t i o n 1, and i n 196k at l o c a t i o n 2, to give estimates of the g l o b a l (sun plus sky) r a d i a t i o n , measured i n cal/cm^/min, and i n f o r m a t i o n on the periods o f daytime cloud cover over the study area. Wind speeds were recorded by a Thornthwaite Four-unit Wind R e g i s t e r System w i t h a D i g i t a l P r i n t o u t Recorder ( F i g . 52) i n 1965 and I966 and i n t e r m i t t e n t l y i n I967 at l o c a t i o n 1. The four anemometers ( F i g . 53) were placed close to and at the height of four s p o r u l a t i n g cankers where there were spore c o l l e c t o r s , to o b t a i n i n f o r m a t i o n on wind speed at the height of spore r e l e a s e . Wind d i r e c t i o n was recorded at one p o i n t c e n t r a l to the study area at a height of k f e e t by a Thornthwaite Wind D i r e c t i o n Recording System, ( F i g . 52) to o b t a i n the p a t t e r n of a i r move-ment over the study area. This wind d i r e c t i o n recorder was s i t u a t e d on the southwest side of a s l i g h t k n o l l exposed to the p r e v a i l i n g southwest winds, and was 60 f e e t from the nearest anemometer. Wind d i r e c t i o n at the canker height was probably s i m i l a r to th a t at the h foot r e c o r d i n g height. Supplementary wind speed and d i r e c t i o n i n f o r m a t i o n during I965 to 1968 was obtained from a M e t e o r o l o g i c a l Branch, Department of Trans-p o r t , recorder operated at k8 f e e t at the Kananaskis S t a t i o n and at a distance of approximately 330 f e e t from the anemometer near canker #2721 on the study area. Time of day reference was made throughout to Mountain Standard Time (M.S.T.). - 139 -R e s u l t s D i u r n a l p e r i o d i c i t y of m e t e o r o l o g i c a l f a c t o r s and microclimate of study l o c a t i o n 1 Most m e t e o r o l o g i c a l f a c t o r s show marked d i u r n a l rhythms, namely l i g h t , a i r temperature and humidity, and wind v e l o c i t y ( c f . F i g s . 58-60, Table XVI). Of these, l i g h t i s the most constant but i t s Inten-s i t y v a r i e s through the presence of haze or cloud. The d i u r n a l tem-perature p a t t e r n r a r e l y departs from a daytime maximum and a nighttime minimum. S i m i l a r l y , r e l a t i v e humidity i s low during the day and high at night, but t h i s rhythm i s o f t e n upset when high h u m i d i t i e s p e r s i s t dur-i n g the daytime, or low h u m i d i t i e s during the night (MacHattie 1966). The d i u r n a l p e r i o d i c i t y of wind i s o f t e n l e s s obvious, but when averaged over r e l a t i v e l y long.periods there i s greater wind v e l o c i t y during the daytime than during the nighttime. Table XVI summarizes the d i u r n a l p a t t e r n of h o u r l y temperature, r e l a t i v e humidity and wind speed f o r two of the s t a t i o n s s i t u a t e d near s p o r u l a t i n g cankers, 0.6 to 1 f t above the ground, f o r the p e r i o d May 17 to J u l y 11, 1966. The temperature and r e -l a t i v e humidity data are compared w i t h records from the standard Bay s t a t i o n s i t u a t e d i n an opening at 4.5 f t above ground and 100 f t from #2712. The wind speed records are compared w i t h those taken at 48 f t at the Kananaskis s t a t i o n . The two s t a t i o n s (#2712 and 2721) represent the extremes of the c o n d i t i o n s recorded 0.6 to 1 f t above the ground at l o c a t i o n 1. M e t e o r o l o g i c a l c o n d i t i o n s at s t a t i o n #2713 were very s i m i -l a r to c o n d i t i o n s recorded at #2712, although those at the l a t t e r were s l i g h t l y more extreme as t h i s s t a t i o n was s i t u a t e d on the edge of an open area. Conditions at #2724 were intermediate between #2713 and #2721, Table XVI. The hourly mean temperature, relative humidity and wind speed at two canker stations, compared with'records from the Bay station for temperature and humidity, and from Kananaskis for wind speed, for the period May 17 to July 11, 1966. Bay #2712 #2721 Ht. of sensor Hour of day Daily (ft) 1 2 3 U 5 6 7 8 9 10 11 12 13 ll* 15 16 17 18 19 20 21 22 23 2U aver TEMPERATURE °C 10.5 9-9 9-7 9. 1 * 9-2 9-7 11.I* 13.5 15.3 16.8 17. •* 18.U 18.8 19.2 19.1 18.8 18.3 17.6 16.3 15.2 13.8 12.8 11.9 11.3 1U.3 0.6 9 . 6 9-1 9 . 0 8 .6 8 . 3 8 .2 9-8 12.1 H*.3 l"l.9 16.1 17.0 17.9 18.3 19.1 18.7 17.7 17.2 16.5 15.1 13.** 12.3 11.3 10.1* 13.5 0 . 6 8 .5 8.2 7 . 8 7.1+ 7 .3 7 .6 9-7 11.5 ll*.2 16.5 17.5 18.2 18.6 18.5 18.8 18.3 17.6 17.0 15.7 13.9 12.5 11.1 10.3 9-2 13.2 RELATIVE HUMIDITY %, Bay 1*.5 77 80 81 82 82 78 70 63 57 51* 51 1*8 1*8 1*7 1*7 1.8 50 51 55 58 61* 69 73 75 63 #2712 1.0 77 80 82 83 85 86 79 68 58 52 1*8 1*5 1*2 1*1 1*0 1*1 U3 1*7 51 53 61 61* 70 71* 61 #2721 1.0 81 82 83 8U 85 86 81 70 59 5"* 1*9 1*7 1*6 1*1* 1*5 1*5 1*7 1*8 52 59 65 71 75 78 61* WIHD SPEED m.p.h. -Kananaskis 1*8.0 3.8 3.9 U.O 3.7 3.5 3.3 3.1* 3.6 5.3 6.1* 7.2 8.0 7.9 8.3 8.7 8.9 8.1* 8.0 7.8 6.8 6.1 5.1 "*.5 3.9 5.8 #2712 1.0 1.1* 1.1* 1.1* 1.1* 1.3 1.3 1.5 1.7 2 . 0 2.1* 2.5 2.6 2.6 2.7 2.8 2.7 2.6 2.3 2.1 1.9 1.7 1.6 l .l* l.l* 1.9 #2721 1.0 1.2 1.2 1.2 1.1 1.1 l . l 1-3 l . l* 1.7 1.8 2.0 2.2 2.3 2.3 2.2 2.1 2.2 1.9 1.8 1.6 1.5 l . l* 1.2 1.1 1.6 H -F-O - l420°C). Unf o r t u n a t e l y , on the night when r e l a t i v e h u m i d i t i e s d i d not exceed horfo and temperatures one foot above the ground d i d not f a l l below l8°C ( J u l y 9/l0, I966), t o t a l numbers of a v a i l a b l e spores were very low. E f f e c t s o f r a i n f a l l F i g . 59 would tend to confirm t h a t there i s l i t t l e or no spore d i s p e r s a l when h u m i d i t i e s remain high or when temperatures remain low. The two main r a i n p e r i o d s , June 12-19 and June 25-29, 19^5, when humid-i t i e s remained high were periods w i t h no d i s p e r s a l , but during dry con-d i t i o n s when the d i u r n a l temperature and humidity p a t t e r n was maintained there was a daytime d i s p e r s a l peak. However,.Fig. 60 which i l l u s t r a t e s two wet periods i n 1966, does not show the same r e l a t i o n s h i p . I n these periods there was considerable d i s p e r s a l during r a i n . The onset o f heavy r a i n on May 30 and 31 increased the spore concentration which on May 31 F i g . 59- Hourly number of aeciospores c o l l e c t e d from Cronartium comandrae canker no. 2516 during the p e r i o d June 7 to J u l y 3, 1965, r e l a t e d to h o u r l y a i r temperature, r e l a -t i v e humidity, r a i n f a l l , incoming r a d i a t i o n and wind speed. - 151 -F i g . 60. Hourly number of aeciospores c o l l e c t e d from Cronartium comandrae canker no. 2721 during the p e r i o d May 29 to June 9> 1966, r e l a t e d to a i r temperature, r e l a t i v e humidity, r a i n f a l l , incoming r a d i a t i o n and wind speed. - 153 -was maintained f o r s e v e r a l hours. I n these cases the e f f e c t s o f r a i n splash c r e a t i n g r a d i a l a i r shock waves w i t h t u r b u l e n t currents and the composite e f f e c t of r a i n d r o p l e t s h i t t i n g the dry spores were important f o r d i s p e r s a l . H i r s t and Stedman (1963) term them " r a i n p u f f " and "r a i n tap" processes. The r e s u l t i n g peak concentrations were o f t e n s e v e r a l times those o c c u r r i n g i n dry weather, 6 of the 9 l a r g e s t d a i l y - h o u r l y maximum canker concentrations i n 1966 were as s o c i a t e d w i t h r a i n , u s u a l l y the onset. The heavy 0.2k i n c h r a i n f a l l t h a t occurred at:1800 hours on May 30, 1966, a time outside the normal peak spore d i s p e r s a l p e r i o d , brought about a 23 and 25. times increase i n the h o u r l y dispersed spore concentration from two cankers, and t h i s was k and 5 times l a r g e r than the 'normal' peak which occurred around Q900 hours on th a t day. Wight r a i n caused s i m i l a r high spore d i s p e r s a l concentrations on some occasions, at a time when i n f e c t i o n c o n d i t i o n s were more favourable and had more chance of p e r s i s t i n g than during the day. L i g h t showers or steady d r i z -z l e d i d not have the same e f f e c t on d i s p e r s a l as heavy rainstorms, pro-bably because there i s l e s s turbulence and shaking of the i n f e c t e d t i s -sues, and because the a e c i a and t h e i r aeciospores g r a d u a l l y become wet thus preventing d i s p e r s a l . A l i g h t shower tended to i n t e r f e r e w i t h the d a i l y d i s p e r s a l rhythm to a c e r t a i n degree (eg. June 9? 1966, at 0700 and at..1200 hours. F i g . 6 0 ) , but o f t e n the normal d i u r n a l p a t t e r n was resumed w i t h i n two or three hours of the shower. With prolonged r a i n the spore concentration i n the a i r was removed, as probably occurred on June 1, 1966 ( F i g . 6 0 ) , when the d i u r n a l p a t t e r n was a b r u p t l y h a l t e d at 0600 hours, but was resumed around 150.0 hours, 7 to 8 hours a f t e r the r a i n ceased. - 154 -G e n e r a l l y a steady or l i g h t r a i n shower reduced d i s p e r s a l , and dry daytime c o n d i t i o n s promoted d i s p e r s a l , however, a heavy r a i n , e s p e c i a l l y w i t h l a r g e d r o p l e t s , at a time when dry mature aeciospores were a v a i l a b l e f o r d i s p e r s a l from the a e c i a increased i n i t i a l l y , the d i s -p e r s a l . A prolonged r a i n reduced the d i s p e r s a l of spores as the a e c i a became too wet. C h a r a c t e r i s t i c a l l y , aeciospores appeared to! hang from the a e c i a i n large aggregated masses a f t e r exposure to long humid condi-t i o n s or f o l l o w i n g long calm periods when few winds were a v a i l a b l e to disperse spores from the canker. E f f e c t of dew Dew occurred-on the ground every night of r e c o r d i n g i n I965 to 1967, at one s i t e i n a r e l a t i v e l y open stand, although dew o n l y l a s t e d f o r 2 or 3 hours on a few n i g h t s . Around mid-summer, on days without r a i n , dew commenced on the average at t h i s s i t e j u s t a f t e r 2200 hours and d i s s i p a t e d by 07*1-0 hours. I n 1965 dew d u r a t i o n was recorded at four s i t e s , two i n open grown f o r e s t and two i n r e l a t i v e l y open exposed s i t e s . The d u r a t i o n of dew v a r i e d considerably from one s i t e to another w i t h i n the f o r e s t . For example, on June 6, the i n i t i a t i o n of dew v a r i e d be-tween I855 and 2245 hours and the d i s s i p a t i o n on June 7 between 0730 and 0905 hours. G e n e r a l l y dew l a s t e d f o r a shorter p e r i o d i n an open grown f o r e s t than i n the open exposed areas. Despite the i n d i v i d u a l s i t e v a r i a t i o n there was no corresponding v a r i a t i o n o f the d i u r n a l d i s p e r s a l p a t t e r n of nearby cankers. On the nights when the i n i t i a t i o n of dew was app r e c i a b l y delayed there was no marked tendency f o r spore d i s p e r s a l to continue, suggesting that other f a c t o r s were more important. A heavy dew tended to delay the d a i l y i n i t i a t i o n of d i s p e r s a l , although how much - 155 -t h i s e f f e c t i s independent of r e l a t i v e humidity and other f a c t o r s i s unknown as on many of these days the r e l a t i v e humidity remained high u n t i l the same hour, although on other occasions, as i s to be expected, the r e l a t i v e humidity of the a i r was decreasing w e l l before dew d i s -appeared. E f f e c t of wind F i g . 58 shows that the main d i u r n a l spore d i s p e r s a l p e r i o d on dry days was c l o s e l y a s s o c i a t e d w i t h the higher daytime wind speeds. . On about 80% of the dry days the maximum d a i l y spore concentration occurred during the p e r i o d of high d a i l y wind speeds; on the other occasions the maximum wind speeds occurred l a t e r i n the day, presumably at a time when the a v a i l a b l e mature spores had already been dispersed. G e n e r a l l y wind speeds of 1.1 mph (1.61 f t / s e c ) were r e q u i r e d f o r considerable i n i t i a l spore d i s p e r s a l to take place from the canker, but spores continued to be dispersed at lower wind speeds. At times during the night when higher wind speeds occurred there was no noticeable- r i s e i n spore d i s p e r s a l from the canker, thus wind speed i s not the sole requirement f o r d i s p e r -s a l . There was a l s o no close c o r r e l a t i o n between high wind speeds and high spore concentrations, as e q u a l l y high concentrations occurred at much lower wind speeds, and high wind speeds r a p i d l y dispersed the spore clouds. A v a i l a b i l i t y of spores I n a d d i t i o n to the e f f e c t s of the v a r i o u s m e t e o r o l o g i c a l f a c -t o r s on spore d i s p e r s a l , the a v a i l a b i l i t y o f spores f o r d i s p e r s a l must-be considered. A v a i l a b i l i t y depends on the stage of aeciospore production - 15.6 -of the i n d i v i d u a l a e c i a l p ustules and on the magnitude of previous d i s -p e r s a l s . More spores are a v a i l a b l e f o r d i s p e r s a l f o l l o w i n g a few days of n o n - d i spersal, a l s o v e r y high concentrations of spore d i s p e r s a l e a r l y i n the day are o f t e n followed by only low concentrations i n otherwise s u i t a b l e d i s p e r s a l c o n d i t i o n s . Large numbers of spores are probably a v a i l a b l e f o r d i s p e r s a l f o r a p e r i o d of only about four weeks, but the p e r i o d of spore production l a s t s two, and i n some years three times t h i s d u r a t i o n ( c f . next s e c t i o n ) . Seasonal spore p e r i o d i c i t y At the beginning of the spore d i s p e r s a l season, towards the end of May, there i s a very sudden r i s e i n the number of spores r e l e a s e d . During t h i s i n i t i a l d i s p e r s a l p e r i o d most of the a e c i a of a canker rup-ture w i t h i n a few days, r e l e a s i n g the mature spores. This high l e v e l of spore r e l e a s e i s maintained f o r two to three weeks t o be followed by a gradual decrease ( F i g . 6 l ) . By mid-July the d a i l y spore r e l e a s e i s at. a low l e v e l but a few spores continue to be r e l e a s e d on most days u n t i l around mid- or l a t e August by which time spore production has ceased and a l l spores have been dislodged from the a e c i a by wind and r a i n a c t i o n . Minor peaks occur throughout the spore production season a f t e r the i n i t i a l high peak due to the rupture of new a e c i a , but these only l a s t a few days and spore concentrations r a r e l y reach the l e v e l a t t a i n e d during the i n i -t i a l spore r e l e a s e p e r i o d . The day to day r e l e a s e of spores i s a f f e c t e d by the seasonal weather c o n d i t i o n s , which may b r i n g about minor d i s p e r s a l peaks independent of the major seasonal peak. I n some seasons the spore r e l e a s e i s more a f f e c t e d by weather c o n d i t i o n s than i n others. F i g . 62 shows the seasonal spore r e l e a s e f o r one t r e e w i t h two b a s a l cankers i n F i g . 6 l . D a i l y number of aeciospores c o l l e c t e d from two Cronartium comandrae cankers during the I966 spore production p e r i o d , p l o t t e d on semi-log s c a l e . 1 - 15? -1,000,000 F i g . 62. D a i l y number of aeciospores c o l l e c t e d from Cronartium comandrae canker no. 2713 during 1965, I966 and 1967, p l o t t e d on semi-log s c a l e . - 158 -- 159 -three d i f f e r e n t years. I n 1965 the lower e f f i c i e n c y spore c o l l e c t o r was i n use, which accounts f o r much of the lower l e v e l of spore concentration i n t h a t year, as the canker appeared to have abundant spores a v a i l a b l e . The beginning of s p o r u l a t i o n was obtained i n two years and the end of s p o r u l a t i o n i n a l l three years. I n I967 s p o r u l a t i o n probably began around June 2nd, as on May 31st prominent s w e l l i n g o f the a e c i a l area o f the cankers was observed but no a e c i a were v i s i b l e . I n 1965 spore production ceased much e a r l i e r . By J u l y 12 s p o r u l a t i o n from the lower of the two cankers had completely ceased and s p o r u l a t i o n from the upper canker ceased a few days l a t e r . The very high concentrations of May 30 - June 1, I966, and August 5 - 7, 19^7 were a s s o c i a t e d w i t h heavy r a i n f a l l which r e l e a s e d large numbers of spores from the a e c i a . Often, p r i o r to the occurrence of r a i n , aeciospores would be hanging from the a e c i a i n large masses but r a i n r a p i d l y dispersed these, and there was o f t e n a r e d u c t i o n of the d i s -persed spore concentration f o l l o w i n g the r a i n y p e r i o d . Spore pr o d u c t i o n from t h i s t r e e was not followed i n I968 but i t would have been very low as only two a e c i a l pustules were produced, and the t r e e was dead through the g i r d l i n g a c t i o n of the fungus by mid-July. AECIOSPORE DISPERSAL FROM A NATURAL POINT SOURCE Methods and M a t e r i a l Various methods were used to e s t a b l i s h the p a t t e r n of aeciospore d i s p e r s a l around a n a t u r a l p o i n t source. I n I967 and 1968, 15 microscope s l i d e s l i g h t l y coated w i t h v a s e l i n e were placed on the ground along eight-r a d i i at 45° . i n t e r v a l s around smalllodgepole pine w i t h abundantly sporu-l a t i n g cankers. On June 23, 1967, a t e s t was run around a t r e e (#2713 at - i6o -l o c a t i o n l ) w i t h two b a s a l cankers. The lower canker extended from the ground to 0 . 6 f e e t , the upper canker extended from 0 . 6 to 1.2 f e e t . The aspect of the lower canker was predominantly south and the upper east, although both completely g i r d l e d the tree and produced spores on a l l as-pects. The circumference of the t r e e at canker height was 0.55 f e e t . From June 20 to June 28, 1968, s i x t e s t s were run around a t r e e (#2689 at l o c a t i o n 3) w i t h a s i n g l e canker extending from 2 . 1 to 3 . 8 f e e t above ground, which g i r d l e d the tree except on the northwest aspect. Spores were produced abundantly from a l l areas of the canker, which had a c i r -cumference at canker height o f 0 .95 f e e t . On the eigh t r a d i i i n I967, s l i d e s were centered at the f o l l o w i n g d i s t a n c e s from the t r e e : l / 4 , l / 2 1, 1 1/2, 2, 2 1/2, 3, k, 5, 6, 7, 8, 9, 10 and 15 f e e t . I n 1968 the same sampling p o i n t s were used, except t h a t the 7 and 9 f o o t p o i n t s were replaced by 12 and 20 f o o t d i s t a n c e s . The s l i d e s were exposed f o r v a r i -ous periods of time (k to 10-g- hours) during the main d a i l y aeciospore d i s p e r s a l p e r i o d , commencing at 0800 hours. Duration of exposure was governed by the t h r e a t of r a i n , which l i m i t e d the t e s t s on c e r t a i n days to l e s s than 8 hours (which was considered to i n c l u d e the peak spore d i s -p e r s a l p e r i o d — 1000 - 1600 hours). A f t e r removal, 13-5 sq cm of each s l i d e were scanned under the microscope f o r deposited spores. A e r i a l shoots of ground v e g e t a t i o n , which would i n t e r c e p t d i s p e r s e d spores, were removed from the t e s t areas. Wind speed, a i r temperature, r e l a t i v e humi-d i t y and r a i n f a l l were recorded rear the cankers f o r the p e r i o d of the t e s t . Wind d i r e c t i o n (and wind speed) was obtained at l o c a t i o n 1 from a recorder kQ f e e t above ground near the study area, and at l o c a t i o n 3 from one at 150 f e e t (operated by the M e t e o r o l o g i c a l Branch, Department - l 6 l -of Transport, i n the Marmot Creek Watershed Research B a s i n ) . An impac-t i o n spore c o l l e c t o r was run on the t e s t area, 9 inches from the t r e e on the east-southeast aspect i n the 1967 t e s t , and c o l l e c t i o n s . f r o m a net-work of p l a s t i c s l i d e holder spore c o l l e c t o r s (see page 175) supplemented the I968 t e s t s . At l o c a t i o n 1 the nearest sources of background spores were from p o o r l y s p o r u l a t i n g cankers 100 f e e t to the southwest and k8 f e e t to the north. At l o c a t i o n 3, the nearest s p o r u l a t i n g cankers were 63 f e e t to the west-southwest and 106 f e e t to the northwest. Therefore, the number of spores from cankers other then the t e s t t r e e s were probably s m a l l and could be ignored. On J u l y 7, 19&8, exposed leaves of ground v e g e t a t i o n growing around a lodgepole pine w i t h a s p o r u l a t i n g canker, centered 3 f e e t above ground, were c o l l e c t e d at l o c a t i o n 3- Leaves were taken near the four main c a r d i n a l p o i n t s at the f o l l o w i n g distances from the t r e e : 1, 2, 5 5 10, 15, 20 and 30 f e e t . The area of each l e a f was measured, and the num-ber of spores present on the surface o f the leaves was counted, to give a gradient o f spore d e p o s i t i o n . Leaves from a number of species were c o l l e c t e d , thus the c a p a b i l i t i e s of leaves to c o l l e c t and r e t a i n spores on t h e i r surfaces during d i f f e r e n t environmental c o n d i t i o n s was not u n i -form. The leaves g e n e r a l l y had the same h o r i z o n t a l o r i e n t a t i o n thus the surface plane of " d e p o s i t i o n was approximately the same. Chances f o r back-ground d i s p e r s a l from other cankers was s m a l l as the t r e e was 63 f e e t from the.nearest s p o r u l a t i n g canker. During the p e r i o d June 9-, to J u l y 1, 1967, p l a s t i c s l i d e holder spore c o l l e c t o r s were placed at a height of- one foo t and at a distance of 10 f e e t on the eigh t c a r d i n a l r a d i i around two f a i r l y i s o l a t e d t r e e s , at - 162 -or near l o c a t i o n 1. One or two c o l l e c t o r s were placed at a d i s t a n c e of 20 f e e t . The canker on t r e e #2719 extended from 3.2 to 7 .9 f e e t above ground and was s p o r u l a t i n g on the north and south aspects, and on t r e e #2710 the canker extended from 0 . 9 to 1.3 f e e t above ground. I n I968, tree #2710 was again used f o r 10 days between June 17 and J u l y k, and t r e e #2689, at l o c a t i o n 3, f o r 9 days between June 2k and J u l y 15. In I968 s l i d e holders were placed at the 5 and 10 f o o t d i s t a n c e s , and around tree #2689 a l s o at the 15 f o o t d i s t a n c e . The s l i d e s were changed d a i l y between 0800 and 0900 hours, and 13.5 sq cm of each s l i d e were scanned f o r deposited spores. Chances f o r background d i s p e r s a l from other s p o r u l a t i n g cankers was s m a l l . Wind d i r e c t i o n and speed, a i r temperature, r e l a t i v e humidity and r a i n f a l l , were recorded near the t e s t t r e e s . R e s u l t s Figure 63 shows the concentration p a t t e r n of spore d i s p e r s a l around the i n f e c t e d t r e e #2713 during a 10^ hour/period June 23, 1967. There was a v e r y marked concentration of spores w i t h i n a few inches of the t r e e w i t h a very r a p i d decrease away from the source i n a l l d i r e c -t i o n s . The mean ho u r l y wind d i r e c t i o n during the p e r i o d was from the southwest, except f o r one hour when i t was from the northwest. The mean wind speed at one f o o t above ground was I.7 mph, and at k8 f e e t 7 mph. From the mean.wind d i r e c t i o n during the p e r i o d one would expect a l a r g e r concentration of spores to be deposited i n the northeast se c t o r , but t h i s was not apparent. The higher concentration i n the southeast sector may have been due to a greater amount of s p o r u l a t i n g a e c i a on t h i s aspect, F i g . 63. P a t t e r n of aeciospore d e p o s i t i o n around Cronartium comandrae canker no.' 2713 during a 10-§- hour p e r i o d on June 23, I967. - 163 -S C A L E 0 - 164 -r a t h e r than to some wind f a c t o r . The predominance of winds from the southwest may w e l l have accounted f o r a l a c k o f spores beyond the 5 foot sampling p o i n t from the t r e e i n the southwest s e c t o r . The. areas along the east-west a x i s , w i t h s l i g h t l y higher concentrations around the 10 to 15 foot distance p o i n t s , may have r e s u l t e d from a wave p a t t e r n of d i s p e r s a l due to gusty or t u r b u l e n t c o n d i t i o n s . L o c a l turbulence i n the a i r , e s p e c i a l l y around the t r e e , was l i k e l y to have caused some d i s -p e r s i o n of spores i n a l l d i r e c t i o n s from the center, but at greater d i s -tances the d i s p e r s a l p a t t e r n was l i k e l y to correspond to the p r e v a i l i n g d i r e c t i o n s of a i r movement during the d i s p e r s a l p e r i o d . To f u r t h e r i n d i c a t e the r a p i d decrease i n concentration, the values f o r each of the sampling p o i n t s beyond the 3-inch p o i n t were con-v e r t e d to a percentage of the concentration at the 3-inch sampling d i s -tance on the same r a d i i . These were then averaged f o r the eigh t r a d i i and the average change of concentration w i t h distance i s shown i n F i g . 64 on a semi-log s c a l e , along w i t h the change f o r the southeast r a d i i , which r e c e i v e d the maximum concentration o f spore d e p o s i t i o n c l o s e t o the t r e e . The decrease i n concentration, much of which i s due to d i f f u s i o n , amounted to n e a r l y 80% i n a distance of 3 inches and to n e a r l y 99% at a distance of 3 f e e t . The southeast r a d i i showed a s l i g h t l y l e s s r a p i d change o f concentration as would be expected from the r a d i i r e c e i v i n g the highest number of deposited spores. F i g . 65 shows the average concentration p a t t e r n of spore d i s -p e r s a l around tre e #2689 during 8 hour periods on June 24, 26 and 28, I96.8, when the mean wind speed was 1.5 mph, 4 f e e t above ground. The p a t t e r n was s i m i l a r to F i g . 63 except t h a t there was l e s s v a r i a t i o n due to the F i g . 6k. The average percentage change of aeciospore d e p o s i t i o n concentration w i t h distance oh eig h t r a d i i around Cronartium comandrae canker no. 2713 on June 23, 19&7, and the percentage change on the southeast r a d i i , p l o t t e d on semi-log s c a l e . D I S T A N C E IN F E E T F i g . 65. P a t t e r n o f aeciospore d e p o s i t i o n around Cronartium comandae canker no. 2689, on three days (080O to 1600 hours) i n June 1968. - 166 -East S C A L E IN FEET 1 0 5 - 167 -averaging of three days of spore d e p o s i t i o n . Again there was a very marked concentration of spores close to the source, even more marked than shown i n the f i g u r e , where the inner c i r c l e shows spore deposi-t i o n s greater than 2 5 0 0 .spores. Depositions at the 3-inch d i s t a n c e on the r a d i i from northwest to northeast a l l averaged more than 13,000, and on the north d e p o s i t i o n was over 25,000 spores. These high con-c e n t r a t i o n s were l a r g e l y the r e s u l t of south and southeast u p v a l l e y winds on June 2k, and the a v a i l a b i l i t y of spores. On the other days fewer spores were a v a i l a b l e f o r d i s p e r s a l , although the wind speeds and d i r e c t i o n were s i m i l a r . F i g . 66 shows the r a p i d decrease i n the con-c e n t r a t i o n of spores f o r the i n d i v i d u a l days p l o t t e d on semi-log s c a l e , w i t h the spore concentration at the 3-inch sampling distance taken as 100$. A l l show the .same steep gradient of d e p o s i t i o n near the source, g i v i n g a hollow curve, s i m i l a r to t h a t shown i n F i g . Gk f o r the I967 t e s t . The decrease i n concentration much of which i s due to d i f f u s i o n of the spore cloud, amounted to n e a r l y 9°$ i n a distance of 1 3/k f e e t on each day, and .to n e a r l y 99$ i n a distance of "6 f e e t on two of the three days. On June 26, 1968, the decrease was more gradual, although there was more than a 98$ decrease at 10 f e e t . The average wind speed on t h i s day was s l i g h t l y higher than on the other two days and could account f o r the f l a t t e r curve.-Number of spores deposited per sq. i n of l e a f at v a r i o u s d i s -tances from a s p o r u l a t i n g canker showed a s i m i l a r r a p i d decrease w i t h i n c r e a s i n g d i stance (Table X V I I ) . Many of the spores were deposited close to the veins of the leaves, and i t was n o t i c e a b l e that more spores were r e t a i n e d on leaves w i t h abundant epidermal h a i r s . D i s r e g a r d i n g the non-F i g . 66. The average percentage change of aeciospore d e p o s i t i o n w i t h d i stance around Cronartium comandrae canker no. 2689, on three days (0800 t o 1600 hours) i n June 1968, p l o t t e d on semi-log s c a l e . - 168 -D I S T A N C E IN F E E T - 169 -u n i f o r m i t y of l e a f sampling surfaces, the spore catch at 10 f e e t was ne a r l y 99% l e s s than at 1 f o o t , and no spores were observed at 20 or 30 f e e t , although the sample leaves were c o l l e c t e d towards the end o f the I968 s p o r u l a t i o n p e r i o d . One would expect some d e p o s i t i o n at distances beyond 15 f e e t due to n a t u r a l d i f f u s i o n . The t o t a l d e p o s i t i o n probably represented spores dispersed over s e v e r a l days and i n d i c a t e s that such surfaces d i d not r e t a i n many spores. Counts o f d a i l y deposits of spores on the p l a s t i c s l i d e holder c o l l e c t o r s 10 f e e t from the cankers gave very low values f o r spores de-p o s i t e d i n 1967. Around one t r e e (#2710), during the nine days of re.-cording, only 8 of the 72 s l i d e s had 6 or more spores deposited on them, and 20% of them had none. There was abundant d i s p e r s a l from t h i s canker as a spore impaction c o l l e c t o r placed a few inches from the canker, w i t h a high c o l l e c t i o n e f f i c i e n c y , c o l l e c t e d a d a i l y average of 13,653 spores during the same p e r i o d . Around the other t r e e (#2719;), during 20 days of r e c o r d i n g , spores were c o l l e c t e d on 9*+ of the l6o d a i l y s l i d e s at the 10-foot d i s t a n c e , but only 15 s l i d e s had 6 or more spores. Trapping of spores on t h e s e . c o l l e c t o r s was much higher i n I968, e s p e c i a l l y around t r e e #2689, probably because of a higher a v a i l a b i l i t y of spores from the cankers, and i n the case of #2689 a r e l e a s e p o i n t averaging 3 f e e t above ground, which would a l l o w greater distance d i s p e r s a l . Table X V I I I shows the average number of aeciospores deposited per sq cm at var i o u s d i s -tances around #2689 during 9 days of c o l l e c t i n g . Again there was a very steep gradient i n the number of spores deposited i n a l l d i r e c t i o n s , w i t h a r e d u c t i o n of between 81.9 and 9*+. 3% from the 5-foot sampling p o i n t to the 15 foot p o i n t . Most winds during the main d a i l y d i s p e r s a l p e r i o d came - 170 -T a b l e X V I I . Number o f a e c i o s p o r e s d e p o s i t e d on n a t u r a l l e a f s u r f a c e s c o l l e c t e d a l o n g t h e f o u r c a r d i n a l , r a d i i a t v a r i o u s d i s -t a n c e s f r o m a s p o r u l a t i n g C r o n a r t i u m comandrae canker on J u l y 7, 1968, a t l o c a t i o n 3-D i s t a n c e L e a f a r e a T o t a l No. o f f r o m canker examined no. o f s p o r e s ( f e e t ) ( s q i n ) s p o r e s p e r s q i n 1 15.78 13,169 834.5 2 8.99 5,117 569.0 5 11.45 1,083 9 4 . 1 10 15.10 153 10.1 15 10.35 58 5-6 20 11.42 0 0 30 14.02 0 0 T a b l e X V I I I . A v e r a g e number o f a e c i o s p o r e s d e p o s i t e d p e r square c e n t i -meter on spo r e c o l l e c t o r c o a t e d s l i d e s a t d i s t a n c e s o f 5 5 10 and 15 f e e t a l o n g e i g h t r a d i i around a s p o r u l a t i n g C r o n a r t i u m comandrae canker a t l o c a t i o n 3, cm 9 days be-tween June 24 and J u l y 15, 1968. D i s t a n c e A v e r a g e number o f s p o r e s p e r s q cm d e p o s i t e d a l o n g each f r o m canker r a d i i ( f e e t ) N NE E SE S sw w NW 5 21 .8 29 .6 19. • 9 21.2 13.9 9 .2 8 .0 14.8 10 12.0 8.9 5. • 9 ' %h 4 . 4 2 .1 2 . 8 2 . 1 15 3 .1 1.7 3. .6 1-9 2.5 0 . 8 0 . 7 1.4 f r o m t h e west and so u t h w e s t , w h i c h a c c o u n t e d f o r t h e l o w e s t s p o r e v a l u e s i n t h e s e d i r e c t i o n , and t h e h i g h e s t i n t h e n o r t h t o s o u t h e a s t s e c t o r ( T a b l e X V I I I ) . - 171 -OTHER. DATA ON DISTANCE OF AECIOSPORE DISPERSAL In an e f f o r t to c o l l e c t some f u r t h e r i n f o r m a t i o n on dis t a n c e of aeciospore d i s p e r s a l under n a t u r a l c o n d i t i o n s , v a r i o u s types o f spore c o l l e c t o r s were run at v a r y i n g distances downwind ( f o r southwest winds) from known sources o f spores. The number of spore c o l l e c t o r s employed at d i f f e r e n t times depended on t h e i r not being r e q u i r e d f o r other aspects of the study, and on the a v a i l a b i l i t y of s p o r u l a t i n g cankers s u f f i c i e n t l y removed from other s p o r u l a t i n g cankers, so that the p o s s i b i l i t i e s of spore d e p o s i t i o n from background cankers were kept to a minimum. During ten days i n J u l y I966, p l a s t i c s l i d e holder spore c o l -l e c t o r s were placed at three heights ( l , 5 and. 10 f e e t ) and at four d i s -tances (15, 25, 50 and 100 f e e t ) downwind from a s p o r u l a t i n g canker one foot above ground. At the same time a r o t o r o d sampler was placed at a distance of 150 f e e t from the canker. A few spores were caught at most distances each day, and at most heighte, but on no occasion were more than 11 spores deposited, i n d i c a t i n g that o n ly s m a l l numbers of spores were c a r r i e d any d i s t a n c e . C o l l e c t i o n s at the one foot height compared w i t h the 5 and 10 foot heights showed a r a t i o . j o f approximately 1.5:1-0 i n d i -c a t i n g that spores can be transported to considerable heights above the r e l e a s e p o i n t . S i m i l a r data obtained from impaction spore c o l l e c t o r s , equip-ped w i t h a s i n g l e s l i d e i n s t e a d of 2h s l i d e s , placed downwind from a b a s a l canker at 25 and 50 foot distances at the one foot l e v e l , showed a decrease of deposited spores between these p o i n t s . During 15 days when both c o l l e c t o r s were operating spores were only deposited at 50 f e e t on one occasion. Catches at 25 feet ranged, on these days, between 0 and - 172 -12 spores. The 25 foot c o l l e c t o r was operated on 7 other days and the catch on these days ranged from 1 to 122 spores. The average catch f o r the 22 days operation at the 25 foot d i s t a n c e was 10 spores, but a s i m i -l a r c o l l e c t o r placed w i t h i n a few inches of the same canker c o l l e c t e d an average of 7,993 spores a day during the same p e r i o d . During the spore d i s p e r s a l p e r i o d i n 1965 to 1967 i n c l u s i v e a H i r s t spore trap was run 34 f e e t north of a s p o r u l a t i n g canker. I n 1966 spores were only c o l l e c t e d on 31 days of the p e r i o d May 19 to August 17, w i t h 15 the highest number of spores c o l l e c t e d i n one day. S i m i l a r l y , i n 1967 spores were only c o l l e c t e d on 10 days of the 43 day sampling p e r i o d between June 26 and August 18, but on s e v e r a l of these days over 100 spores were c o l l e c t e d . Rotorod samplers were run i n 1967, 50 and 200 f e e t north of a s p o r u l a t i n g canker f o r l 6 days. Spores were only c o l l e c t e d on the 50 foot sampler on 3 days, and on the 200 f o o t sampler on 2 days, d e s p i t e the f a c t that t h i s type of sampler samples a i r at an average r a t e of 60 l i t e r s per'minute. In 1968 a r o t o r o d sampler was run at a p o i n t 100 f e e t away from s e v e r a l s p o r u l a t i n g cankers, and another at a distance of 725 f e e t from the nearest canker. The samplers were run f o r 13 days between June 17 and J u l y 4. Spores were only c o l l e c t e d on the 100 f o o t sampler on 2 days, and none were c o l l e c t e d at a distance of 725 f e e t . Weekly r e c o r d i n g p o l l e n samplers were operated i n 1965 and 1967 at v a r i o u s distances from s p o r u l a t i n g cankers during the spore d i s p e r s a l p e r i o d . A few spores were c o l l e c t e d i n 1965 when they were operated at distances no greater than 25 f e e t , but i n 1967 no spores were c o l l e c t e d when operated at dist a n c e s of 150 and 725 f e e t from the nearest cankers. - 173 -AECIOSPORE DISPERSAL EXPERIMENTS FROM POINT SOURCES M a t e r i a l s and Methods Ten experimental t e s t s were c a r r i e d out w i t h aeciospores l i b e r -ated from a r t i f i c i a l spore-ejectors p o s i t i o n e d one and f i v e f e e t above ground l e v e l , to e s t a b l i s h the concentration p a t t e r n and distance, of d i s -p e r s a l under the process o f d i f f u s i o n . I n eig h t experiments spores were re l e a s e d at f i v e f e e t and i n two experiments at one foot above ground. V a s e l i n e coated s l i d e s were set up i n p l a s t i c holders at v a r y i n g d i s t a n c e s from the r e l e a s e p o i n t to c o l l e c t spores. A l l experiments were c a r r i e d out over a f l a t short grass area, surrounded by sm a l l t r e e s and b u i l d i n g s at the Kananaskis Forest Experiment S t a t i o n . These experiments were c a r r i e d out over a plane surface i n the open r a t h e r than w i t h i n a f o r e s t stand, to s i m p l i f y the con d i t i o n s under which the process of spore d i f -f u s i o n takes p l a c e . I n most experiments 3 gms of f r e s h or stored a e c i o -spores were l i b e r a t e d when winds came from the southwest. For l i b e r a t i o n the spores were placed i n a spore-ejector ( F i g . 67) of s i m i l a r , design.to t h a t described by Gregory et a l . (1961), except t h a t the diaphragm was held between two pieces of brass gauge, and the lower f u n n e l stem was attached d i r e c t l y by some k-0 f e e t of rubber tubing to the a i r flow r e g u l a -t i n g u n i t ( F i g . 68), s i t u a t e d away from the experimental s i t e . When a i r was forced through the diaphragm, s i n g l e spores were c a r r i e d out through the e x i t tube of the spore-ejector i n the form of spore clouds. The e x i t tube of the spore-ejector was d i r e c t e d towards the m i d l i n e o f the tr a p p i n g s l i d e network, so th a t the spores would be re l e a s e d i n t o the normal flow of wind during the experiment. The r a t e of spore r e l e a s e was c o n t r o l l e d by the a i r compressor v a l v e , and u s u a l l y spores were r e l e a s e d F i g . 67. Spore-ejector used f o r l i b e r a t i n g aeciospores i n d i s p e r s a l experiments from p o i n t source. F i g . 68. Compressor u n i t w i t h connecting rubber tubing, f o r r e g u l a -t i n g a i r flow f o r r e l e a s e of aeciospores i n d i s p e r s a l experiments. F i g . 69. W i n d - d i r e c t i o n a l ^ p l a s t i c s l i d e holder spore c o l l e c t o r s (without microscope s l i d e s ) used f o r c o l l e c t i n g a e c i o -spores i n d i s p e r s a l experiments and from n a t u r a l sources. - 174 -- 175 -under a pressure of 10 pounds per square i n c h . To c o l l e c t spores from the spore cloud at a large number of po i n t s downwind from the re l e a s e source, a simple inexpensive wind-d i r e c t i o n a l spore c o l l e c t o r was developed, and 113 of these were used to c o l l e c t spores during each experiment. Each c o l l e c t o r c o n s i s t e d of a standard 2.5 x 7-5 cm microscope s l i d e , w i t h one side l i g h t l y coated w i t h v a s e l i n e , held i n a plastic holder. The p l a s t i c holder ( F i g . 69) c o n s i s t e d of four pieces of a c r y l i c p l a s t i c . A piece to hold the s l i d e , a stem attached to a piece of p l a s t i c tubing and a vane to maintain the holder f a c i n g i n t o the wind. The c o l l e c t o r was placed on a sm a l l aluminum rod which had a brass t i p and washer upon which the c o l l e c t o r turned f r e e l y i n response to wind. The holder was i n c l i n e d at 1+5° to the h o r i z o n t a l as Gregory and Stedman (1953) had found that the c o l l e c t i o n e f f i c i e n c y of a s l i d e i n c l i n e d at 45° v a r i e d l e s s than i f a s l i d e was o r i e n t e d v e r t i c a l l y or h o r i z o n t a l l y . The i n c l i n e d s l i d e was l e a s t e f f i c i e n t at low wind speeds, but was b e t t e r f o r l a r g e r than f o r smaller spores.. Others (Har-r i n g t o n et a l . 1959> Hyre 1950; Ogden and Raynor i960) have al s o shown that the catch i s greater w i t h s l i d e s at a. "+5° angle. A l s o the uniform' o r i e n t a t i o n of the w i n d - d i r e c t i o n a l holders w i t h respect to a i r flow en-sures c o m p a r a b i l i t y of samples (Ogden and Raynor i 9 6 0 ) . Experimental arrangement The spore c o l l e c t o r s were set .out i n a network at three heights, over an angle of 90° and a radius of 150 f e e t , w i t h samplers on the mid-l i n e up to 200 f e e t from the r e l e a s e p o i n t ( F i g s . 70, 71 and 7 2 ) . A l s o on the m i d - l i n e a ro t o r o d sampler (Perkins 1957) was set out at 200 or F i g . 70. P o r t i o n of spore c o l l e c t o r network, w i t h c o l l e c t o r s on d i f f e r e n t r a d i i at v a r i o u s distances from r e l e a s e p o i n t . Wind d i r e c t i o n vane and wind speed anemometer can be seen to the l e f t o f the stand f o r the spor e - e j e c t o r . Note spore c o l l e c t o r s at 5 and 10 f e e t on the masts. F i g . 71. P o r t i o n of spore c o l l e c t o r network w i t h c o l l e c t o r s on d i f f e r e n t r a d i i at v a r i o u s distances from spore-ejector at r e l e a s e p o i n t . Wind speed anemometer to l e f t o f spore-ejector. - 176 -F i g . 72. Diagram of p o i n t source aeciospore d i s p e r s a l experiment sampling g r i d , w i t h 113 sampling c o l l e c t o r s l a i d out f o r southwest winds. Spore c o l l e c t o r s were a l s o l o c a t e d at the 300 and k-00 foot distances along the m i d - l i n e . - 177 -© © © © © © © © © © 0 • © # © * © • • . • © *• • • • ' : > © • © • © 0 25 50 I I I F E E T • Spore release point • Single spore collector at 1 foot © M a s t with spore collectors at 1, 5 and 10 feet - 178 -300 f e e t , and an impaction spore c o l l e c t o r (Powell and Morf 1967) at k-00 f e e t , to augment the p l a s t i c spore c o l l e c t o r network. The spore c o l -l e c t o r s were set out f o r a southwest wind- (the p r e v a i l i n g daytime wind d i r e c t i o n during the p e r i o d of the experiments) so that the arc of 90° was centered on a northeast l i n e from the r e l e a s e p o i n t . C o l l e c t o r s were placed on nine r a d i i from the r e l e a s e p o i n t set at 11.25° i n t e r v a l s , w i t h a c o l l e c t o r on each r a d i i at 5, 10, 20, 35, 50, 75, 100 and 150 f e e t from the r e l e a s e p o i n t at 1 foot above ground ( F i g . 72). On the 20, 50, 100 and 150 foot arcs c o l l e c t o r s were a l s o placed at the 5 and 10 foot l e v e l s above ground on a l t e r n a t e r a d i i f o r c o l l e c t i o n of spores at higher levels. Maximum c o l l e c t i o n distances from the r e l e a s e p o i n t were l i m i t e d to 150 f e e t except on the m i d - l i n e by the s i z e of the experimental area. Wind speed was recorded at the 1, 5 and 10 foot l e v e l s i n the center o f the experimental layout, and at 1 or 5 f e e t at the r e l e a s e p o i n t , by C a s e l l a t o t a l i z i n g cup anemometers of the Sheppard type. Wind d i r e c t i o n , a i r temperature and humidity were al s o recorded during the r e l e a s e p e r i o d . Although the experimental s i t e was s e l e c t e d as the most s u i t -able area a v a i l a b l e , the presence of b u i l d i n g s and tre e s to the windward of the r e l e a s e p o i n t undoubtedly added the f u r t h e r c o m p l i c a t i o n of down-draught and downwash e f f e c t s , of the type described by Hawkins and Non-hebel (1955), both e f f e c t s tended to b r i n g spores to ground l e v e l more q u i c k l y than i n unobstructed flow. However, the e f f e c t s from the r e -lease heights o f the t e s t s were probably s m a l l , and s i m i l a r e f f e c t s would be experienced i n f o r e s t c l e a r i n g s or -along f o r e s t margins, thus t h i s e r r o r was of l i t t l e importance. - 179 -Experimental procedure I n conducting the experiment the prepared microscope s l i d e s were placed i n the holders of the c o l l e c t o r s when favourable southwest winds were i n d i c a t e d . The spores were placed i n the spore - e j e c t o r , and the compressor was run w i t h the v a l v e closed. At a s e l e c t e d time, when winds were favourable f o r the c o l l e c t o r layout, spore l i b e r a t i o n was commenced by s l o w l y opening the compressor v a l v e u n t i l the d e s i r e d r a t e of l i b e r a t i o n was obtained. At the same time the four t o t a l i z i n g wind anemometers were s t a r t e d . A f t e r making c e r t a i n that a l l spores i n s i d e the spore-ejector had been l i b e r a t e d the time was again noted, and the t o t a l i z i n g wind anemometers were stopped and read to give the mean wind speed during the p e r i o d of the d i s p e r s a l experiment. The records of the continuously r e c o r d i n g wind d i r e c t i o n and hygrothermograph recorders were a l s o noted. Table XIX summarizes the time and m e t e o r o l o g i c a l c o n d i t i o n s f o r each of the ten experiments, numbers I to X, and i n d i c a t e s the height of spore r e l e a s e . Some spore r e l e a s e i n e v i t a b l y took place i n gusty c o n d i t i o n s which caused d i s p e r s a l away from the m i d - l i n e . With a s h i f t o f wind d i r e c t i o n the r e l e a s e of spores was h a l t e d u n t i l the wind was once again predominatly from the southwest. The experiments were only run on c l e a r days during the hours around noon which corresponds to the optimum co n d i t i o n s and n a t u r a l p e r i o d of spore d i s p e r s a l . A f t e r completion of the d i s p e r s a l experiment the l a b e l l e d s l i d e s were c a r e f u l l y removed from t h e i r r e s p e c t i v e holders and kept i n a dust-proof s l i d e box u n t i l counted. The chance of n a t u r a l l y d ispersed spores being c a r r i e d i n t o the experimental area during the experiments was very s m a l l , as the nearest s p o r u l a t i n g i n f e c t e d t r e e s were over 600 f e e t away from the Table XIX. P a r t i c u l a r s of experiments on d i s p e r s i o n of Cronartium comandrae aeciospores from a p o i n t source. Expt. no. Date (June 1966) Time of s t a r t (MST) Duration (min.) Spores f r e s h / dry Height of l i b e r a t i o n ( f t . ) Mean wind speed at rel e a s e height ( f t / s e c . ) Dominant wind d i r e c t i o n Temper-ature ( ° c ) R e l a t i v e humidity I 8 11.1+9 4 D 1 5.6 SW 16 55 I I 21 13.47. 30 D 5 7-9 SSW 21 30 I I I 22 15.25 30 D 5 11.1 SW(S) 19 36 IV 27 13.47 13 D 5 9-8 SSW 21 . 37 V 27 14.32 13 F / D 5 20.6 SSW 21 37 VI 27 15.52 11 D 5 13.3 SSW 20 40 V I I 28 14.22 4 D 1 8.3 SSW 20 46 V I I I 28 16.00 7 D 5 12.7 SSW 20 4l IX 29 13.57 12 D 5 6.5 ssw-wsw 22 30 X 30 14.10 30 F 5 5.3 ssw-wsw 22 32 S i t e : Grass square, Kananaskis Forest Experiment S t a t i o n . Trap height and p o s i t i o n f o r a l l experiments: 1 f t above ground on 9 r a d i i , 11.25 degrees apart, at 5, 10,. 20, 35, 50, 75, 100, 150 f t from source. 5 and 10 f t above ground on 5 r a d i i , 22.5 degrees apart, at 20, 50, 100 and 150 f t from source. Spore quantity: 3 gms. i n a l l experiments, except Expt. 1 ( l gm) and Expt. V (5 gms). - 181 -r e l e a s e p o i n t , and considerable f o r e s t growth intervened which would i n t e r c e p t most spores c a r r i e d by a i r currents close to the ground. A n a l y s i s of data Spore d e p o s i t i o n from the spore cloud was counted on a 13-5 sq cm area of the exposed p o r t i o n of the s l i d e by making 2 mm wide t r a v e r s e s of the s l i d e under the microscope, or on a t h i r d of t h i s area by counting every t h i r d t r a v e r s e . Comparison of the two methods of spore counting i n d i c a t e d that the method of scanning only a t h i r d of the area gave a f a i r estimate of the t o t a l catch on the t o t a l exposed area of the s l i d e . A l l counts were adjusted f o r a 3 gm spore r e l e a s e q u a n t i t y . The spore c o l l e c t o r s undoubtedly had a r e l a t i v e l y s m a l l impaction e f f i c i e n c y , and t h e r e f o r e o f f e r e d an underestimate of spore cloud concentration. They were not r e p r e s e n t a t i v e of n a t u r a l surfaces, but there was l i t t l e e v i -dence of spore blow-off because of the s t i c k y c o ating. The t o t a l counts were p l o t t e d on s c a l e diagrams of the sampling area and i s o p l e t h s of spore concentration drawn f o r the three l e v e l s of t r a p p i n g . P a t t e r n s of d i s p e r s a l do not always show a smooth unimodal d i s t r i b u t i o n because changes i n wind d i r e c t i o n a s s o c i a t e d w i t h a few large scale eddies during the short sampling p e r i o d r e s u l t i n multimodal patt e r n s covering d i f f e r e n t angular s e c t o r s . For t h i s reason m i d - l i n e data alone are o f t e n inadequate and t h e r e f o r e averages f o r v a r i o u s sector s i z e s (22*5, ^5 and 90°) have been used which represent more a c c u r a t e l y the t y p i c a l h o r i z o n t a l plume d i s p e r s a l p a t t e r n . V e r t i c a l p r o f i l e s of m i d - l i n e and sector data were constructed to show change of concentration w i t h distance under the d i f -f e r e n t wind speed co n d i t i o n s and l e v e l s of spore r e l e a s e . V e r t i c a l com-parisons between d i f f e r e n t heights introduces a p o s s i b l e e r r o r since - 182 -e f f i c i e n c y of spore catch i s a f u n c t i o n of height. A l s o the use of the mean wind speed during the r e l e a s e p e r i o d was a p o s s i b l e source of e r r o r p a r t i c u l a r l y at the one foot l e v e l , since the e f f i c i e n c y of spore c o l l e c -t i o n on the c o l l e c t o r s would vary r a p i d l y w i t h wind speed and e s p e c i a l l y at low speeds. Measurements were made of s i z e s of spores deposited at d i f f e r e n t d i s t a n c e s and heights from the r e l e a s e p o i n t to e s t a b l i s h whether there was any c o r r e l a t i o n between spore s i z e and d i s t a n c e or height of d i s p e r s a l , and between f r e s h and stored spores used i n the t e s t s . As simultaneous measurements of cloud concentration and spore d e p o s i t i o n on the. ground were not taken, and the volume of a i r sampled by the spore c o l l e c t o r s was unknown, i t was not p o s s i b l e to estimate a c c u r a t e l y the.amount of d e p o s i t i o n that occurred i n the experimental area. However, a numerical r a t i o of spores c o l l e c t e d at i n t e r v a l s from the source was c a l c u l a t e d g e o m e t r i c a l l y , assuming t h a t a l l other f a c t o r s a f f e c t i n g d i s p e r s a l and d e p o s i t i o n were constant. A geometrical approach i s not too s a t i s f a c t o r y (Gregory 1961), but under the c o n d i t i o n s of the present experiment s e r i e s t h i s d i d a f f o r d an estimate of the number of spores expected at greater distances from the source. I f the chances f o r spore c o l l e c t i o n were the same f o r a l l distances w i t h i n the sampling area, one would expect the concentration of the spore cloud to decrease i n inverse p r o p o r t i o n to t h e square of the d i s t a n c e from the r e l e a s e p o i n t . G e o m e t r i c a l l y the cone shape best describes the expansion of a cloud of spores. I n cross s e c t i o n the cone i s not c i r c u l a r , but i s greater i n the h o r i z o n t a l or l a t e r a l dimension. I f a pyramid base i s assumed i n s t e a d of a cone base the proportions of the b a s a l areas at any distance from the - 183 -r e l e a s e p o i n t w i l l remain the same, thus l i t t l e e r r o r i s introduced. To f i n d the cross s e c t i o n a l area of the pyramid at each sampling distance through which the spore cloud passed i t was assumed t h a t i n each t e s t the v e r t i c a l d i s p e r s i o n was l i m i t e d to 15° from the h o r i z o n t a l , and l a t -e r a l d i s p e r s i o n was l i m i t e d to 30° on e i t h e r side of. the m i d - l i n e . The v e r t i c a l d i s p e r s i o n of 15° was chosen as spores were c o l l e c t e d at 5 f e e t above the r e l e a s e p o i n t at a 20 foot distance i n a l l t e s t s from a 5 foot-r e l e a s e p o i n t . Changing the angle of l a t e r a l d i s p e r s i o n to other l i m i t s would not change the proportions o f the areas at each d i s t a n c e , these would remain constant, t h e r e f o r e the angle of l a t e r a l d i s p e r s i o n was not important. The cross s e c t i o n a l areas at 20, 50, 100 and 150 f e e t from the r e l e a s e p o i n t , were c a l c u l a t e d from the equation: Spore r e l e a s e height + the arc f o r 15° x the arc f o r 60° = area sq f t , e.g. at 20 f e e t the cross s e c t i o n a l area = (5 + 40rf) x 40rt = 214.53 sq f t . The areas at 24 6 the four sampling distances were reduced to rough p r o p o r t i o n s as f o l l o w s : 1 : 4.4 : 15.2 : 32.4. These distances were chosen as they i n c l u d e d spore c o l l e c t i o n s at three heights on f i v e r a d i i 22.5° apart. Spore c o l l e c t i o n s on the other four r a d i i at 1 foot above ground were not inc l u d e d i n the a n a l y s i s . Two methods of est i m a t i n g the expected catches at the v a r i o u s distances were employed, usi n g the observed spore counts: A) by accepting the t o t a l number of spores caught at 20 f e e t as a reference p o i n t the expected catch at the f u r t h e r distances could be c a l c u l a t e d by d i v i d i n g the t o t a l 20 f e e t catch by the proper r a t i o f r a c t i o n , or B) d i s t r i b u t e the t o t a l number of spores caught- at the four distances i n in v e r s e p r o p o r t i o n to the cross s e c t i o n a l areas at the various d i s t a n c e s . These methods were used to analyse the catches f o r a l l r e l e a s e s at 5 f e e t . - 184 -R e s u l t s R e s u l t s of the ten experiments are summarized i n Tables XX and XXI, g i v i n g the t o t a l catch.at each distance and height. Complete r e s u l t s f o r one t y p i c a l experiment are given f o r the spore catch at the one foo t height at each sampling p o i n t , i n Table XXII. Concentration p a t t e r n s Figures 73 and 74 i l l u s t r a t e the conc e n t r a t i o n p a t t e r n s at three heights f o r re l e a s e s from the one and f i v e ' f o o t l e v e l s i n two separate experiments. The decrease i n spore concentration was very e v i -dent being most marked i n the one foot r e l e a s e s . The p o i n t of maximum concentration one foo t above ground was f u r t h e r from the source w i t h a f i v e foot r e l e a s e than a one foot . Large d i f f e r e n c e s were evident i n concentration p a t t e r n s from one experiment to another which could be r e l a t e d to wind speed and turbulence d i f f e r e n c e s . Wind d i r e c t i o n s h i f t s or eddies of v a r y i n g s i z e s during the experiment tended to d i s -perse the spores by d i f f u s i o n unevenly i n the tr a p p i n g s e c t o r . Mean wind d i r e c t i o n was always close to the m i d - l i n e , but gustiness could d i s t o r t the average p i c t u r e . Wilson and Baker (1946) found a s i m i l a r v a r i a t i o n , but when the r e l e a s e p e r i o d was increased to 30 minutes or more, the r e s u l t i n g d i s t r i b u t i o n s were found to resemble more n e a r l y t h a t of normal p r o b a b i l i t y . A composite concentration p a t t e r n f o r a l l the experiments r e l e a s e d from the f i v e foot l e v e l i s shown i n F i g . 75 f o r the three heights. Change of concentration w i t h distance V e r t i c a l p r o f i l e s of concentration are shown i n F i g . 76 f o r - 1 8 5 -Table XX. T o t a l number of spores trapped at one foot above ground at each distance on a l l r a d i i . Expt. Distance from source ( f e e t ) no. 5 10 20 35 50 75 100 150 1* 17,7^8 I I 1, 377 I I I 438 IV 33 V 46 VI 3 V I I * 8,010 V I I I " 15 IX 108 X 194 *Spores r e l e a s e d 2 ,736 324 27 1,566 612 159 552 586 210 57 231 96 60 ' 59 58 58 99 54 6,894 1,392 252 82 216 221 627 207 87 246 536 383 at 1 f o o t , a l l others 0 0 0 0 84 5 4 37 70 89 81 9 27 18 0 0 47. 7 5 25 51 18 3 0 126 18 9 9 131 82 44 • 33 66 ' 51 21 12 198 77 45 19 at 5 f e e t . Table XXI. T o t a l number of spores trapped at 5 and 10 f e e t above ground at 20, 50, 100 and 150 f e e t on f i v e r a d i i . Expt. 5 feet above ground 10 f e e t above ground no. Distance from source ( f e e t ) 20 50 100 150 20 50 100 • 150 I * 36 0 0 0 9 0 0 0 I I 126 21 17 14 26 6 1 0 I I I 450 134 37 10 237 49 10 5 IV 75 21 0 0 21 21 3 0 V 232:! 34 2 0 79 38 14 2 VI . 63 33 6 0 36 3 6 3 V I I * 132 45 15 9 30 18 15 v 9 V I I I 239 53 10 5 4o 22 18 4 IX 213 21 3 15 171 45 15 3 X 300 101 8 14 122 113 32 9 *Spores released . at 1 foot, a l l others at 5 f e e t . F i g . 73- Aeciospore concentration p a t t e r n s at three sampling heights f o r a spore r e l e a s e one foot above ground (Ex-periment V I I ) . 1 FOOT 5 FEET 10 FEET i i i i i i 0 10 20 30 40 50 F E E T F i g . 7k. Aeciospore concentration patterns at three sampling heights f o r a spore r e l e a s e f i v e f e e t above ground (Experiment X ) . F i g . 75- Composite aeciospore concentration p a t t e r n s at three sampling heights f o r a l l e i g h t spore r e l e a s e s f i v e f e e t above ground (Experiments I I - V I , V I I I - X ) . 0 F i g . 76. V e r t i c a l p r o f i l e s of aeciospore concentration p a t t e r n s along the m i d - l i n e of the sampling network f o r two spore r e l e a s e s at one foot (Experiments I and V I I ) , and two spore r e l e a s e s f i v e f e e t above ground (Experiments V and X ) . - 189 -Exp. I lO - i DISTANCE FROM SOURCE (FEET) - 190 -Table XXII. R e s u l t s of Experiment X, showing number of spores trapped on an area of 13.5 sq cm at the one foot l e v e l at each sampling p o i n t . Angle to wind Distance from source ( f e e t ) (°) 5 10 20 35 50 75 100 150 200 - 4 5 . 0 8 6 14 19 12 3 0 0 -33.75 22 20 48 30 11 4 3 0 -22 .5 13 31 41 34 26 4 4 4 -11.25 8 36 l 8 l i l l 53 19 11 0 0 11 22 120 73 39 24 8 5 0 +11.25 69 28 4o 69 26 7 4 2 +22. 5 15 36 44. 24 20 5 9 1 +33-75 26 51 10 9 6 8 6 5 +45.0 22 16 42 14 5 3 0 2 T o t a l 194 246 536 383 198 77 45 19 -. - l i n e p r o f i l e s f o r four experiments, and i n F i g . 77 f o r v e r t i c a l pro-f i l e s encompassing v a r i o u s sector angles o f the experimental area f o r Experiment V I I I . Experiments I and V I I , the two t e s t s w i t h r e l e a s e of spores at one fo o t , both show a maximum concentration at the f i v e f o o t d i s t a n c e , the c l o s e s t sampling d i s t a n c e , w i t h a very r a p i d r e d u c t i o n at greater d i s t a n c e s from the r e l e a s e source ( F i g . 7 6 ) . I n Experiment I , no spores were c o l l e c t e d more than 35 f e e t from the source and upward d i s p e r s i o n was l e s s than i n Experiment V I I , when spores were dispersed to a height o f 10 f e e t w i t h i n a distance of 20 f e e t . The other e x p e r i -ments a l s o show considerable v a r i a t i o n of the concentration along the mi d - l i n e . G e n e r a l l y the maximum concentration sampled occurred at a dist a n c e of 20 f e e t and at a height o f 5 f e e t w i t h a f i v e foot r e l e a s e p o i n t ( F i g . 7 8 ) . In Experiments I I and X the maximum concentration at the 20 foot distance was at the one foot height. I n both these t e s t s the wind speed was l e s s and f r e s h spores were dispersed i n Experiment X. F i g . 77. V e r t i c a l p r o f i l e s of aeciospore concentration p a t t e r n s along the m i d - l i n e and f o r H-5° and 90° sectors of the sampling network, f o r one spore r e l e a s e at f i v e f e e t above ground (Experiment V I I I ) . - 191 -EXPERIMENT VIII 0 5 10 20 35 50 75 100 DISTANCE FROM SOURCE (FEET) F i g . 78. Composite v e r t i c a l p r o f i l e s of aeciospore concentration p a t t e r n s along the m i d - l i n e and f o r and 90° sectors of the sampling network, f o r a l l eight spore r e l e a s e s f i v e f e e t above ground (Experiments I I - V I , V I I I - X ) . D I STANCE FROM S O U R C E (FEET) - 193 -In Experiment I I , when the mean wind speed was only 7.9 f t / s e c , higher concentrations were deposited at the 5 and 10 foot d i s t a n c e s than at the 20 f o o t , whereas a l l other 5 foot r e l e a s e t e s t s gave higher con-c e n t r a t i o n s at the 20 foot distances and the 1 foot height, as shown by the composite v e r t i c a l p r o f i l e of F i g . 78. R e l a t i v e l y high concentrations were obtained i n Experiments I I I , V, IX and X, at the 10 foot height at the 20 foot d i s t a n c e , i l l u s t r a t i n g that spores may be c a r r i e d upwards by a i r turbulence, and transported beyond the experimental area before being deposited. Experiment V, which had the highest mean wind speed (20.6 f t / sec), shows t h i s p o s s i b i l i t y best. S i z e of spore apparently had l i t t l e e f f e c t on d i s p e r s a l p a t t e r n s , measurements of 10 spores c o l l e c t e d at 5, 20 and 50 f e e t from the source i n each experiment gave no evidence that l a r g e r spores were deposited c l o s e r to the source or that smaller spores were deposited f u r t h e r away. V a r i a t i o n i n spore s i z e and i t s e f f e c t on r a t e of spore f a l l , t h e r e f o r e , could not be s a i d to have any e f f e c t at the windspeeds of the experiments. L a t e r a l spread of spores extended to, 1+5° on e i t h e r side of the mean wind d i r e c t i o n at distances of 10 and 20 f e e t i n a l l experiments except Experiments TV and VI, which showed only 22.5° or 33-75° l a t e r a l spread on one or both sides of the mean. This was probably a r e s u l t of the i n f l u e n c e of wind f l u c t u a t i o n s . Sreeramulu and Ramalingam (1961) found t h a t l a t e r a l spread v a r i e d depending on time of day, mean wind speed, and d u r a t i o n of spore l i b e r a t i o n . I n t h e i r cases lateralj'spore spread extended to 30° on e i t h e r side of the mean wind a x i s , and around noon and e a r l y afternoon, the time of my experiments, extended to 50° and 70°. L a t e r a l spread i n some of my experiments extended w e l l beyond - 19k -k5°, as was i l l u s t r a t e d by some of the high spore deposits on the 45° r a d i i from the m i d - l i n e , e.g., 753 spores at 10 f e e t i n Experiment V I I , 105 spores at 10 f e e t i n Experiment IX. Wilson and Baker (1946) found th a t at low-wind v e l o c i t i e s d i s p e r s i o n at a given distance from the source was f r e q u e n t l y much greater than at medium v e l o c i t i e s , probably because of more v a r i a b i l i t y i n wind d i r e c t i o n at lower than at higher v e l o c i t i e s . I n the ten experiments spores were only c o l l e c t e d at the 200 foot d i stance three times w i t h the p l a s t i c s l i d e holder c o l l e c t o r , and twice more by the more e f f i c i e n t r o t o r o d sampler, which was placed at t h i s distance f o r three t e s t s . With the r o t o r o d sampler placed at 300 f e e t spores were c o l l e c t e d during three of f i v e tests.- An impaction spore c o l l e c t o r placed at 400 f e e t c o l l e c t e d spores (2) o n l y during one t e s t . The l a c k of spores c o l l e c t e d at greater d i s t a n c e s from the source and the r a p i d decreases of concentration were misleading. Each sampling p o i n t only c o l l e c t e d spores whose f l i g h t path l a y through the sm a l l v e r t i c a l area- taken up by the i n c l i n e d s l i d e . Due to d i f f u s i o n i n the v e r t i c a l and h o r i z o n t a l planes, the concentration of the- spore cloud must have decreased approximately, w i t h the square of the distance from the source. Therefore, the observed r a p i d decrease of the spore concen-t r a t i o n may have been due l a r g e l y to d i f f u s i o n and not have represented d e p l e t i o n of the spore cloud by d e p o s i t i o n . To o b t a i n an estimate of the expected decrease of the sampled spore concentration w i t h d i s t a n c e , due to d i f f u s i o n , two methods were employed, accepting the t o t a l spore catch at four d i s t a n c e s , or the catch at 20 f e e t , as reference p o i n t s (Table X X I I l ) . - 195 -Table X X I I I . Comparison of the observed and expected concentra-t i o n of spores at four d i s t a n c e s from the 5 foot r e l e a s e p o i n t during ei g h t t e s t s . Experiment Type of Spore concentration at each no. data^'^ sampling distance ( f e e t ) 20 50 100 150 I I Observed 764 111 51 Expected A 764 174 50 24 Expected B 580 272 79 18 I I I Observed 1273 253 128 24 Expected A 1273 289- 84 39 Expected B 1023 481 i 4 o 32 IV Observed 327 79 3 0 Expected A 327 74 22 10 Expected B 250 117 34 8 V Observed 618 198 36 45 Expected A 618 141 41 19 Expected B 548 257 74 17 VI Observed 198 87 15 0 Expected A 198 45 13 6 Expected B 183 86 25 6 V I I I Observed 495 206 72 42 Expected A 495 113 33 15 Expected B 498 234 68 15 IX Observed 477 132 39 30 Expected A 477 108 31 . 14 Expected B 4 l 4 195 56 13 X Observed 683 316 85 35 Expected A 683 155 45 21 Expected B 684 321 93 21 Average Observed 6o4 173 50 28 Expected A 6o4 137 4o 19 Expected B 523 245 71 16 -^Observed values are t o t a l s of spores c o l l e c t e d at three heights and on f i v e r a d i i at each d i s t a n c e . ^Expected values are estimates of spores c o l l e c t e d at each d i s t a n c e , A) accepting catch at 20 f e e t and assuming r a t i o of 1 : 4 . 4 : 1 5 . 2 : 3 2 . 4 , and B) t a k i n g t o t a l catch at a l l d i stances and d i s t r i b u t i n g the t o t a l spores i n inv e r s e p r o p o r t i o n to the cross s e c t i o n a l areas at each di s t a n c e . - 196 -Inl.Experiments V, V I I I , IX and X, the observed counts at s e v e r a l distances were higher than those estimated by method A, which assumes an accurate measure of the spore concentration at 20 f e e t . Experiments I I I , IV and VI, a l l showed a lower t o t a l at the 150 foot distance than the expected, which may have been due to greater v e r t i c a l d i s p e r s i o n . Experiments I I and V showed higher observed t o t a l s at 15O than 100 f e e t . Based on method B the expected values at 20 f e e t were g e n e r a l l y much lower than the ob-served values, except i n Experiments VI, V I I I and X. Table X X I I I i n d i c a t e s that much of the observed decrease i n spore concentration w i t h distance was due to d i f f u s i o n of the spore cloud and not to d e p o s i t i o n . I t was d i f f i c u l t to i n t e r p r e t the estimated and observed values. The d i f f e r e n c e i n the two methods f o r o b t a i n i n g expected values was l a r g e l y a problem of r e d i s t r i b u t i n g the data, assuming accuracy f o r some of the observed v a l u e s . Method A, on an average, showed th a t the observed values at 50, 100 and 150 f e e t were too high, but method B i n d i c a t e d t h a t observed values were too low at 20, 50 and 100 f e e t . Probably there was considerable v a r i a t i o n and e r r o r i n the observed values, making spore d i f f u s i o n estimates based on these questionable. At low wind speeds the e f f i c i e n c y of the c o l l e c t o r was low and the catch t h e r e f o r e was sma l l , thus the e r r o r of e s t i m a t i o n becomes great. 'There could have been momentary large v a r i a t i o n s i n spore concentration between adjacent sampling areas due to changes i n turbu-lence and s i z e of eddies, or f l u c t u a t i o n s i n wind v e l o c i t y which a f f e c t e d the v e r t i c a l and l a t e r a l d i s p e r s i o n from one distance to another. The number of spores passing through a u n i t area would t h e r e f o r e have v a r i e d g r e a t l y over the sampling network. With an increase i n wind v e l o c i t y the expected degree of d i s p e r s i o n would decrease and the amount of d e p o s i t i o n - 197 -would be reduced. I n unstable a i r on c l e a r days, spore clouds may move i n a s e r i e s of great loops (Waggoner I965I and c e r t a i n sampling p o i n t s may have been skipped which could have accounted f o r p o i n t s c l o s e r to the source having lower spore concentrations (Gregory 1968). RATE OF FALL OF AECIOSPORES IM CALM AIR The distances that spores are c a r r i e d i n d i s p e r s a l depend on the a l t i t u d e reached by spores i n the a i r currents, the r a t e of spore f a l l under the i n f l u e n c e of g r a v i t y , the v e l o c i t y , azimuth and d u r a t i o n of winds, and the v e r t i c a l mass exchange brought about under t u r b u l e n t movement. I f the r a t e of f a l l of a spore i n calm a i r i s determined, the t h e o r e t i c a l d i s p e r s a l distance f o r a spore l i b e r a t e d at a given height, or c a r r i e d to a c e r t a i n a l t i t u d e can be c a l c u l a t e d f o r any mean wind speed. More important, the v e l o c i t y of f a l l is- an important f a c t o r i n determining the f l i g h t path of a spore i n atmospheric turbulence. M a t e r i a l s and Methods Various types of c y l i n d e r s f o r measuring r a t e o f . V f a l l of spores or p o l l e n g r a i n s have been employed by McCubbin (1918a),' Ukkelberg (1933), Durham (19I+6), Weinhold (1955), and others. In the present study the r a t e of f a l l of aeciospores was mea-sured i n a p l a s t i c c y l i n d e r , 3m long and 23 cm i n diameter. The c y l i n d e r was grounded and coated w i t h t e f l o n powder to reduce s t a t i c e f f e c t s . The upper end of the c y l i n d e r was covered w i t h a t i g h t l y - f i t t i n g a c r y l i c p l a s -t i c d i s c w i t h a center hole f o r r e l e a s e of spores. The hole was covered - 198 -w i t h a p l a s t i c s t r i p before and a f t e r spore r e l e a s e to minimize turbulence w i t h i n the c y l i n d e r . The upper end of the c y l i n d e r was attached to the c e i l i n g a l l o w i n g the c y l i n d e r to hang f r e e l y i n a room held at constant temperature (22°C, 30% R.H.) w i t h minimal a i r movement i n the experimental room. The lower end of the c y l i n d e r c o n s i s t e d of a t i g h t l y - f i t t i n g s h a l -low d i s h w i t h a f a l s e bottom made from 0.64 cm t h i c k a c r y l i c p l a s t i c . I n the base of the d i s h an opening 5 cm x 1.5 cm was cut to all o w f r e e f a l l of aeciospores on to an exposed v a s e l i n e coated s l i d e . A s l i d e c a r r i e r 56 cm long was constructed to hold eighteen 2.5 x 7-5 cm microscope s l i d e s . The s l i d e c a r r i e r was i n s e r t e d through the openings of the f a l s e bottom of the c y l i n d e r , passing d i r e c t l y through the center of the c y l i n d e r under the spore o u t l e t . During the f a l l of aeciospores each s l i d e of the s e r i e s was exposed beneath the spore o u t l e t f o r 15 seconds. The changing of s l i d e s r e q u i r e d l e s s than a second. Before each t r i a l the c y l i n d e r was c a r e f u l l y washed w i t h water to remove a l l spores from the w a l l and base. The c y l i n d e r was then d r i e d , f r e s h l y coated w i t h t e f l o n powder and allowed to s t a b i l i z e i n the environment of the experimental room. A c o n t r o l s l i d e was exposed i n the c y l i n d e r before each t r i a l t o determine whether i t was fre e of spores.. The aeciospores used i n the experiments were e i t h e r f r e s h spores or had been stored f o r known periods of time, the samples being c a r e f u l l y screened to remove extraneous m a t e r i a l and to reduce the num-ber and s i z e of any spore clumps present. A measured amount o f spores was released i n t o the c y l i n d e r by i n v e r t i n g a 2 ml v i a l c o n t a i n i n g the spores over the open hole at the top of the c y l i n d e r . A l l spores c o l -l e c t e d on the s l i d e s t h e r e f o r e had f a l l e n 3 meters. Traverses of the complete exposed p o r t i o n of each s l i d e were made under the microscope to - 199 -count the number of s i n g l e spores c o l l e c t e d during each exposure p e r i o d . The number of clumps of spores deposited on each s l i d e were al s o noted. Larger clumps tended to occur on the e a r l i e r exposed s l i d e s , but unfor-t u n a t e l y estimates of the s i z e of clumps' were not kept. The diameters of 20 spores were measured on the second and every second or t h i r d s l i d e t h e r e a f t e r , of each s e r i e s , to check f o r any r e l a t i o n s h i p between the s i z e of spore and r a t e of f a l l . One t e s t was a l s o c a r r i e d out w i t h each s l i d e exposed f o r 30 seconds to check whether a l l spores were being de-p o s i t e d i n the 4 min 15 sec d u r a t i o n of the other t e s t s . No spores were c o l l e c t e d from K min 30 sec onwards, thus the exposure d u r a t i o n was s a t i s -f a c t o r y . T r i a l s were al s o c a r r i e d out w i t h wet and dry spores to check f o r any marked e f f e c t of moisture content on the r a t e of f a l l . A c o l -l e c t i o n of spores was d i v i d e d i n t o two equal weight u n i t s . One u n i t was r e l e a s e d immediately i n the c y l i n d e r . The other was allowed to take up moisture:'.in a saturated atmosphere f o r h8 hours, was reweighed and r e -leased i n the c y l i n d e r . An increase i n weight, f o l l o w i n g exposure to a saturated atmosphere, was recorded. Diameter measurements of spores were als o made from the wet and dry samples immediately a f t e r r e l e a s e i n the c y l i n d e r . The formula and method used to c a l c u l a t e the r a t e of f a l l f o l -lowed that used by Ukkelberg (1933)- This formula c a l c u l a t e d the average number of seconds r e q u i r e d f o r s i n g l e spores to f a l l . The formula was Zfx/N, where Z= summation, f = frequency i n a c l a s s (number of spores deposited on each s l i d e during each exposure i n t e r v a l ) , x =. c l a s s center (mean of each exposure period) and N = t o t a l number of spores deposited. - 200 -By using the c l a s s center, the assumption was made that the spores were deposited uniformly, throughout each exposure p e r i o d , a s i t u a t i o n which probably d i d not occur. The s l i g h t e r r o r introduced, however, would have l i t t l e e f f e c t on the mean as the v a r i a t i o n s would be i n both d i r e c -t i o n s . The r a t e of f a l l as expressed i n centimeters per second was ob-t a i n e d from the f o l l o w i n g formula: R = D/T, where D = distance f a l l e n ( and T = average time i n seconds r e q u i r e d to f a l l the given d i s t a n c e . R e s u l t s Considerable v a r i a t i o n was found i n the time r e q u i r e d f o r s i n -gle aeciospores to f a l l the length of the c y l i n d e r . Table XXIV shows the percentage of s i n g l e aeciospores deposited 3 meters from p o i n t of l i b e r a t i o n on g l a s s s l i d e s exposed s u c c e s s i v e l y f o r 15 second periods during ten i n d i v i d u a l r a t e of f a l l t r i a l s , and the average of these t r i a l s . The r a t e of f a l l of over 55,000 i n d i v i d u a l aeciospores was measured. I n three t r i a l s a few spores f e l l 3 meters i n 15 seconds or l e s s , w h i l e other spores-required over k minutes to f a l l that, d i s t a n c e . On average 82% of the aeciospores f e l l w i t h i n 2 minutes and 97% w i t h i n 3 minutes. The average number of seconds r e q u i r e d f o r the s i n g l e spores of the ten d i f f e r e n t t r i a l s to f a l l 3 meters, and the average r a t e of f a l l are shown i n Table XXV. By averaging the mean r a t e s of f a l l one obtains an average v e l o c i t y of f a l l f o r aeciospores of 3-64 cm per second, however, the range of the i n d i v i d u a l t e s t s i s considerable (2.79 to 5.10 cm per sec). Much of t h i s v a r i a t i o n between i n d i v i d u a l t e s t s can be a t t r i b u t e d to the degree of spore clumping i n the t e s t sample and to i t s a s s o c i a t e d mass .subsidence, which was more obvious i n some t e s t s Table XXIV. Percentages of i n d i v i d u a l t e s t s of aeciospores of Cronartium comandrae deposited 3 meters from p o i n t of l i b e r a t i o n on glass s l i d e s exposed s u c c e s s i v e l y f o r 15 second periods i n a closed c y l i n d e r . Test Exposure periods (no. of seconds a f t e r spores were l i b e r a t e d ) no. 0 - 16- 31- 46- 61- 76- 9 1 - 106- 121- 136- 151- 166- 181- 196- 211- 226- 2 4 l -15 30 45 6o 75 90 105 120 135 150 I65 180 195 210 225 240 255 1 0 . 3 2 .3 35-5 19-5 2 . 1 5-3 9- •9 7 .1 6 .2 5 .8 2.6 1.8 1.1 0 . 3 • 0 . 3 0 . 0 0 . 0 2 0 . 0 0 . 1 0 . 8 7.9 13.4 13.8 17. ,2 14.8 10.9 7-5 5-0 2-5 2 .6 1.5 0 . 6 ' 0 . 7 0 . 5 3 0 . 0 0 .2 i o . 8 16.7 23.4 4 . 3 5. ,7 12.9 9 . 8 4 . 9 3-1 3 - 1 1.8 1.1 1.7 0. 2 0 . 2 4 0 . 0 1.1 7-3 10.8 28.5 2 0 . 6 12. ,1 6 .3 3 . 8 2 .0 1.6 2.7 1.2 0 . 8 0 .4 0. 5 0 . 5 5 0 .5 11.1 2 0 . 4 15.9 8 . 8 13-7 11. • 7 7.0 3 .0 2.5 1.8 0 . 9 1.0 0 .5 0 . 3 0 . 5 0 . 3 6 0 . 0 0 .2 1.2 36.2 21.5 16.0 10. .2 3-9 3 . 8 1.9 1.8 1.2 6 . 8 0 . 6 o.h 0 . 4 0 . 2 7 0 . 0 0 .0 0.7 3-5 21 .1 2 5 . 8 12. ,1 10.9 6.0 7-0 5-2 2-9 1.4 1.5 1.5 0 . 3 0 . 3 8 0 . 1 0 . 3 1.4 19.1 1.8 19.2 22. • 7 15.8 .9-9 3-6 2.3 1.1 0 . 7 1.2 0 . 6 0. 1 0 . 1 9 0 . 0 18.3 36.4 9-4 6.6 7-4 9-• 3 4 . 9 4 . 0 2 .1 0 .5 0 .7 0 .2 0 . 0 0 . 1 0 . 1 0 . 1 10 0 . 0 7-9 18.8 4 . 2 12.8 21.3 14. ,2 6.5 4 . 9 2 . 1 3 .1 1.6 1.7 0 . 6 0 .2 0 . 0 0 . 1 Average 0 . 1 4 . 1 13-3 14.3 i 4 . o 14.7 12. • 5 9 . 0 6.2 3-9 2.7 1.8 1.3 0 . 8 0 . 6 0 . 3 0 . 2 - 202 -Table XXV. Average r a t e of f a l l i n s t i l l a i r of ten aeciospore t e s t s Test T o t a l no. Aver. no. seconds Aver, r a t e of f a l l no. of spores to f a l l 3 m. (cm/sec.) 1 3,278 74.12 4 .04 2 6,909 107.39 2.79 3 M 7 8 92.34 3.24 1+ 5,509 85.60 3.50 5 11,910 72.51 M 3 6 4,674 78 .46 3.82 7 3,789 102.53 2.92 8 3,144 96.43 3-11 9 6,947 58.81 5.10 10 ^,575 8 0 . 4 i 3-73 Mean — 84.90 . 3 . 6 4 • than i n others. On being r e l e a s e d at the top of the c y l i n d e r i t was no t i c e a b l e that some spores f e l l more r a p i d l y than others i n i t i a l l y , but as the spores f e l l f u r t h e r the mass of spores became more dis p e r s e d and the f a l l speed of i n d i v i d u a l spores was more uniform throughout the t e s t . By the time t h i s f a l l r a t e became more uniform some spores were a t h i r d or more of the way down the c y l i n d e r . The number of spore clumps de-p o s i t e d on the i n d i v i d u a l s l i d e s i n the ten t e s t s i s shown i n Table XXVI. No clumps occurred on any s l i d e s exposed a f t e r 2 min 15 sec. There was no way of knowing how many i n d i v i d u a l spores had broken away from clumps during the f a l l p e r i o d and t h e r e f o r e had an excessive f a l l r a t e f o r a p o r t i o n of the p e r i o d . However, some samples had very few clumps and these t e s t s , e.g. 7 and 8 (Table XXV), probably give a b e t t e r estimate of the true r a t e of f a l l . Test numbers 1, 5 and 9, a l l had numerous clumps, w i t h most being deposited on the e a r l y exposed s l i d e s , which i n d i c a t e d t h a t they were probably l a r g e r clumps which may w e l l have created a higher degree of mass subsidence. These t e s t s a l l had double maxima i n t h e i r - 203 -Table XXVI. Number of aeciospore clumps deposited 3 meters from p o i n t of l i b e r a t i o n on g l a s s s l i d e s exposed s u c c e s s i v e l y f o r 15 second periods i n a closed c y l i n d e r . Test no. 0 - 16- 31- - 46- 61- 76- 91- 106- 121- T o t a l no. 15 30 45 60 75 90 105 120 135 of clumps 1 0 4 22 9 2 l 0 0 0 38 .2 0 0 2 6 4 5 4 4 1 26 3 0 1 0 6 8 4 2 0 0 21 4 0 0 5 5 5 0 0 0 0 15 5 1 17 15 13 4 2 1 0 0 53 6 0 0 3 0 1 0 0 0 0 4 7 0 0 0 1 2 0 0 0 0 : 3 8 0 0 0 7 0 ' 0 1 0 0 8 9 0 7 29 8 3 3 0 0 0 50-. 10 0 4 7 4 3 0 0 0 0 18 rate ! of f a l l (Table XXIV), and had the highest avera^ le r a t e of f a l l of the t e s t s e r i e s (Table XXV). . Test no. 10 a l s o had a doub l e maxima wit! the f i r s t peak o c c u r r i n g w i t h i n 45 sec of spore : l i b e r a t i o n (Table XXV) Two other t e s t s had double maxima but these occurred much l a t e r a f t e r the r e l e a s e . I f the average r a t e of f a l l f o r t e s t s w i t h e a r l y double maxima (nos. 1, 5 3 9 and 10) i s omitted, then the remaining- t e s t s show an average f a l l r a t e of 3-23 cm/sec (range 2.79 -' 3 - 8 2 ) , which i s pro-bably much c l o s e r to the true r a t e , although i n i t i a l mass subsidence probably s t i l l caused t h i s to be an overestimate. The one t e s t w i t h s l i d e s exposed f o r 30 sec i n t e r v a l s gave an average f a l l r a t e of 3-06 cm/ sec. Other f a c t o r s may cause v a r i a t i o n s between t e s t s . There may 1 •: have been s l i g h t d i f f e r e n c e s i n the room environmental c o n d i t i o n s , un-avoidable a i r currents w i t h i n the c y l i n d e r , •-or v a r i a t i o n s i n spore s i z e or spore moisture content. Measurements of length and width of spores deposited on the second, f o u r t h , s i x t h and seventh s l i d e s of each t r i a l - 204 -s e r i e s showed l i t t l e o v e r a l l v a r i a t i o n , although smaller spores g e n e r a l l y took longer to f a l l . The spores f o r the ten t e s t s were c o l l e c t e d from i n -fec t e d t r e e s , maintained i n the greenhouse, and kept f o r periods from one hour to two days before being r e l e a s e d , thus v a r y i n g degrees o f spore dry i n g were experienced which explained some of the t e s t v a r i a t i o n . To e x p l a i n the e f f e c t of t h i s v a r i a t i o n a separate experiment was run w i t h dry and saturated wet spores, o r i g i n a t i n g from the same source. Table XXVII gives the average r a t e of f a l l of the dry and wet spores i n two t e s t s . The r a t e of f a l l of the dry spores (average 3.46 cm/sec) was probably an overestimate, but the wet spores f e l l about 2.7 times f a s t e r . There was considerable clumping of the wet spores and mass subsidence was evident, but the change of spore f a l l r a t e w i t h v a r i a t i o n i n spore moisture content was i l l u s t r a t e d . Measurement of the l e n g t h and width of 10-15 spores de-p o s i t e d on the second, s i x t h , t e nth and fourteenth s l i d e during the f i r s t dry-wet t e s t showed some v a r i a t i o n (Table X X V I I I ) , w i t h smaller spores g e n e r a l l y t a k i n g longer to f a l l . Table XXVII. Average r a t e of f a l l i n s t i l l a i r of dry and wet aeciospores during two t e s t s . Test Spore Aver. no. seconds Aver, r a t e of no. c o n d i t i o n to f a l l 3 m. f a l l (cm/sec.) 1 Dry 80.91 3.52 2 Dry 88.41 3-39 1 Wet 28.84 10.09 2 Wet 32.60 9,20 - 205 -Table XXVIII. Average length and width (u) of aeciospores deposited on s l i d e s during the dry and wet spore t e s t s of r a t e of f a l l . S l i d e Dry Wet no. l e n g t h width l e n g t h width 2 62.4 21.1 55.9 28.9 6 61.5 22.4 55.7 28.1 10 55-5 19-4 49.1 28.5 14 55-5 19-6 45.0 29.1 AECIOSPORE DISPERSAL DISCUSSION M e t e o r o l o g i c a l f a c t o r s and spore p e r i o d i c i t y D i u r n a l p e r i o d i c i t y i n airborne organisms was f i r s t observed by P i e r r e M iquel i n the l a s t quarter of the nineteenth century i n France, from h i s d a i l y counts of b a c t e r i a and molds (Miquel 1878-1899). Since 1950 there has been renewed i n t e r e s t due l a r g e l y to the development o f the H i r s t spore trap ( H i r s t 1952) and other continuous a i r samplers. The d i u r n a l p e r i o d i c i t y of various components of the a i r f u n g a l spora have now been reported by many workers (Adams 1964; Cammack 1955; Carter and Banyer 1964; Cole 1966; Gregory 1952, I 9 6 I ; Gregory and H i r s t 1957; Gregory and Sreeramulu 1958; Hamilton 1959; Harvey 1967; H i r s t 1953; Kramer e_t a l . 1963, 1964; Lukezic and K a i s e r 1966; M i l l s I967; Pady et a l . 1962, 1965; Panzer et a l . 1957; Pathak and Pady I965; Pawsey 1964; R i c h and Waggoner 1962; Shanmuganathan and Arulpragasam 1966; Sreeramulu 1959, 1962, 1963; Sreeramulu and Seshavatarum 1962; Sreeramulu and V i t t a l 1966; Van A r s d e l 1967; Waggoner and Taylor I 9 5 8 ) . Gregory (1961) d i v i d e d the spore d i u r n a l p a t t e r n s i n t o f i v e types: b a c t e r i a l (with two maxima and - 206 -minima), no c t u r n a l , forenoon, afternoon and evening. G e n e r a l l y the pat-terns w i t h C. comandrae aeciospores f a l l i n t o a"forenoon" or "afternoon" p a t t e r n . The l a t t e r p a t t e r n i s t y p i c a l of the m a j o r i t y of the daytime dry spore forms, and the former group inc l u d e s a few crop-pathogenic f u n g i (Gregory 1961). These patte r n s show a peak spore concentration between 1000 and 1600 hours, although the weather of a p a r t i c u l a r day may d i s t u r b the normal rhythm. A review of the l i t e r a t u r e showed l i t t l e comparable informa t i o n on the d i u r n a l p e r i o d i c i t y of r u s t spores of i n d i v i d u a l spe-c i e s , u s u a l l y the data were grouped under 'uredospores' and 'ba s i d i o -spores', sometimes w i t h an i n d i c a t i o n of p o s s i b l e sources. Wo in f o r m a t i o n was noted on the d i r u n a l p e r i o d i c i t y of r u s t aeciospores. H i r s t (1953)5 i n h i s r e p o r t s of the a i r - s p o r a at Rothamsted Experimental S t a t i o n i n 1951 and 1952, s t a t e d that "no aecidiospores were recognized". The pre-sent study i s t h e r e f o r e the f i r s t f o r t h i s r u s t spore s t a t e . Information on r u s t uredospores g e n e r a l l y show an afternoon p a t t e r n (Adams I96U-; Cammack 1955; Hamilton 1959; H i r s t 1953; Pady et a l . 1965; Sreeramulu 1959; a n ( i Powell, unpublished data f o r Cronartium comandrae) . B a s i d i o -spores, as ,a group, show a predominantly n o c t u r n a l p a t t e r n (Adams 1964; Gregory I 9 6 I ; Gregory and H i r s t 1957; H i r s t 1953; Kramer et a l . 1963; Pawsey 1964; Shanmuganathan and Arulpragasam I966; Sreeramulu and Seshavataram 1962; Van A r s d e l 196?), although few rep o r t s have been ap-p l i e d to i n d i v i d u a l species of Basidiomycetes., and fewer s t i l l to r u s t species, eg. P u c c i n i a malvacearum Mont. (Carter and Banyer 1964), Cronartium r i b i c o l a (Van A r s d e l 1967)• Other studies a l s o found evidence of a double or secondary peak i n the a i r spora concentration (Gregory and Stedman 1958; Harvey - 207 -1967; Pady et a l . 1965; Pathak and Pady 1965; Pawsey 1964; R i c h and Waggoner 1962). In some cases t h i s was a p e r s i s t e n t f e a t u r e , but i n others occurred s p o r a d i c a l l y or through some combination of environ-mental f a c t o r s . Most of t h i s work concerned the d i u r n a l p e r i o d i c i t y of Cladosporium spores, where a peak was recorded i n the forenoon, and a second smaller peak i n the afternoon or e a r l y evening. This p e c u l i a r d i u r n a l cycle was f i r s t discussed by R i c h and Waggoner (1962) who a s c r i b e d the phenomenon to the cy c l e of atmospheric turbulence, whereby mature spores become airborne w i t h the i n c r e a s i n g morning turbulence to produce a forenoon peak. During the afternoon or e a r l y evening the turbulence lessens a l l o w i n g the spores to s e t t l e and give r i s e to a secondary peak. The midday minor minimum was explained by the spore source being depleted as there was only a s i n g l e d a i l y spore crop and because the concentration of spores i n the atmosphere was lowered. I t i s not known i f r u s t s produce a s i n g l e d a i l y crop o f aeciospores, but the atmospheric turbulence c y c l e may help to e x p l a i n the o c c a s i o n a l evidence of a secondary peak w i t h aeciospores of C_. comandrae. I t i s more l i k e l y t h a t , because the spores were c o l l e c t e d close to the source, a combination of i n t e r r e l a t e d f a c t o r s was i n v o l v e d i n d i s r u p t i n g the t y p i c a l d i u r n a l p a t t e r n . The apparent close r e l a t i o n s h i p of spore d i s p e r s a l p e r i o d -i c i t y w i t h temperature and r e l a t i v e humidity i s not unexpected, i n t h a t i t i s t y p i c a l of most components of dry weather a i r spora. The l a t t e r show an increase i n spore concentration g e n e r a l l y c o i n c i d i n g w i t h the s t a r t of atmospheric turbulence (a r a p i d change i n temperature, humidity and wind c o n d i t i o n s ) . Turbulence g e n e r a l l y increases from s u n r i s e to - 208 -around noon and then f a l l s again, and i t seems l i k e l y t h a t turbulence i s a more important f a c t o r than temperature and humidity a c t i n g s e p a r a t e l y f o r d a i l y spore d i s p e r s a l , although the l a t t e r are c o n t r i -b u t i n g f a c t o r s to turbulence. Turbulent c o n d i t i o n s were as s o c i a t e d w i t h a l l peak aeciospore concentrations whether they occurred i n the forenoon or afternoon, on dry sunny days or ass o c i a t e d w i t h the onset of heavy r a i n showers or storms. Lukezic and K a i s e r (1966) showed a h i g h l y s i g n i f i c a n t c o r r e l a t i o n between Fusarium spore concentration and atmospheric turbulence. Holmes and Bassett. (1963) showed a s i m i l a r r e l a t i o n s h i p w i t h d i u r n a l ragweed p o l l e n d i s p e r s a l studies at Ottawa, and observed an evening r i s e i n p o l l e n count on about h a l f the days, which may have been as s o c i a t e d w i t h p o l l e n s e t t l i n g f o l l o w i n g a decrease i n atmospheric turbulence. M i l l s (1967) showed th a t turbulence was the main disseminating agent f o r U s t i l a g o avenae (Pers.) R o s t r . when wind v e l o c i t i e s were low. The v a r y i n g e f f e c t of r a i n , has been noted by other workers. H i r s t (1953) observed that l i g h t r a i n s reduced the concentration of Cladosporium spores, but t h a t a thunderstorm increased concentration. Ainsworth (1952) and Gregory (195*0 a l s o found a r a p i d increase i n Cladosporium spore concentration during r a i n showers, but Hamilton (1959) found an appreciable decrease. R i c h and Waggoner (1962) found an increase i n the Cladosporium spore concentration w i t h the heavy r a i n showers of unstable c o n d i t i o n s , but the concentration was not increased by l i g h t r a i n and d r i z z l e a s s o c i a t e d w i t h s t a b i l i t y i n the atmosphere. H i r s t and Stedman (1963) reported that the t r a n s i e n t increase i n spore concentration of many spore types a s s o c i a t e d w i t h - 209 -onset of r a i n was d i s c e r n i b l e i n about h a l f of the r a i n p e r i o d s . Shanmu-ganathan and Arulpragasam (1966) noted a sharp increase i n b a s i d i o s p o r e s of Exobasidium vexans Massee, e i t h e r during, or immediately a f t e r heavy afternoon thunderstorms. I f the r a i n was prolonged the concentration de-creased a f t e r the i n i t i a l onset due to removal of spores from the a i r , however, a steady d r i z z l e had l i t t l e e f f e c t . J a r v i s ('1962) found that r a i n showers were f r e q u e n t l y a s s o c i a t e d w i t h high spore concentrations of B o t r y t i s c i n e r e a F r . i n otherwise u n s u i t a b l e c o n d i t i o n s at nig h t and dur-ing the day. M i l l s (1967) reported that w i t h some r a i n periods accom-panied by increased winds there was a marked increase i n numbers of U s t i l a g o avenae spores caught, but not w i t h other r a i n showers which were accompanied by a smaller increase i n wind. Sreeramulu and V i t t a l (1966) found that prolonged r a i n f a l l reduced the incidence of U s t i l a g i n o i d e a v i r e n s (Cke.) Tak. spores. From the v a r y i n g evidence, and that of the present study, i t would appear t h a t gusty heavy rainstorms w i t h t h e i r as-so c i a t e d turbulence tend to increase spore concentrations by c a r r y i n g more spores a l o f t through u p d r a f t s , a f t e r j a r r i n g the i n f e c t e d t i s s u e s which r e l e a s e s the spores, or through r a i n s plash. On the-.other.liand, l i g h t gentle r a i n s tend to wash the spores out of the atmosphere and wet the i n f e c t e d surfaces, which e f f e c t i v e l y prevents removal of spores- i n t o the a i r . Weickmann (1963) pointed out that heavy storms w i t h high r a i n -f a l l r a t e s are a s s o c i a t e d w i t h atmospheric c i r c u l a t i o n s of a high degree of o r g a n i z a t i o n and p e r s i s t e n t updrafts, whereas weak storms l a c k the organized c i r c u l a t i o n and t h e i r updrafts are more t r a n s i e n t . Davies (1959) demonstrated t h a t wind-driven water d r o p l e t s detach 6 to 335 times more Cladosporium spores than are detached by dry a i r moving at the same speeds, - 210 -and that the greatest e f f e c t was noted from wind speeds of 0.7 to 2.0 m/sec- H i r s t and Stedman (1963) showed that the l a r g e r the r a i n drop s i z e the l a r g e r the number of spores l i b e r a t e d i n t o the a i r through me-ch a n i c a l shaking and by r a d i a l a i r movements. Chamberlain (1967) repor-ted t hat the e f f i c i e n c y of capture by raindrops i s high f o r p a r t i c l e s of about 20u or higher, a l s o he c a l c u l a t e d (1953) t h a t a r a i n f a l l i n t e n s i t y of 2 mm/hr w i l l e f f e c t i v e l y reduce a 30M- p a r t i c l e c oncentration by about 65% a f t e r 30 minutes and by n e a r l y 90% a f t e r one hour. The f i e l d r e s u l t s of H i r s t (l953j 1959) i n d i c a t e d an even more r a p i d removal of spores from the a i r during a 30 minute 0.95 mm r a i n f a l l . Counts of p o l l e n and.spores of U s t i l a g o , Cladosporium, A l t e r n a r i a and Erysiphe, before, during and a f t e r the thunderstorm, i n d i c a t e d the spore counts a f t e r r a i n were r e -duced to l / 2 5 , l/4, l / 5 , l/4 and l / l l r e s p e c t i v e l y of the counts before r a i n , and that during the 30 minute r a i n , counts were increased between 9 and hh times ( H i r s t 1959). McDonald (1962) c a l c u l a t e d t h a t the "wash-out p r o b a b i l i t i e s " f o r p o l l e n w i t h a diameter of 20-26u, and a r a i n f a l l of 1 mm, was 99% f° r raindrop diameters of 0.2 mm, and 72% f o r diameters of 1.0 mm. The corresponding c o l l e c t i o n e f f i c i e n c e s v a r i e d between 65-71% f o r 0.2 mm and 84-87% f o r 1 mm raindrops because of d i f f e r e n c e s i n p o l l e n d e n s i t i e s . Dingle and Gatz (1966) reported t h a t heavy convective r a i n brought about a r a p i d decrease i n the c o n c e n t r a t i o n of p o l l e n . The e f f e c t of dew was minimal as i t only tended to delay the d a i l y i n i t i a l d i s p e r s a l of aeciospores. During periods of dew formation, wind v e l o c i t y and turbulence are l i k e l y to be very low, thus i f any spores are r e l e a s e d , and evidence suggests that they are not g e n e r a l l y dispersed under these c o n d i t i o n s , d e p o s i t i o n i s l i k e l y to take place - 211 -w i t h i n a meter of t h e i r p o i n t o f r e l e a s e by sedimentation. Studies of e f f e c t of dew have u s u a l l y teen undertaken i n a s s o c i a t i o n w i t h spores r e q u i r i n g wet periods f o r d i s p e r s a l (Moore 1958; H i r s t and Stedman I962; Carter 1963). For most dry-spore f u n g i , wind i s an important f a c t o r i n spore r e l e a s e , as they have no s p e c i a l active- mechanism by which they are l i -berated. Once the spores become exposed the number of spores shed at any time should depend on the v a r y i n g ease w i t h which spores are detached from preformed spore masses ( H i r s t 1953> 1959)- P a r t i c l e s of 30n diameter are not e a s i l y removed by a i r movements along (Chamberlain 1967), as any surface i s surrounded by an a i r l a y e r known as the laminar or surface boundary l a y e r i n which there i s no turbulence, and i n which a i r flows i n streamlines p a r a l l e l to the nearest surface (Gregory I96I; T y l d e s l e y 1967). Any spore l i b e r a t e d i n t o t h i s surface boundary l a y e r w i l l s i n k to the surface by sedimentation. The major e f f e c t of wind i s t h a t of g e t t i n g the p a s s i v e l y l i b e r a t e d spores away from the surface where they are formed across the boundary l a y e r of non-turbulent f l o w i n g a i r i n t o the general c i r c u l a t i o n of t u r b u l e n t a i r . The thickness of the laminar boundary l a y e r v a r i e s w i t h the wind speed and w i t h the roughness of the adjacent surface, thus the higher the wind speed the more e a s i l y spores can be pic k e d up or blown o f f the surface by eddies which force t h e i r way to the surface d i s -r u p t i n g the laminar l a y e r . R i c h and Waggoner (1962) showed that repeated j a r r i n g r a p i d l y depletes a spore source, thus any increase of wind v e l o c i t y which mechanically moves the source w i l l b r i n g about a r a p i d d e p l e t i o n of the mature detached spores. An i n i t i a l gusty or higher wind speed pro-b a b l y produces a greater spore concentration than an i n i t i a l slower wind - 212 -v e l o c i t y , as a higher percentage of the spores would be p a s s i v e l y l i b e r a t e d from the a e c i a at the outset of stronger winds than by weaker winds. H i r s t (1959) s t a t e d t h a t "there i s l i t t l e evidence t h a t spore concentrations are increased i n p r o p o r t i o n to i n c r e a s i n g wind speed, probably because spore clouds are r a p i d l y dispersed by high winds". Both Zoberi (1961) and Smith (1966) showed a d i r e c t r e l a t i o n s h i p between wind speed and spore l i b e r a t i o n from experimental t e s t s , and Zoberi a l s o showed th a t dry a i r was more e f f e c t i v e than damp a i r i n d i s p e r s i n g spores. As mentioned ear-l i e r , M i l l s (1967) i n d i c a t e d t h a t r a i n accompanied by high winds brought about higher concentrations but low wind v e l o c i t i e s d i d not, and the data of other workers suggests the same r e l a t i o n s h i p of t h i s combination of" f a c t o r s . Sreeramulu (1962) was able to show th a t high concentrations of U s t i l a g o nuda (Jens.) R o s t r . spores o f t e n coincided w i t h high wind v e l o c i -t i e s . However, as spores can a l s o be dispersed by l i g h t winds, considera-b l y stronger winds o c c u r r i n g l a t e r are u n l i k e l y to app r e c i a b l y increase the concentrations of airborne spores, as was noted by J a r v i s (1962). Shanmuganathan and Arulpragasam (1966) i n d i c a t e d a negative c o r r e l a t i o n between wind speed and Exobasidium vexans spore concentration, which i s the reverse of the expected p h y s i c a l r e l a t i o n s h i p . Hamilton (1959) has als o reported a decrease i n concentration of some spores w i t h increased wind. One feature that should be borne i n mind i s that the e f f i c i e n c y of t r a p p i n g v a r i e s over the range of wind v e l o c i t y ( H i r s t 1953) and i t i s d i f f i c u l t to compare adequately the performance of spores of d i f f e r e n t species i n r e l a t i o n to wind v e l o c i t y , as spores vary c o n s i d e r a b l y i n t h e i r impaction e f f i c i e n c y (Gregory 1961). - 213 -Spore d i s p e r s a l In the present study no aeciospores were c o l l e c t e d at distances greater than 400 f e e t from the source on pine. Samplers operated at a distance of 725 f e e t during two seasons f a i l e d to c o l l e c t a s i n g l e spore;. Wo doubt aeciospores were c a r r i e d greater distances than 400 f e e t on some occasions due to d i f f u s i o n of the spore cloud, but a s u i t a b l e sampling network, free from background spore contamination, was not set up i n the study area. Pennington and S n e l l ( c i t e d i n Spaulding 1922) trapped the smaller C. r i b i c o l a aeciospore up to a distance of 550 f e e t , and on another occasion Pennington ( c i t e d i n Spaulding 1922) caught aeciospores up to 1200 f e e t from pines. Tubeuf (19OI) s t a t e d t h a t C. r i b i c o l a aeciospores spread the disease up to 500 meters or more. S n e l l ( c i t e d i n Spaulding 1922) provided evidence that aeciospores of C. r i b i c o l a were c a r r i e d at l e a s t 7 m i l e s from pines on the mainland to i n f e c t Ribes on i s l a n d s o f f the Wew Hampshire coast. There are s e v e r a l r e p o r t s of Ribes i n f e c t e d at distances greater than a mile from the nearest known i n f e c t e d pine (Mielke 1943; Pennington 1924, 1925; S n e l l 1920; Spaulding 1922). S e v e r a l workers used Lycopodium or fu n g a l spores f o r e x p e r i -mental d i s p e r s a l s tudies u s i n g an array of samplers to measure the d i s -t r i b u t i o n of spore concentration from a p o i n t source (Gregory et a l . I 9 6 I ; Hodgson 1949; Sreeramulu and Ramalingam I 9 6 I ; Stepanov 1935; Wilson and Baker' 1946), and others used ragweed p o l l e n (Dingle et a l . 1959; Hewson and Dingle 1956; Ogden et a l . I963, 1964, 1966; Raynor and Ogden 1965; Raynor et a l . 1966). D i s p e r s i o n studies have also been c a r r i e d out u s i n g gaseous or p a r t i c u l a t e r e l e a s e s ( A l l i s o n et a l . 1968; Chamberlain 1953; Hay and Pasqui11 1959; Record and Cramer I958; Singer and Smith 1966). - 21k -Some of the spore d i s p e r s i o n studies were conducted p r i m a r i l y to t e s t d i s -p e r s i o n t h e o r i e s , and these showed that the concentration decrease w i t h distance from source resembles the decrease meteorologists have p r e d i c t e d from a i r b o r n e m a t e r i a l d i f f u s i n g from a p o i n t or l i n e source (Gregory 1°A5, I 9 6 I ; Waggoner 1965; and o t h e r s ) . When spores are re l e a s e d continuously from a p o i n t source the spore cloud i s s i m i l a r to th a t of a plume of smoke, which takes the form of a h o r i z o n t a l cone.with i t s apex r e s t i n g at the poi n t o f spore l i b e r a t i o n and i t s base o r i e n t e d i n the d i r e c t i o n o f the mean wind. As the spore concentration moves downwind i n the t u r b u l e n t a i r i t d i f f u s e s h o r i z o n t a l l y and v e r t i c a l l y , and at any p o i n t downwind i t s diameter i s , on the average, p r o p o r t i o n a l to the.distance i t has t r a v e l l e d . Thus the c o n i c a l form i s achieved, except that i n cross sec-t i o n i t has the shape of an e l l i p s e / , since the t u r b u l e n t d i s p e r s a l i s smaller i n the v e r t i c a l d i r e c t i o n than i n the h o r i z o n t a l one. Concentra-t i o n s remain greater around the h o r i z o n t a l a x i s of the "cone" than nearer i t s surface and concentrations become l e s s and l e s s w i t h distance from the source. As the plume spreads v e r t i c a l l y i t s l a t e r a l spread w i l l be a f f e c t e d by the systematic v a r i a t i o n of wind d i r e c t i o n w i t h height above ground ( P a s q u i l l I961). Ge n e r a l l y i n nature spores are r e l e a s e d near ground l e v e l thus the base of the "cone" drags along the surface l o s i n g spores s t e a d i l y by va r i o u s types of d e p o s i t i o n as i t proceeds. Various formulae (Chamberlain 1953; Gregory 19"+5, I 9 6 I ; Gregory and Stedman 1953; P a s q u i l l I 9 6 I , 1962; Waggoner 1952, 1965) have been developed to describe d i s p e r s a l or d i f f u s i o n of spores and other p a r t i c u l a t e matter from a p o i n t or l i n e source under a number of daytime c o n d i t i o n s . The d i f f u s i o n the-o r i e s do not apply at night because the v e r t i c a l d i f f u s i v i t y goes to - 215 -zero under temperature i n v e r s i o n c o n d i t i o n s . Evidence from eddy d i f f u s i o n theory p r e d i c t s r a p i d depostion near the source, and corresponds to ob-served disease gradients- and experiments of spore d e p o s i t i o n . I n the f o r -mulae of Gregory (1945) no account was taken of spore s i z e , as i t was assumed th a t the r a t e of f a l l of spores i n s t i l l a i r was of l i t t l e account i n r e l a t i o n to the movements which occur w i t h i n the eddies of a body of a i r i n t u r b u l e n t motion. Later Gregory (1952) admits that the v e l o c i t y of f a l l of spores has some i n f l u e n c e on the t o t a l d i s t ance of dissemina-t i o n , however, the various d i f f u s i o n formulae methods have not yet been f u l l y adapted to in c l u d e the e f f e c t of g r a v i t a t i o n a l s e t t l i n g and de-p o s i t i o n ( T y l d e s l e y 1967). Recently t h i s p a r a b o l i c equation of d i f f u s i o n was solved by an e l e c t r o n i c analog computer which showed the e f f e c t of d e p l e t i o n of spore concentrations downwind from a l i n e source (Brock 1962). Other numerical methods,"Jusing d i g i t a l computers are now used i n which s e t -t l i n g and d e p o s i t i o n can be accounted f o r ( T y l d e s l e y 1967). Schrodter ( i 9 6 0 ) s t r o n g l y c r i t i c i z e s the neglect of r a t e of spore f a l l as a f a c t o r i n a e r i a l spore d i s p e r s i o n formulae. He says "the v e l o c i t y of f a l l i s an extremely important f a c t o r i n determining the range of f l i g h t and cannot be neglected i n the problem of dissemination':' as "the 1 g r a v i t a t i o n a l f a l l i s always going on" f o r "even i n t u r b u l e n t a i r when the net movement of the p a r t i c l e i s upward g r a v i t a t i o n a l f a l l continues". H i r s t (1959) s t a t e d that the o v e r a l l e f f e c t of sedimentation was important only i n calm c o n d i t i o n s or i n s h e l t e r e d p l a c e s . Gregory (1961) reported that at windspeeds below 2 m/sec sedimentation under g r a v i t y p l a y s a more important p a r t i n the spore deposition than t u r b u l e n t flow over the surface. T y l d e s l e y (1967) reasoned t h a t the turbulence of the " f r i c t i o n a l l a y e r " which ascends to 500-1000 m (Sutton 1953), b r i n g s about v e r t i c a l a i r - 216 -f l u c t u a t i o n s of the order of 10 cm/sec which i s at l e a s t as great as the f a l l speed of la r g e p o l l e n . Therefore the d i s o r g a n i z e d v e r t i c a l motion of the a i r e f f e c t i v e l y i n h i b i t s : ' s e t t l i n g and the a i r spora i s e s s e n t i a l l y i n suspension under the t h e r m a l l y unstable conditions of midday. Ludlam (1967) s t a t e d t h a t i f the " f a l l - s p e e d i s greater than any f r e q u e n t l y r e c u r r e n t upward a i r speed at the time and l e v e l of; i n j e c t i o n , the par-t i c l e s r e t u r n to the surface w i t h i n a distance not many times the height at which they are i n j e c t e d " . Waggoner (1965) concluded t h a t e x t r a c t i o n of spores from a spore cloud could be equal to or greater than the r a t e corresponding to t h e i r v e l o c i t y i n s t i l l a i r , and that e x t r a c t i o n was most r a p i d when a source was embedded i n a stand of p l a n t s . V e l o c i t i e s of f a l l have been calculated f o r s p h e r i c a l spores using Stokes' Law, and'Falck (1927), McCubbin (1944) and Schrodter (1954) c a l c u l a t e d a r e l a t i o n s h i p between s p h e r i c a l and other shaped spores. Schrodter (1954) used F a l c k ' s formulae to derive values f o r v a r i o u s groups of spores. T y p i c a l v e l o c i t i e s f o r these spore.groups f a l l between l e s s than 1 mm/sec to .7 cm/sec. Chamberlain (1966, 1967) gave the f o l l o w i n g values f o r 20, 30 and 50M- diameter spheres of u n i t d e n s i t y , 1.2, 2.7 and 7.1 cm/sec r e s p e c t i v e l y . Stepanov (1935) obtained a value of 2.78 cm/sec fo r e l l i p s o i d a l spores o f Helminthosporium sativum Pammel, King & Bakke, measuring 68 x 24u, and t h a t a c y l i n d e r of such dimensions would approxi-mately equal the volume of a sphere of 40u diameter, which would have an expected t e r m i n a l v e l o c i t y of about 4.8 cm/see, considerably f a s t e r than t h a t a c t u a l l y recorded f o r the e l l i p s o i d a l spore (Gregory 1945). The aeciospore of C. comandrae i s not s p h e r i c a l or e l l i p s o i d a l and the spore i s not of equal d e n s i t y throughout, the " t a i l " being considerably l e s s - 217 -dense. Ignoring the " t a i l " p o r t i o n of the spore, i t s s i z e would be approximately 23 x 3k\i and thus would act s i m i l a r l y to a sphere of 28p of equal d e n s i t y throughout. The average v e l o c i t y o f 3.23 cm/sec ob-t a i n e d i n the more r e l i a b l e t e s t s of the experiments i s t h e r e f o r e probably an overestimate of the order of 0.5 cm/sec, e s p e c i a l l y when one adds the i n f l u e n c e of the " t a i l " , which gives a bu l k d e n s i t y of l e s s than u n i t y . The overestimate i s p a r t l y due to v a r i a b i l i t y between t e s t s and the pro-blem of minimizing the e f f e c t of spore clumping and mass subsidence. More accurate r e s u l t s would probably be obtained by r e l e a s i n g a smaller spore sample i n t o the c y l i n d e r . Others have explained the e f f e c t of a number of fa c t o r s on v e l o c i t y of f a l l which could account f o r some of the range of v e l o c i t i e s found i n the present study. Weinhold (1955) has shown t h a t ' v a r i a t i o n s i n the r a t e of f a l l occur w i t h s l i g h t changes i n temperature and r e l a t i v e humidity. F a l c k (1930) s t a t e d that the speed of f a l l i n g of dry spores d i f f e r e d from that of f r e s h spores because the l o s s o f water caused changes i n both weight and shape. S i m i l a r l y , B u l l e r (1922) showed that spores f a l l i n g i n dry a i r r a p i d l y l o s t v e l o c i t y owing to desiccation, and completely a i r - d r i e d spores were seen to f a l l o n l y o n e - t h i r d as f a s t as f u l l y t u r g i d spores, which corresponds approximately to the d i f f e r e n c e between dry and wet C. comandrae spores. A l s o , the storage of spores f o r long periods would probably a f f e c t the a b i l i t y of the spores to respond to r e l a t i v e humidity changes, as i n d i c a t e d i n the t e s t s o f Weinhold (1955) w i t h uredospores of F u c c i n i a graminis t r i t i c i E r i k s s . & Henn. Harrington and Metzger (1963) have shown that ragweed p o l l e n w i l l take up moisture at h u m i d i t i e s above 52%, i n c r e a s i n g t h e i r d e n s i t y which a f f e c t s t h e i r t e r m i n a l v e l o c i t y . Gregory (1945) s t a t e d t h a t "exact p r e d i c t i o n of the - 218 -t e r m i n a l v e l o c i t y of a spore i s hindered by the d i f f i c u l t y of determining i t s d e n s i t y (which ... depends on i t s h y d r a t i o n ) , and by d e v i a t i o n from the i d e a l smooth s p h e r i c a l form". Chamberlain (1966) a t t r i b u t e d the v a r i a t i o n i n reported values of the t e r m i n a l v e l o c i t y of Lycopodium spores to s m a l l v a r i a t i o n s i n s i z e of spore used. The l a r g e r the average d i a -meter the greater the t e r m i n a l v e l o c i t y . Chamberlain (1967) a l s o mentions the phenomenon of buLk sedimentation which may take e f f e c t close to the source before t u r b u l e n t d i s p e r s i o n has taken e f f e c t on the cloud of r e -leased spores and may operate to give an excessive apparent v e l o c i t y , of d e p o s i t i o n . This may a f f e c t the v e l o c i t i e s of f a l l i n some s t i l l a i r experimental t e s t s , and he suggests i t was p o s s i b l y the reason f o r Gregory et a l . (1961) f i n d i n g a decreased v e l o c i t y of f a l l w i t h d i s t a n c e downwind where the r e l e a s e d Lycopodium spores had undergone considerable d i f f u s i o n . Christensen (l^kh), McCubbin (1918a) and Ukkleberg (1933) have determined the t h e o r e t i c a l d i s p e r s a l distances of spores based on the h o r i z o n t a l wind speed, a l t i t u d e a t t a i n e d , and r a t e of vel o c i t y . . o f f a l l . Such c a l c u l a t i o n s , however, have l i t t l e p r a c t i c a l value and no t h e o r e t i -c a l v alue, as they do not consider upward movements of a i r a s s o c i a t e d w i t h atmospheric turbulence. Near the ground the s t r u c t u r e of turbulence changes s y s t e m a t i c a l l y w i t h height ( P a s q u i l l 1961), and i s composed of separate bodies — bubbles, s h e l l s or c e l l s — of a i r which may move i n p r e d i c t a b l e paths, and als o s p e c i f i c l o c a l thermal updraft and downdraft patt e r n s e x i s t i n a s s o c i a t i o n w i t h l o c a l topographic or v e g e t a t i o n a l f e a -tures (Cone 1961, 1962;'Ludlam and Scorer 1953; Yates 1953). These s t r u c -t u r a l atmospheric features may show marked d i u r n a l v a r i a t i o n , and because of t h i s , random spore d i f f u s i o n is. u n l i k e l y to occur over great distances - 219 -(Van A r s d e l I967, Waggoner I 9 6 5 ) . Van A r s d e l (1965, 1967) was able to show the p e r s i s t e n c e of such atmospheric features at ni g h t when con d i t i o n s are more s t a b l e than under the more t u r b u l e n t conditions of the day. From t h i s he was able to p r e d i c t the movement of spore clouds, and to r e l a t e t h i s to the h i s t o r y of Cronartium r i b i c o l a r u s t i n f e c t i o n over a p e r i o d of 20 years. A l l the experiments and sampling of the present study supply evidence to support the f i n d i n g s of others (Buchanan and •Kimmey 1938; Gregory 19*+5> 1952, I 9 6 I ; Gregory et a l . I 9 6 I ; Sreeramulu and Ramalingam I 9 6 I ; Stepanov 1935; Wilson and Baker 19*+6), t h a t under normal c o n d i t i o n s a s u b s t a n t i a l percentage of the spores l i b e r a t e d near the ground w i l l be deposited w i t h i n a short distance of the source. Gregory (1952, and c o r r e c t i o n 1958) s t a t e d that "observations and theory agree that f o r spores or p o l l e n l i b e r a t e d near the ground under normal c o n d i t i o n s of turbulence, 90% °f "the spores w i l l be deposited w i t h i n 100 m of the source ... and under r e l a t i v e l y calm c o n d i t i o n s " spores would be deposi-ted over "a s t i l l more l i m i t e d range". L a t e r he c a l c u l a t e d (1961) t h a t a spore cloud r e l e a s e d at 0 . 1 , 1 and 10 m above the ground would be de-p l e t e d by 90, 70 and <10% i n 100 m of t r a v e l , which i n d i c a t e s the con-sequence of e l e v a t i o n of source. S i m i l a r l y , Waggoner (1965) c a l c u l a t e d the maximum spore concentration on the ground would occur 3 3 22 and kO m downwind from sources that are 1, 6 and 10 m above the ground, as the maximum concentration i s not found on the ground next to the source when the source i s elevated. A l s o , when the source i s elevated, there i s l e s s r a p i d d e p l e t i o n of the spore cloud. With r e l e a s e of C. comandrae aecio-spores from sources 1 and 5 f e e t above ground, maximum spore concentrations - 220 -were r e s p e c t i v e l y at the 5 and 20 foot sampling p o i n t s from the source, although the a c t u a l peaks may have occurred at distances other than where the sampling p o i n t s were placed. From the t e s t s of n a t u r a l d i s p e r -s a l patterns around a spore source one to two f e e t above the ground i n the f o r e s t , the spore concentration i n a l l d i r e c t i o n s from the source gave very s i m i l a r steep gradients of d e p o s i t i o n . Generally, d e p o s i t i o n at 6 f e e t was l e s s than 1% of the concentration at the f i r s t sampling p o i n t (3 inches) and reductions of 90% occurred w i t h i n 2 f e e t . Wind speeds during the sampling averaged 2.20 to 2..4'9 f t / s e c , and spore concen-t r a t i o n s were s l i g h t l y greater downwind from the source along the path of the supposed p r e v a i l i n g wind during the p e r i o d , but the p a t t e r n of d i s p e r s a l around the source i n d i c a t e d that the wind d i r e c t i o n f l u c t u a t e d widely. Some evidence e x i s t s of the gradient of d i s p e r s a l of aeciospores of F u c c i n i a graminis, although these spores are considerably smaller (17 x 20u) than those of_C. comandrae. Lambert (1929) reported except-i o n a l l y steep gradients o f spore r e d u c t i o n from source. Spores were trapped f o r a p e r i o d of 20 hours at 3, 6 and 23 f e e t from an i n f e c t e d barberry hedge, and gave counts of 160,000, 33,000 and 210 r e s p e c t i v e l y . On a few days spores were trapped up to a mile from the bushes. Johnson and Dickson (1919) reported i n f e c t i o n gradients from b a r b e r r y bushes, and showed that the percentage of stems i n f e c t e d was 100% at 15 f e e t , 30% at 90 f e e t , 10% at 175-200 f e e t and 1% or l e s s a f t e r 300 f e e t . These steep gradients o f d e p o s i t i o n were o f t e n i n t e r p r e t e d as spores disseminated over only a short distance (Gregory 1968). This i n f e r e n c e may sometimes be j u s t i f i e d , but Gregory i n d i c a t e d that steep gradients may not always represent r a p i d d e p o s i t i o n near the source, as d i f f u s i o n of the spore cloud - 221 -should be considered. Spore d i s p e r s a l i n a i r i s mainly c o n t r o l l e d by eddy d i f f u s i o n , t h e r e f o r e i f no d e p o s i t i o n took place the spore concentra-t i o n would v a r y approximately i n p r o p o r t i o n to the d i s t a n c e . Spore cloud and surface d e p o s i t i o n are i n c o n s t a n t l y r e l a t e d , and unless the t o t a l e f f e c t i v e spore emission i s known, no estimates of the number of spores c a r r i e d beyond the sampling area can be made (Gregory 1968). There i s much evidence from a i r c r a f t and other p o i n t s of c o l l e c t i o n remotely r e -moved from source areas, t h a t p o l l e n and spores of r u s t f u n g i and other p l a n t pathogens are transported i n the atmosphere over considerable d i s -tances, i n some cases thousands of miles ( C r a i g i e 1945; D i l l o n Weston 1929; H i r s t et a l . 1967a,b; K e l l y et a l . 1951; MacLachlan 1935; Pady 1954; Stak-man'and Christensen 1946; Stakman et a l . 1923). How can we solve the d i l -emma of r e c o n c i l i n g the experimental evidence of apparent r a p i d d e p o s i t i o n near the source w i t h spore concentrations c o l l e c t e d i n the atmosphere, even over the oceans? The s o l u t i o n appears to r e s t w i t h t h a t f r a c t i o n of spores which escapes d e p o s i t i o n near the source and which i s c a r r i e d up by mechanical or thermal turbulence to greater heights i n the atmosphere (Gregory 1963). Gregory (1962) suggests that t h i s f r a c t i o n may commonly be of the order of 10$. Expt. V i n t h i s study gave some evidence t h a t spores were probably escaping from the source area. Spores l i b e r a t e d on warm dry days when turbulence i s at a maximum, stand much.more chance of t r a v e l l i n g long distances than those dispersed at nig h t or i n wet condi-t i o n s , and the smaller the spore the greater the chances f o r long d i s t a n c e d i s p e r s a l . Waggoner (1965) i n d i c a t e d that spores o f t e n t r a v e l i n a s e r i e s of great loops on a c l e a r t u r b u l e n t day, and th a t towards sunset the spores would tend to be l o f t e d . - 222 -Schrodter (1954, i960) showed that spores of approximately 20 n diameter and w i t h a v e l o c i t y of f a l l of approximately 1 cm/sec could be-c a r r i e d 16 km under- a wind v e l o c i t y of 2 m/sec and a mass exchange of 10 gm/cm sec. Gregory (1962), however, obtained values c o n s i d e r a b l y l e s s than Schrodter's u s i n g Chamberlain's (1953) m o d i f i c a t i o n to adapt to h i s . theory (Gregory 1961), only o b t a i n i n g a probable f l i g h t range (di s t a n c e at which 50% of spores l i b e r a t e d are s t i l l i n suspension) of 50 m f o r large spores l i b e r a t e d i n normal turbulence at a l i b e r a t i o n height o f 0.1 m, and only 4 m i n low turbulence. More r e c e n t l y , Chamberlain (1966) c a l c u l a t e d that 40-50% of the p a r t i c l e s of 30n diameter r e l e a s e d at 0.5 m above ground i n n e u t r a l c o n d i t i o n s could be transported 1 km, and 60% i n unstable c o n d i t i o n s . I f the same p a r t i c l e s are r e l e a s e d at.10 m t h e i r median range i s close to 10 km. Recently, H i r s t and h i s colleagues ( H i r s t and Hurst I967, H i r s t et a l . 1967a, 1967b) have been able to assess long distance spore t r a n s p o r t by measurements made from a i r c r a f t over the sea around B r i t a i n , but they have been unable to t e s t the v i a b i l i t y of the spores c o l l e c t e d . Schrodter (i960) a l s o discussed the a l t i t u d e which spores of var i o u s s i z e s could a t t a i n i n t h e i r f l i g h t . Spores of approxi-mately 20u wereviunlikely to a t t a i n a l t i t u d e s of more than 100 m unless extreme mass exchange or turbulence occurred. On June 30, 1965, an op-p o r t u n i t y was taken to expose microscope s l i d e s during f l i g h t s i n f a i r l y s t a b l e low tu r b u l e n t conditions i n an area to.the west and northwest of Edmonton at heights between 500 and 1500 f e e t . No C. comandrae aeciospores were observed on the s l i d e s , but smaller r u s t spores were present. Although there was no evidence a v a i l a b l e to i n d i c a t e the presence of aeciospores i n the a i r below the sampling height, the presence of only s m a l l e r - s i z e d - 223 -spores at these l e v e l s may support the data of Schrodter (i960) that the l a r g e r spores are u n l i k e l y to be c a r r i e d at such sampling heights under normal turbulence. However, Davidson ( c i t e d i n Mielke 19^3) reported c o l l e c t i n g C. r i b i c o l a aeciospores at each 1,000 f o o t l e v e l up to 5,000 f e e t (the highest l e v e l at which exposures were made) above a h e a v i l y i n f e c t e d area i n B r i t i s h Columbia, but no inf o r m a t i o n on the atmospheric conditions was given. Schrodter (i960) c a l c u l a t e d t h a t the f l i g h t dura-t i o n of la r g e spores at sm a l l mass exchange values near the ground i s but a few minutes. For a spore of approximately 20u, a f a l l v e l o c i t y of 1 cm/ sec, and a mass exchange of 10 gm/cm sec, f l i g h t d u r a t i o n i s 2 l/k hours, and the same spore w i t h a mass exchange of 50 gm/cm sec i s 11 hours. The dur a t i o n of f l i g h t and a l t i t u d e a t t a i n e d i s s i g n i f i c a n t from an epidemiolo-g i c a l p o i n t of view i n connection w i t h the problem of v i a b i l i t y of spores to be discussed i n a later s e c t i o n . H i r s t and Hurst (1967) r e p o r t t h a t the midsummer f r e e z i n g l e v e l over the B r i t i s h I s l e s may be as low as 1 km and s i m i l a r l e v e l s could be expected i n the t e s t area. Spores may be sub-j e c t e d to considerable d e s i c c a t i o n , a l s o i n t e n s i t y of l i g h t i s increased above the surface, both important f a c t o r s a f f e c t i n g the v i a b i l i t y . H i r s t and Hurst (1967) a l s o reported that the er o s i o n at the base of v e r t i c a l spore p r o f i l e s seemed u s u a l a f t e r long t r a v e l over both land and sea, thus spores c a r r i e d highest during a c t i v e convection w i l l have the best chance of being transported f u r t h e r , and be subjected to-convection the next day. Spore d i s p e r s a l not only i n v o l v e s f l i g h t but als o l a n d i n g or de p o s i t i o n . G e n e r a l l y speaking,, the same forces t h a t are r e s p o n s i b l e f o r t r a n s p o r t a t i o n through the a i r are a l s o d e c i s i v e f o r d e p o s i t i o n of spores. The p r i n c i p l e mechanisms i n v o l v e d are sedimentation under the i n f l u e n c e of - 22k -g r a v i t y , impaction, i n c l u d i n g t u r b u l e n t d e p o s i t i o n , and r a i n wash-out, a f a c t o r discussed above. Sedimentation i s only important i n the absence of turbulence, or at wind speeds below 2 m/sec (Gregory 1961) where i t p l a y s a more important p a r t i n spore d e p o s i t i o n than t u r b u l e n t a i r flow over the surface. D e p o s i t i o n by impaction plays the great e s t r o l e i n the l a n d i n g of s i n g l e spores. When a i r flows past an ob s t a c l e , the flow streamlines d i v i d e to pass e i t h e r s i d e , but p a r t i c l e s i n the a i r tend to move by t h e i r own momentum towards the objec t before they are i n t u r n de-f l e c t e d by the wind, f l o w i n g around the obs t a c l e before being impacted on the windward-side of the o b s t a c l e . G e n e r a l l y the l a r g e r the p a r t i c l e the greater the chance of impaction, as the e f f i c i e n c y of impaction i n -creases d i r e c t l y w i t h spore s i z e and wind v e l o c i t y , and i n v e r s e l y w i t h the width of the ob s t a c l e . A number of t h e o r e t i c a l i n v e s t i g a t i o n s of impaction e f f i c i e n c y on c y l i n d e r s , spheres and other shapes have been summarized by Fuchs (196*+) and Green and Lane (196*+), and experimental i n -v e s t i g a t i o n s by Chamberlain (1966), Gregory (1961), .Gregory et a l . (1961) and Gregory and Stedman (1953)- According to Gregory (1952) a spore s i z e of 10n, the most frequent diameter of spores dispersed by wind, represents a compromise between the c o n f l i c t i n g requirements o f d i s p e r s a l and depo-s i t i o n . The large-spored l e a f - and stem-pathogens appear as s p e c i a l i z e d impactors. Johnstone et a l . (l9*+9) pointed out that the a b i l i t y o f a p a r t i c l e to penetrate among close v e g e t a t i o n i s the inverse of i t s im-p a c t i o n e f f i c i e n c y . I n close v e g e t a t i o n a high impaction e f f i c i e n c y , such as that of C. comandrae aeciospores, would reduce the chance of a spore g e t t i n g very f a r from i t s p o i n t of r e l e a s e . I n the f o r e s t the canopy r e s t r i c t s the exchange of a i r , and as Raynor ( i n Chamberlain 1967) has - 225 -pointed out, some spores w i l l be l o s t by impaction i n the canopy, but the e f f e c t of sedimentation i n the comparatively s t i l l a i r below the cano-py i s more important. One f a c t o r a s s o c i a t e d w i t h r a i n or l e a f wetness, important f o r d e p o s i t i o n , was that a wet surface tended to a s s i s t i n the r e t e n t i o n of spores at high wind v e l o c i t i e s (Chamberlain 1967), but there was l i t t l e d i f f e r e n c e at low v e l o c i t i e s where sedimentation was more im-portant than impaction. A wet or s t i c k y surface probably reduces rebound or bounce-off.- Blow-off a f t e r d e p o s i t i o n had been shown to be n i l or n e g l i g i b l e even under high wind v e l o c i t i e s . Bagnold (i960) a l s o showed that p a r t i c l e s l e s s than about 50p. diameter are not r e a d i l y removed from surfaces because 'they are imbedded w i t h i n the v i s c o u s boundary l a y e r o f a i r flow which p r o t e c t s them from the b u f f e t i n g o f t u r b u l e n t eddies. How do the f i n d i n g s of the response of a spore r e l e a s e d i n the open r e l a t e to what happens to the spore t h a t i s r e l e a s e d i n the f o r e s t stand? The r a p i d d e p l e t i o n of the spore cloud as i t t r a v e l s from the source w i l l be even f a s t e r i n the f o r e s t . Below the canopy, wind speeds are g r e a t l y reduced, which w i l l tend to reduce the d i s t a n c e the spore cloud can t r a v e l . Secondly, turbulence i s reduced and spores w i l l tend to s e t t l e out of the cloud, or spores w i l l sediment d i r e c t l y due to ab-sence of any considerable a i r movement. D e p l e t i o n w i l l then be v e r y r a p i d . T h i r d l y , the r e l a t i v e l y l a r ge spores w i l l impact on the narrow needles and other v e g e t a t i v e objects which they encounter w i t h i n the stand, f u r t h e r d e p l e t i n g the spore load. The narrow c o n i f e r needles w i l l act as b e t t e r impactors of the l a r g e spores than the s m a l l spores ( l e s s than 5u), and b e t t e r than deciduous broad leaves i n the f o r e s t stand. In a stand w i t h an open trunk space w i t h a high canopy the spores w i l l - 226 -encounter l e s s f o l i a g e and d e p l e t i o n should be slower'. These h y p o t h e t i -c a l features of the d e p l e t i o n of spores i n a f o r e s t have been borne out by Ogden et a l . (1964, 1966) and Raynor et a l . (1966) i n t h e i r recent s t u -d i e s of the d i f f u s i o n of p o l l e n emitted i n s t a n t a n e o u s l y from sources at various distances and at s e v e r a l heights upwind of a f o r e s t edge, and by studies by A l l i s o n et a l . (1968) who r e l e a s e d f l u o r e s c e n t p a r t i c l e t r a c e r s w i t h i n a f o r e s t . Raynor et a l . (1966) found t h a t l e s s than 1$ of the p o l l e n r e l e a s e d i n t o the f o r e s t was s t i l l air-borne at 100 m, and that the sample r e l e a s e d from a lower l e v e l was depleted f i r s t . They als o found t h a t only 50$ of the p o l l e n r e l e a s e d k-0 m from the f o r e s t edge ever reached the f o i s t , but 90$ of t h a t r e l e a s e d 10 m away reached the f o r e s t . The p o l l e n was depleted f a s t e r w i t h a lower wind speed than w i t h a higher o n e , v i n d i c a t i n g that d e p o s i t i o n was more important than impaction. W i t h i n the f o r e s t , the great decrease i n wind speed slows the forward t r a n s p o r t of the p a r t i c u l a t e cloud to the p o i n t where g r a v i t a t i o n a l s e t t l i n g removes the bulk of the m a t e r i a l . A l l i s o n et a l . (1968) reported that a f l u o r e s c e n t p a r t i c l e cloud moved at l / l O of the outside wind speed immediately above the canopy of a dense " t r o p i c a l type" deciduous f o r e s t . One i n t e r e s t i n g f i n d i n g of t h e i r work was that the d i r e c t i o n of cloud movement w i t h i n the f o r e s t was, on the average, s i g n i f i c a n t l y to the l e f t o f the e x t e r n a l wind by about 23°, and a s i m i l a r r e l a t i o n s h i p was shown by smoke p u f f s . Wilson and Baker (1946), who s t u d i e d the spread of S c l e r o t i n i a t a x a Ader. & Ruh. i n orchards, showed that t r a v e l of spores through the branches of a t r e e increased v e r t i c a l d i s p e r s i o n when com-pared w i t h t r a v e l across unobstructed t e r r a i n . Raynor et a l . (1966) n o t i c e d that at the edge of the f o r e s t , spread occurred i n the v e r t i c a l - 227 -as w e l l as the h o r i z o n t a l . A l l i s o n et a l . (1968) a l s o noted t h a t v e r t i -c a l d i f f u s i o n was r a p i d w i t h i n the f o r e s t i n terms of downwind d i s t a n c e . I f spores are c a r r i e d from a f o r e s t stand i n t o a c l e a r i n g or out of the f o r e s t , then the reverse e f f e c t s w i l l occur, w i t h spores be-ing c a r r i e d over.greater distances or depleted l e s s r a p i d l y because of the increase of wind v e l o c i t y and turbulence. I n f a c t , i f thermals occur through heating of the c l e a r i n g or open ground, spores may a c t u a l l y , be c a r r i e d on updrafts to considerable heights before being deposited. - 228 -AECIOSPORE GERMINATION Germination i s the i n i t i a l stage i n the development of a fungus mycelium from the spore, u t i l i z i n g i t s stored reserves f o r metabolism. Spores w i l l germinate i n water or on very simple media, i f other e n v i r -onmental conditions are s a t i s f i e d . The germination process i n v o l v e s mor-p h o l o g i c a l and p h y s i o l o g i c a l changes w i t h i n the spore w a l l which transform the spore from a stage of low to one of high metabolic a c t i v i t y , and ex-t e r n a l changes i n v o l v i n g p r o t r u s i o n and e l o n g a t i o n of the germ tube from the spore w a l l . The process of germination i s enhanced or l i m i t e d by v a r i o u s ranges or combinations of environmental conditons. These environ-mental conditions i n c l u d e the f a c t o r s of temperature, humidity, l i g h t and form, type and hydrogen i o n concentration of substrate media. Each of these f a c t o r s i n f l u e n c e s germination i n s e v e r a l important ways i n c l u d i n g the e f f e c t upon percentage germination, the time taken to germinate, the r a t e of germ tube elongation or growth, and the k i n d or form of germina-t i o n . Spores g e n e r a l l y germinate over a range of values f o r each f a c t o r . I t i s not always p o s s i b l e to s t a t e the optimum value w i t h p r e c i s i o n be-cause of the i n t e r a c t i o n of s e v e r a l environmental f a c t o r s together, and because of the i n f l u e n c e of other v a r i a b l e s . These v a r i a b l e s i n c l u d e the • previous h i s t o r y of the spores, t h e i r age and inherent v a r i a b i l i t y , as w e l l as other f a c t o r s l a r g e l y connected w i t h spore handling and germina-t i o n techniques. Many studies have been made of the germination requirements f o r the urediospore and b a s i d i o s p o r e s t a t e s of r u s t f u n g i , but u n t i l very r e -c e n t l y , l i t t l e i n f o r m a t i o n was a v a i l a b l e f o r the germination requirements - 229 -of r u s t aeciospores of f o r e s t pathogens and then r a r e l y more than tem-perature requirements (Doran 1919, 1922; Fergus 1959, H i r t 1937; Mains 1916; Siggers I9I+7; Spaulding 1922; Van A r s d e l et a l . I956). Since the commencement of t h i s study, some in f o r m a t i o n on the requirements f o r ger-mination of Cronartium spp. aeciospores has appeared (Anderson and French I965; Nighswander and Patton I965; Peterson I968; P o w e l l and Morf I966; Roncadori and Matthews I966; Walkinshaw I965, I968; Walkinshaw e t a l . 1967), but only very r e c e n t l y , has any in f o r m a t i o n been pu b l i s h e d f o r C. comandrae ( K r e b i l l 1968c). The study on Peridermium s t a l a c t i f o r m e and P. h a r k n e s s i i of the Cronartium c o l e o s p o r i o i d e s complex (Powell and Morf I966) was a companion study to much of the present i n v e s t i g a t i o n on C_. comandrae, a l -though only i n v o l v i n g temperature and pH requirements f o r aeciospore ger-mination. Wo studies have followed aeciospore germination on a day to day b a s i s , although Doran (1922) and Spaulding (1929) r e p o r t on C. r i b i c o l a , and K r e b i l l (1968c) on C_._ comandrae spore v i a b i l i t y at infrequent i n t e r -v a l s i n a season. This s e c t i o n of the study, f i r s t i n v e s t i g a t e s , through l a b o r a -t o r y t e s t s , v a r i o u s environmental requirements f o r germination and assesses the e f f e c t of other v a r i a b l e s on aeciospore germination, and secondly, f o l l o w s the day to day v a r i a t i o n s i n aeciospore germination from i n d i v i -d u a l a e c i a l cankers and pustules throughout the spore production p e r i o d . - 230 -FACTORS AFFECTING AECIOSPORE GERMINATION Methods and M a t e r i a l s Spore m a t e r i a l and methods of handling Dry aeciospores were c o l l e c t e d by brushing them from i n d i v i -d u a l s p o r u l a t i n g a e c i a l p ustules of the r u s t on lodgepole pine from a wide area i n the F o o t h i l l s and Rocky Mountains area of southwest A l b e r t a be-tween Robb (53°13'N, ll6°58'W) and Beaver Mines (1+9°28'N, 11H-°12'W). Where p o s s i b l e , spores were s e l e c t e d from r e c e n t l y ruptured a e c i a to give samples of uniform age, and w i t h l e s s contamination. C o l l e c t i o n areas were a l l made i n mountain v a l l e y s between 3,500 and -6,000 f t . e l e v a t i o n . Most of the t e s t m a t e r i a l was c o l l e c t e d i n the v i c i n i t y of l o c a t i o n s 1, 2 and 3 ( F i g . 38). I n d i v i d u a l spore samples were screened through s t e r i l i z e d 100-and 200-mesh sieves to remove extraneous m a t e r i a l . A l l c o l l e c t i o n s were t e s t e d f o r v i a b i l i t y by germinating on water agar before being used or placed i n storage. Spores c o l l e c t e d i n I963, 1964 and I965 were stored f o r periods of from 1 day to 2 years i n s m a l l glass v i a l s plugged w i t h cotton. The spore v i a l s were kept continuously i n a d e s i c c a t o r over a saturated calcium c h l o r i d e s o l u t i o n , at a 5°C temperature. I n l a t e r years the calcium c h l o r i d e was not used f o r d r y i n g as i t was found t h a t spore v i a b i l i t y was reduced by t h i s method. B e t t e r v i a b i l i t y was main-t a i n e d from 1966 onwards by s t o r i n g spores at a temperature close to 0°C w i t h a high humidity. Good to f a i r germination was obtained from spores stored f o r two months, but germination was u s u a l l y poor a f t e r s i x to nine months storage. Unless otherwise s p e c i f i e d , the reported r e s u l t s were - 231 -obtained from f r e s h spores, or from spores stored f o r l e s s than 10 days. Methods f o r e x p l o r a t o r y experiments During 1963 and I96U v a r i o u s experiments were c a r r i e d out of an e x p l o r a t o r y nature, to e s t a b l i s h techniques f o r handling and main t a i n -i n g germination t e s t s , and f i n d i n g s u i t a b l e media and conditions f o r ger-mination, as few g u i d e l i n e s were a v a i l a b l e i n the l i t e r a t u r e f o r t h i s r u s t spore s t a t e . I n t e s t i n g f o r the e f f e c t of each f a c t o r i t i s important that a l l other f a c t o r s i n v o l v e d approach t h e i r optimum l e v e l and t h e r e f o r e are not l i m i t i n g . To ensure that the co n d i t i o n s f o r r e g u l a r t e s t s would be s a t i s f a c t o r y , s e v e r a l e x p l o r a t o r y t e s t s were c a r r i e d out w i t h temperature, l i g h t , humidity and pH conditions,' and f u r t h e r t e s t s were c a r r i e d out to f i n d a s u i t a b l e non-stimulatory- medium f o r germination. The time r e q u i r e d f o r spores to i n i t i a t e germination, and the p e r i o d r e q u i r e d f o r the ma-j o r i t y of v i a b l e spores to germinate was al s o i n v e s t i g a t e d , so th a t t e s t s of s u f f i c i e n t d u r a t i o n could be e s t a b l i s h e d . I n i t i a l germination t e s t s were c a r r i e d out usi n g a s l i g h t mo-d i f i c a t i o n of Ri g h t e r ' s (1939) germination method which enables a l a r g e number of samples to be t e s t e d under uniform c o n d i t i o n s . Glass p e t r i plates' were s t e r i l i z e d and a s m a l l amount of p a r a f i n wax was melted i n the p l a t e . On coding a s e r i e s o f germination chambers (holes) were pun-ched i n the wax surface w i t h a s t e r i l i z e d cork borer. The holes were arranged i n a s e r i e s of eig h t numbered.rows each w i t h f i v e l e t t e r e d h oles. The experiment was f i r s t designed to place each spore sample r e p l i c a at random i n the p l a t e but t h i s was d i f f i c u l t f o r ease of op e r a t i o n and f i v e r e p l i c a s of eig h t samples were e v e n t u a l l y used w i t h each sample i n a - 232 -numbered row. D i s t i l l e d water was placed i n each hole and spores were seeded according to a set p l a n . The p l a t e s were then placed i n d e s i c c a -t o r s i n c o n t r o l l e d temperature incu b a t o r s . A high humidity l e v e l was maintained-by p l a c i n g water i n the bottom of the d e s i c c a t o r s . This me-thod became too cumbersome when la r g e numbers were i n v o l v e d , or when informati o n was r e q u i r e d at set i n t e r v a l s . Counting of the ko samples sometimes took over an hour which kept the samples out of the t e s t e n v i r -onment too long. The spores a l s o tended to arrange themselves around the water meniscus i n the hole which made counting d i f f i c u l t . The spores nearest the circumference of the hole germinated more r a p i d l y than those nearer the center, thus t h i s method was u n r e l i a b l e f o r determination of percentage germination at set i n t e r v a l s . I n l a t e r t e s t s s m a l l 5~cm d i a -meter p l a s t i c p e t r i p l a t e s were used f o r germination t e s t s i n which spores were seeded onto a l a y e r of d i s t i l l e d water or a l a y e r of d i s t i l l e d water poured' onto a 2$ Bacto-plus 2$ Malt agar. Percentage germination was ex-tremely low on such media and o f t e n the spores appeared to become saturated, l o s e t h e i r c o l o r and s i n k to the bottom of the water w i t h no germination. Subsequently the use of a 0.3-0.5$ D i f c o Bacto water agar was found to improve germination. This medium gave s a t i s f a c t o r y r e s u l t s and was used i n a l l t e s t s unless otherwise st a t e d . Use of an agar medium obviated s i n k -i n g , post d i s p e r s a l clumping of spores, and spore counting was e a s i e r on a s o l i d surface. I t was found t h a t l i g h t and dark had no s i g n i f i c a n t e f f e c t on germination, and f r e e water or a saturated atmosphere appeared to be a germination requirement. Temperature t e s t s showed some germination over the range 5 to 30°C w i t h the optimum close to 15°C. A t r a c e of germina-t i o n was evident a f t e r 1-l/k hours, and most germination had taken place - 233 -by 2k hours, w i t h l i t t l e increase a f t e r k8 hours and n i l a f t e r 96 hours. The germ tubes g e n e r a l l y grew away from the surface of the medium dur-i n g the f i r s t few hours of growth, but e v e n t u a l l y the germ tubes made contact w i t h the medium w i t h l y s i s o f t e n o c c u r r i n g . At t h i s time the contents of the germ tube were concentrated towards the t i p . Germination was tested at 20°C on a b a s i c 0.3% water agar med-ium (pH 6.5), the pH of which was adjusted through a d d i t i o n of a number of b u f f e r e d and unbuffered s o l u t i o n s , s i n g l y or i n combination, to f i n d s u i t a b l e non-toxic media f o r t e s t i n g f o r e f f e c t of pH. The b a s i c water agar medium was prepared and s t e r i l i z e d without the s o l u t i o n s , which were added i n v a r i o u s concentrations to the melted b a s a l medium j u s t p r i o r to pouring p l a t e s . The s o l u t i o n s used i n c l u d e d a c e t i c a c i d , calcium carbonate, calcium hydroxide, l a c t i c a c i d , potassium a c i d p h t h a l a t e , potassium hy-droxide, potassium phosphate, sodium hydroxide and sodium phosphate. Wo germination occurred at pH k w i t h any media, but some occurred w i t h a pH approaching 9- Sodium appeared to i n h i b i t germination, and potassium phos-phate was unfavourable except at pH 6. L a c t i c a c i d was the l e a s t favour-able of the acids used i n the pH range 5 to 6, but no s i g n i f i c a n t d i f -ferences were shown i n the s o l u t i o n s used i n the a l k a l i n e pH range. Cer-t a i n media were u n s u i t a b l e because they f a i l e d to maintain s t a b i l i t y of pH over p e r i o d of t e s t i n g . C e r t a i n s o l u t i o n s were a l s o t e s t e d f o r t h e i r m o l a r i t y e f f e c t on germination percentage and on germ tube length. Gen-e r a l l y germination improves w i t h lower m o l a r i t y (a m o l a r i t y of 0.01 or l e s s w i t h most b u f f e r s ) and germ tube e l o n g a t i o n was greater. Best ger-mination i n a l l these t e s t s occurred over the pH range 5>5 to 7.5, suggesting a gener.allmedium w i t h a pH i n t h i s range would be s u i t a b l e f o r - 234 -r o u t i n e germination t e s t s and f o r t e s t s f o r other f a c t o r s so that the hydrogen i o n concentration would not be l i m i t i n g . As the side e f f e c t s of the b u f f e r s were unknown, a bu f f e r e d s o l u t i o n was not used f o r the r e -gular germination t e s t s . The technique developed f o r evenly d i s p e r s i n g a known q u a n t i t y of spores over a t e s t substrate was described e a r l i e r (Powell and Morf I966). U n i f o r m i t y of spore d i s p e r s a l d e n s i t y i s recognized as a neces-s i t y f o r q u a n t i t a t i v e e v a l u a t i o n of b i o l o g i c a l responses. The number of spores to be seeded onto the t e s t substrate was c o n t r o l l e d by a d j u s t i n g the a i r flow from a pressure pump w i t h a bleeder v a l v e and by a timer a t -tached to the pump. Time t e s t s v a r y i n g between 2 and 8 seconds gave spore d e n s i t i e s between 300 and 1600 per sq cm. The average percentage ger-mination f o r three t e s t s (30 f i e l d s at each time i n t e r v a l ) was not s i g -n i f i c a n t l y d i f f e r e n t over t h i s range of spore d e n s i t i e s , but any e f f e c t of d e n s i t y on the r a t e of germination was not i n v e s t i g a t e d . For the re g u l a r t e s t s spores were seeded at a uniform d e n s i t y of 300 - 500 spores per sq cm. '. Methods f o r subsequent experiments A 0.3 - 0.5$ D i f c o Bacto water agar was used i n a l l t e s t s unless otherwise s p e c i f i e d . Ten ml of agar was poured from a Cornwall continuous p i p e t t o r syringe i n t o each 5-cm diameter p l a s t i c p e t r i p l a t e which was maintained, p r i o r to use, i n a w a l k - i n r e f r i g e r a t o r at 3 - 4°C and at 75$ r e l a t i v e humidity. Before seeding the spores, a l l water agar p l a t e s were e q u i l i b r a t e d at the re q u i r e d temperature f o r the subsequent germination experiment, thus a v o i d i n g long e q u i l i b r i u m periods during the test,:: Hand-l i n g of p l a t e s at temperatures other than those of the t e s t c o n d i t i o n s was - 235 -kept to a minimum. This was als o the case when intermediate observations were made during a t e s t ; p l a t e s were removed from the t e s t c o n d i t i o n s , observed and counted, and returned immediately before another p l a t e was counted. During peak per i o d s , i n order to o b t a i n intermediate informa-t i o n , o f t e n at ho u r l y i n t e r v a l s , two or three people were employed i n the counting, the' p l a t e s being observed i n the same sequence, m a i n t a i n i n g a constant time f a c t o r between observations.' The spores were incubated i n the dark (other than i n the l i g h t experiments) at v a r i o u s c o n t r o l l e d tem-peratures over the range 0° to 35°C (1"1°C). U s u a l l y a s e r i e s at 5°C i n -t e r v a l s from 5 "to 30°C was used. The e f f e c t of humidity on germination was t e s t e d i n an appara-tus described by Scharpf (1964), where the humidity of the t e s t chamber was maintained by passing a i r through a container of d i s t i l l e d - d e i o n i z e d water or a glycerine-water mixture which allowed accurate humidity a d j u s t -ment. A Honeywell r e l a t i v e humidity i n d i c a t o r (Minneapolis-Honeywell Regulator Co., Minneapolis, Minnesota), accurate t o t 1% r e l a t i v e humidity, was used to check the humidity l e v e l of the a i r w i t h i n the chamber, over the v a r i o u s glycerine-water mixtures used. The apparatus was allowed to s t a b i l i z e before the experiment was c a r r i e d out. The t e s t spores were placed on a dry s l i d e h e l d i n the chamber and were exposed f o r 24 to 48 hours under the t e s t humidity and at a constant temperature of 15°C, the whole apparatus being run w i t h i n an incubator. F o l l o w i n g negative r e -s u l t s the spores were seeded onto water agar i n a a p e t r i p l a t e f o r 24 hours, or placed on f i l t e r paper saturated w i t h a 1% s i u t i o n o f 2,3, 5~"triphenyl t e t r a z o l i u m c h l o r i d e ( A l l i e d Chemical Corp., New York, N. Y.) f o r 24 hours or longer i n the dark (as t e t r a z o l i u m i s l i g h t - s e n s i t i v e ) to t e s t 236 -f o r v i a b i l i t y . As a f u r t h e r check on the r e s u l t s w i t h the above method, a s e r i e s of aqueous sucrose s o l u t i o n s was prepared at concentrations of 10, 30, 50 and 100$. These concentrations gave t h e o r e t i c a l r e l a t i v e h umidities of 99-85, 99-4-3, 99-04 and 98.03$ i n s m a l l sealed humidity chambers at 20°C (Clayton 1942). Spores from two separate samples were brushed onto two cover glasses attached to a microscope s l i d e . The s l i d e was then h e l d on supports j u s t above the water or sucrose s o l u t i o n i n small sealed g l a s s j a r s , which were placed i n the dark i n a 20°C incuba-t o r f o r 48 hours. For a c o n t r o l , drops of d i s t i l l e d water were placed on the cover glasses and spores were brushed onto the water surface. The v i a b i l i t y of spores g i v i n g negative r e s u l t s was t e s t e d as above, on com-p l e t i o m o f the t e s t . The e f f e c t of hyd r a t i o n of .spores i n a saturated humidity f o l -lowing storage was i n v e s t i g a t e d p r i o r to t h e i r use i n germination t e s t s . Hydration was accomplished by p l a c i n g v i a l s of spores or dry spores on a microscope s l i d e , f o r 24 hours, on the s h e l f of a d e s i c c a t o r c o n t a i n i n g d i s t i l l e d - d e i o n i z e d water, placed i n an incubator at 15°C. Won-hydrated spores served as c o n t r o l . Many spores are reported to s w e l l p r i o r to ger-mination. This was i n v e s t i g a t e d w i t h both f r e s h and stored aeciospores. The same sample of spores were measured i n the dry c o n d i t i o n , immediately when placed' on water or Czapek-Dox agar, and a f t e r 1 - l - l / 2 hours and l a t e r i n t e r v a l s on the media. Measurements were made w i t h a micrometer at a.50x power. Hydrated stored spores were al s o measured by the same method to see whether spore s w e l l i n g had occurred during h y d r a t i o n . To t e s t the e f f e c t of l i g h t on germination two experiments were set up. I n one, samples of spore c o l l e c t i o n s were d i v i d e d i n t o three - 237 -p o r t i o n s , p r i o r t o b e i n g seeded on w a t e r agar p l a t e s . One p l a t e was k e p t i n t h e d a r k a t 15°C, t h e second i n a 15°C i n c u b a t o r w i t h a ' d a y l i g h t ' f l u o r e s c e n t lamp as l i g h t s o u r c e , and t h e t h i r d k e p t o u t s i d e under n a t u r a l l i g h t c o n d i t i o n s , w i t h a r e c o r d i n g h y g r o t h e r m o g r a p h n e a r b y . I n t h e s e -cond t e s t , t h e " e f f e c t o f v a r i o u s c o l o r e d l i g h t w a v e l e n g t h bands on g e r -m i n a t i o n were compared. A s e r i e s o f G e n e r a l E l e c t r i c 18 i n c h l o n g , 1 i n c h d i a m e t e r , 15-watt f l u o r e s c e n t lamps was s e t up i n i n c u b a t o r s k e p t a t 15°C, w i t h one i n c u b a t o r m a i n t a i n e d w i t h o u t a l i g h t s o u r c e as a c o n t r o l . The lamps were mounted about 12 cm above t h e t e s t p l a t e s w h i c h were p l a c e d d i r e c t l y b e n e a t h t h e c e n t e r p o r t i o n o f t h e lamp f o r 2k h o u r s , where even l i g h t i n t e n s i t y was m a i n t a i n e d . S p e c t r a l e m i s s i o n c u r v e s p r o v i d e d b y G e n e r a l E l e c t r i c Lamp D i v i s i o n showed t h a t b l a c k l i g h t f l u o r e s c e n t lamps emit 250 and k&0 m\i w i t h an.energy peak a t 350 mu; b l u e between 325 and 665 mLi w i t h peak a t 525 mi-1; p i n k between 525 and 750+ mu w i t h peak a t 615 mp; and ' d a y l i g h t ' between 320 and 750+ mp w i t h peaks a t 475 and 575 m u . . The i n t e n s i t y o f t h e l i g h t a t t h e l e v e l o f t h e t e s t p l a t e s was measured i n f o o t c a n d l e s ( f t - c . ) b y a S e k o n i c S t u d i o E x p o s u r e M e t e r (Brockway Camera C o r p o r a t i o n , New Y o r k ) . The r e s p e c t i v e f t - c . v a l u e s were b l a c k — 5 f t - c ; b l u e — 80 f t - c ; g r e e n — 175 f t - c ; p i n k — 75 f t - c ; and ' d a y l i g h t ' — 125 f t - c . The i n t e n s i t i e s o f l i g h t measured a r e n o t com-p a r a b l e as l i g h t s e n s o r s r e s p o n d d i f f e r e n t l y as t h e s p e c t r a l w a v e l e n g t h d i s t r i b u t i o n o f i n c i d e n t l i g h t changes ( F e d e r e r and Tanner I 9 6 6 ) , and no c o r r e c t i o n s were a t t e m p t e d . F o r hydrogen i o n c o n c e n t r a t i o n t e s t s o v e r t h e range pH 3 t o 10, t h e d e s i r e d pH o f t h e agar media was o b t a i n e d b y a d d i n g a p p r o p r i a t e amounts o f p o t a s s i u m a c i d p h t h a l a t e , p o t a s s i u m p h o s p h a t e , p o t a s s i u m d i h y d r o g e n - 238 -phosphate o r b o r i c a c i d b u f f e r s , and sodium h y d r o x i d e , sodium p h o s p h a t e , p o t a s s i u m h y d r o x i d e and h y d r o c h l o r i c a c i d s o l u t i o n s t o t h e m e l t e d b a s a l agar media j u s t p r i o r t o p o u r i n g p l a t e s . T h i s method p r o d u c e d f i r m l y s e t media a t a l l pH v a l u e s u s e d . I n one "test s e r i e s o v e r t h e pH range k t o 8, two o r t h r e e media o f d i f f e r e n t c o m b i n a t i o n s were used i n t h e e x p e r i m e n t s f o r each pH v a l u e . I n most t e s t s t h e c o n t r o l medium was a 0 . 3 $ w a t e r agar (pH 6 . 8 - 7 - 0 ) , b u t i n one t e s t a 2$ Czapek-Dox agar was used (pH 7 - 0 ) . Measurements o f t h e a d j u s t e d media, b e f o r e and a f t e r t h e e x p e r i m e n t s , w i t h a Beckman pH meter, showed l i t t l e pH change d u r i n g t h e e x p e r i m e n t ( l e s s t h a t t 0 . 2 ) . The r e s u l t i n g end pH o f t h e media was used i n a l l c a s e s . I n I967, when s e v e r a l p o o r g e r m i n a t i o n r e s u l t s were o b t a i n e d i n t h e d a i l y g e r m i n a t i o n t e s t s , ah e x p e r i m e n t was s e t up t o i n v e s t i g a t e t h e e f f e c t o f some r e a d i l y a v a i l a b l e c o m m e r c i a l l y p u r i f i e d agar and b r o t h media on g e r m i n a t i o n , number o f germ tub e s and germ tube l e n g t h . Ten m edia were p r e p a r e d , w h i c h i n c l u d e d B a c t o w a t e r a g a r , p o t a t o d e x t r o s e agar, d e x t r o s e agar, m a l t e x t r a c t agar and b r o t h , Czapek-Dox b r o t h , n u t r i e n t agar and y e a s t e x t r a c t ( T a b l e X X X I V ) . The media were p r e p a r e d a c c o r d i n g t o procedures' g i v e n b y t h e m a n u f a c t u r e r s ( D i f c o L a b o r a t o r i e s , I n c . , D e t r o i t , M i c h i g a n , f o r a l l media e x c e p t Czapek-Dox b r o t h — F i s h e r S c i e n t i f i c , F a i r Lawn, New J e r s e y ) , e x c e p t t h a t t h e agar c o n t e n t was sometimes v a r i e d , and 1$ and i n one case 3-5$ agar was added t o t h e b r o t h s t o f i r m l y s o l i d i f y t h e media. I n 1968 t h e g e r m i n a t i v e r e s p o n s e s o f s p o r e s t o s u c r o s e as a c a r b o h y d r a t e s o u r c e o v e r t h e range 1 t o 40$ were compared w i t h g e r m i n a t i o n on a s t a n d a r d B a c t o w a t e r agar, f o l l o w i n g t h e s u p e r i o r r e s u l t s o b t a i n e d w i t h a Czapek-Dox b r o t h agar, w h i c h i t s e l f c o n t a i n s 3$ s u c r o s e . I n 1966 t h e e f f e c t o f h o s t l e a f e x t r a c t s on g e r m i n a t i o n was - 239 -t e s t e d by adding 5 to 25 gm of chopped Geocaulon and Comandra l e a f m a t e r i a l to 100 ml of 0.25% water agar. The l e a f m a t e r i a l was e i t h e r added p r i o r to heating of the water agar medium or added as the agar cooled but before pouring and s o l i d i f i c a t i o n o f t h e . p l a t e s took p l a c e . I n 1967 the t e s t was repeated by adding 5 gm of chopped leaves to 100 ml of 0.3% water agar when the agar was c o o l . The pH of the l e a f e x t r a c t s and water agar was recorded i n both years. To check that the pH of the host leaves v a r i e d l i t t l e throughout the season and that the values were s i m i l a r to those used i n the media, measurements were made of the pH of the expressed l e a f j u i c e of the p l a n t s at r e g u l a r i n t e r v a l s , by methods s i m i l a r to those used by Hurd-Karrer (1939)- Tbe e f f e c t of the presence of host leaves, was a l s o i n v e s t i g a t e d , by p l a c i n g host leaves on the medium surface. I n two t e s t s 3 mm diameter holes were punched i n the leaves; i n another a f i n e spray mist was a p p l i e d to l i g h t l y cover the leaves w i t h moisture to see i f t h i s would enhance germination. Both young and o l d Comandra leaves were used i n one t e s t . I n a l l t e s t s to i n v e s t i g a t e the e f f e c t of any f a c t o r or t r e a t -ment, one or two c o n t r o l p l a t e s from each spore sample were employed. Samples of spores from two to ten cankers, u s u a l l y f i v e , were used f o r each s e r i e s of treatments. I n most treatments each t e s t was repeated at l e a s t twice, and i n some cases four or more times. U s u a l l y the spore samples used f o r comparative treatments were of the same age, c o l l e c t e d the same hour of the day from one l o c a t i o n , and had been subjected to i d e n t i c a l storage and p r e p a r a t i o n c o n d i t i o n s . When spores of d i f f e r e n t c o l l e c t i o n times or storage conditions were used, t h i s i s i n d i c a t e d i n the t e x t . Germination percentage f o r each treatment was the average of - 2k0 -a l l samples used ( u n l e s s one was e l i m i n a t e d because o f o b v i o u s c o n t a m i n a -t i o n o r because a n o t h e r f a c t o r a f f e c t e d t h e a c c u r a c y o f t h e g e r m i n a t i o n p e r c e n t a g e ) . A t l e a s t 300 s p o r e s were counted i n each sample, t h e count b e i n g o b t a i n e d b y o b s e r v i n g each spore' i n a number o f randomly p l a c e d 50x power m i c r o s c o p e f i e l d s . When no g e r m i n a t i o n was i n d i c a t e d b y t h i s method o f c o u n t i n g , t h e whole a r e a o f t h e medium w i t h s p o r e s was scanned as o c c a s i o n a l l y a few g e r m i n a t e d s p o r e s c o u l d be fo u n d o r a con t a m i n a n t had a f f e c t e d g e r m i n a t i o n i n a d e f i n i t e s e c t o r o f t h e medium. A spor e was c o n s i d e r e d t o have g e r m i n a t e d when t h e germ t u b e l e n g t h was e q u a l t o o r l o n g e r t h a n h a l f t h e min o r d i a m e t e r o f t h e s p o r e . I n as many t r e a t m e n t s as p o s s i b l e , t h e number o f germ t u b e s p e r s p o r e , t h e l e n g t h o f t h e germ t u b e s , t h e f r e q u e n c y and t y p e o f b r a n c h i n g , and t h e o c c u r r e n c e o f p l a s -m o p t y s i s o r o t h e r o b s e r v e d f a c t o r s a f f e c t i n g g e r m i n a t i o n were r e c o r d e d . A compromise was n e c e s s a r y between t h e number o f s p o r e s measured p e r sam-p l e and t r e a t m e n t and t h e ti m e i n v o l v e d i n measurements, b u t g e n e r a l l y a t l e a s t 10 s p o r e s s e l e c t e d a t random i n each sample were measured t o o b t a i n t h e average number o f germ t u b e s o r average l e n g t h o f l o n g e s t o r a l l germ t u b e s . O b s e r v a t i o n s on g e r m i n a t i o n p e r c e n t a g e and germ t u b e growth were made a t v a r i o u s i n t e r v a l s between 1 and 168 h o u r s , b u t t h e i n t e r -v a l s o f 6 and 2k h o u r s were t h o s e n o r m a l l y used. R e s u l t s E f f e c t o f t e m p e r a t u r e on g e r m i n a t i o n A s e r i e s o f t e s t s was c a r r i e d o u t t o e s t a b l i s h t h e range and optimum t e m p e r a t u r e s f o r g e r m i n a t i o n . I n I965 t h r e e samples, a l l showing g o o d - g e r m i n a t i o n , were t e s t e d a t 5°C i n t e r v a l s f r o m 5 t o 30°C ( F i g . 79). F i g . 79- E f f e c t o f t e m p e r a t u r e on p e r c e n t g e r m i n a t i o n o f t h r e e C r o n a r t i u m comandrae a e c i o s p o r e samples a f t e r 2k hours on w a t e r agar (pH 6.8). F i g . 80. E f f e c t o f t e m p e r a t u r e on C r o n a r t i u m comandrae a e c i o s p o r e g e r m i n a t i o n . A verage p e r c e n t g e r m i n a t i o n o f f i v e s e r i e s and v a r i o u s numbers o f r e p l i c a s w i t h t h r e e t o f i v e samples each, a f t e r 2k hours on w a t e r a g a r . - 2kl -- 2k2 -G e r m i n a t i o n o c c u r r e d o v e r t h e range 5 t o 25°C, b u t was much r e d u c e d a t 25°C. Optimum g e r m i n a t i o n o c c u r r e d a t 15°C, and t h e p e r c e n t a g e g e r m i n a -t i o n was s t i l l r e l a t i v e l y h i g h a t 10°C. I n I966 and 1967 f u r t h e r t e s t s were r u n t o c o n f i r m t h e optimum t e m p e r a t u r e and t h e upper and l o w e r l i m i t s o f g e r m i n a t i o n w i t h g r e a t e r p r e c i s i o n . G e n e r a l l y t h e l a t e r t e s t s gave much lower- g e r m i n a t i o n p e r c e n t a g e s b u t t h e p a t t e r n was s i m i l a r w i t h an optimum c l o s e t o 15°C. F i g . 80 shows t h e average p e r c e n t a g e g e r m i n a t i o n o f f i v e s e p a r a t e s e r i e s o f . t e s t s . T e s t s e r i e s 1 i s t h e average o f s i x r e p l i c a s each w i t h k o r 5 samples, f o r t h e t e m p e r a t u r e s 5, 10, 15, 17, 19, 22 and 28°C. The g e r m i n a t i o n p e r c e n t a g e s were l o w e r a t t e m p e r a t u r e s above 15°C i n d i c a t i n g t h a t t h e optimum t e m p e r a t u r e f o r g e r m i n a t i o n may be a l i t t l e b e l o w 15°C as t h e r e was l e s s r e d u c t i o n a t 10° t h a n a t 17°C. T e s t s e r i e s 2, w i t h two r e p l i c a s each u s i n g 5 samples o v e r t h e tempera-t u r e range 1 t o 30 °C, gave some g e r m i n a t i o n a t 30°C, b u t o n l y a t r a c e a t 1°C. G e r m i n a t i o n a t 30°C was not c o n s i s t e n t as o n l y f i v e samples gave any g e r m i n a t i o n a t t h i s t e m p e r a t u r e . T e s t s e r i e s 3, w i t h two r e p l i c a s , and 2 o r 3 samples each, o v e r t h e range 2 t o 30°C, T e s t s e r i e s k, a s i n g l e t e s t w i t h k samples o v e r t h e range 5 t o 30°C, and T e s t s e r i e s 5, a s i n g l e t e s t w i t h k samples o v e r t h e range 5 t o 20°C, show t h e same p e r c e n t a g e g e r m i n a t i o n p a t t e r n . A l t h o u g h i n th e v a r i o u s s e r i e s ( F i g s . 79 and 80) some g e r m i n a t i o n was r e c o r d e d o v e r t h e range 1 t o 30°C, ge r m i n a -t i o n was g e n e r a l l y poor ( < 10$) o u t s i d e t h e range 5 t o 22°C. The average number o f germ t u b e s p e r spor e w h i c h d e v e l o p e d a t th e v a r i o u s t e m p e r a t u r e s i s shown f o r f o u r t e m p e r a t u r e s e r i e s i n T a b l e XXIX. I n a l l s e r i e s t h e maximum number o f germ t u b e s o c c u r r e d a t 15°C, u s u a l l y w i t h a marked r e d u c t i o n a t t e m p e r a t u r e s above and below, w i t h - 2h3 -T a b l e XXIX. Average number o f germ tub e s p e r s p o r e , and t h e range o f t h e a v e r a g e f o r f o u r t e s t s e r i e s a t v a r i o u s t e m p e r a t u r e s . Temperature °C 5 10 15 20 25 30 A v e r . no. o f jerm t u b e s / s p o r e 1.5 1.5 1-9 1.3 1.2 l . l * Range o f the average i n t h e i n d i v i d u a l s e r i e s 1.1 - 2.7 1.1 - 2.7 1.5 - 2.7 1.1 - 1.7 1.1 - l.k 1.1* * D a t a f r o m o n l y one s e r i e s f ewer b e i n g p r o d u c e d a t t h e h i g h e r t e m p e r a t u r e s . The h i g h r a n g e s , shown i n T a b l e XXIX, a l l o c c u r r e d i n one s e r i e s , when t h e r e was no d i f f e r e n c e i n t h e number o f germ t u b e s p r o d u c e d a t 5, 10 and 15°C, b u t t h e u s u a l d e c r e a s e a t h i g h e r t e m p e r a t u r e s . The average o f numerous o t h e r t e s t s , when s p o r e s were t e s t e d o n l y a t 15°C, was 2.0 germ t u b e s p e r s p o r e , a f i g u r e v e r y c l o s e t o t h a t shown i n T a b l e XXIX f o r t h i s t e m p e r a t u r e . One t o s i x germ t u b e s were p r o d u c e d p e r s p o r e , a l l emerging a t t h e same.time t h r o u g h t h e spore w a l l , b u t t h e germ t u b e s e l o n g a t e d a t u n e q u a l r a t e s , and i t was uncommon f o r more t h a n one germ tube t o d e v e l o p t o any l e n g t h . What d e t e r m i n e s w h i c h germ tube w i l l d e v e l o p i s unknown, b u t t h e two n u c l e i o f t h e s p o r e a r e , a l m o s t w i t h o u t e x c e p t i o n , p r e s e n t i n t h e d e v e l o p e d germ t u b e . The average l e n g t h o f the l o n g e s t germ tube i s shown f o r t h r e e samples i n ' F i g . 8l, a l o n g w i t h t h e average l e n g t h o f a l l germ t u b e s p e r spor e a f t e r 2k hours a t t e m p e r a t u r e s o v e r t h e range 5 "to 25°C. The a v e r -age l e n g t h o f t h e l o n g e s t germ t u b e was g r e a t e s t a t 15°C, w i t h l i t t l e d i f f e r e n c e between l e n g t h s a t t h e o t h e r t e m p e r a t u r e s , a l t h o u g h a t t h e F i g . 8 l . Average l e n g t h o f germ t u b e s o f t h r e e C r o n a r t i u m comandrae a e c i o s p o r e samples g e r m i n a t e d on w a t e r a g a r (pH 6.8) after 2k hours a t v a r i o u s t e m p e r a t u r e s . ( a ) L e n g t h o f l o n g e s t germ t u b e p e r s p o r e . (b) L e n g t h o f a l l germ t u b e s p e r s p o r e . - ¥ T S -- 24-5 -extremes (5 and 25°C) t h e y a r e g e n e r a l l y s l i g h t l y s h o r t e r . When t h e l e n g t h s o f a l l germ t u b e s a r e c o n s i d e r e d t h e r e l a t i v e p o s i t i o n s a r e l i t -t l e changed ( F i g . 8 l ) , because g e n e r a l l y o n l y one germ tube d e v e l o p s t o any l e n g t h . The degree o f germ tube b r a n c h i n g v a r i e d l i t t l e w i t h tem-perature.. U s u a l l y t h e germ tube was i r r e g u l a r l y b r a n c h e d w i t h most o f t h e b r a n c h i n g o c c u r r i n g towards t h e t i p o f t h e germ t u b e . Branches were g e n e r a l l y s h o r t a t a l l t e m p e r a t u r e s , r a r e l y more t h a n 50M- l o n g . There was c o n s i d e r a b l e v a r i a t i o n i n t h e l e n g t h o f t h e l o n g e s t germ t u b e a t 15°C i n t h e v a r i o u s t e s t s e r i e s , b u t a t o t h e r t e m p e r a t u r e s g r o w t h a f t e r 2k hours v a r i e d l e s s . The average l e n g t h a t 15°C r a n g e d f r o m 300u t o 713 u5 w i t h an average v a l u e f o r t h e v a r i o u s t e m p e r a t u r e t e s t s o f around 400u. Germ tube e l o n g a t i o n g e n e r a l l y c o n t i n u e d f o r 96 hours i n v i t r o o r u n t i l p l a s m o p t y s i s o f t h e germ tube o c c u r r e d . Most growth o c c u r s i n t h e f i r s t 2k hours and growth beyond t h i s p e r i o d i s much s l o w e r . R u p t u r e o f t h e germ tube o c c u r r e d a t t h e g r o w i n g t i p and t h e p r o t o p l a s m s p i l l e d o n t o t h e agar media. On some o c c a s i o n s n e a r l y h a l f t h e germ t u b e s p l a s m o p t y z e d , and t h i s phenomenon was more common a t t h e optimum and s u p r a o p t i m a l tem-p e r a t u r e s . The i n f l u e n c e o f t e m p e r a t u r e on t h e r a t e o f g e r m i n a t i o n i s shown i n F i g s . 82 and 83 f r o m two s e p a r a t e s e r i e s o f t e s t s . The e f f e c t was t e s t e d o v e r t h e t e m p e r a t u r e range 2 t o 30°C, w i t h c o u n t s and o b s e r v a t i o n s made a t one o r two h o u r l y i n t e r v a l s up t o 8 hours w i t h a f i n a l count a f t e r 2k h o u r s . I n b o t h s e r i e s , b e t t e r i n i t i a l g e r m i n a t i o n o c c u r r e d a t 20°C t h a n a t o t h e r t e m p e r a t u r e s a f t e r 1 hour, and t h i s p o s i t i o n was r e t a i n e d a t 2 h o u r s . S l o w e s t g e r m i n a t i o n o c c u r r e d a t 2 and 25°C w i t h o t h e r tempera-t u r e s i n t e r m e d i a t e . The g e n e r a l p e r c e n t a g e l e v e l o f t h e f i n a l g e r m i n a t i o n F i g . 82. I n f l u e n c e o f v a r i o u s t e m p e r a t u r e s on r a t e o f C r o n a r t i u m comandrae a e c i o s p o r e g e r m i n a t i o n a f t e r v a r i o u s t i m e i n t e r v a l s on w a t e r a g a r . F i g . 83. Average r a t e o f g e r m i n a t i o n o f t h r e e C r o n a r t i u m comandrae a e c i o s p o r e samples a t d i f f e r e n t t e m p e r a t u r e s and a f t e r v a r i o u s t i m e i n t e r v a l s on w a t e r a g a r . - 2hG -loo-. 60-, Temperature T IME ( H O U R S ) - 21+7 -was u s u a l l y r e a c h e d a f t e r k t o 5 h o u r s , w i t h v e r y l i t t l e i n c r e a s e i n g e r -m i n a t i o n p e r c e n t a g e a t 2k h o u r s . T h i s was not t h e case w i t h germ t u b e growth, w h i c h c o n t i n u e d t o i n c r e a s e up t o 2k hours ( F i g . 8U). I n i t i a l germ tube growth, as i n o t h e r t e s t s , was f a s t e s t a t 20°C f o l l o w e d c l o s e l y b y g r o wth a t 10, 15 and 25°C, a l t h o u g h growth a t 25°C tend e d t o sl o w down q u i c k l y . Growth a t 2 and 5°C was a p p r e c i a b l y s l o w e r , a l t h o u g h f i n a l g r o w t h a t 5°C was s i m i l a r t o t h a t a t 10 and 20°C. The growth c u r v e t e n d e d t o l e v e l o f f a f t e r about 6 h o u r s , a l t h o u g h growth a t 25°C had l e v e l l e d o f f e a r l i e r . The h o u r l y r a t e o f germ tube growth a f t e r t h e f i r s t 6 hours was l e s s t h a n h a l f t h a t o f t h e e a r l i e r p e r i o d , and re m a i n e d f a i r l y c o n s t a n t i n t h i s l a t e r p e r i o d . Measurements o f germ t u b e s a f t e r k8 and 72 h o u r s growth i n d i c a t e d l i t t l e i n c r e a s e i n l e n g t h f r o m t h a t a t 2k h o u r s . O f t e n p l a s m o p t y s i s o f germ t u b e s o c c u r r e d when i n c o n t a c t w i t h t h e media, w h i c h e f f e c t i v e l y p r e v e n t e d any f u r t h e r growth. A l s o , t h e c o n t e n t s o f t h e germ tube t e n d e d t o c o l l e c t a t t h e t i p o f t h e tube and for m a v e s i c l e t y p e s t r u c t u r e ( F i g s . 28-30) w h i c h p r o b a b l y p e r f o r m s t h e f u n c t i o n o f an ap-p r e s s o r i u m f r o m w h i c h an i n f e c t i o n peg would e v e n t u a l l y be p r o d u c e d . E f f e c t o f h u m i d i t y on g e r m i n a t i o n Seven s e p a r a t e t e s t s were r u n a t a r e l a t i v e h u m i d i t y o f 100$ and one t e s t a t 97$, t o e s t a b l i s h whether g e r m i n a t i o n w o u l d o c c u r i n t h e absence o f a l i q u i d medium. I n a l l t e s t s no g e r m i n a t i o n was o b t a i n e d . To check t h a t v i a b l e s p o r e s were used i n t h e t e s t s , t h e s p o r e s were su b s e -q u e n t l y seeded o n t o a w a t e r agar p e t r i p l a t e and/or t e s t e d w i t h a t e t r a -zolium. c h l o r i d e s o l u t i o n . I n f i v e o f t h e t e s t s some g e r m i n a t i o n (up t o 20$) was o b t a i n e d on t h e p l a t e s , and s i m i l a r p e r c e n t a g e s o f t h e s p o r e s t u r n e d v a r i o u s shades o f r e d i n t h e t e t r a z o l i u m t e s t s — t h e degree o f c o l o r b e i n g F i g . 8k. Average l e n g t h o f t h e l o n g e s t germ tube f r o m t h r e e t o f i v e C r o n a r t i u m comandrae a e c i o s p o r e samples g e r m i n a t e d on w a t e r agar a t d i f f e r e n t t e m p e r a t u r e s and a f t e r v a r i -ous t i m e i n t e r v a l s . - 248 -360-i 0 4 8 12 16 20 24 TIME (HOURS) - 2k9 -l a r g e l y due t o t h e t i m e t h e s p o r e s were i n c o n t a c t w i t h t h e s o l u t i o n , p r o v i n g t h a t t h e t e s t s p o r e s had been v i a b l e . R e s u l t s o f t h e t e s t s w i t h v a r i o u s s u c r o s e s o l u t i o n s s i m i l a r l y gave no g e r m i n a t i o n . However, i f d e p o s i t s o f w a t e r were p r e s e n t on t h e t e s t s l i d e t h r o u g h c o n d e n s a t i o n t h e n g e r m i n a t i o n would t a k e p l a c e . As i t i s v e r y d i f f i c u l t t o measure and m a i n t a i n w i t h c e r t a i n t y a r e l a t i v e h u m i d i t y o f 100%, t h e c o n c l u s i o n r e a c h e d f r o m t h e above t e s t s i s t h a t no g e r m i n a t i o n t o o k p l a c e b e l o w 100%, and i t i s d o u b t f u l i f any g e r m i n a t i o n o c c u r r e d i n a s a t u r a t e d 100% r e l a -t i v e h u m i d i t y , b u t i f w a t e r was p r e s e n t g e r m i n a t i o n o c c u r r e d . S w e l l i n g o f s p o r e s on l i q u i d media T a b l e XXX shows t h a t a l l a e c i o s p o r e samples s w e l l e d when p l a c e d on a Czapek-Dox a g a r . S i m i l a r r e s u l t s were o b t a i n e d on w a t e r a g a r . The ' measurements i n d i c a t e d an i n c r e a s e i n b o t h a e c i o s p o r e l e n g t h and w i d t h , though t h e p r o p o r t i o n a l i n c r e a s e was g r e a t e r i n t h e w i d t h d i m e n s i o n . The w i d t h o f f r e s h s p o r e s i n t h e d r y s t a t e was g r e a t e r t h a n s t o r e d s p o r e s , b u t i f s t o r e d s p o r e s r e m a i n v i a b l e , as was t h e case w i t h Sample 3, t h e immediate i n c r e a s e when p l a c e d on agar was s i m i l a r . However, t h e r e was no s i m i l a r i n c r e a s e i n dead s t o r e d s p o r e s (Samples h and 5 )5 t h e i n c r e a s e i n w i d t h o n l y amounted t o about 35% o f t h e i n c r e a s e o f v i a b l e s t o r e d s p o r e s , a l t h o u g h on w a t e r agar t h e i n c r e a s e , was c l o s e r t o 65%. That t h e w i d t h s i z e i n c r e a s e t o o k p l a c e a l m o s t i m m e d i a t e l y was shown b y t h e l i t t l e o r no i n c r e a s e a t t h e 1 - 1-1/2 hour measurements. Sample 3 was a l s o measured a t 2 and 3 hour i n t e r v a l s as g e r m i n a t i o n was l a t e r i n t h i s sample, and showed v i r t u a l l y no s i z e change. Samples k and 5 showed no change when measured a f t e r 3 hours nor a f t e r 3 days, t h u s t h e l o w e r i n c r e a s e was not - 250 -Table XXX. Average length and width ( i n microns) of 25 aeciospores from 5 f r e s h or stored samples. Measurements were made on dry spores and when spores were placed on a Czapek-Dox agar medium. Spore sample Spore c o n d i t i o n Dry When placed on agar A f t e r 1 - Its-hours on agar no. length (n) width (n) l e n g t h (n) width (n) l e n g t h (n) width M 1 f r e s h 56 26 65 37 72 - 37 2 f r e s h 6k 30 70 37 — — 3 stored 2 months 6o 20 66 36 70 36 k stored ik months 58 20 65 26 6k 28 5 stored 26 months* 59 21 63 27 *50 spores measured j u s t a case of slow absorption. As a f u r t h e r check o f the s w e l l i n g d i f -ference between v i a b l e and non-viable spores, a p o r t i o n of v i a b l e spores, from Sample 3 was exposed to p l u s 30°C temperature f o r a few hours and then placed on Czapek-Dox agar and measured a f t e r 1 hour and a f t e r 3 days.' Average measurements were 6k\i long and 28u wide, values t y p i c a l f o r non-v i a b l e spores, and no germination occurred. The degree of s w e l l i n g was th e r e f o r e a s s o c i a t e d w i t h a spore being v i a b l e or non-viable. The i n -crease i n length was more v a r i a b l e as i n nature the ' t a i l ' i s qu i t e v a r i -able i n length, and i t i s p o s s i b l e that most s i z e increase occurred w i t h i n the main spore r a t h e r than i n the ' t a i l ' . This aspect was not observed separately. I f non-viable dry spores were hydrated p r i o r to p l a c i n g on a l i q u i d medium, no s w e l l i n g occurred, but v i a b l e spores d i d begin s w e l l i n g during hydration. The t a k i n g i n of moisture appeared to be a requirement - 251 -f o r g e r m i n a t i o n t o a c t i v a t e mechanisms w i t h i n t h e s p o r e , f o r s w e l l i n g ceased when germ tube emergence began, and no germ t u b e s appeared w i t h -o u t c o n s i d e r a b l e p r i o r s w e l l i n g . F o l l o w i n g germ tube emergence, spo r e d i m e n s i o n s d e c r e a s e d b y a few m i c r o n s , i n d i c a t i n g t h a t some s p o r e c o n t e n t s had moved out i n t o t h e germ t u b e . E f f e c t o f h y d r a t i o n o f s p o r e s on g e r m i n a t i o n S i x samples o f d r y s p o r e s were s t o r e d f o r 5, 6,17 and 38 days a t -k°C. A p o r t i o n o f each sample was p l a c e d i n a s a t u r a t e d h u m i d i t y f o r 2k h o u r s f o l l o w i n g s t o r a g e t o a l l o w h y d r a t i o n t o t a k e p l a c e . T h i s p o r t i o n and a c o n t r o l sample o f d r y s p o r e s were t h e n seeded o n t o p e t r i p l a t e s o f Czapek-Dox agar o r O.jfo w a t e r agar and g e r m i n a t e d i n t h e d a r k a t 15°C f o r 2k h o u r s . T a b l e XXXI g i v e s t h e average g e r m i n a t i o n p e r c e n t - . ages o f t h e 6 samples f o r t h e i n d i v i d u a l days o f t e s t i n g , f o l l o w e d b y t h e g e r m i n a t i o n r a n g e . I n i t i a l l y , h y d r a t i o n o f s p o r e s improved t h e g e r -m i n a t i o n by 7 t o 10% compared w i t h s t o r e d n o n - h y d r a t e d s p o r e s . A s i m i l a r i n c r e a s e (9-1% f o r 5 days and 6.9% f o r 6 days) was r e c o r d e d on t h e w a t e r a g a r , a l t h o u g h t h e a c t u a l g e r m i n a t i o n p e r c e n t a g e s on t h i s medium were l o w e r . F o l l o w i n g 17 days s t o r a g e , t h e r e was no improvement i n g e r m i n a -t i o n , i n f a c t a d e c r e a s e , and t h i s became more marked a f t e r 38 days when the g e r m i n a t i o n p e r c e n t a g e o f h y d r a t e d s p o r e s was l e s s t h a n h a l f t h a t o f d r y s p o r e s . T h i s i s shown v e r y w e l l b y t h e range i n p e r c e n t a g e g e r -m i n a t i o n o f t h e s i x samples. - 252 -Table XXXI. Average germination percentages f o r spores stored f o r various periods of time at -k°C, and then germinated, dry or a f t e r 24 hours h y d r a t i o n i n a saturated humidity, on Czapek-Dox agar at 15°C. Days o f Dry non- Hydrated storage hydrated spores spores 5 85.1(68.8-95.1) 94.6(91.3-98.1) 6 71.3(61.9-80.6) 78.7(61.2-95.4) 17 72.6(49.0-85.9) 65.3(32.6-82.5) 38 67.9(50.3-87.9) 30.2(16.7-38.8) E f f e c t of l i g h t on germination Table XXXII gives the r e s u l t s of three s e r i e s of germination t e s t s i n the dark and l i g h t . I n a l l three s e r i e s ' l i g h t ' was obtained from a 15-watt ' d a y l i g h t ' f l u o r e s c e n t lamp, and i n one t e s t n a t u r a l l i g h t was a l s o used. The t e s t s showed no s i g n i f i c a n t d i f f e r e n c e between dark and ' d a y l i g h t ' i n two s e r i e s , but i n S e r i e s 2, two of the 8 samples showed higher percentages i n the l i g h t than the corresponding ones i n the dark. The t e s t w i t h n a t u r a l l i g h t was s l i g h t l y lower i n germination than the two c o n t r o l l e d c o n d i t i o n s . This may have r e s u l t e d from the n a t u r a l tem-perature f l u c t u a t i o n s , but even i n t h i s t e s t o n ly two of the ei g h t samples gave germination percentages markedly d i f f e r e n t from the other t e s t s . There was no appreciable d i f f e r e n c e i n the lengths of the longest germ tube under the three conditions (Table X X X I l ) , and the number of germ tubes per spore and the form of branching was s i m i l a r . L i g h t and dark t h e r e f o r e appeared to have no s i g n i f i c a n t e f f e c t on germination. I n 1966 and 1967 various t e s t s were run usi n g a s e r i e s of colored l i g h t lamps, and compared w i t h control t e s t s run i n the dark. S u f f i c i e n t - 253 -T a b l e X X X I I . E f f e c t o f l i g h t and d a r k c o n d i t i o n s on p e r c e n t g e r m i n a -t i o n and germ tube g r o w t h o f t h r e e s e r i e s o f a e c i o s p o r e . samples, g e r m i n a t e d on Czapek-Dox agar a t 15°C o r under f l u c t u a t i n g o u t s i d e t e m p e r a t u r e s f o r 2k h o u r s . S e r i e s Wo. o f samples Dark ' D a y l i g h t ' N a t u r a l l i g h t G e r m i n a t i o n % 67.6 52.5 86.1 66.2 1+0.0 82.7 59-6 Average l e n g t h o f l o n g e s t germ tube (u) 1+81 569 . 1+66 575 518 - - -Temperature °C 15 15 7 - 2 6 T a b l e X X X I I I . E f f e c t o f d a r k and t h r e e c o l o r e d l i g h t wave bands on p e r c e n t g e r m i n a t i o n and germ tube g r o w t h o f t h r e e s e r i e s o f a e c i o s p o r e samples, g e r m i n a t e d a t 15°C on Czapek-Dox agar f o r 2k h o u r s . S e r i e s 1 2 3 1 and 3 Wo. o f r e p l i c a s 3 2 3 3 T o t a l no. o f samples 17 18 15 11 G e r m i n a t i o n % Average Peak wave l e n g t h ' o f l o n g e s t band germ tube (u) ( m i l l i c r o n s ) D ark 75-9 75-2 33.3 236 Green l i g h t 74.5 39-8 233 525 B l u e l i g h t 81.9 89.6 1+2.9 252 kko P i n k l i g h t 77-6 83.9 (365) 1 615 •'-Data f r o m o n l y one t e s t w i t h 5 samples - 254 -i n c u b a t o r s w i t h a d a p t o r s f o r l i g h t lamps were n o t a v a i l a b l e t o t e s t t h e e f f e c t o f the d i f f e r e n t c o l o r l i g h t wave bands t o g e t h e r . I n I967 one s e r i e s was r u n u s i n g t h r e e c o l o r s and t h e c o n t r o l , b u t i n a l l o t h e r s e r i e s o n l y two and t h e d a r k c o n t r o l were a v a i l a b l e . T a b l e X X X I I I summarizes t h e t h r e e s e r i e s made i n I967, S e r i e s 1 and 3 were r u n t h r e e t i m e s , and S e r i e s 2 twice.- I n a l l t h r e e s e r i e s t h e b l u e l i g h t gave s l i g h t l y b e t t e r g e r m i n a t i o n t h a n t h e others used, b u t t h e p e r c e n t a g e g e r m i n a t i o n o f t h e c o l o r e d l i g h t s was not m a r k e d l y d i f f e r e n t f r o m t h e d a r k . c o n t r o l . The re s u l t s s u g g e s t e d t h a t t h e b l u e l i g h t s p e c t r u m (440p) s l i g h t l y f a v o u r s g e r m i n a t i o n , b u t t h e r e appeared t o be no g r a d i e n t f r o m low m i l l i c r o n v a l u e s t o h i g h , as i n t h e one comparable s e r i e s , t h e p i n k s p e c t r u m (615P) gave 3% g r e a t e r g e r m i n a t i o n t h a n t h e g r e e n (5251-0. There was l i t t l e d i f f e r e n c e i n t h e l e n g t h o f t h e l o n g e s t germ t u b e between d a r k and b l u e and g r e e n l i g h t . I n S e r i e s 1 when p i n k l i g h t was compared w i t h t h e o t h e r s , germ tubes, a v e r a g e d 75M- l o n g e r under t h e p i n k l i g h t t h a n under b l u e and d a r k , w i t h t h e g r e a t e s t d i f f e r e n c e (l50u.) between, p i n k and g r e e n . The t e s t s made i n 1966 were l e s s s a t i s f a c t o r y m a i n l y b e c a u s e p e r c e n t g e r m i n a t i o n s were low. I n a l l t h e s e t e s t s t h e same f o u r s p o r e samples were used and g e r m i n a t e d on w a t e r agar a t 15°C. The f o l l o w i n g c o l o r s were used — ' d a y l i g h t ' , g r e e n , b l u e , p i n k and b l a c k — , two a t a ti m e w i t h a d a r k c o n t r o l , and a l l p o s s i b l e c o m b i n a t i o n s were r e p e a t e d t w i c e , w h i c h i n v o l v e d a t o t a l o f 20 t e s t s . The d a r k c o n t r o l gave t h e h i g h e s t g e r m i n a t i o n p e r c e n t a g e h a l f t h e t i m e , s i m i l a r l y each o f t h e c o l o r e d l i g h t s was h i g h e s t f o r h a l f o f t h e i r t e s t s , e x c e p t b l u e , w h i c h was o n l y e q u a l t o t h e h i g h e s t i n one o f i t s f o u r t e s t s . T h i s was t h e o p p o s i t e o f t h e 1967 t e s t s , b u t t h e range o f t h e t o t a l p e r c e n t a g e s was - 255 -so s m a l l i n t h e I966 t e s t s t h a t i t was d o u b t f u l i f t h i s d i f f e r e n c e was s i g n i f i c a n t . ( i n t h e f o u r common t e s t s t h e average p e r c e n t a g e g e r m i n a -t i o n f o r b l u e l i g h t was w i t h i n jf0 o f t h e o t h e r l i g h t c o n d i t i o n s ) . E f f e c t o f hydrogen i o n c o n c e n t r a t i o n oh g e r m i n a t i o n A 1966 t e s t o v e r t h e pH range 3 t o 10 u s i n g t h r e e f r e s h s p o r e c o l l e c t i o n s i n d i c a t e d t h a t g e r m i n a t i o n a f t e r 24 hours a t 15°C o c c u r r e d o v e r t h e range 5 t o 8, b u t g e r m i n a t i o n was v e r y low a t t h e extreme v a l u e s ( F i g . 85). Where two media were t e s t e d a t one pH v a l u e (k - 8), t h e media g i v i n g t h e h i g h e r p e r c e n t a g e g e r m i n a t i o n was used . Optimum c o n d i -t i o n s o c c u r r e d a t pH 6. G e r m i n a t i o n a t 20°C on t h e same pH medi a s e r i e s gave l e s s t h a n h a l f t h e p e r c e n t a g e g e r m i n a t i o n r e c o r d e d a t 15°C. A t e s t s e r i e s was r u n i n 1968 o v e r t h e pH range 4.5 t o 8 and used o n l y combina-t i o n s o f p o t a s s i u m a c i d p h t h a l a t e , p o t a s s i u m d i h y d r o g e n p h o s p h a t e and sodium h y d r o x i d e w i t h a Czapek-Dox agar medium. Spores f r o m two c o l l e c -t i o n s s t o r e d f o r k and 6 weeks a t 0°C, were t e s t e d a t 5, 10, 15, 20 and 25°C. Me d i a w i t h pH i n t e r v a l s o f a p p r o x i m a t e l y 0.5 u n i t s were u s e d t o i n d i c a t e b e t t e r t h e optimum pH f o r g e r m i n a t i o n . F i g . 86 shows t h e g e r -m i n a t i o n p e r c e n t a g e o v e r t h e pH range t e s t e d f o r s p o r e s i n c u b a t e d a t 5, 15 and 25°C. Optimum g e r m i n a t i o n o c c u r r e d a t pH 6.6 a t 5 and 15°C, b u t was c l o s e r t o pH 6.1 a t 25°C, t h i s was a l s o t h e case a t 10 and 20°C. A t pH 4.5 g e r m i n a t i o n o n l y o c c u r r e d a t 10 and 15°C. A t t h e o t h e r pH extreme (8.0) some g e r m i n a t i o n o c c u r r e d a t a l l t e m p e r a t u r e s e x c e p t 25°C w h i c h a l s o r e c o r d e d o n l y a t r a c e o f g e r m i n a t i o n a t pH 7.5 and 7.7. A 1964 s e r i e s o f t e s t s , u s i n g s p o r e s s t o r e d f o r s i x months g e r m i n a t e d a t 15°C on u n b u f f e r e d and b u f f e r e d media, gave g e r m i n a t i o n o v e r t h e range 5-1 t o 8.8 w i t h optimum g e r m i n a t i o n between pH 6.6 and 7-3- Wo g e r m i n a t i o n o c c u r r e d on med i a w i t h F i g . 85. E f f e c t o f hydrogen i o n c o n c e n t r a t i o n on g e r m i n a t i o n o f C r o n a r t i u m comandrae a e c i o s p o r e s a f t e r 2k h o u r s a t 15 and 20°C. F i g . 86. E f f e c t o f hydrogen i o n c o n c e n t r a t i o n on g e r m i n a t i o n o f C r o n a r t i u m comandrae a e c i o s p o r e s a f t e r 2k h o u r s a t 5, 15 and 25°C Spore samples were s t o r e d f o r k and 6 weeks a t 0°C p r i o r t o u s e . - 257 -a pH between 4.0 and 5-0 i n t h i s t e s t s e r i e s . I n a l l t e s t s e r i e s germ tube l e n g t h was much r e d u c e d on t h e b u f f e r e d pH media compared w i t h t h e c o n t r o l u n a d j u s t e d agar media (pH 6.7 - 7-0), and g e n e r a l l y t h e p e r c e n t -age g e r m i n a t i o n was l o w e r . S i m i l a r l y , t h e average l e n g t h o f germ tub e s i n a l l t e s t s was r e d u c e d b y 50%, o r more, a t t h e pH extremes f o r g e r -m i n a t i o n . E f f e c t o f s u b s t r a t e on g e r m i n a t i o n a. V a r i o u s agar media T a b l e XXXIV i n d i c a t e s t h a t t h e r e was c o n s i d e r a b l e range i n t h e e f f e c t o f t h e v a r i o u s agar media on a e c i o s p o r e g e r m i n a t i o n . I n t h e ex-p e r i m e n t o f June 21, I967, seven s p o r e samples were used, and on J u l y 4, t h r e e samples f r o m t h e same c a n k e r s as o f t h e e a r l i e r d a t e . I t was no-t i c e a b l e t h a t t h e aver a g e p e r c e n t a g e s were r e d u c e d a t t h e l a t e r d a t e , T a b l e XXXIV. Average p e r c e n t a g e and range o f g e r m i n a t i o n o f two s e r i e s o f a e c i o s p o r e s a f t e r 24 hours on d i f f e r e n t media a t 15°C. Me d i a June 21, 1967 J u l y 1+, 1967 7 samples 3 samples Average % % Range Average % % Range Czapek-Dox b r o t h + % agar 93-3 87.5-98.3 63.5 51.5-73.4 Czapek-Dox b r o t h + 3 .5% agar 86.0 50.2-97.7 46.1+ 27.3-75.3 P o t a t o d e x t r o s e agar 50.3 15.1-89.8 35-3 22.0-61.3 D e x t r o s e + 1% agar 35.1 8.2-78.1+, 30.9 IO.O-56.I N u t r i e n t agar 52.3 20.8-90.1+ 18.2 9.2-32.3 Water agar 0.5% 36.9 5.0-81.5 27.7 6.9-63.5 Water agar 2% 44.7 2.8-91.1+ 15.8 9.0-28.2 Y e a s t e x t r a c t + 1% §ar 16.5 l+.5-!+1.6 11.1+ 1.0-21.0 M a l t e x t r a c t agar 13.6 0.3-1+3.8 1.7 1.6- 1.9 M a l t e x t r a c t b r o t h + 1.5% agar 1.1+ 0 . 0 - 5.9 1.4 0.7- 2.5 - 258 -and t h i s was even more marked i f o n l y t h e t h r e e samples were compared. The p e r c e n t a g e g e r m i n a t i o n showed t h e same t r e n d on b o t h d a t e s w i t h r e -s u l t s on t h e two Czapek-Dox media much s u p e r i o r , f o l l o w e d by t h e two on t h e d e x t r o s e media. R e s u l t s on t h e y e a s t and m a l t e x t r a c t media were p o o r e r t h a n on t h e w a t e r a g a r . The range o f g e r m i n a t i o n p e r c e n t a g e s showed t h e g r e a t v a r i a t i o n i n r e s p o n s e o f t h e v a r i o u s c a n k e r s , and t h i s r e s p o n s e was n o t always c o n s t a n t w i t h r e g a r d t o t h e media. I n a subsequent t e s t seven o f t h e media ( T a b l e XXXV) were used t o t e s t t h r e e s p o r e samples f o r t h e e f f e c t o f t h e media on t h e number o f germ t u b e s p e r s p o r e and t h e a v e r a g e l e n g t h o f t h e l o n g e s t germ tube a f t e r 2k hours a t 15°C. Ten o r more s p o r e s were o b s e r v e d i n each sample a t each t r e a t m e n t . The average number o f germ t u b e s p e r s p o r e s v a r i e d between 1.5 and 2.0 on t h e d i f f e r e n t media, w i t h t h e l o w e s t number on t h e m a l t e x t r a c t and p o t a t o d e x t r o s e a g a r s and t h e h i g h e s t on t h e d e x t r o s e and Czapek-Dox a g a r s . The a v e r a g e l e n g t h o f t h e l o n g e s t germ t u b e v a r i e d between 95 and 286u, w i t h t h e l o n g e s t o c c u r r i n g on t h e w a t e r agar c l o s e l y f o l l o w e d b y t h o s e on t h e d e x t r o s e and p o t a t o d e x t r o s e a g a r s and t h e s h o r t e s t ones on t h e n u t r i e n t a g a r . I t i s o f i n t e r e s t , t h a t when t h e i n d i v i d u a l samples were compared on a l l media, t h e sample h a v i n g t h e l e a s t number o f germ t u b e s had t h e h i g h e s t average l e n g t h , and t h e sample w i t h t h e h i g h e s t average number o f germ t u b e s t h e s h o r t e s t average l e n g t h o f t h e l o n g e s t germ t u b e . G e n e r a l l y o n l y one germ t u b e d e v e l o p e d t o any l e n g t h and two germ t u b e s f r o m one s p o r e were r a r e l y o f e q u a l l e n g t h . When t h e l e n g t h s o f a l l germ t u b e s were c o n s i d e r e d t h e same r e l a t i v e p o s i t i o n s were r e t a i n e d between samples and on t h e v a r i o u s media, a l t h o u g h t h e t o t a l g r o w t h on t h e w a t e r agar was i n c r e a s e d ( T a b l e XXXV"). - 259 -T a b l e XXXV. Average number o f germ t u b e s and l e n g t h s o f germ t u b e s p e r s p o r e f o r t h r e e samples on seven d i f f e r e n t media a f t e r 24 hours a t 15°C. A v e r . no. A v e r , l e n g t h (u) A v e r , l e n g t h (u) M e d i a o f germ o f l o n g e s t germ o f a l l germ t u b e s / s p o r e t u b e / s p o r e t u b e s / s p o r e Czapek-Dox b r o t h + 1% agar 2.0 215 228 P o t a t o d e x t r o s e agar 1.7 244 264 D e x t r o s e + 1% agar 2.0 272 283 N u t r i e n t agar 1.9 95 119 Water agar 0 .5% 1-9 • 286 348 Y e a s t e x t r a c t + 1% agar 1 .8 154 172 M a l t e x t r a c t agar 1.5 149 163 I n f u r t h e r t e s t s t h e e f f e c t o f 0.5% w a t e r agar and Czapek-Dox agar were compared. P e r c e n t a g e g e r m i n a t i o n was always b e t t e r on Czapek-Dox a t a l l l e v e l s o f g e r m i n a t i o n . There was no s i g n i f i c a n t d i f f e r e n c e i n t h e average number o f germ tub e s p e r s p o r e f r o m i n d i v i d u a l samples on t h e two media, b u t t h e d i f f e r e n c e i n t h e l e n g t h o f t h e l o n g e s t germ tube was c o n s i d e r a b l e . I n f o u r t e s t s , germ tube l e n g t h on w a t e r agar a v e r a g e d 3 l 8 p , and on Czapek-Dox agar l67P, a f t e r 24 h o u r s . b. A d d i t i o n o f s u c r o s e t o media Two t e s t s were c a r r i e d out w i t h v a r i o u s c o n c e n t r a t i o n s o f s u -c r o s e i n t r o d u c e d i n t o agar media t o g i v e an a d d i t i o n a l c a r b o h y d r a t e s o u r c e f o r g e r m i n a t i o n . I n one s e r i e s a range o f s u c r o s e s o l u t i o n s f r o m 1 t o 40% was added t o B a c t o w a t e r agar (pH 7 .0) and gave pH v a l u e s f r o m 7-2 t o 7 . 4 . G e r m i n a t i o n p e r c e n t a g e v a r i e d l i t t l e f r o m t h a t o f t h e c o n t r o l between 1 and 30%, b u t g e r m i n a t i o n i n a 40% s u c r o s e medium was o n l y a t r a c e . The i n c r e a s e d o s m o t i c c o n c e n t r a t i o n a t 40% t h e r e f o r e m a r k e d l y r e d u c e d g e r m i n a t i o n , b u t c o n c e n t r a t i o n s up t o 30% d i d n o t a f f e c t t h e - 260 -g e r m i n a t i o n p e r c e n t a g e . .A second s e r i e s o f 8 m e d i a o v e r a range o f s u -c r o s e between 3 and 6.1$ i n c o r p o r a t e d i n t o a Czapek-Dox agar (pH 7.2-7-4) a l s o gave v e r y s i m i l a r g e r m i n a t i o n p e r c e n t a g e s ( r a n g e 20.0 t o 23.6$). The a d d i t i o n o f s u c r o s e i n t h e above t e s t s d i d not appear t o i n c r e a s e g e r -m i n a t i o n , a l t h o u g h t h e t e s t s comparing Czapek-Dox agar (3$ s u c r o s e ) w i t h w a t e r agar i n t h e p r e v i o u s s e c t i o n gave s u p e r i o r g e r m i n a t i o n p e r c e n t a g e f o r t h e f o r m e r medium. c. P r e s e n c e o f h o s t l e a v e s on, o r l e a f e x t r a c t s i n t h e media P e r c e n t a g e g e r m i n a t i o n i n t h r e e out o f f o u r t e s t s e r i e s r u n o v e r a p e r i o d o f 9 days i n 1966 w i t h t h e same k t o 6 spore samples 'were p o o r e r on Comandra l e a f e x t r a c t agar t h a n on t h e c o n t r o l agar (0.25$ w a t e r a g a r ) . I n t h e o t h e r s e r i e s , l e a f e x t r a c t s , w h i c h were added t o t h e agar as i t c o o l e d , gave an i n c r e a s e d g e r m i n a t i o n , b u t l e a f e x t r a c t s t h a t were h e a t e d w i t h t h e agar gave a d e c r e a s e i n g e r m i n a t i o n . I n two o f t h e f o u r t e s t s e r i e s w i t h Geocaulon l e a f e x t r a c t , some g e r m i n a t i o n p e r c e n t a g e s were h i g h e r t h a n on t h e c o n t r o l a g a r . I n a t h i r d s e r i e s , g e r m i n a t i o n on a 'heated' ...leaf e x t r a c t agar was s l i g h t l y h i g h e r , b u t was l o w e r on an agar w h i c h had t h e l e a f m a t e r i a l added as i t c o o l e d , i n con-t r a s t t o t h e two p r e v i o u s t e s t s e r i e s . I n t h e f o u r t h s e r i e s , g e r m i n a t i o n p e r c e n t a g e s were a l i t t l e l o w e r t h a n on t h e w a t e r a g a r , b u t much h i g h e r t h a n on Comandra l e a f e x t r a c t a g a r . I n a l l o f t h e s e r i e s t h e r e was l i t -t l e d i f f e r e n c e i n t h e g e r m i n a t i o n p e r c e n t a g e f o r G e o c a u l o n l e a f e x t r a c t s h e a t e d w i t h t h e agar o r added as t h e agar c o o l e d . I n one 1966 s e r i e s d i f -f e r e n t q u a n t i t i e s o f l e a f m a t e r i a l were added t o t h e agar o v e r t h e range 5 t o 25 ;.gm'' p e r 100 m l o f agar, w i t h b e s t g e r m i n a t i o n r e s u l t s on a 15 gm c o n c e n t r a t i o n . I n I967 one t e s t s e r i e s was r u n w i t h 5 gmsof l e a f m a t e r i a l - 261 -i n 100 m l o f 0.3% w a t e r a g a r , t h e m a t e r i a l b e i n g added as t h e agar c o o l e d . G e r m i n a t i o n on t h e l e a f e x t r a c t s was s l i g h t l y l o w e r t h a n on t h e w a t e r agar, and was p o o r e s t on t h e Comandra l e a f e x t r a c t a g a r . However, t h e average l e n g t h o f t h e l o n g e s t germ tube was l o n g e s t on t h e Comandra (M+2u) and s h o r t e s t on t h e Geocaulon (29*+p), w i t h t h e c o n t r o l w a t e r agar i n t e r -m e d i a t e (358M-) - There was no d i f f e r e n c e i n t h e number o f germ t u b e s p e r s p o r e o r t h e f o r m o f b r a n c h i n g between t h e l e a f e x t r a c t a g a r s and t h e w a t e r a g a r . The pH o f t h e l e a f e x t r a c t a g a r s i n t h e v a r i o u s s e r i e s was • i n t h e range 5-2 t o 5 . 9 , whereas t h e w a t e r agar had a pH around 6 . 8 , a v a l u e much c l o s e r t o t h e pH optimum. The pH o f e x p r e s s e d l e a f j u i c e s o f a l t e r n a t e h o s t s t a k e n f r o m d i f f e r e n t l o c a t i o n s a t v a r i o u s t i m e s i n t h e s e a s o n and i n d i f f e r e n t y e a r s , gave v a l u e s f o r Comandra l e a v e s r a n g i n g f r o m 5.0 t o 5 I + 3 " a r i b f o r G e o c a u l o n f r o m 5-4 t o 6 . 0 , v a l u e s s i m i l a r t o t h e e x t r a c t media. There was no t r e n d f o r l e a f pH's t o change i n one d i r e c -t i o n d u r i n g t h e s e a s o n a t a p a r t i c u l a r l o c a t i o n . When s p o r e s were g e r m i n a t e d i n 3 mm d i a m e t e r h o l e s c u t i n young Comandra l e a v e s p l a c e d on 2.5% w a t e r agar and compared w i t h t h e c o n t r o l , g e r m i n a t i o n p e r c e n t a g e s were improved i n t h e case o f two samples, b u t were s i m i l a r f o r t h r e e o t h e r samples. R e s u l t s w i t h G e o c a u l o n l e a v e s gave one sample s i m i l a r and one sample l e s s g e r m i n a t i o n w i t h i n t h e h o l e s . No d i f f e r e n c e was n o t e d between g e r m i n a t i o n around t h e edges and c e n t e r o f t h e h o l e . I n t h e t e s t when p e r cent g e r m i n a t i o n was compared on t h e s u r -f a c e o f a l t e r n a t e h o s t l e a v e s and on w a t e r agar near t h e l e a v e s , g e r m i n a -t i o n was always e q u a l o r h i g h e r on t h e w a t e r a g a r , under b o t h t h e d r y and m o i s t - m i s t c o n d i t i o n s o f t h e e x p e r i m e n t . I t i s not- known whether s u f f i c i e n t - 262 -m o i s t u r e f o r s p o r e g e r m i n a t i o n was a v a i l a b l e on t h e l e a v e s , f o r t h e d r y and m o i s t c o n d i t i o n s o f t h e t e s t gave no s i g n i f i c a n t d i f f e r e n c e s , and use o f o l d and young l e a v e s gave no d i f f e r e n c e s . These t e s t p l a t e s were r e -t a i n e d and by t h r e e weeks u r e d i a l development and u r e d i o s p o r e p r o d u c t i o n was r e c o r d e d on t h e Comandra l e a v e s , b u t no such development o c c u r r e d on the G e o c a u l o n l e a v e s , w h i c h were m a r k e d l y d i s c o l o r e d and p r o b a b l y de-n a t u r e d . D i s c u s s i o n Many w o r k e r s have r e p o r t e d on some o f t h e g e r m i n a t i o n c h a r a c -t e r i s t i c s o f t h e a e c i o s p o r e s o f C r o n a r t i u m ( P e r i d e r m i u m ) r u s t s . The op-timum t e m p e r a t u r e f o r g e r m i n a t i o n o f C_. comandrae a e c i o s p o r e s ( l 5 ° C ) was h i g h e r t h a n t h o s e r e p o r t e d b y Doran (1919), McKenzie (I9I+2) and E r e b i l l (1968c), s i m i l a r t o t h o s e f o u n d b y H i r t (1937), Nighswander and P a t t o n ( I 9 6 5 ) , Van A r s d e l e t a l . (1956), P o w e l l and M o r f (1966) and P e t e r s o n (1968), and l o w e r t h a n t h o s e f o u n d b y S i g g a r s (19I+7), K l i n g s t r B m (1963) and R o n c a d o r i and Matthews ( 1966) . The maximum g e r m i n a t i o n t e m p e r a t u r e (28-30°C) was s i m i l a r t o t h o s e r e p o r t e d b y S i g g a r s (191+7) and R o n c a d o r i and Matthews (1966) f o r C. f u s i f o r m e , b y P o w e l l and Mo r f (1966) f o r P. h a r k n e s s i i , b y H i r t (1937) and Van A r s d e l e_t a l . (1956) f o r £. r i b i c o l a , Nighswander and P a t t o n (1965) f o r C_. quercuum, and P e t e r s o n (1968) f o r P. f i l a m e n t o s u m , b u t K r e b i l l (1968c) o b t a i n e d some g e r m i n a t i o n a t 33°C w i t h h a l f h i s C_. comandrae samples. There a r e no r e p o r t s o f C r o n a r t i u m a e c i o -s p o r e g e r m i n a t i o n a t t e m p e r a t u r e s b e l o w 3°C, as found i n t h e p r e s e n t s t u d y w i t h C. comandrae. C. comandrae t h e r e f o r e a p p e a r s . t o have a w i d e r tempera-t u r e range f o r g e r m i n a t i o n t h a n most o t h e r C r o n a r t i u m s p e c i e s . The r e p o r t s - 263 -f o r t h e v a r i o u s C r o n a r t i u m s p e c i e s may r e p r e s e n t s p e c i f i c d i f f e r e n c e s , b u t r a c i a l and g e o g r a p h i c v a r i a t i o n may o c c u r , and t h e p o s s i b l e impor-t a n c e o f f a c t o r s s u c h as t e m p e r a t u r e a d a p t a t i o n and s p o r e m a t e r i a l o f d i f f e r e n t ages, s u g g e s t s c a u t i o n i n s e t t i n g d e f i n i t e t e m p e r a t u r e l i m i t s f o r g e r m i n a t i o n o f i n d i v i d u a l s p e c i e s . Some e v i d e n c e was o b t a i n e d t h a t s p o r e s m a t u r i n g d u r i n g c o o l weather g e r m i n a t e d a t l o w e r t e m p e r a t u r e s t h a n s p o r e s m a t u r i n g d u r i n g warmer we a t h e r . A l s o , i f s p o r e s had been s t o r e d , even f o r a few days, t h e i r t e m p e r a t u r e range and optimum f o r g e r m i n a t i o n was changed ( P o w e l l and M o r f 1 9 6 6 ) , t h u s s p o r e h i s t o r y i s a f a c t o r t o be c o n s i d e r e d . I n t h e p r e s e n t s t u d y the l o n g e s t germ t u b e s were o b t a i n e d a t the optimum t e m p e r a t u r e (15°C) w i t h s h o r t e r germ tube l e n g t h s a t t h e extreme t e m p e r a t u r e s f o r g e r m i n a t i o n . H i r t ( 1 9 3 7 ) , S i g g a r s ( 1 9 ^ 7 ) , A n d e r s o n and F r e n c h ( 1 9 6 5 ) , Nighswander and P a t t o n ( 1 9 6 5 ) , P e t e r s o n ( 1 9 6 8 ) and K r e b i l l ( 1 9 6 8 c ) a l s o o b s e r v e d r e d u c e d growth i n t h e range 2 5 - 2 9°C. Nighswander and P a t t o n ( 1965) r e p o r t e d r e d u c e d g r o w t h a t 8°C, and a l s o n o t e d t h a t t h e r a t e o f growth was g r e a t e s t a t 20°C, a l t h o u g h t h e optimum g e r m i n a t i o n a t k, 8 and 16 hours o c c u r r e d a t l 6°C. T h i s compares f a v o u r -a b l y w i t h t h e p r e s e n t s t u d y where i n i t i a l g r o w t h was most r a p i d a t 20°C. The minimum p e r i o d r e q u i r e d f o r g e r m i n a t i o n o f a e c i o s p o r e s was 1-l/k - 2 h o u r s , s l i g h t l y f a s t e r t h a n r e p o r t s b y S i g g a r s (19^7) f o r C. f u s i f o r m e , s i m i l a r t o t h o s e f o r P. h a r k n e s s i i ( P o w e l l and M o r f 1966) and C. f u s i f o r m e (Walkinshaw e t a l . 1 9 6 7 ) , b u t m a r k e d l y f a s t e r t h a n r e p o r t s f o r C. r i b i c o l a (Doran 1919, 1 9 2 2 ) , P. p i n i ( K l i n g s t r o m 1963) and P. s t a l a c t i f o r m e ( P o w e l l and Mo r f 1 9 6 6 ). K r e b i l l ( 1 9 6 8 c ) a l s o n o t e d t h a t most v i a b l e C. comandrae s p o r e s g e r m i n a t e d w i t h i n 3 h o u r s . I t was n o t e d - 264 -i n t h e p r e s e n t s t u d y t h a t f r e s h s p o r e s g e n e r a l l y g e r m i n a t e f a s t e r t h a n s t o r e d s p o r e s , and t h i s c o u l d a c c o u n t f o r some o f t h e wide v a r i a t i o n r e -p o r t e d i n t h e l i t e r a t u r e . Germ tu b e e l o n g a t i o n was r a p i d f o r t h e f i r s t 8 hours o f g e r m i n a t i o n , much s l o w e r u n t i l about 2k hours w i t h o n l y a s m a l l f u r t h e r i n c r e a s e t o 72 o r 96 h o u r s . A n d e r s o n and F r e n c h (1965) showed a s i m i l a r p a t t e r n o f e l o n g a t i o n f o r C. quercuum a t optimum t e m p e r a t u r e s . The average and maximum germ tube l e n g t h s were s i m i l a r t o t h o s e r e p o r t e d f o r C..comandrae ( K r e b i l l 1968c), C. f u s i f o r m e (Walkinshaw e t a l . 1967), C. quercuum ( A n d e r s o n and F r e n c h I965), P. s t a l a c t i f o r m e ( P o w e l l and M o r f 1966) and t h e Coronado r a c e o f P. f i l a m e n t o s u m ( P e t e r s o n 1968), a l t h o u g h t h e r e was a c o n s i d e r a b l e range i n t h e average v a l u e s as was shown f o r t h e s e p a r a t e s e r i e s o f t h e p r e s e n t s t u d y (300 - 715M-) - A l t h o u g h 1 t o 6 germ tube s ( a v e r a g e 2) were p r o d u c e d p e r s p o r e , o n l y one u s u a l l y d e v e l o p e d t o any l e n g t h . The d e v e l o p e d germ tub e s were b i - n u c l e a t e , n o n - s e p t a t e and had m u l t i p l e i r r e g u l a r b r a n c h e s . T h i s type- o f germ tube development i s t y p i c a l o f t h e h e t e r o e c i o u s group o f C r o n a r t i u m r u s t s ( H i r a t s u k a 1968; H i r a t s u k a e t a l . I966; P o w e l l and M o r f 1966). The germ t u b e s o f t h e au-t o e c i o u s group a r e much s h o r t e r and have s e p t a f o r m a t i o n w h i c h d i v i d e s t h e germ t u b e i n t o two t o f o u r u n i n u c l e a t e c e l l s f r o m w h i c h one s h o r t b r a n c h may d e v e l o p . F r e e w a t e r was n e c e s s a r y f o r g e r m i n a t i o n t o t a k e p l a c e as no g e r m i n a t i o n was o b t a i n e d i n a s a t u r a t e d a i r o r a t a r e l a t i v e h u m i d i t y b e l o w 100$. S c h e i n (l964) doubted whether most s p o r e s w o u l d g e r m i n a t e a t a h u m i d i t y b e l o w s a t u r a t i o n and s u g g e s t e d t h a t a l l r e p o r t s p r o b a b l y o c c u r r e d i n f r e e w a t e r and n o t i n near s a t u r a t e d atmospheres. S w e l l i n g o f s p o r e s was shown t o t a k e p l a c e i m m e d i a t e l y when s p o r e s were p l a c e d on - 265 -a l i q u i d media, and was a p r e r e q u i s i t e f o r i n i t i a t i o n o f a germ t u b e . Many sp o r e s are low i n m o i s t u r e a t m a t u r i t y and must be h y d r a t e d b e f o r e development can resume, as s w e l l i n g appears t o r e p r e s e n t an i n t r i n s i c p a r t o f the growth p r o c e s s and ceases when germ tube emergence b e g i n s . T h i s r e q u i r e m e n t i s p a r t i c u l a r l y p r e v a l e n t among a i r b o r n e s p o r e s ( A l l e n I965), and t h e a e c i o s p o r e s o f C. comandrae a r e no e x c e p t i o n . G o t t l i e b (1950) s u g g e s t e d t h a t dead s p o r e s do not s w e l l and t h a t a b s o r p t i o n v a r i e s w i t h t h e v i a b i l i t y o f t h e s p o r e . The p r e s e n t s t u d y shows t h a t n o n - v i a b l e s p o r e s do s w e l l i n i t i a l l y b u t n o t as much as v i a b l e s p o r e s , and a s i m i l a r p o s i t i o n was r e p o r t e d b y Barnes and P a r k e r (1966). I t seems l o g i c a l t h a t d r y s p o r e s , whether v i a b l e o r no t , w o u l d t a k e on w a t e r i n i t i a l l y because o f t h e i r low m o i s t u r e c o n t e n t , b u t t h a t o n l y v i a b l e s p o r e s w h i c h a r e dependent on m o i s t u r e f o r i n i t i a t i o n o f a c t i v e m e t a b o l i s m w o u l d c o n t i n u e t o s w e l l . U n l i k e t h e s t u d y o f T r i c h o d e r m a s p o r e s ( B a r n e s and P a r k e r I966), s w e l l i n g was completed a l m o s t i m m e d i a t e l y w i t h l i t t l e change a t o t h e r i n -t e r v a l s b e f o r e germ tu b e emergence. G e r m i n a t i o n o f s p o r e s s t o r e d f o r ' s h o r t p e r i o d s was s l i g h t l y i mproved i f s p o r e s were h y d r a t e d i n a s a t u r a t e d atmosphere, p r i o r t o b e i n g t e s t e d on a l i q u i d media. R o n c a d o r i and Mat-thews (1966) a l s o f o u n d t h a t g e r m i n a t i o n o f s t o r e d C_. f u s i f o r m e a e c i o s p o r e s was i n c r e a s e d f o l l o w i n g h y d r a t i o n . R o w e l l (1956) and Sharp and S m i t h (1957) have c a u t i o n e d t h a t r u s t s p o r e s a r e v e r y s e n s i t i v e t o h y d r a t i o n f o l -l o w i n g s t o r a g e and sp o r e c o n t e n t s may be r u p t u r e d c a u s i n g damage t o t h e c e l l i f h y d r a t i o n i s t o o r a p i d . C_. comandrae a e c i o s p o r e s do n o t appear t o be l i g h t s e n s i t i v e f o r g e r m i n a t i o n , as sp o r e s g e r m i n a t e d e q u a l l y w e l l i n the d a r k and i n d i f -f u s e d n a t u r a l and a r t i f i c i a l l i g h t . K r e b i l l (1968c) a l s o f o u n d t h i s , b u t - 266 -he o b t a i n e d no g e r m i n a t i o n i n d i r e c t s u n l i g h t . Hart. (1926) and R o b i n s o n (1914) f o u n d t h a t a e c i o s p o r e s o f Melampsora l i n i and P u c c i n i a poarum r e s -p e c t i v e l y , g e r m i n a t e e q u a l l y w e l l i n t h e d a r k and i n t h e l i g h t . The v a r i o u s c o l o r e d l i g h t w a v e l e n g t h s had no s i g n i f i c a n t i n d i v i d u a l e f f e c t s , a l t h o u g h i n t h e I967 s e r i e s b l u e l i g h t gave a s l i g h t l y b e t t e r g e r m i n a t i o n p e r c e n t a g e t h a n t h e o t h e r c o l o r l i g h t s . Sussman (1965) r e p o r t e d t h a t b l u e l i g h t was s t i m u l a t i n g f o r g e r m i n a t i o n o f many f u n g i , a l t h o u g h d i f -f e r e n t s p o r e s t a t e s o f a s i n g l e o r g a n i s m may r e s p o n d d i f f e r e n t l y t o l i g h t . The u r e d i o s p o r e s o f C_. r i b i c o l a a r e r e p o r t e d t o r e q u i r e l i g h t f o r g e r m i n -a t i o n b u t t e l i o s p o r e s do n o t . The o n l y i n f o r m a t i o n on t h e r e s p o n s e o f C r o n a r t i u m s p o r e s t o hydrogen i o n c o n c e n t r a t i o n s appears t o be t h e companion s t u d y on P. h a r k n e s s i i and P. s t a l a c t i f o r m e ( P o w e l l and M o r f 1966), and ones by Wal-k i n s h a w (1965) f o r C_. f u s i f o r m e , and a b r i e f m e n t i o n b y P e t e r s o n (1968) f o r r a c e s o f P. f ilamentosum. The range o f pH o v e r w h i c h C. comandrae a e c i o s p o r e s would g e r m i n a t e were v e r y s i m i l a r t o P. h a r k n e s s i i and P. s t a l a c t i f o r m e . W alkinshaw o b t a i n e d good g e r m i n a t i o n o f £. f u s i f o r m e o v e r t h e range 4.5 t o 7-0, and r e d u c e d g e r m i n a t i o n a t 3-0 and 8.5- P e t e r s o n m e n t i o n e d r e d u c e d g e r m i n a t i o n f o r P. f i l a m e n t o s u m a t pH 8.0 and a l m o s t none a t pH 8.3- Optimum c o n d i t i o n s f o r £. comandrae g e r m i n a t i o n o c c u r r e d a t about pH 6.5 and 15°C, a l t h o u g h d a t a f r o m a number o f t e s t s i n d i c a t e d a r e l a t i v e l y b r o a d pH optimum between 6.0 and 7-0, w i t h optimum f o r s t o r e d s p o r e s c l o s e r t o t h e a l k a l i n e r a n g e . C o n d i t i o n s f o r g e r m i n a t i o n e x t e n d e d f u r t h e r i n t o t h e a c i d range t h a n i n t o t h e a l k a l i n e , w h i c h i s g e n e r a l f o r most f u n g i ( G o t t l i e b 1950; Sussman 1965). G e r m i n a t i o n a t t h e sub- and s u p r a o p t i m a l pH's was b e t t e r on t h e Czapek-Dox medium t h a n on w a t e r agar, - 267 -s u g g e s t i n g t h a t t h e g r o w th media may s t r o n g l y i n f l u e n c e t h e c a r d i n a l pH v a l u e s f o r g e r m i n a t i o n . S i m i l a r l y v a r i o u s b u f f e r systems p r o d u c e d d i f -f e r e n t e f f e c t s , as was shown i n t h e e x p l o r a t o r y t e s t s and b y Walkinshaw (1965). F l e n t j e (1959) s t a t e s t h a t "the s p o r e s o f t h e m a j o r i t y o f a i r -b orne f u n g i appear t o g e r m i n a t e s a t i s f a c t o r i l y i n d i s t i l l e d w a t e r i n d i -e a t i n g t h a t no s p e c i a l g e r m i n a t i o n s t i m u l u s o t h e r t h a n w a t e r i s r e q u i r e d " . A r t h u r (1929), e a r l i e r f o u n d no r e p o r t s i n t h e l i t e r a t u r e where r u s t spore g e r m i n a t i o n was b e n e f i t e d b y n u t r i e n t s o r t i s s u e e x t r a c t s , e x c e p t t h a t sugar promoted g e r m i n a t i o n o f p y e n i o s p o r e s . However, t h e m o i s t u r e f i l m on any p l a n t s u r f a c e i n e v i t a b l y c o n t a i n s s u b s t a n c e s i n s o l u t i o n r e -l e a s e d f r o m t h e u n d e r l y i n g p l a n t t i s s u e s w h i c h may i n f l u e n c e s p o r e g e r -m i n a t i o n . Because o f t h i s , t h e use o f an e x t r a c t o r d e c o c t i o n o f t h e n a t u r a l s u b s t r a t e o f an o r g a n i s m as a medium f o r i t s g e r m i n a t i o n i s a common p r a c t i c e . As l o n g ago as ±°,0k K l e b a h n found t h a t C. r i b i c o l a a e c i o s p o r e s gave b e t t e r g e r m i n a t i o n on R i b e s - d e c o c t i o n agar t h a n w a t e r . More r e c e n t l y Van A r s d e l e_t a l . (1956) r e p e a t e d K l e b a h n ' s f i n d i n g . Nighswander and P a t t o n (1965) and R o n c a d o r i and Matthews (1966) have o b t a i n e d b e t t e r g e r m i n a t i o n o f C_. quercuum and C_. f u s i f o r m e on a Quercus l e a f d e c o c t i o n . I n t h e p r e s e n t s t u d y , however, t h e a d d i t i o n o f a Coman-d r a l e a f e x t r a c t t o t h e media d i d not i n c r e a s e g e r m i n a t i o n , i n f a c t i t d e c r e a s e d i t . There was some e v i d e n c e t h a t G e o c a u l o n l e a f e x t r a c t i n -c r e a s e d g e r m i n a t i o n a l t h o u g h t h e r e s p o n s e was n o t c o n s i s t e n t . Why t h e r e was no r e s p o n s e w i t h a Comandra e x t r a c t b u t some w i t h G e o c a u l o n i s unknown. To f u r t h e r c o m p l i c a t e t h e p i c t u r e , i n a n o t h e r e x p e r i m e n t , no g e r m i n a t i o n was o b t a i n e d on ' s t r a i g h t ' G e o c a u l o n l e a v e s and i n f e c t i o n e x p e r i m e n t s i n - 268 -t h e greenhouse p r o v e d u n s u c c e s s f u l , b u t t h e r e was nev e r any d i f f i c u l t y o b t a i n i n g g e r m i n a t i o n and i n f e c t i o n on Comandra l e a v e s . A l s o i n n a t u r e , i n t h e a r e a f r o m w h i c h t h e e x p e r i m e n t a l G e o c a u l o n l e a v e s and p l a n t s were c o l l e c t e d , i n f e c t e d p l a n t s have o n l y been found on two o c c a s i o n s i n f o u r y e a r s o f s e a r c h i n g , s u g g e s t i n g t h a t t h e Geo c a u l o n p l a n t s i n t h i s a r e a have a h i g h degree o f r e s i s t a n c e t o t h e pathoge n . Of t h e o t h e r s u b s t r a t e s t e s t e d i n t h e p r e s e n t s t u d y t h e g e r -m i n a t i o n r e s p o n s e t o Czapek-Dox medium was not s u r p r i s i n g when i t i s c o n s i d e r e d t h a t t h i s medium has a s u c r o s e b a s e . S i m i l a r l y , t h e d e x t r o s e medium enhanced g e r m i n a b i l i t y , f o r i t i s known t h a t s m a l l amounts o f carbo n f r o m sugar compounds a r e i n c o r p o r a t e d i n t o t h e c o n s t i t u e n t s o f sp o r e s and may a i d g e r m i n a t i o n m e t a b o l i s m ( A l l e n 1965). The r e l a t i v e l y p o o r g e r m i n a t i o n o f a e c i o s p o r e s i n t h e p r e s e n c e o f y e a s t and m a l t e x t r a c t s was u n e x p e c t e d . The pH o f t h e m a l t e x t r a c t agar (4 .6) and t h e m a l t ex-t r a c t b r o t h ( 4 . 7 ) were o u t s i d e t h e normalppH range f o r g e r m i n a t i o n , w h i c h may have been a f a c t o r , b u t t h i s was n o t t h e case w i t h t h e y e a s t e x t r a c t ( 6 . 6 ) . I t may i n d i c a t e t h a t d e r i v a t i v e s o f t h e v i t a m i n B complex, w h i c h a r e u s u a l l y n e c e s s a r y f o r growth, a r e not r e q u i r e d t o t h e same e x t e n t f o r g e r m i n a t i o n . A s i m i l a r r e s p o n s e i s s u g g e s t e d f o r t e l i o s p o r e s o f U s t i l a g o t r i t i c i ( P e r s . ) R o s t r . ' b y Graham ( i 9 6 0 ) and b y Mandels and Darby (1953) who fo u n d t h a t y e a s t e x t r a c t s f a i l e d t o s u p p o r t g e r m i n a t i o n o f M y r o t h e c i u m v e r r u c a r i a . Graham a l s o found t h a t a s u c r o s e medium g r e a t l y i n c r e a s e d g e r m i n a t i o n and t h a t o r g a n i c and i n o r g a n i c s a l t s r e d u c e g e r m i n a t i o n . The s e p a r a t e s u c r o s e t e s t s o f t h e p r e s e n t s t u d y d i d n o t appear t o i n c r e a s e g e r m i n a t i o n , b u t a t a c o n c e n t r a t i o n o f kO'fo s u c r o s e , g e r m i n a t i o n was m a r k e d l y r e d u c e d . T h i s may have been i n r e s p o n s e t o t h e i n c r e a s e d o s m o t i c concen-- 269 -t r a t i o n o r p o s s i b l y due t o t h e m o i s t u r e a c t i v i t y l i m i t , i . e . t h e e f f e c t i v e c o n c e n t r a t i o n o f w a t e r i n t h e s u b s t r a t e . A 40% s u c r o s e s o l u t i o n would have a m o i s t u r e a c t i v i t y o f about 96% ( B e e r s 1957) o r a t h e o r e t i c a l r e -l a t i v e h u m i d i t y o f 99.2% a t 20°C ( C l a y t o n 1942), w h i c h c o u l d be s u f f i c i e n t -l y low t o l i m i t g e r m i n a t i o n . B e e r s (1957) showed t h a t many b a c t e r i a i n s u c r o s e have a m o i s t u r e l i m i t f o r g e r m i n a t i o n o f between 9 6 . 3 and 97-6%. The g e n e r a l c o n c l u s i o n drawn f r o m t h e l i t e r a t u r e , c o n c e r n i n g t h e p h y s i o l o g y o f r u s t s p o r e g e r m i n a t i o n , i s t h a t v a r i o u s added compounds, w i t h t h e p o s s i b l e e x c e p t i o n o f s u g a r s , c o n t r i b u t e r e l a t i v e l y T i t t l e t o t h e i r o v e r a l l m e t a b o l i s m ( A l l e n 1965). A t m a t u r i t y r u s t s p o r e s c o n t a i n a l l t h e e s s e n t i a l c o n s t i t u e n t s f o r g e r m i n a t i o n and growth o f a s h o r t germ t u b e . G e r m i n a t i o n appears t o i n v o l v e m a i n l y an u p t a k e o f w a t e r , d i s s o l u -t i o n o f t h e p o r e i n t h e spor e w a l l , and s y n t h e s i s o f new c e l l w a l l ma-t e r i a l f o r germ tube emergence (Walkinshaw e t a l . 1967). E v i d e n c e i n d i -c a t e s t h a t r u s t s p o r e s u t i l i z e t h e i r r e s e r v e s o f f a t t y a c i d s and p o l y -a l c o h o l s as t h e i r i n i t i a l s u b s t r a t e f o r g e r m i n a t i o n , b u t t h e r o l e o f f a t t y a c i d s and p o l y a l c o h o l s i s n o t c l e a r ( A l l e n 1965)• T u l l o c h and Ledingham (1962) r e p o r t e d t h a t t h e f a t t y a c i d s c i s - 9 , 1 0 - E p o x y - o c t a d e c a n o i c (39.1%), p a l m i t i c (21.6%) and l i n o l e n i c (12.1%) were t h e most i m p o r t a n t components o f C. comandrae a e c i o s p o r e o i l s . S i m i l a r p e r c e n t a g e s o f t h e s e a c i d s were r e p o r t e d f o r t h e p i n e r u s t s C_. r i b i c o l a and P. s t a l a c t i f o r m e . L a t e r , T u l l o c h (1964) showed t h a t t h e r e was a marked d e c r e a s e i n t h e c o n t e n t o f e p o x y - a c i d d u r i n g s t o r a g e and i n c u b a t i o n o f C r o n a r t i u m s p o r e s , and o t h e r r u s t s ( T u l l o c h 19.63), and t h a t t h e e p o x y - a c i d was c o n v e r t e d t o t h r e o 9, 1 0 - d i h y d r o x y o c t a d e c a n o i c a c i d . T u l l o c h (1963) b e l i e v e d t h a t as m o i s t con-d i t i o n s were r e q u i r e d f o r g e r m i n a t i o n some o f t h e epoxy a c i d w o u l d be - 270 -h y d r o l y z e d t o d i h y d r o x y a c i d b e f o r e g e r m i n a t i o n o c c u r r e d , b u t i t was not-known how t h i s w o u l d a f f e c t g e r m i n a t i o n . Walkinshaw (1968) i n d i c a t e d t h a t l o s s o f g e r m i n a t i o n appeared t o be a f u n c t i o n o f w a t e r u p t a k e . O t h e r s have shown a s i m i l a r d e c r e a s e i n f a t t y a c i d c o n t e n t d u r i n g g e r m i n a t i o n o f r u s t s p o r e s w i t h a change-over f r o m a l i p i d t o a c a r b o h y d r a t e m e t a b o l -i s m ( C a l t r i d e r e t a l . 1963; F r e a r and Jo h n s o n I 9 6 I ; G o t t l i e b and C a l t r i d e r 1962; Shu e t a l . 1954). R e c e n t l y , Walkinshaw e t a l . (1967) i n d i c a t e d , f r o m e l e c t r o n m i c r o s c o p y s t u d i e s , t h a t t h e r e was a d e c r e a s e i n t h e number . and s i z e o f l i p i d b o d i e s i n germ t u b e s compared w i t h q u i e s c e n t s p o r e s , w h i c h p r o v i d e s f u r t h e r e v i d e n c e o f l i p i d u t i l i z a t i o n . R e i s e n e r et' a l . (1961) s u g g e s t e d t h a t e x t e r n a l l y s u p p l i e d s h o r t - c h a i n f a t t y a c i d m i ght be e x p e c t e d t o s t i m u l a t e g e r m i n a t i o n b y i n c r e a s i n g t h e s u p p l y o f c a r b o n u n i t s . Walkinshaw (1965) a l s o s u g g e s t e d t h a t l o n g - c h a i n f a t t y a c i d s may a c c e l e r a t e g e r m i n a t i o n o f C. f u s i f o r m e a e c i o s p o r e s , and Walkinshaw and S c h e l d (1965) f u r t h e r s u g g e s t e d t h a t c e r t a i n endogenous s u b s t r a t e s , e s -p e c i a l l y o l e i c a c i d , may p a r t i a l l y r e p l e n i s h t h e s p o r e r e s e r v e s d e p l e t e d t h r o u g h a g i n g , as an a d d i t i o n o f o l e i c a c i d improves t h e g e r m i n a t i o n p e r -centage . . DAILY AECIOSPORE GERMINATION Methods and M a t e r i a l s A e c i o s p o r e samples were c o l l e c t e d d a i l y between 0800 and 0830 hours t h r o u g h o u t t h e s p o r u l a t i o n p e r i o d f r o m a number o f marked c a n k e r s a t l o c a t i o n 1 d u r i n g t h e y e a r s I965, 1966 and 1967, t o f o l l o w ' d a y t o day-changes i n p e r c e n t a g e s p o r e g e r m i n a t i o n . I n I965 s p o r e s were c o l l e c t e d ) - 271 -f r o m any p a r t o f t h e s p o r u l a t i n g a e c i a l c a n k e r , b u t i n I966 and 1967 s p o r e s were c o l l e c t e d f r o m i n d i v i d u a l l y marked p u s t u l e s t o g i v e u n i f o r m i t y o f s p o r e sample s o u r c e . I n a d d i t i o n , i n 1966 and 1967, a number o f marked p u s t u l e s (up t o 9 o n o n e t r e e ) were sampled d a i l y on two t r e e s t o i n d i c a t e i n d i v i d u a l p u s t u l e v a r i a t i o n w i t h i n an a e c i a l c a n k e r . On t h e s e t r e e s , i f t h e p u s t u l e l a b e l l e d 'A' ceased t o p r o d u c e s p o r e s , t h e 'A' marker was moved t o a n o t h e r p r o d u c i n g p u s t u l e t o o b t a i n g e r m i n a t i o n d a t a f o r t h e whole o f t h e a e c i o s p o r e p r o d u c t i o n p e r i o d , and t o g i v e d a t a t o compare w i t h 1965. The s p o r e s were c o l l e c t e d f r o m t h e a e c i a l p u s t u l e s on t h e p o i n t o f a f i n e n e e d l e , w h i c h was s t e r i l i z e d between each s a m p l i n g b y p l a c i n g i n a l c o h o l . The s p o r e s were d i s p e r s e d i m m e d i a t e l y onto t h e s u r f a c e o f 0.3% D i f c o B a c t o w a t e r agar h e l d i n 5~cm d i a m e t e r p l a s t i c p e t r i p l a t e s , w h i c h were c a r r i e d i n t o t h e f i e l d i n a s u i t a b l e c o n t a i n e r . O n l y .5 t o 15 m i n u t e s e l a p s e d between c o l l e c t i o n o f s p o r e s and pla c e m e n t o f seeded p l a t e s i n an i n c u b a t o r , where t h e s p o r e s were i n c u b a t e d i n t h e d a r k a t 15°C f o r 2k hours t o e s t a b l i s h t h e d a i l y s p o r e g e r m i n a t i o n p e r c e n t a g e . An i n c u b a t i o n p e r i o d o f 2k h o u r s , was s u f f i c i e n t t o a l l o w a l l v i a b l e s p o r e s t o g e r m i n a t e , and 15°C was s u f f i c i e n t l y c l o s e t o t h e optimum t e m p e r a t u r e f o r g e r m i n a t i o n ( s e e e a r l i e r s e c t i o n ) . E v e r y e f f o r t was made t o conduct t h e g e r m i n a t i o n t e s t s under as u n i f o r m c o n d i t i o n s as p o s s i b l e t h r o u g h o u t t h e t h r e e y e a r s . O n l y t h r e e d i f f e r e n t m a n u f a c t u r e r s l o t s o f B a c t o agar •were used, and s u f f i c i e n t p l a t e s f o r t h r e e t o f i v e days were p r e p a r e d a t a t i m e u s i n g d i s t i l l e d and d e i o n i z e d w a t e r . Agar p l a t e s s t o r e d f o r two t o f o u r days a t k°C gave r e s u l t s n ot s i g n i f i c a n t l y d i f f e r e n t from t h o s e s t o r e d f o r one day. Any d i f f e r e n c e t h a t e x i s t e d between t h e medium b a t c h e s was p r o b a b l y not an i m p o r t a n t f a c t o r c a u s i n g v a r i a t i o n i n ge r m i n a -t i o n . - 272 -The same c r i t e r i a f o r e s t a b l i s h i n g g e r m i n a t i o n and t h e same method o f c o u n t i n g t h e p e r c e n t a g e g e r m i n a t i o n were used i n t h e s e d a i l y t e s t s , e x c e p t t h a t a t t e m p t s were made t o a v o i d a r e a s o f t h e medium where-l a r g e clumps o f s p o r e s had been d e p o s i t e d . I n t h e s e a r e a s i t was d i f -f i c u l t t o count g e r m i n a t i o n , and o f t e n some s p o r e s were not i n c o n t a c t w i t h t h e medium. Clumping may cause s e l f - i n h i b i t i o n ( A l l e n 1955) o r s e l f - s t i m u l a t i o n (Manners 1966), t h u s , b y a v o i d i n g a r e a s o f sp o r e clumps, f u r t h e r p o s s i b l e causes o f g e r m i n a t i o n v a r i a t i o n were m i n i m i z e d . I t i s unknown whether s e l f - i n h i b i t o r s o r s t i m u l a t o r s a r e p r e s e n t i n a e c i o s p o r e s o f C_. comandrae, b u t i t was o f t e n o b s e r v e d t h a t b e t t e r g e r m i n a t i o n o c -c u r r e d i n t h e a r e a o f a spore clump. W i l s o n (1958) f o u n d no s e l f - i n h i b i -t o r s p r e s e n t i n u r e d o s p o r e s o f C_. r i b i c o l a , b u t t h e r e a r e no r e p o r t s f o r a e c i o s p o r e s , a l t h o u g h Walkinshaw (1965) showed t h a t c e r t a i n f a t t y a c i d s , p r o b a b l y p r e s e n t i n s p o r e s , were i n h i b i t o r y t o C. f u s i f o r m e a e c i o s p o r e s . A f t e r c o u n t i n g , t h e t e s t p l a t e s were k e p t a t room t e m p e r a t u r e f o r a few days t o a l l o w c o l o n i e s o f any a s s o c i a t e d f u n g i , y e a s t o r b a c -t e r i a t o grow. . Any a s s o c i a t e d m i c r o o r g a n i s m s w h i c h grew were i s o l a t e d f r o m t h e agar s u r f a c e s , c u l t u r e d and i d e n t i f i e d , i n o r d e r t h a t t h e i r p o s -s i b l e r o l e as a co n t a m i n a n t , a b l e t o re d u c e o r s t i m u l a t e a e c i o s p o r e g e r -m i n a t i o n c o u l d be a s s e s s e d . R e s u l t s and D i s c u s s i o n F i g u r e 87 i n d i c a t e s t h e average d a i l y g e r m i n a t i o n , o f f i v e t o seven c a n k e r s , d u r i n g t h e main s p o r e p r o d u c t i o n p e r i o d i n 19^5, 1966 and I967. C o n s i d e r a b l e v a r i a t i o n o c c u r r e d i n t h e d a i l y p e r c e n t a g e g e r m i n a t i o n between y e a r s , g e n e r a l l y t h e r e was a much l o w e r g e r m i n a t i o n p e r c e n t a g e d u r i n g t h e second h a l f o f t h e p r o d u c t i o n p e r i o d . I n 1965 a r e l a t i v e l y F i g . 87. A v e r a g e d a i l y p e r c e n t g e r m i n a t i o n o f f i v e t o seven C r o n a r t i u m comandrae a e c i o s p o r e samples a f t e r 2k h o u r s on w a t e r agar a t 15°C, f o r t h e y e a r s 1965, 1966 and 1967. - 273 -- 274 -high percentage was obtained f o r about a month, but i n the other years such high l e v e l s of germination were obtained f o r o n l y short p e r i o d s . An average of 50$ germination was only reached on two days i n 1967. That the p a t t e r n of d a i l y germination f o r i n d i v i d u a l cankers i n the three years was s i m i l a r i s shown i n F i g . 88, u n f o r t u n a t e l y the f u l l d u r a t i o n of spore production was not sampled f o r canker no. 7 ( t r e e #2728) i n I965. I n I965 and 1966 f o r canker no. 1 ( t r e e #2722), 59 and 50$ of the days r e s p e c t i v e l y , gave a germination percentage of over 50$, but i n I967 only 11$ of the days, and only 39$ of the days gave a germination per-centage of over 10$. I n 1966 f o r canker no. 7, 71$ of the days, had a germination percentage of over 50$ and the lowest day f o r the. whole season was l8$, but i n 1967 only 23$ of the days had a germination above 50$. The v a r i a t i o n i n percent germination among the seven cankers sampled i n I965, the best year f o r germination, : i s shown i n F i g . 89. A l l cankers i n d i c a t e d a high germination percentage during'the f i r s t h a l f o f the spore production p e r i o d , and there was a good r e l a t i o n s h i p between the cankers i n day to day v a r i a t i o n . Even i n the l a t t e r h a l f o f the produc-t i o n p e r i o d there was s t i l l a close r e l a t i o n s h i p i n the p a t t e r n of d a i l y v a r i a t i o n , although the range of percentage germination was considerable. The seven cankers sampled i n 1967 a l s o showed a close agreement i n the day to day p a t t e r n of germination v a r i a t i o n , but agreement was l e s s mar-ked i n 1966, w i t h fewer days showing a s i m i l a r germination trend. V a r i a t i o n was a l s o considerable i n the d a i l y per cent germina-t i o n between the pustules of an i n d i v i d u a l canker ( F i g s . 90 and 91) , but there was u s u a l l y a considerable c o r r e l a t i o n between the germination trend of i n d i v i d u a l p u s t u l e s , as i s shown i n the f l u c t u a t i o n s of June 5 - 8 , and F i g . 88. D a i l y p e r ce n t g e r m i n a t i o n o f a e c i o s p o r e s f r o m two C r o n a r t i u m comandrae c a n k e r s f o r t h e y e a r s I965, I966 and I 9 6 7 . F i g . 89. V a r i a t i o n i n t h e d a i l y p e r cent g e r m i n a t i o n o f a e c i o -s p o r e s f r o m seven C r o n a r t i u m comandrae c a n k e r s i n 1965. F i g . 90. D a i l y p e r cent g e r m i n a t i o n o f a e c i o s p o r e s f r o m f i v e i n d i v i d u a l a e c i a o f a C r o n a r t i u m comandrae canker d u r i n g t h e I966 s p o r u l a t i o n p e r i o d . G E R M I N A T I O N % - LLZ -F i g . 91- D a i l y p e r c e n t g e r m i n a t i o n o f a e c i o s p o r e s f r o m f i v e i n d i v i d u a l a e c i a o f a C r o n a r t i u m comandrae c a n k e r d u r i n g t h e 1967 s p o r u l a t i o n p e r i o d . G E R M I N A T I O N % " 8^3 -- 279 -J u l y 2 - 7 , 1966, and t h e p e r c e n t a g e s on June 13, 21 and 28, I967. O t h e r s (Doran 1919, 1922; K r e b i l l 1968c; S p a u l d i n g 1922) have n o t e d t h a t g e r m i n a t i o n o f C r o n a r t i u m a e c i o s p o r e s was more v i g o r o u s e a r l y i n t h e season, d i m i n i s h i n g as t h e seaso n advanced. T h i s r e d u c t i o n i n spore v i a b i l i t y may be due t o an i n h e r e n t p h y s i o l o g i c a l change, t o t h e e f f e c t s o f t h e env i r o n m e n t , e s p e c i a l l y l i g h t i n t e n s i t y , o r t o t h e i n -c r e a s e d amount o f c o n t a m i n a t i o n b y a s s o c i a t e d m i c r o o r g a n i s m s on t h e c a n k e r . C e r t a i n f a c t o r s o b v i o u s l y a f f e c t e d s p o r e g e r m i n a t i o n o n . c e r t a i n d ays, b u t t h e r e was no c o n s i s t e n t r e l a t i o n s h i p between v a r i a t i o n s i n d a i l y s p o r e g e r m i n a t i o n and accompanying weather c o n d i t i o n s . A r e a s o n f o r g e r m i n a t i o n p e r c e n t a g e s b e i n g h i g h e r i n I965 t h a n i n 1966 and 1967, c o u l d be due t o t h e method o f s a m p l i n g . I n 1965 s p o r e s were t a k e n f o r a l o n g e r p e r i o d f r o m p u s t u l e s w i t h abundant s p o r e s and n o t u n t i l l a t e r i n t h e season were p u s t u l e s sampled w h i c h had p r a c t i c a l l y ceased p r o d u c -t i o n , as c o u l d have o c c u r r e d w i t h t h e i n d i v i d u a l p u s t u l e s a m p l i n g o f I966 and 1967. M a t u r i t y o f s p o r e s w o u l d o b v i o u s l y have some e f f e c t on g e r m i n a t i o n p e r c e n t a g e , on some o c c a s i o n s a number o f immature s p o r e s ..were n o t e d , b u t t h i s was t h e e x c e p t i o n . On t h e o c c a s i o n s i n 1965 when immature s p o r e s were n o t e d i n t h e t e s t sample, t h e p e r c e n t a g e g e r m i n a t i o n was l o w e r t h a n on a d j a c e n t days, b u t u s u a l l y b y no more t h a n 20$. The f l u c t u a t i o n s towards t h e end o f t h e spor e p r o d u c t i o n . s e a s o n i n 1965 were o f t e n caused b y a t e s t sample b e i n g t a k e n f r o m a new l y opened pus-t u l e on a canker w h i c h gave r e s u l t s a t y p i c a l o f t h e g e n e r a l g e r m i n a t i o n t r e n d , e.g. canker no. 2 on June 25 and 27-D a i l y s p o r e samples were t a k e n f r o m t h e p u s t u l e s b e f o r e 0830 hours t h u s any s p o r e s p r o d u c e d d u r i n g d a r k n e s s had not been exposed t o h i g h d a y t i m e t e m p e r a t u r e s o r t o l o n g p e r i o d s o f h i g h l i g h t i n t e n s i t y - 280 -w h i c h would a f f e c t t h e i r v i a b i l i t y ( c f . n e x t s e c t i o n ) . Many o f t h e spo r e s p r o d u c e d d u r i n g d a y l i g h t hours on t h e p r e v i o u s day would have been d i s p e r s e d , so s p o r e s w h i c h had been exposed t o h i g h t e m p e r a t u r e s and h i g h l i g h t i n t e n s i t i e s would g e n e r a l l y o n l y f o r m a m i n o r i t y o f t h e s p o r e sam-p l e . On a mor n i n g f o l l o w i n g a day o f low spor e d i s p e r s a l t h i s would not be t h e case, and a h i g h e r p e r c e n t a g e o f t h e sample may have had i t s v i a b i l i t y a f f e c t e d t h r o u g h p r o l o n g e d e x p o s u r e . I n t h e f i e l d , u n d i s p e r s e d s p o r e s t e n d t o l o s e t h e i r c o l o r o v e r a p e r i o d o f a few weeks o r even days, t u r n i n g f r o m a b r i g h t y e l l o w - o r a n g e t o a g r a y c o l o r . T h i s change i n spo r e c o l o r , w h i c h has been r e p o r t e d a l s o f o r C. f u s i f o r m e ( S i g g a r s 19+7; Walkinshaw 1968), i s a s s o c i a t e d w i t h a l o s s o f v i a b i l i t y . Immature s p o r e s change c o l o r r a p i d l y . Doran (1922) n o t e d t h a t i f a e c i o s p o r e s o f C_. r i b i c o l a were d e t a c h e d when immature t h e y f a i l e d t o d e v e l o p and g e r m i n a t e . Wet s p o r e s change c o l o r f a s t e r t h a n d r y s p o r e s and t e n d t o s h r i v e l r a p i d l y upon d r y i n g . The r a p i d s h r i v e l -l i n g was n o t e d f o r o t h e r f u n g i b y Ogawa and McCain ( i960) . The c o l o r change i s p r o b a b l y a s s o c i a t e d w i t h p h y s i o l o g i c a l changes w i t h i n t h e s p o r e b r o u g h t about b y p r o l o n g e d e x p o s u r e t o l i g h t , h e a t and m o i s t u r e . S t o r e d s p o r e s a l s o t e n d t o undergo t h e same c o l o r change ( M i e l k e 1955), b u t a t a much r e d u c e d r a t e . S p o r e s w h i c h have l o s t t h e i r c o l o r s w e l l o n l y s l i g h t -l y when p l a c e d on w a t e r , a f u r t h e r i n d i c a t i o n o f n o n - v i a b i l i t y . The e f -f e c t o f d i r e c t s u n l i g h t and o t h e r f a c t o r s on spore v i a b i l i t y i s d i s c u s s e d f u r t h e r , i n a l a t e r s e c t i o n . D u r i n g t h e t e s t s i t appeared t h a t s p o r e s t a k e n f r o m p r o t e c t e d a e c i a o f a canker gave b e t t e r r e s u l t s t h a n t h o s e t a k e n f r o m exposed a e c i a . To f u r t h e r t e s t t h i s v i e w , on June 25, 1965 s p o r e s were t a k e n f r o m - 281 -p r o t e c t e d a e c i a under t h e b a r k and fr o m u n p r o t e c t e d a e c i a f u l l y exposed on t h r e e c a n k e r s . Samples from one canker showed no s i g n i f i c a n t d i f f e r -ence, b u t t h e p r o t e c t e d s p o r e s on t h e o t h e r two c a n k e r s gave an i n c r e a s e d g e r m i n a t i o n o f 8k and 30% s u g g e s t i n g t h a t e x p o s u r e t o l i g h t and contam-i n a t i o n b y m i c r o o r g a n i s m s c o u l d be a c o n t r i b u t i n g f a c t o r t o r e d u c t i o n o f spor e g e r m i n a t i o n . Wo c o n t a m i n a t i o n was p r e s e n t i n t h e p r o t e c t e d samples, b u t b a c t e r i a were p r e s e n t i n t h e two u n p r o t e c t e d samples w i t h r e d u c e d g e r m i n a t i o n . A t o t h e r t i m e s c o n t a m i n a n t s o f t e n r e d u c e d t h e g e r m i n a t i o n p e r c e n t a g e . On a few o c c a s i o n s g e r m i n a t i o n was c l o s e t o 100$ i n a l l a r e a s o f t h e p l a t e e x c e p t where b a c t e r i a , y e a s t o r i m p e r f e c t fungus c o l o n i e s ( u s u a l l y P e n i c i l l i u m spp. and/or C l a d o s p o r i u m spp.) became e s t a b l i s h e d , where o n l y 0 - 10$ g e r m i n a t i o n o c c u r r e d . Most p l a t e s c o n t a i n e d some con-t a m i n a n t s , e i t h e r d i r e c t l y f r o m t h e s p o r e s o r f r o m t h e g e n e r a l a i r - s p o r a w h i c h c o u l d e n t e r t h e p l a t e a t t i m e o f s p o r e s e e d i n g . I n 1965 v e r y few ' c l e a n ' p l a t e s were o b s e r v e d , t h e s e g e n e r a l l y o c c u r r e d a t t h e b e g i n n i n g o f t h e seas o n o r when p r o t e c t e d s p o r e s were sampled. There was some i n -d i c a t i o n o f a b u i l d up o f m i c r o o r g a n i s m s i n a s s o c i a t i o n w i t h t h e s p o r e s as t h e p r o d u c t i o n p e r i o d p r o g r e s s e d , and t h i s c o n t a m i n a t i o n may have con-t r i b u t e d m a r k e d l y t o t h e g e n e r a l d e c l i n e o f s p o r e v i a b i l i t y i n t h e second h a l f o f t h e p r o d u c t i o n p e r i o d . The h i g h g e r m i n a t i o n p e r c e n t a g e o f t h e f i r s t h a l f n e v e r o c c u r r e d d u r i n g t h e second h a l f o f t h e p e r i o d , even f r o m r e l a t i v e l y f r e s h p u s t u l e s . Doran (1922) and o t h e r s n o t e d t h a t s p o r e s g e r m i n a t e b e t t e r when t h e media are not c o n t a m i n a t e d b y o t h e r f u n g i s p o r e s . C l a d o s p o r i u m was a p r o m i n e n t fungus w h i c h i n h i b i t e d g e r m i n a t i o n o f t e l i o -s p o r e s o f P u c c i n i a malvacearum (Doran 1922), and v a r i o u s b a c t e r i a i n h i b i t e d g e r m i n a t i o n o f t h e u r e d o s p o r e s o f P u c c i n i a g r a m i n i s t r i t i c i ( F r e n c h - e t a l . - 282 -1964). Both o f these microorganism groups were very common contaminants i n the present study. On s e v e r a l occasions there was a marked r e d u c t i o n i n germina-t i o n percentage when the a e c i a l p u s t u l e s were wet. Reduction of 80 - 95$ was not uncommon i n 1965. The a c t u a l w e t t i n g may i t s e l f a f f e c t the spore v i a b i l i t y through i n t e r n a l changes. K l i n g s t r o m (1963) found that germin-a t i o n of Peridermium p i n i aeciospores was poor or non-existent when spores were c o l l e c t e d during r a i n y weather. Manners (1950) a l s o found t h a t wet r u s t spores germinated very p o o r l y . Walkinshaw (1968) found t h a t C_. fusiforme aeciospores remained quiescent under c o o l dry co n d i t i o n s i n the f i e l d , but l o s t v i a b i l i t y under moist c o n d i t i o n s (Walkinshaw e_t a l . 1967) • A f u r t h e r cause o f germination r e d u c t i o n a s s o c i a t e d w i t h w e t t i n g may be due to contamination r e s u l t i n g from stem flow, during r a i n , which washes microorganisms from the bark i n t o the p u s t u l e . Such may. have occurred on June 18, 1965, f o l l o w i n g a 1.64 i n c h r a i n f a l l which soaked the bark. .A l i g h t shower would not s i m i l a r l y a f f e c t the bark. The o r i e n t a t i o n of the p u s t u l e to the p r e v a i l i n g shower or storm d i r e c t i o n i s important and would r e s u l t i n some pus t u l e s remaining dry, w h i l e others became wet. A check of the microorganisms, u s u a l l y present on he a l t h y pine bark and on the a e c i a l canker, was c a r r i e d out i n J u l y I967 by shaking s m a l l pieces of each m a t e r i a l i n d i s t i l l e d water f o r s e v e r a l hours, s t r e a k i n g the s o l -u t i o n on agar p l a t e s , then i s o l a t i n g , c u l t u r i n g , and i d e n t i f y i n g the or-ganisms which developed. Almost twice as many species were present on the h e a l t h y bark as on the a e c i a l p u s t u l e zone. However, any of these organisms r e g u l a r l y c o l o n i z i n g the caulosphere (Garner 1967) could p l a y an important r o l e as aeciospore contaminants. - 283 -AECIOSPORE V I A B I L I T Y The p r o b l e m o f m a i n t a i n i n g ' s p o r e v i a b i l i t y o v e r l o n g p e r i o d s , n e c e s s a r y because o f t h e b r i e f a n n u a l o c c u r r e n c e o f each s p o r e s t a t e , i s one o f t h e l i m i t i n g f a c t o r s i n s t u d i e s on r u s t s p o r e b i o l o g y and p a t h -o g e n i c i t y . D u r i n g t h e e a r l y p e r i o d o f t h i s s t u d y i t became o b v i o u s t h a t we were d e a l i n g w i t h an o r g a n i s m whose a e c i o s p o r e s t a t e o n l y r e t a i n e d i t s v i a b i l i t y w i t h d i f f i c u l t y , even s p o r e s s t o r e d f o r a few days showed g r e a t v a r i a t i o n i n g e r m i n a t i o n p e r c e n t a g e . F o r example, on J u l y 20, 1964, s p o r e s were t e s t e d f r o m 102 r e c e n t c o l l e c t i o n s , and t h e p e r c e n t g e r m i n a -t i o n v a r i e d f r o m 0 t o 70, w i t h j u s t o v e r h a l f t h e samples r e c o r d i n g 1% o r l e s s g e r m i n a t i o n , and t h e average o f a L l samples was o n l y 8%. T h i s was i n marked c o n t r a s t t o c o l l e c t i o n s o f P e r i d e r m i u m h a r k n e s s i i ( a v e r a g e 76%), and t o a l e s s e r e x t e n t t o P. s t a l a c t i f o r m e (39%) s p o r e s w h i c h had been c o l l e c t e d o v e r t h e same p e r i o d and k e p t under i d e n t i c a l c o n d i t i o n s . I n some cases g e r m i n a t i o n o f C_. comandrae samples had been r e d u c e d f r o m t h e i n i t i a l g e r m i n a t i o n p e r c e n t a g e by o v e r 60% i n 8 days. S p o r e s c o l l e c t e d f r o m t h e same canker a t w e e k l y i n t e r v a l s were most v a r i a b l e , and g e r m i n a -t i o n was g e n e r a l l y t o o low t o be s a t i s f a c t o r y f o r t e s t s t o e s t a b l i s h r e -q u i r e m e n t s . T h e . e f f e c t o f v a r i o u s e n v i r o n m e n t a l f a c t o r s on a e c i o s p o r e v i a -b i l i t y was i n v e s t i g a t e d , i n c l u d i n g t h e e f f e c t o f v a r i o u s s t o r a g e c o n d i t i o n s . Methods and M a t e r i a l s S p o r e s were c o l l e c t e d , p r e p a r e d f o r s t o r a g e and g e r m i n a t e d b y t h e same methods as t h o s e d e s c r i b e d i n t h e s e c t i o n on s p o r e g e r m i n a t i o n , u n l e s s o t h e r w i s e s p e c i f i e d . - 284 -The e f f e c t o f t e m p e r a t u r e on s p o r e v i a b i l i t y - was t e s t e d i n a number o f e x p e r i m e n t s u s i n g v a r i o u s t e m p e r a t u r e s and t r e a t m e n t s . I n one t e s t , f i v e d r y samples o f s p o r e s were d i v i d e d i n t o s i x t r e a t m e n t groups w h i c h were f u r t h e r d i v i d e d i n t o s i x v i a l s . A f u r t h e r s m a l l q u a n t i t y o f s p o r e s f r o m each sample was t e s t e d i m m e d i a t e l y t o g i v e an i n i t i a l c o n t r o l g e r m i n a t i o n p e r c e n t a g e . The r e m a i n i n g s p o r e s o f each sample were sub-j e c t e d t o s i x s t o r a g e t r e a t m e n t s : at- - 2 , 5, 10, 15, 25 and 30°C. Spores f r o m one v i a l o f each treatment- were t e s t e d f o r p e r c e n t a g e g e r m i n a t i o n a t t h e f o l l o w i n g i n t e r v a l s , 1, 2, 4, 8, l 8 and 55 days. Samples o f s p o r e s w h i c h had shown z e r o o r o n l y a t r a c e o f g e r m i n a t i o n on w a t e r agar, on t h e l 8 t h day i n t r e a t m e n t s 15, 25 and 30°C were h y d r a t e d on t h e 29th day f o r t h r e e days i n a s a t u r a t e d h u m i d i t y and.then g e r m i n a t e d on Czapek's agar f o r 24 hours a t 15°C. I n a second t e s t , s p o r e s f r o m t h r e e samples were s t o r e d f o r 4 days a t -10, 2, 15, 20 and 25°C b e f o r e b e i n g g e r m i n a t e d on w a t e r agar a t 2, 15, 20 and 25°C. I n a t h i r d t e s t , s p o r e s f r o m f i v e samples were s t o r e d a t 15°C f o r p e r i o d s o f 4 t o 6 days and t e s t e d o v e r t h e range 5 bo 30°C. I n a f o u r t h t e s t , s p o r e s f r o m t h r e e samples were s t o r e d a t - 4 , 15 and 40°C, and t e s t e d a f t e r t h e f o l l o w i n g i n t e r v a l s o f s t o r a g e : l / 4 , l / 2 , 1, 2 and 6 h o u r s . Spores were a l s o r e g u l a r l y s t o r e d at- a t e m p e r a t u r e o f -2 o r 4°C, and t h e s e p r o v i d e d some i n f o r m a t i o n on spore l o n g e v i t y under t h e s e s t o r a g e c o n d i t i o n s . A f u r t h e r t e s t s e r i e s , i n an e f f o r t t o r e t a i n s p o r e v i a b i l i t y f o r a l o n g e r p e r i o d , i n v o l v e d . r a p i d c o o l i n g o f s p o r e s b y two s u b z e r o f r e e z i n g methods; i n a d e e p f r e e z e r c h e s t a t -20°C, and b y u l t r a - r a p i d d r o p l e t f r e e z i n g i n l i q u i d n i t r o g e n ( -196°C). The v i a l s o f s p o r e s r a p i d l y c o o l e d i n l i q u i d n i t r o g e n were f r o z e n b y imm e r s i n g t h e lo w e r h a l f o f t h e v i a l i n l i q u i d n i t r o g e n h e l d i n - 285 -a v e s s e l . T h i r t y - f i v e to f o r t y samples were given each r a p i d c o o l i n g treatment. F o l l o w i n g r a p i d c o o l i n g the l i q u i d n i t r o g e n treatments were stored i n a -20°C f r e e z e r along w i t h the f r e e z e r treatment spores. These spores were t e s t e d a f t e r 7 to 18 days and a f t e r 18 months. P r i o r to t e s t i n g f o r germination h a l f the l i q u i d n i t r o g e n treatment samples were given a r a p i d warming treatment by p l a c i n g the v i a l s i n a water bath at 38-4o°C f o r one minute. The other h a l f were allowed to warm s l o w l y at 15°C by p l a c i n g them d i r e c t l y on water agar. At the 18 months t e s t i n g h a l f of each sample from the l i q u i d n i t r o g e n and f r e e z e r treatments were hydrated f o r 2h hours i n a saturated atmosphere, to see i f germination percentage was increased by t h i s procedure. I n a l l the above t e s t s germination percentages of a l l samples were obtained p r i o r to treatment, and the time between c o l l e c t i n g and placement i n a treatment was kept to a few minutes. The e f f e c t of humidity on spore v i a b i l i t y was i n v e s t i g a t e d i n two t e s t s by d i v i d i n g spore samples i n t o three p o r t i o n s , plus a s m a l l amount which was t e s t e d immediately to serve as a c o n t r o l . Each of the three p o r t i o n s was then d i v i d e d i n t o s i x or more v i a l s , and each group of v i a l s was stored i n one of three treatments. Spores were subjected to the f o l l o w i n g humidity c o n d i t i o n s : ( l ) i n dry a i r , (2) i n dry a i r over a saturated calcium c h l o r i d e s o l u t i o n , ( 3 ) . i n a saturated atmosphere over d i s t i l l e d and d e i o n i z e d water. A l l were maintained i n d e s i c c a t o r s held i n an incubator at a temperature of 2°C. One v i a l from each t r e a t -ment was t e s t e d f o r germination at the f o l l o w i n g time i n t e r v a l s , 1, 2, k and 8 days, and one or other of the t e s t s at l6, 18, 23, 27, 3^ or 55 days. The e f f e c t of d i f f e r e n t humidity storage c o n d i t i o n s on number of germ tubes per spore and l e n g t h of germ tubes was checked a f t e r 2, k, 8 - 286 -and 23 days i n one t e s t and a f t e r l6 days i n t h e o t h e r . The e f f e c t o f n a t u r a l l i g h t on spo r e v i a b i l i t y was t e s t e d on f o u r days, t h r e e on c l e a r sunny days and one on a c l o u d y day. . Samples o f s p o r e s f r o m t h r e e t o f i v e c a n k e r s were c o l l e c t e d on t h r e e o c c a s i o n s p r i o r t o s u n r i s e f r o m f r e s h o r unexposed a e c i a l p u s t u l e s . On t h e o t h e r o c c a s i o n (June 23, 1967) s p o r e s were c o l l e c t e d w i t h i n one hour a f t e r s u n r i s e , t h u s t h e y had been b r i e f l y exposed t o s u n l i g h t . A p o r t i o n o f each sample was seeded o n t o w a t e r agar i m m e d i a t e l y t o o b t a i n an i n i t i a l g e r m i n a t i o n p e r c e n t a g e . The r e m a i n d e r o f each sample was p l a c e d i n a d r y 5 cm p l a s t i c p e t r i p l a t e t h a t was p l a c e d , w i t h t h e l i d o f f , i n a s h a l l o w c a r d b o a r d box. The box was t h e n exposed t o f u l l l i g h t , t h e box b e i n g t u r n e d so t h a t i t was o r i e n t a t e d towards t h e sun a t a l l t i m e s . The box p r o v i d e d some p r o t e c t i o n f r o m t h e wi n d p r e v e n t i n g t h e s p o r e s f r o m b e i n g d i s p e r s e d . A few s p o r e s f r o m each sample were seeded o n t o t h e t e s t g e r m i n a t i o n media a t i n t e r v a l s of. one hour f o r about 12 h o u r s , w i t h t h e a i d o f a camel h a i r b r u s h . I n t h e f i r s t t e s t s p o r e s were checked o n l y e v e r y t h r e e h o u r s f o r 15 h o u r s . I n one t e s t , a s m a l l sample o f s p o r e s was k e p t i n t h e d a r k i n an i n c u b a t o r a t 15°C f o r t h e d u r a t i o n o f t h e e x p e r i m e n t , and t e s t e d a t t h e same t i m e as t h e l a s t l i g h t exposed sample. Temperature and r e l a t i v e h u m i d i t y d a t a were o b t a i n e d d u r i n g t h e p e r i o d o f t h e t e s t s from a hy g r o t h e r m o g r a p h exposed a d j a c e n t t o t h e box a t t h e same h e i g h t . Wo a t t e m p t was made t o r e c o r d t e m p e r a t u r e s o f t h e s p o r e s w i t h i n t h e p e t r i p l a t e s and box. Inco m i n g r a d i a t i o n and an e s t i -mate o f t h e degree o f c l o u d i n e s s was r e c o r d e d on an a c t i n o g r a p h o p e r a t e d a t a s i t e a p p r o x i m a t e l y l/k m i l e away. - 287 -R e s u l t s E f f e c t o f t e m p e r a t u r e on spor e v i a b i l i t y F i g . 92 shows t h e change i n p e r c e n t a g e g e r m i n a t i o n o f s p o r e s s t o r e d f o r s h o r t p e r i o d s a t t h r e e t e m p e r a t u r e s . L o s s o f sp o r e v i a b i l i t y was most r a p i d a t kO°C, w i t h l e s s t h a n 1$ o f t h e s p o r e s g e r m i n a t i n g a f t e r o n l y 30 m i n u t e s e x p o s u r e a t t h i s t e m p e r a t u r e , s u g g e s t i n g t h a t s u c h h i g h t e m p e r a t u r e s a r e l e t h a l f o r s p o r e v i a b i l i t y . S p o r e s exposed a t kO°C a l s o had b l e a c h e d and appeared s m a l l e r a f t e r o n l y 30 m i n u t e s . A l l t r e a t m e n t s showed a marked i n i t i a l l o s s o f v i a b i l i t y , b u t i t was l e s s • n o t i c e a b l e a t -k°C. T h i s i n i t i a l l o s s may have been due t o t h e 10 t o 15 m i n u t e i n t e r v a l between c o l l e c t i o n , h a n d l i n g and plac e m e n t i n s t o r a g e , a l t h o u g h a i r t e m p e r a t u r e s a t t h e t i m e o f c o l l e c t i o n were o n l y l6°C. Throughout t h e 6 hour d u r a t i o n o f t h e t e s t , t h e r e was a s l i g h t r e d u c t i o n i n g e r m i n a t i o n i n t h e -k and 15°C t r e a t m e n t , w i t h a h i g h e r o v e r a l l g e r m i n -a t i o n f r o m t h e -U°C t r e a t m e n t . The e f f e c t o f t e m p e r a t u r e on v i a b i l i t y was f u r t h e r i n v e s t i g a t e d b y u s i n g s i x t e m p e r a t u r e t r e a t m e n t s o v e r t h e range -k° t o 30°C f o r a p e r i o d o f 55 days ( T a b l e X X X V I ) . The r e s u l t s showed a g e r m i n a t i o n r e d u c t i o n g r a d i e n t ' w i t h most r a p i d r e d u c t i o n a t t h e h i g h e r t e m p e r a t u r e s and l e a s t r e d u c t i o n a t t h e lo w e r t e m p e r a t u r e s . A l l t r e a t m e n t s showed a marked r e d u c t i o n a f t e r one day o f s t o r a g e , and a f t e r two days o n l y a t r a c e o f g e r m i n a t i o n was o b t a i n e d f r o m t h e 25 and 30°C t r e a t m e n t s , and none a t t h e f o u r day i n t e r v a l s . A f t e r k days o f s t o r a g e o n l y t h e -k and 5°C t r e a t m e n t s r e t a i n e d more t h a n 10$ o f t h e i n i t i a l g e r -m i n a t i o n , and t h e s e two t r e a t m e n t s a c t u a l l y r e t a i n e d more t h a n 10$ o f the i n i t i a l g e r m i n a t i o n a f t e r 55 days, b u t a l l o t h e r s were r e d u c e d t o z e r o o r a t r a c e . On t h e 29th day o f s t o r a g e samples o f t h e 15, 25 and 30°C F i g . 92. A v e r a g e p e r c e n t g e r m i n a t i o n o f t h r e e C r o n a r t i u m comandrae a e c i o s p o r e samples s t o r e d a t t h r e e t e m p e r a t u r e s , and g e r -m i n a t e d on w a t e r agar a t 15°C a f t e r v a r i o u s h o u r s o f s t o r a g e . - 289 -T a b l e XXXVI. Average g e r m i n a t i o n p e r c e n t a g e s o f f i v e a e c i o s p o r e sam-p l e s s t o r e d a t s i x t e m p e r a t u r e s and g e r m i n a t e d on w a t e r agar a t 15°C a f t e r v a r i o u s i n t e r v a l s o f s t o r a g e . Days o f S t o r a g e t e m p e r a t u r e s (°C) s t o r a g e -4 5 10 15 25 30 1 32.7% . 22.4% 8.1% 3-9% 2.5% 2.3% 2 2 6 . 8 19.4 6 .3 2 .1 0 . 2 0 . 0 4 18 .6 15.5 4 . 4 1.3 0 . 0 0 . 0 8 15.6 14.6 3-5 1.1 0 . 0 0 . 0 18 11.0 10.3 2 . 1 0 . 3 0 . 0 0 . 0 55 8 .1 6.5 0 . 5 0 . 2 Average i n i t i a l g e r m i n a t i o n o f 5 samples - 51-9% t r e a t m e n t s were h y d r a t e d f o r t h r e e days and t h e n t e s t e d on t h e more nu-t r i t i o u s Czapek-Dox a g a r . S p o r e s f r o m t h e 25 and 30°C t r e a t m e n t s f a i l e d t o r e s p o n d and gave no g e r m i n a t i o n , b u t t h e g e r m i n a t i o n o f t h e 15°C t r e a t -ment was improv e d f r o m t h e t r a c e r e c o r d e d a f t e r 18 days. T h i s showed t h a t a f a i r p r o p o r t i o n o f t h e s p o r e s a t t h i s t r e a t m e n t had r e t a i n e d t h e i r v i a b i l i t y , b u t t h a t a h y d r a t i o n p e r i o d was n e c e s s a r y f o r most o f them t o g e r m i n a t e . A f t e r 55 days s t o r a g e a l l t r e a t m e n t s were t e s t e d on b o t h a w a t e r agar ( T a b l e X X X V l ) and a Czapek agar, and t h e p e r c e n t a g e g e r m i n a t i o n s f o r t h e -4 and 5°C t r e a t m e n t s were improved on t h e Czapek agar i n a l m o s t a l l samples. O n l y two samples gave s l i g h t l y improved g e r m i n a t i o n f o r t h e 10 and 15°C t r e a t m e n t s , as most s p o r e s i n t h e s e t r e a t m e n t s were a l r e a d y n o n - v i a b l e . The o t h e r l e s s comprehensive t e s t s w i t h s t o r a g e o f s p o r e s a t t e m p e r a t u r e s between -10° and 25°C f o r p e r i o d s o f o n l y 4 t o 6 days con-f i r m e d t h e f i n d i n g s o f t h e above t e s t s . The r e s u l t s f o r t h r e e t e s t s a r e summarized i n T a b l e X X X V I I . I n one t e s t t h e h i g h i n i t i a l g e r m i n a t i o n per-' centage was m a i n t a i n e d a f t e r 4 days s t o r a g e a t -4 and 2°C, was m a r k e d l y r e d u c e d a t 15°C, and was r e d u c e d t o o n l y a t r a c e a t 20 and 25°C. I n t h e - 290 -T a b l e X X X V I I . Average g e r m i n a t i o n p e r c e n t a g e s o f a e c i o s p o r e samples s t o r e d f o r f o u r t o s i x days a t v a r i o u s t e m p e r a t u r e s and g e r m i n a t e d on w a t e r agar a t 15°C. Average Days o f Wo. o f i n i t i a l S t o r a g e t e m p e r a t u r e s s t o r a g e samples g e r m i n a t i o n (°C) % -k 2 15 20 25 h 3 75.5 79-7 72.5 19-1+. 0 . 7 O.k h 3 6 6 . 1 . 8 . 8 6 2 55-0 8 . 8 o t h e r t e s t , s t o r a g e o f sp o r e s f o r k t o 6 days a t 15°C r e d u c e d t h e i n i t i a l g e r m i n a t i o n b y o v e r $0%. I n l o n g t e r m s t o r a g e t e s t s a t -k and 2°C, s e v e r a l s p o r e samples r e t a i n e d some v i a b i l i t y f o r 9 months, b u t g e n e r a l l y a f a i r p e r c e n t a g e g e r -m i n a t i o n (>1 0 % ) was o n l y m a i n t a i n e d f o r 2 t o 3 months. An o c c a s i o n a l sample r e t a i n e d a t r a c e o f g e r m i n a t i o n f o r 20 o r 21 months, t h e s e s p o r e s had u s u a l l y been s t o r e d a t -4°C, b u t a few a t 2°C. A g r e a t number o f m i c r o o r g a n i s m s have been i s o l a t e d f r o m n o n - v i a b l e s t o r e d s p o r e s . The con-t a m i n a n t s were l a r g e l y P e n i c i l l i u m and C l a d o s p o r i u m spp. T a b l e X X X V I I I summarizes t h e average g e r m i n a t i o n p e r c e n t a g e o f spore samples r a p i d l y c o o l e d i n l i q u i d n i t r o g e n and i n a f r e e z e r p r i o r t o s t o r a g e i n a -20°C f r e e z e r f o r p e r i o d s o f 7 t o 18 days. The l i q u i d n i t r o g e n c o o l e d s p o r e s were thawed b y p l a c i n g d i r e c t l y on 15°C w a t e r a g a r , o r g i v e n a p r i o r t e m p e r a t u r e t r e a t m e n t a t 38°C f o r 1 m i n u t e b e f o r e seed-i n g . P e r c e n t a g e g e r m i n a t i o n was r e d u c e d under a l l s t o r a g e and t h a w i n g t r e a t m e n t s compared w i t h t h e p e r c e n t a g e g e r m i n a t i o n a t time o f s t o r a g e . Thawing s p o r e s r a p i d l y a t 38°C appeared t o improve g e r m i n a t i o n s l i g h t l y . - 291 -T a b l e X X X V I I I . Average p e r c e n t a g e g e r m i n a t i o n o f a number o f spor e samples g i v e n two methods o f r a p i d c o o l i n g and s t o r e d a t -20°C f o r p e r i o d s between 7 and 18 days, and t h e n g e r m i n a t e d on wa t e r agar a t 15°C f o r 24 h o u r s . A p o r -t i o n o f t h e l i q u i d n i t r o g e n c o o l e d s p o r e s was r a p i d l y thawed f o r one minut e a t 38°C p r i o r t o s e e d i n g on t h e aga r . No. o f No. o f days A v e r a g e g e r m i n a t i o n p e r c e n t a g e samples s t o r a g e a t I n i t i a l C o o l e d C o o l e d i n C o o l e d i n -20°C g e r m i n a t i o n a t -20°C l i q u i d LN + 38°C n i t r o g e n t h a w i n g (LN) -196°C 11 7 2 0 . 9 4 . 8 6.0 12.2 k 10 39-4 8.6 7-7 6 .1 8 Ik 2 0 . 0 5-3 7-9 13-4 12 16 - 18 24.4 3.3 4 . 9 9 .2 The e x p e r i m e n t was r e p e a t e d a f t e r t h e s p o r e samples had been s t o r e d f o r 18 months a t -20°C. G e r m i n a t i o n p e r c e n t a g e s were p o o r . O n l y one sample o u t o f 35 g i v e n t h e l i q u i d n i t r o g e n t r e a t m e n t p r i o r t o s t o r a g e , gave 10$ g e r m i n a t i o n a f t e r 18 months and two o t h e r s a t r a c e . The sample r e t a i n i n g a degree o f v i a b i l i t y , and g i v e n t h e t h a w i n g t r e a t m e n t a t k0°C gave a s l i g h t l y r e d u c e d g e r m i n a t i o n p e r c e n t a g e (6$) compared w i t h t h e p o r t i o n o f sample thawed on 15°C w a t e r a g a r . G e r m i n a t i o n p e r c e n t a g e s f o r f r e e z e r c o o l e d samples were b e t t e r , 9 samples out o f 38 samples gave g e r m i n a t i o n between 1 and 22$, and k o t h e r s gave some g e r m i n a t i o n . One group o f 4 samples gave s i m i l a r p e r c e n t a g e s a f t e r 10 days (3-17$) and 18 months s t o r a g e ( 4 - l 4 $ ) a t -20°C, b u t o t h e r samples, g i v i n g some g e r m i n a t i o n a t l 8 months, were much r e d u c e d f r o m t h e p e r c e n t a g e s o f t h e s h o r t e r p e r i o d o f s t o r a g e . H y d r a t i o n o f b o t h l i q u i d n i t r o g e n and f r e e z e r t r e a t e d s p o r e s f o r 1 t o 5 days d i d not improve g e r m i n a t i o n a f t e r 18 months s t o r a g e . - 292 -P e r c e n t a g e s were m a r k e d l y r e d u c e d f o r f r e e z e r t r e a t e d s p o r e s , b u t were s i m i l a r f o r t h e one good l i q u i d n i t r o g e n sample. E f f e c t o f h u m i d i t y on s p o r e v i a b i l i t y F i g . 93 i n d i c a t e s t h e change i n p e r c e n t g e r m i n a t i o n o f s p o r e s s t o r e d f o r v a r y i n g p e r i o d s of. t i m e under u l t r a - d r y , d r y o r humid c o n d i t i o n s a t 2°C. When th e s p o r e s were s t o r e d i n a d e s i c c a t o r o f c a l -c ium c h l o r i d e , w h i c h p r o d u c e s a s u p r a - d r y c o n d i t i o n , s p o r e s l o s t t h e i r v i a b i l i t y v e r y r a p i d l y , t h e r e b e i n g a r e d u c t i o n f r o m t h e i n i t i a l g e r m i n a -t i o n p e r c e n t a g e o f o v e r k0% on t h e f i r s t day. S t o r a g e i n a s a t u r a t e d e n v ironment a c t u a l l y i n c r e a s e d g e r m i n a t i o n a f t e r one day's s t o r a g e , w h i c h was not u n e x p e c t e d , as i t has been shown e l s e w h e r e i n t h i s s t u d y t h a t h y d r a t i o n improves g e r m i n a t i o n s l i g h t l y . P e r c e n t a g e g e r m i n a t i o n was m a i n t a i n e d a t a h i g h e r l e v e l t h r o u g h o u t t h e e x p e r i m e n t under s a t u r a t e d c o n d i t i o n s t h a n under d r y s t o r a g e c o n d i t i o n s , a l t h o u g h t h e r a t e o f r e -d u c t i o n was s i m i l a r , and was o n l y o f f s e t b y t h e i n i t i a l i n c r e a s e under s a t u r a t e d c o n d i t i o n s . A l t h o u g h s p o r e s s t o r e d under s a t u r a t e d c o n d i t i o n s appeared t o r e t a i n a h i g h e r degree o f v i a b i l i t y l o n g e r , t h e y were more l i a b l e t o damage fr o m c o n t a m i n a t i o n , as t h e s e c o n d i t i o n s t e n d e d t o f a v o u r g r o w t h o f c o n t a m i n a n t s . I n a second t e s t , s i m i l a r o v e r a l l r e s u l t s were o b t a i n e d t o t h o s e i n F i g . 93. S t o r a g e o v e r c a l c i u m c h l o r i d e however, r e d u c e d g e r m i n a t i o n t o z e r o w i t h i n 2k h o u r s , and g e r m i n a t i o n p e r c e n t a g e s under t h e o t h e r t r e a t m e n t s were n o t c o n s i s t e n t , a l t h o u g h t h e y were u s u a l l y h i g h e r under s a t u r a t e d t h a n d r y c o n d i t i o n s . F o u r days o f s t o r a g e i n s a -t u r a t e d c o n d i t i o n s gave b e t t e r g e r m i n a t i o n t h a n 1 o r 2 days s t o r a g e o r t h a n t h e i n i t i a l g e r m i n a t i o n . T h i s h i g h e r p e r c e n t a g e g e r m i n a t i o n was m a i n t a i n e d a t t h e 8 and l 6 day i n t e r v a l s f o r two o f t h e t h r e e samples, b u t F i g - 93- A v e r a g e p e r c e n t g e r m i n a t i o n o f f i v e C r o n a r t i u m comandrae a e c i o s p o r e samples s t o r e d i n a wet, d r y o r u l t r a d r y atmosphere, and g e r m i n a t e d on w a t e r agar a t 15°C a f t e r v a r i o u s i n t e r v a l s o f s t o r a g e . - 293 -70-! - 29*+ -a t t h e 27 day i n t e r v a l g e r m i n a t i o n p e r c e n t a g e s under t h e s a t u r a t e d t r e a t -ment were l o w e r t h a n t h o s e o f t h e d r y t r e a t m e n t . T h i s sudden r e d u c t i o n under s a t u r a t e d c o n d i t i o n s appeared t o he due t o c o n t a m i n a t i o n . T a b l e XXXIX g i v e s t h e average number o f germ t u b e s p e r s p o r e and t h e average l e n g t h o f t h e l o n g e s t germ t u b e a f t e r v a r i o u s i n t e r v a l s o f s t o r a g e under t h e t h r e e dry-humid t r e a t m e n t s . There was a t e n d e n c y f o r i n c r e a s e d s t o r a g e t o g i v e a l a r g e r number o f germ t u b e s p e r s p o r e , however, t h e l e n g t h o f t h e l o n g e s t germ tube was not a f f e c t e d b y s t o r a g e , e x c e p t t h a t t h e s a t u r a t e d t r e a t m e n t r e d u c e d g r o w t h a f t e r 23 days s t o r a g e . O b s e r v a t i o n s o f s p o r e s showed t h a t i f s p o r e s became wet, t h e y r a p i d l y l o s t t h e i r n a t u r a l c o l o r and v i a b i l i t y . T a b l e XXXIX. Av e r a g e number o f germ tub e s p e r s p o r e and average l e n g t h o f l o n g e s t germ tube i n t h r e e dry-humid t r e a t m e n t s a f t e r v a r i o u s i n t e r v a l s o f s t o r a g e . Days o f No. o f No. o f germ t u b e s L e n g t h o f l o n g e s t s t o r a g e samples p e r spo r e germ-tubes (u) D r y C a C l 2 S a t u r a t e d D r y C a C l 2 S a t u r a t e d 2 5 1.1 1.1 1.1 •1+16 1+36 ' koh k 2 1.3 1 .5 1 1.5 375 500 1 318 8 3 1.6 1.5 2.1 1+12 352 365 23 5 1.5 1 . 7 2 1.9 1+62 363 2 208 16 3 1.8 2.0 284 — 183 O n l y 2 germ t u b e s ^ O n l y 6 germ t u b e s E f f e c t o f l i g h t on sp o r e v i a b i l i t y F i g . 9I+ shows t h e r e d u c t i o n i n p e r c e n t a g e g e r m i n a t i o n f o l l o w -i n g e x p o s u r e o f s p o r e s t o l i g h t . The t e s t r u n on June l 6 , 1966, was o n l y checked a t t h r e e h o u r l y i n t e r v a l s , and g e r m i n a t i o n may w e l l have F i g . 9k. E f f e c t o f exposure o f C r o n a r t i u m comandrae a e c i o s p o r e s t o d i r e c t l i g h t f o r v a r y i n g p e r i o d s o f t i m e on c l e a r sunny days (June l 6 , and J u l y 5, 19&6; June 23, 1967), and on a c l o u d y day (June l 6 , 1967). - 295 -No. of CLEAR DAY samples — June 16/66 5 — July 5/66 2 — June 23 /67 3 CLOUDY DAY — June 16/67 1 4 6 8 H O U R S OF E X P O S U R E TO L I G H T 10 ~l 12 - 296 -been r e d u c e d t o z e r o p r i o r t o s i x hours e x p o s u r e . The sample was o b t a i n e d f r o m a p u s t u l e under t h e b a r k , t h u s t h e s p o r e s had not p r e v i o u s l y been exposed t o l i g h t . The o t h e r samples were c o l l e c t i o n s f r o m ' r e l a t i v e l y f r e s h p u s t u l e s and had p r e s u m a b l y been exposed t o some l i g h t w h i c h may c o n t r i b u t e t o t h e lo w e r i n i t i a l p e r c e n t g e r m i n a t i o n f o r t h e s e t h r e e sam-p l e s . A l s o t h e sample t e s t e d on June 23, 1967, was c o l l e c t e d j u s t a f t e r s u n r i s e , w h i c h may c o n t r i b u t e t o t h e l o w e r i n i t i a l g e r m i n a t i o n o f t h i s sample. G e r m i n a t i o n p e r c e n t a g e was r e d u c e d r a p i d l y on t h e c l e a r d ays, b e i n g r e d u c e d t o z e r o i n 5 o r 6 h o u r s . Spores exposed on a c l o u d y day r e t a i n e d t h e i r g e r m i n a t i o n f o r a t l e a s t 11 h o u r s , a l t h o u g h t h e r e appeared t o be a f a i r l y s t e a d y l i n e a r r e d u c t i o n i n g e r m i n a t i o n t h r o u g h o u t t h e p e r i o d . The maximum t e m p e r a t u r e on t h i s day was 5 t o 9°C l o w e r , and hu-m i d i t y was 35$ h i g h e r t h a n on t h e c l e a r days. The i n c o m i n g r a d i a t i o n on th e c l e a r day i n I967 between k a.m. and noon was o v e r t h r e e t i m e s as much as on t h e c l o u d y day. The l e n g t h o f t i m e t h e s p o r e s had been ex-posed on t h e c l o u d y day had no e f f e c t on t h e number o f germ t u b e s p r o d u c e d , o r t h e average l e n g t h o f germ t u b e s when t h e s p o r e s had g e r m i n a t e d . I n t h e t e s t on J u l y 5, 1966, when a sample o f s p o r e s was k e p t i n t h e d a r k f o r t h e d u r a t i o n o f t h e l i g h t e x p e r i m e n t , some g e r m i n a t i o n (2$) was o b t a i n e d a f t e r t w e l v e hours whereas i n t h e l i g h t p o r t i o n o f t h e t e s t no g e r m i n a t i o n was r e c o r d e d a f t e r t h e f i f t h h our. T h e r e f o r e , l i g h t appeared t o be r e s p o n s i b l e f o r a l a r g e r p r o p o r t i o n o f t h e r e d u c t i o n i n g e r m i n a t i o n t h a n t e m p e r a t u r e i n t h i s t e s t , as t h e 15°C e x p e r i e n c e d by t h e d a r k sample was a s i m i l a r t e m p e r a t u r e t o t h e average t e m p e r a t u r e f o r t h e f i r s t 8 hours o f t h e l i g h t t e s t . U n f o r t u n a t e l y no d a r k c o n t r o l s were employed i n t h e o t h e r t e s t s . - 297 -D i s c u s s i o n A r e v i e w o f t h e l i t e r a t u r e , and p a r t i c u l a r l y t h e summary t r e a t i s e s ( G o t t l i e b 1950; Cochrane i960), i n d i c a t e s t h a t s p o r e l o n g e v i t y g e n e r a l l y i n c r e a s e s as t e m p e r a t u r e d e c r e a s e s t o 0°C, and t h e l o s s o f v i a -b i l i t y i s e s p e c i a l l y r a p i d above t h e maximum t e m p e r a t u r e f o r germ tube f o r m a t i o n . I n t h e p r e s e n t . s t u d y t e m p e r a t u r e s o f 25°C and above were f o u n d t o be l e t h a l t o s p o r e s i n a v e r y s h o r t t i m e , w h i c h s u p p o r t s t h e above s t a t e m e n t ( G o t t l i e b 1950). I n a l l t e s t s t h e g e r m i n a t i o n o f s p o r e s m a i n t a i n e d a t t h e s e h i g h t e m p e r a t u r e s was r e d u c e d t o l e s s t h a n 1% i n 1 t o k days, and a t kO°C w i t h i n an hou r . . A t t h e i n t e r m e d i a t e t e m p e r a t u r e s (10-15°C) spore v i a b i l i t y was r e d u c e d t o 1% i n 10 t o 30 days b u t germ i n a -t i o n c o u l d s t i l l be improved f o l l o w i n g h y d r a t i o n . V i a b i l i t y a t t h e l o w e r t e m p e r a t u r e s was r e t a i n e d f o r a l o n g e r p e r i o d , f o r a t l e a s t 270 days and an o c c a s i o n a l t r a c e f o r 21 months a t -k and 2°C, and f o r a t l e a s t 18 months a t -20°C. R a p i d c o o l i n g i n l i q u i d n i t r o g e n (-196°C) and s t o r a g e a t -20°C p r e s e r v e d some v i a b i l i t y f o r 17 months, a l t h o u g h r e s u l t s were not c o n s i s t e n t . K l i n g s t r 8 m (1963) f o u n d t h a t 5 hours s t o r a g e o f d r y P e r i d e r m i u m p i n i a e c i o s p o r e s . a t 38°C had no e f f e c t on v i a b i l i t y , b u t a t e m p e r a t u r e o f 1+6°C was l e t h a l . Damp sp o r e s l o s t t h e i r v i a b i l i t y a t 38°C when p l a c e d on w a t e r agar, and Walkinshaw (1968) n o t e d a s i m i l a r s i t u a t i o n with.C_. f u s i -forme s t o r e d d r y o r i n a wat e r s u s p e n s i o n a t 37°C. Doran (1922) f o u n d t h a t s t o r a g e t e m p e r a t u r e s o f 7, 15 and 23°C had no e f f e c t on l o n g e v i t y o f C. r i b i c o l a , a l t h o u g h he r e p o r t e d no a e c i o s p o r e s g e r m i n a t e d a f t e r 8 weeks. G r a v a t t and T a y l o r ( c i t e d i n S p a u l d i n g 1922) found t h a t v i a b i l i t y o f C. r i b i c o l a a e c i o s p o r e s was l e s s t h a n 1% a f t e r a month's s t o r a g e i n a r e f r i g e r a t o r o r out o f d o o r s , b u t Y o r k ( c i t e d i n S p a u l d i n g 1922) found - 298 -t h a t some s p o r e s s t o r e d o u t o f doors i n t h e shade r e t a i n e d t h e i r v i a b i l -i t y f o r 75 days. K r e b i l l (1968c) exposed C. comandrae a e c i o s p o r e s on Comandra s h o o t s i n t h e greenhouse, where t e m p e r a t u r e s between 10 and 21°C were m a i n t a i n e d , and o b t a i n e d some g e r m i n a t i o n a f t e r 3 weeks, b u t n o t a f t e r k weeks e x p o s u r e . S i g g a r s (19I+7) s t o r e d C_. f u s i f o r m e s p o r e s a t below 10°C and was a b l e t o r e t a i n a h i g h l e v e l o f v i a b i l i t y f o r k-Y t o 76 days, and some were s u c c e s s f u l l y used f o r i n o c u l a t i o n a f t e r 211 d a y s . F e r g u s (1959) w o r k i n g w i t h C o l e o s p o r i u m s o l i d a g i n i s (Schw.) Thuem. a e c i o -s p o r e s o b t a i n e d no g e r m i n a t i o n a f t e r h hours s t o r a g e a t 35 and kO°C, b u t o b t a i n e d some a f t e r 120 days a t -17°C. Walkinshaw (1965) found l i t t l e change i n g e r m i n a t i o n o f C_. f u s i f o r m e s p o r e s s t o r e d a t 1 t o h°C d u r i n g t h e f i r s t 30 days, t h e n a g r a d u a l d e c l i n e . L i g h t l e (1955), K l i n g s t r B m (1963) and B r a m l e t t and Redman (1963) were a b l e t o r e t a i n t h e v i a b i l i t y o f v a r i o u s C r o n a r t i u m ( P e r i d e r m i u m ) s p o r e s f o r o v e r a y e a r a t k t o 5°C. S i m i l a r l y M i e l k e (1955) found t h a t s t o r a g e a t 5 t o 7°C p r o l o n g e d v i a b i l -i t y o f s e v e r a l C r o n a r t i u m ( P e r i d e r m i u m ) s p e c i e s , and t h a t t h e n a t u r a l spore c o l o r was m a i n t a i n e d l o n g e r . N a t u r a l c o l o r r e t e n t i o n i s a s s o c i a t e d w i t h v i a b i l i t y as no spor e t h a t had l o s t i t s c o l o r e v e r g e r m i n a t e d . S i g g a r s (19V7),. S p a u l d i n g (1922), F e r g u s (1959) and Walkinshaw (1968) a l l o b s e r v e d t h i s v i a b i l i t y - c o l o r change r e l a t i o n s h i p . S c h e i n (1962) r e p o r t e d t h a t r r e d o s p o r e s o f Uromyces p h a s e o l i (Reb.) W i n t . r e t a i n e d kCffo v i a b i l i t y a f t e r 670 days s t o r a g e a t -60°C, b u t s p o r e s s t o r e d a t -13 t o - l 6°C l o s t a l l v i a b i l i t y a f t e r 2 t o 5 months. Some g e r m i n a t i o n was ob-t a i n e d w i t h C. comandrae s p o r e s s t o r e d f o r l 8 months a t -20°C, and' a few s p o r e s g e r m i n a t e d f o l l o w i n g c o o l i n g a t -196°C and s t o r a g e a t -20°C. Why t h e r e were p o o r e r r e s u l t s w i t h t h e l i q u i d n i t r o g e n was unknown as - 299 -a f t e r 7 t o 18 days s t o r a g e t h e g e r m i n a t i o n f o l l o w i n g l i q u i d n i t r o g e n t r e a t m e n t was s l i g h t l y b e t t e r on average t h a n c o o l i n g and s t o r a g e a t -20°C, and e s p e c i a l l y i f l i q u i d n i t r o g e n t r e a t e d s p o r e s were thawed r a p i d l y a t 38°C. Goos e t a l . (1967), L i n e (1963) and o t h e r s , have a l l r e p o r t e d t h a t s t o r i n g s p o r e s i n l i q u i d n i t r o g e n was a more p r o m i s i n g method o f s t o r a g e t h a n f r e e z e - d r y i n g p r e s e r v a t i o n , a l t h o u g h t h e i r p e r i o d s o f s t o r a g e were r e l a t i v e l y s h o r t . I f s p o r e s c o u l d have been r e t a i n e d c o n t i n u o u s l y a t a te m p e r a t u r e c l o s e t o -196°C r e s u l t s m i ght have been more p r o m i s i n g i n th e p r e s e n t s t u d y , b u t a l i q u i d n i t r o g e n r e f r i g e r a t o r was not a v a i l a b l e . Goos e t a l . (1967) and L o e g e r i n g and Harmon (1962) found t h a t r a p i d thaw-i n g f o l l o w i n g s t o r a g e gave b e t t e r r e s u l t s t h a n s l o w t h a w i n g , and t h i s was s u p p o r t e d i n most t e s t s a f t e r 7 t o 18 days b u t not a f t e r l 8 months s t o r a g e . R e h y d r a t i o n o f s p o r e s f o l l o w i n g 18 months o f s t o r a g e r e d u c e d t h e g e r m i n a -t i o n p e r c e n t a g e o f b o t h l i q u i d n i t r o g e n and f r e e z e r t r e a t e d s p o r e s . L i n e (1963) r e p o r t e d t h a t s p o r e s s t o r e d i n l i q u i d n i t r o g e n d i d not r e q u i r e h y d r a t i o n b u t t h o s e s t o r e d b y t h e f r e e z e - d r y i n g p r o c e s s d i d . R o n c a d o r i and Matthews (1966) a l s o f o u n d t h a t h y d r a t i o n i n c r e a s e d g e r m i n a t i o n o f f r e e z e - d r i e d and c a l c i u m c h l o r i d e d r i e d C_. f u s i f o r m e s p o r e s , and o b t a i n e d some g e r m i n a t i o n b y b o t h s t o r a g e methods a f t e r 77 weeks, a l t h o u g h g e r m i n -a t i o n o f s p o r e s s t o r e d b y t h e l a t t e r method was t h e n v e r y low. H y d r a t i o n had a pronounced e f f e c t on Uromyces p h a s e o l i s p o r e s s t o r e d a t -60°C f o r 21 months ( S c h e i n 1962). P r e s e n t s t u d i e s i n d i c a t e d t h a t v i a b i l i t y was l o s t f a i r l y r a -p i d l y when s p o r e s were s t o r e d o v e r c a l c i u m c h l o r i d e , no g e r m i n a t i o n b e i n g o b t a i n e d a f t e r 23 d a y s . F e r g u s (1959) f o u n d t h a t C o l e o s p o r i u m s o l i d a g i n i s a e c i o s p o r e s had a maximum l o n g e v i t y o f lk3 days when s t o r e d o v e r c a l c i u m c h l o r i d e a t 8°C, some ko days l o n g e r t h a n when s t o r e d o v e r w a t e r . - 300 -Roncadori and Matthews (1966) r e p o r t no adverse e f f e c t s of storage over calcium c h l o r i d e f o r ho to 50 days, and obtained some germination of C. fusiforme a f t e r 77 weeks at 5°C. Others ( L i g h t l e 1955; Walkinshaw I965) have d r i e d spores over calcium c h l o r i d e f o r 48 hours but have then removed them from the d r y i n g agent. L i g h t l e (1955) found t h a t use of calcium c h l o r i d e reduced percentage germination of P. h a r k n e s s i i more than a i r - d r i e d or non d r i e d treatments a f t e r 14 months. Use of any c a l -cium c h l o r i d e d r y i n g w i t h C_. comandrae spores appeared to be d e t r i m e n t a l to v i a b i l i t y . Doran (1922) s t a t e d that C. r i b i c o l a spores, stored i n moist a i r , r e t a i n e d t h e i r v i a b i l i t y longer than those stored i n dry a i r , - and Line (1963) found the optimum r e l a t i v e humidity f o r P u c c i n i a graminis var. t r i t i c i spores was 50$. Schein and Rotem (1965), working w i t h ure-dospores of Uromyces p h a s e o l i , found t h a t g e r m i n a b i l i t y was i n v e r s e l y p r o p o r t i o n a l to r e l a t i v e humidity with no i n d i c a t i o n of an optimum r e l a -t i v e humidity of near 50$ f o r spore l o n g e v i t y . Storage of dry spores at higher h u m i d i t i e s , i n the present study, appeared to be s l i g h t l y more favourable over the i n i t i a l storage p e r i o d , or at l e a s t u n t i l any con-taminants i n t e r f e r e d . However, i f spore samples were stored wet, v i a -b i l i t y was r a p i d l y l o s t , s i m i l a r l y , i f wet spore samples were d r i e d and stored,' some i r r e v e r s i b l e process appeared to have taken place as v i a -b i l i t y was not r e s t o r e d . Fergus (1959) noted that contaminating f u n g i were encountered at higher h u m i d i t i e s i n l o n g e v i t y t e s t s . There are very few r e p o r t s of the e f f e c t o f l i g h t on v i a b i l i t y of Cronartium or other r u s t aeciospores. I n the present study d i r e c t s u n l i g h t reduced germination to l e s s than 50$ of i n i t i a l germination i n - 301 -1 t o 3 hours and t o z e r o i n 5 t o 6 h o u r s . D u r i n g t h i s p e r i o d a spo r e c o l o r change was most n o t i c e a b l e . P e n n i n g t o n ( c i t e d i n S p a u l d i n g 1922) a l s o r e p o r t e d a d e c r e a s e o f 50 t o 75% v i a b i l i t y o f C. r i b i c o l a a e c i o -s p o r e s a f t e r 3 hours exposure t o s u n l i g h t and f o u n d o n l y 0.05% v i a b l e a f t e r h o u r s . As men t i o n e d e a r l i e r , K r e b i l l (1968c) was u n a b l e t o ob-t a i n any g e r m i n a t i o n ' o f C. comandrae i n d i r e c t s u n l i g h t , and McCubbin ( l9 l8b) a l s o n o t e d t h a t an expo s u r e o f 10 t o 15 m i n u t e s t o f u l l s u n l i g h t was s u f f i c i e n t t o i n h i b i t , g e r m i n a t i o n o f C_. r i b i c o l a a e c i o s p o r e s . G r a v a t t and T a y l o r ( c i t e d i n S p a u l d i n g 1922) found t h a t s t o r i n g C_. r i b i c o l a a e c i o -s p o r e s i n a c o o l , d a r k environment s t i m u l a t e d g e r m i n a t i o n and p r o l o n g e d v i a b i l i t y . Hwang. (19^2) d e m o n s t r a t e d a d i r e c t r e l a t i o n s h i p between l i g h t i n t e n s i t y and t h e speed w i t h w h i c h P u c c i n i a r u s t u r e d o s p o r e s l o s t t h e i r v i a b i l i t y . I t i s d i f f i c u l t t o s e p a r a t e t h e f a c t o r s o f l i g h t i n t e n s i t y and t e m p e r a t u r e and t h e i r i n d i v i d u a l e f f e c t on s p o r e v i a b i l i t y , b u t t h e p r e s e n t s t u d y g i v e s some e v i d e n c e o f t h e in d e p e n d e n t e f f e c t o f l i g h t . On t h e c l o u d y e x p e r i m e n t a l day when t h e h e a t i n g e f f e c t o f t h e sun was m i n i m a l , l i g h t s t i l l a d v e r s e l y a f f e c t e d v i a b i l i t y , a l t h o u g h t h e r a t e o f v i a b i l i t y l o s s was r e t a r d e d . A l s o , s p o r e s s t o r e d i n t h e l i g h t i n a c o o l e n v ironment (5°C) l o s t t h e i r v i a b i l i t y q u i c k e r t h a n t h o s e s t o r e d a t t h e same t e m p e r a t u r e i n t h e d a r k . . - 302 -SUMMARY AND CONCLUSIONS The comandra b l i s t e r r u s t , C r o n a r t i u m comandrae, i s f a r more w i d e s p r e a d and damaging t o n a t i v e h a r d p i n e s i n w e s t e r n Canada t h a n was p r e v i o u s l y r e a l i z e d . The r u s t can be f o u n d i n most young and mature P i n u s c o n t o r t a and P. b a n k s i a n a s t a n d s t h r o u g h o u t t h e a r e a , o c c u r r i n g i n l o c a l e p i d e m i c s e s p e c i a l l y when c l o s e t o p a t c h e s o f Comandra u m b e l l a t a s s p . p a l l i d a i n s o u t h e r n a r e a s o r G e o c a u l o n l i v i d u m i n n o r t h e r n a r e a s . I n w e s t e r n Canada i t s d i s t r i b u t i o n e x t e n d s t o t h e p i n e l i m i t s i n t h e n o r t h , and p r o b a b l y i n t o t h e f o r e s t - t u n d r a s e c t i o n o f t h e b o r e a l f o r e s t (Rowe 1959) where G. l i v i d u m o c c u r s , s i m i l a r l y i t e x t e n d s a c r o s s t h e p r a i r i e s on C_. u m b e l l a t a . The damage caused by t h e r u s t has r e c e n t l y become o f economic i m p o r t a n c e on h a r d p i n e p l a n t a t i o n s w h i c h have been e s t a b l i s h e d i n l o c a t i o n s where t h e a l t e r n a t e h o s t s o c c u r . I n s u c h a r e a s , t h i s endemic r u s t no l o n g e r e x i s t s i n a s t a b l e community o f h o s t , p a r a s i t e and e n v i r o n -ment as i t does i n n a t i v e s t a n d s , and t h u s e p i d e m i c s can be e x p e c t e d . T h i s s h o u l d a c t as a w a r n i n g f o r f o r e s t management p r a c t i c e s I n w e s t e r n N o r t h A m e r i c a , f o r a l t h o u g h t h e a l t e r n a t e h o s t s a r e w i d e s p r e a d i n t h e i r d i s t r i b u t i o n , t h e i r a u t e c o l o g i c a l r e q u i r e m e n t s a r e q u i t e r e s t r i c t i v e . I f a r e a s f a v o u r a b l e f o r t h e a l t e r n a t e h o s t s can be a v o i d e d as p i n e p l a n t i n g s i t e s , t h e i n c i d e n c e o f t h e d i s e a s e c o u l d be k e p t t o a minimum. E r a d i -c a t i o n o f t h e a l t e r n a t e h o s t s i s not p r a c t i c a l b ecause o f t h e i r p e r e n n i a l r h i z o m a t o u s n a t u r e . I n n a t i v e s t a n d s , e s p e c i a l l y young o v e r s t o c k e d s t a n d s , t h e r u s t a c t s as a b e n e f i c i a l , b i o l o g i c a l , t h i n n i n g a g e n t . I f t h e p i n e stems a r e i n f e c t e d when young, under about 20 y e a r s , t h e r u s t i s a b l e t o g i r d l e t h e stem w i t h i n a few y e a r s t h e r e b y k i l l i n g t h e t r e e . I f t h e stem - 303 -i s n o t i n f e c t e d u n t i l t h e t r e e i s o l d e r t h e r u s t w i l l t a k e l o n g e r t o g i r -d l e t h e t r e e and t h e canker can a c t as a s o u r c e o f i n o c u l u m f o r many y e a r s I n most cases i n f e c t i o n was i n i t i a t e d t h r o u g h t h e n e e d l e s , e i t h e r d i r e c t l y i n t o t h e stem n e e d l e b e a r i n g t i s s u e s o r i n t o n e e d l e b e a r i n g b r a n c h e s . I n t h e l a t t e r t h e r u s t m y c e l i u m grew down t h e b r a n c h , and i f c l o s e enough t o the stem, was a b l e t o i n v a d e and i n f e c t t h e stem b e f o r e t h e b r a n c h was g i r d l e d and k i l l e d . Many stem c a n k e r s were t h e r e f o r e c e n t e r e d around t h e base s o f b r a n c h e s w h i c h a c t e d as t h e avenue o f i n f e c t i o n , and r a d i a t e d i n zones f r o m t h i s c e n t e r . U s u a l l y t h e canker d e v e l o p e d f a s t e r l o n g i t u d i n -a l l y i n t h e stem t h a n l a t e r a l l y , t h u s a canker was o f t e n t h r e e t i m e s as l o n g as i t was w i d e . The main p e r i o d o f a e c i o s p o r e p r o d u c t i o n o c c u r r e d f r o m l a t e May t o mid J u l y , b u t v a r i e d i n i t s d u r a t i o n d e p e n d i n g on weather c o n d i t i o n s . The e a r l i e s t o c c u r r e n c e r e c o r d e d o f a e c i o s p o r e s was May 13 and t h e l a t e s t on September 1. I n one y e a r a s m a l l second p e r i o d o f p r o d u c t i o n was ob-s e r v e d a t one l o c a t i o n d u r i n g a warm p e r i o d i n l a t e September and O c t o b e r , and o c c u r r e d i n t h e p y c n i a l zone o f t h e c u r r e n t y e a r . G e n e r a l l y t h e a e c i a p r o d u c t i o n o c c u r r e d o v e r t h e zone o f t h e canker w h i c h p r o d u c e d p y c n i a and p y c n i o s p o r e s t h e p r e v i o u s y e a r . On each canker t h e p e r i p h e r a l zone was o c c u p i e d b y t h e a d v a n c i n g r u s t m y c e l i u m and t h e p y c n i a l a r e a . The a e c i a l zone was w i t h i n t h e p y c n i a l zone and t h e c e n t e r o f t h e canker c o n s i s t e d o f o l d e r zones where t h e b a r k was rough, c r a c k e d and dead. The a e c i a l zone on a canker may be s e v e r a l i n c h e s wide t h u s v e r y l a r g e numbers o f a e c i o s p o r e s were p r o d u c e d each y e a r i f t h e whole a e c i a l zone r e m a i n e d p r o -d u c t i v e . There were many, i n d i v i d u a l a e c i a w h i c h a t t i m e s appeared t o c o a l e s c e t o f o r m s e v e r a l i r r e g u l a r a e c i a . These a e c i a v a r i e d i n s i z e b u t - 304 -an-.average aecium was a b l e t o p r o d u c e a e c i o s p o r e s f o r a p e r i o d o f 35 t o 50 days. As a l l a e c i a d i d not mature a t t h e same t i m e , an i n d i v i d u a l canker c o u l d p r o d u c e a e c i o s p o r e s f o r up t o 95 days. G e n e r a l l y t h e a e c i a c l o s e r t o t h e o l d a e c i a l zone p r o d u c e d a e c i o s p o r e s f i r s t and f o r a l o n g e r p e r i o d as t h e y were u s u a l l y t h e l a r g e r a e c i a . . A e c i a a d j a c e n t t o t h e p y c n i a l zone may not i n i t i a t e s p o r u l a t i o n u n t i l two weeks o r more a f t e r t h e more c e n t r a l a e c i a . A e c i a on t h i c k - b a r k e d t r e e s may a l s o be l a t e r t h a n on t h i n - b a r k e d t r e e s , b u t t e n d e d t o p r o d u c e a e c i o s p o r e s f o r a l o n g e r p e r i o d . A number o f f a c t o r s p r e v e n t e d a r e g u l a r a n n u a l i n c r e a s e i n t h e number o f a e c i a p r o d u c e d on a canker and t h e subsequent i n c r e a s e and d i s -p e r s a l o f a e c i o s p o r e s . I n most c a s e s , a e c i a l p r o d u c t i o n was l i m i t e d b y t h e a c t i v i t y o f o t h e r f u n g i , i n s e c t s and r o d e n t s on t h e c a n k e r , and by r e s i n o s i s i n t h e t r e e . Rodent damage and a s s o c i a t e d r e s i n p r o d u c t i o n p r o -b a b l y most a f f e c t e d a e c i o s p o r e p r o d u c t i o n . F r e s h r o d e n t damage o c c u r r e d on 72$ o f t h e c a n k e r s under o b s e r v a t i o n , o v e r a wide a r e a o f s outhwest A l b e r t a f r o m 1966 t o 1968. A t many o f t h e l o c a t i o n s i t was d i f f i c u l t t o f i n d an a c t i v e c a n k er t h a t had e s c a p e d r o d e n t damage, and most o f t h o s e t h a t had escaped were on v e r y s m a l l t r e e s i n open a r e a s where r o d e n t popu-l a t i o n s , e s p e c i a l l y s q u i r r e l s , were l o w e r . Other undamaged c a n k e r s were m a i n l y i n a c t i v e and a p p a r e n t l y no l o n g e r a t t r a c t i v e t o t h e r o d e n t s . The r o d e n t s , w h i c h i n c l u d e d h a r e s , p o r c u p i n e s , p o s s i b l y chipmunks and m i c e , were a t t r a c t e d l a r g e l y t o t h e p y c n i a l zone o f t h e c a n k e r , b u t a l s o r e -moved t h e c u r r e n t a e c i a l zone. Rodent gnawing t o o k p l a c e a t any t i m e o f t h e y e a r , b u t r e a c h e d a peak i n l a t e w i n t e r and e a r l y s p r i n g , o c c u r r i n g a n n u a l l y on some c a n k e r s . On many o c c a s i o n s t h e whole o f t h e p y c n i a l and - 305 -a e c i a l zones were removed; as a r e s u l t c a n k e r s were n o n - s p o r u l a t i n g , and t h e r u s t m y c e l i u m o n l y d e v e l o p e d i n i s o l a t e d p o c k e t s o r p a t c h e s around t h e p e r i p h e r y o f t h e canker where t h e a d v a n c i n g m y c e l i u m had n o t been r e -moved. The r o d e n t chewing a l s o t e n d e d t o h a s t e n t h e g i r d l i n g o f t h e t r e e a d v a n c i n g i t s d e a t h b y a y e a r o r two. R e s u l t i n g f r o m t h e r o d e n t damage l a r g e amounts o f r e s i n were p r o d u c e d , w h i c h , w i t h t h e r e s i n o s i s p r o d u c e d by t h e t r e e i n r e s p o n s e t o t h e r u s t , formed a c r u s t o v e r p a r t s o f t h e canker p r e v e n t i n g a e c i o s p o r e d i s p e r s a l on o c c a s i o n s . The r e s i n a l s o i m p r e g n a t e d t h e b a r k and o u t e r sapwood. S i x t y - f o u r m i c r o f l o r a l o r g a n i s m s were i s o l a t e d f r o m a e c i o s p o r e c o l l e c t i o n s o r d i r e c t l y f r o m t h e a e c i a l zone o f t h e c a n k e r . T h i s i n c l u d e d 8 b a c t e r i a and 56 f u n g i , i n c l u d i n g 5 y e a s t s o r y e a s t - l i k e f u n g i . Many o f t h e s e o r g a n i s m s h e l p e d t o r e d u c e t h e numbers o f v i a b l e a e c i o s p o r e s t h a t were p r o d u c e d , and many o t h e r s p r o b a b l y p l a y no such r o l e b e i n g s i m p l y members o f t h e a i r s p o r a d e p o s i t e d by chance on t h e c a n k e r s . Two or g a n i s m s a p p e a r e d t o p l a y a r e g u l a r r o l e i n r e d u c i n g a e c i o s p o r e p r o d u c t i o n ; t h e p u r p l e mold, T u b e r c u l i n a maxima, and an u n d e s c r i b e d d a r k g r e e n mold, C l a -d o s p o r i u m t a x . sp. 1. T u b e r c u l i n a was f o u n d p a r a s i t i c on t h e r u s t c a n k e r , most a b u n d a n t l y i n t h e a e c i a l zone, b u t a l s o i n t h e p y c n i a l zone. Char-a c t e r i s t i c a l l y , T. maxima k i l l e d t h e c a n k e r e d b a r k a f t e r i n v a s i o n and g r e a t l y r e d u c e d o r e n t i r e l y i n h i b i t e d t h e p r o d u c t i o n o f a e c i o s p o r e s where i t oc-c u r r e d . I t a l s o t e n d e d t o i n d u c e more r e s i n o s i s t h a n d i d t h e r u s t a l o n e . T u b e r c u l i n a had n o t been f o u n d on t h e C r o n a r t i u m ( P e r i d e r m i u m ) r u s t s i n A l -b e r t a u n t i l t h i s s t u d y , a l t h o u g h known on some r u s t s f r o m B r i t i s h C o l u m b i a and t h e n o r t h w e s t U n i t e d S t a t e s . T. maxima has now been r e c o r d e d on a l l C r o n a r t i u m ( P e r i d e r m i u m ) r u s t s i n w e s t e r n Canada, a l t h o u g h t h e r e have been - 306 -no c o l l e c t i o n s i n Saskatchewan, M a n i t o b a and t h e Yukon. T u b e r c u l i n a was p r e s e n t on 13 t o 2k% o f a l l c a n k e r s o b s e r v e d i n any one y e a r i n t h i s s t u d y , and a t some l o c a t i o n s t h e i n c i d e n c e was c l o s e t o 50%. T h i s p e r c e n t a g e w o u l d have been even h i g h e r i f o n l y a c t i v e c a n k e r s were c o n s i d e r e d , as no T u b e r c u l i n a i n f e c t i o n was e v e r n o t e d on i n a c t i v e c a n k e r s . I n each y e a r a p p r o x i m a t e l y a t h i r d o f t h e c a n k e r s were c l a s s i f i e d as i n a c t i v e . Many cases were o b s e r v e d where t h e p o t e n t i a l a e c i a l zone was w h o l l y i n f e c t e d by T u b e r c u l i n a . U s u a l l y , t h e f o l l o w i n g y e a r , t h e s e c a n k e r s were e n t i r e l y i n a c t i v e . Sometimes o n l y a p e r c e n t a g e o f t h e aecia! zone was i n f e c t e d one y e a r , and t h e f o l l o w i n g y e a r a e c i o s p o r e s were p r o d u c e d , b u t t h e r e was always a r e d u c t i o n i n t h e amount o f a e c i a l p r o d u c t i o n . T u b e r c u l i n a was t h e r e f o r e a f a i r l y s l o w i n v a d e r o f C_. comandrae, b u t once e s t a b l i s h e d i n a l o c a t i o n i t was an e f f e c t i v e b i o l o g i c a l c o n t r o l agent o f t h e c a n k e r . C l a d o s p o r i u m t a x . sp. 1 d i d not p l a y such an i m p o r t a n t r o l e i n d e s t r o y i n g t h e i n f e c t e d t i s s u e s o f t h e c a n k e r , b u t may a f f e c t t h e v i a b i l i t y o f a e c i o s p o r e s upon w h i c h i t may be p a r a s i t i c . C l a d o s p o r i u m t a x . sp. 1 o f t e n o v e r r a n t h e whole s p o r u l a t i n g s u r f a c e o f t h e c a n k e r , b u t u n l i k e T u b e r c u l i n a , f a i l e d t o i n a c t i v a t e t h e c anker and was o f t e n p r e s e n t f o r a number o f y e a r s . I t s i n c i d e n c e was about h a l f t h a t o f T u b e r c u l i n a , and n e a r l y h a l f t h e C l a d o s p o r i u m i n f e c t i o n s o c c u r r e d on T u b e r c u l i n a i n f e c t e d c a n k e r s . O t h e r o r g a n i s m s t h a t were o b s e r v e d i n f e c t i n g t h e a c t i v e c a n k e r , b u t o n l y i n f r e q u e n t l y , were F u s a r i u m sp., M o n o c i I l i u m sp., C o n i o t h y r i u m o l i v a c e u m , S c l erophoma p i t h y o p h i l a and a number o f P e n i c i l l i u m spp. A few s e c o n d a r y o r g a n i s m s , i n c l u d i n g L a c h n e l l u l a a r i d a , were p r e s e n t on o l d e r p o r t i o n s o f c a n k e r s . A l t e r n a r i a t e n u i s , E p i c o c c u m nigrum, S p o r o -bolomyces, R h o d o t o r u l a and s e v e r a l b a c t e r i a , were o c c a s i o n a l l y i s o l a t e d f r o m t h e c u r r e n t y e a r a e c i a l zone, and more f r e q u e n t l y f r o m a e c i o s p o r e s , - 307 -b u t t h e i r r o l e , i f any, was unknown. A l a r g e number o f i n s e c t s , m i t e s and s p i d e r s (117 s p e c i e s ) were c o l l e c t e d o r r e a r e d f r o m C. comandrae c a n k e r s . A t l e a s t t h r e e o f t h e s e appeared t o be t r u e m y c e t o b i o n t s w h i c h were dependent on t h e r u s t f o r t he development o f p a r t o r a l l o f t h e i r l i f e c y c l e . These were a n i t i d u l i d b e e t l e , E p u r a e a o b l i q u u s , an u n i d e n t i f i e d c e c i d o m y i i d a e f l y , and a d r o s o p h i l i d f l y , P a r a c a c o x e n u s g u t t a t u s . F o r t h e two named s p e c i e s t h i s r e p r e s e n t e d t h e f i r s t t i m e a n y t h i n g had been known o f t h e i r h a b i t a t o r immature s t a g e s . E p u r a e a a d u l t s were p r e s e n t on t h e a e c i a l c a n k e r f r o m t h e b e g i n n i n g o f a e c i a s p o r u l a t i o n , and even b e f o r e , as b e e t l e s were f o u n d i n u n r u p t u r e d a e c i a . The b e e t l e s , and l a t e r t h e l a r v a e , p r e s e n t f r o m m i d-June onwards, f e d e x c l u s i v e l y on t h e a e c i o s p o r e s d u r i n g t h i s p e r i o d and consumed l a r g e q u a n t i t i e s o f t h e s p o r e s , so t h a t t h e canker became a mass o f f a e c a l p e l l e t s l o d g e d i n c r e v i c e s o f t h e canker and a t t h e base o f t h e t r e e . W i t h t h e appearance o f p y c n i a l d r o p s t h e l a r v a e a l s o f e d e x t e n s i v e l y on t h e s e , moving f r o m one t o a n o t h e r . A f t e r August l a r v a e were not common on t h e c a n k e r s and a p p a r e n t l y o v e r w i n t e r e d i n s o i l o r d u f f . The l a r v a e o f t h e two d i p t e r a s p e c i e s a l s o f e d e x t e n s i v e l y on t h e two spo r e s t a t e s c a u s i n g some damage t o t h e b a r k . A d u l t P a r a c a c o x e n u s were r e a r e d f r o m c a n k e r s and were a l s o t r a p p e d w h i l e v i s i t i n g p y c n i a l d r o p s . I t i s l i k e l y t h a t t h e eggs were l a i d i n t h e p y c n i a l zone. P r o b a b l y t h e u n i d e n t i f i e d c e c i d o m y i i d a e had a s i m i l a r l i f e c y c l e . The i n c i d e n c e o f i n s e c t damage o f one k i n d and a n o t h e r t o t h e canker o r t h e a e c i o s p o r e s r a n g e d f r o m 32 t o 59$ i n any one y e a r . A t many l o c a t i o n s l e v e l s o f o v e r 60$ were r e c o r d e d e v e r y y e a r . Much o f t h i s f l u c -t u a t i o n depended on t h e age o f t h e t r e e s and t h e a s s o c i a t e d r o d e n t damage, - 308 -f o r t h e l a t t e r t e n d e d t o d e s t r o y t h e i n s e c t s and much o f t h e s u i t a b l e h a b i t a t . The i n s e c t s p r o b a b l y c a u s i n g most damage t o t h e a c t i v e canker zones, were t h e moth, D i o r y c t r i a spp., and t h e w e e v i l s , C y l i n d r o c o p t u r u s d e l e o n i and P i s s o d e s s c h w a r z i . The two w e e v i l s were p r o b a b l y o n l y s e c o n d -a r y i n s e c t s w h i c h a t t a c k e d t r e e s a l r e a d y weakened by t h e r u s t , b u t i n so d o i n g t h e y s e v e r e l y s c o r e d t h e sapwood f o r t h e i r f e e d i n g and p u p a l cham-b e r s , t h e r e b y h a s t e n i n g t h e d e a t h o f t h e t r e e by one o r more y e a r s . The D i o r y c t r i a l a r v a e f e d v e r y e x t e n s i v e l y on t h e b a r k and o u t e r sapwood d e s -t r o y i n g much o f t h e t i s s u e s i n f e c t e d by t h e r u s t ; t h e i r c h a r a c t e r i s t i c f r a s s m a t e r i a l hung i n l a r g e l o o s e l y h e l d a g g r e g a t e s on t h e s u r f a c e o f t h e c a n k e r . Other L e p i d o p t e r a l a r v a e , L a s p r e y r e s i a sp. and C o l e o t e c h n i t e s ( R e c u r v a r i a ) spp., a l s o caused some damage t o t h e c a n k e r . S e v e r a l o t h e r s p e c i e s were c o n s i s t e n t l y r e a r e d f r o m c a n k e r s and p r o b a b l y p l a y e d some r o l e i n d e s t r o y i n g t h e c a n k e r , b u t t h e y were p r o b a b l y m y c e t o p h i l e s o r even mycetoxenes, w h i c h c o u l d use t h e ca n k e r b u t were not dependent upon i t . Among t h e s e s p e c i e s were t h e c o l e o p t e r a , A t h e t a , E r n o b i u s , C o r t i c a r i a and o t h e r L a t h r i d i i d a e , v a r i o u s d i p t e r a , i n c l u d i n g B r a d y s i a spp., c o l l e m b o l a , p s o c o p t e r a , C i n a r a a p h i d s p e c i e s , and v a r i o u s m i t e s i n c l u d i n g s p e c i e s o f A n d r o l a e l a p s , H i s t i o g a s t e r and a new s p e c i e s o f C e r a t o z e t e s . Many s p e c i e s o f hymenoptera were a l s o r e a r e d , b u t most o f t h e s e were p r o b a b l y p a r a s i t e s o f t h e o t h e r i n s e c t s p e c i e s p r e s e n t i n t h e c a n k e r s . T o g e t h e r , t h e s e b i o l o g i c a l a g e n t s p r e v e n t e d t h e p r o d u c t i o n o f a l a r g e volume o f a e c i a l m a t e r i a l . The r o d e n t s were r e s p o n s i b l e f o r gnawing about 30$ o f t h e p o t e n t i a l a e c i a l p r o d u c i n g t i s s u e s i n any one y e a r , and t h i s p e r c e n t a g e w o u l d be much h i g h e r i n s t a n d s where t h e r o d e n t p o p u l a t i o n was abundant, o r when o t h e r s o u r c e s o f f o o d were r e s t r i c t e d . The m i c r o -- 309 -f l o r a l a g e n t s , e s p e c i a l l y T u b e r c u l i n a , d e s t r o y e d p o t e n t i a l a e c i a l p r o d u c i n g t i s s u e s , and were a l s o p r o m i n e n t c o n t a m i n a n t s r e d u c i n g a e c i o s p o r e , : . v i a b i l i t y , and p r o b a b l y a c c o u n t e d f o r a f u r t h e r 5 t o 10% r e d u c t i o n . Some o f t h e i n -s e c t s , n o t a b l y D i o r y c t r i a spp. and some o f t h e c o l e o p t e r a , d e s t r o y e d po-t e n t i a l a e c i a l p r o d u c i n g t i s s u e s , and many i n s e c t s a t e t h e a e c i o s p o r e s t h a t were p r o d u c e d o r e f f e c t i v e l y p r e v e n t e d t h e i r d i s p e r s a l . These• m i c r o -f a u n a l agents p r o b a b l y a c c o u n t e d f o r a n o t h e r 10% r e d u c t i o n i n t h e amount o f a e c i a o r a e c i o s p o r e s p r o d u c e d . R e s i n o s i s i n t e r f e r e d w i t h p r o d u c t i o n o f a e c i a when t h e b a r k became i m p r e g n a t e d w i t h r e s i n , and t h e f l o w o f r e s i n on t h e s u r f a c e o f t h e canker p r e v e n t e d r u p t u r e o f a e c i a and d i s p e r -s a l o f many a e c i o s p o r e s . R e d u c t i o n o f a e c i a l p r o d u c t i o n f r o m r e s i n damage p r o b a b l y a c c o u n t e d f o r a f u r t h e r 5%. Thus i n any one y e a r i n t h e s t u d y a r e a , p r o b a b l y 50 t o 55% o f t h e p o t e n t i a l a e c i a l m a t e r i a l was d e s t r o y e d b y t h e s e b i o l o g i c a l a g e n t s . I n some y e a r s and a t some l o c a t i o n s t h i s p e r c e n t a g e was much h i g h e r . Rodent damage was o b s e r v e d a t a l l 23 l o c a -t i o n s under o b s e r v a t i o n d u r i n g t h e s t u d y , T u b e r c u l i n a was n o t o b s e r v e d a t s i x l o c a t i o n s and no i n s e c t damage was r e c o r d e d a t one l o c a t i o n , b u t r e s i n o s i s was common everywhere. D e s p i t e t h i s enormous r e d u c t i o n , v i a b l e a e c i o s p o r e s were s t i l l p r o d u c e d and d i s p e r s e d i n v e r y l a r g e numbers. There was a sudden r i s e i n t h e d a i l y s p o r e r e l e a s e i n l a t e May when t h e m a j o r i t y o f mature a e c i a r u p t u r e d s i m u l t a n e o u s l y . T h i s h i g h l e v e l o f spor e d i s p e r s a l was m a i n t a i n e d f o r two o r t h r e e weeks, f o l l o w e d b y a g r a d u a l d e c r e a s e t o a low l e v e l b y mid- J u l y w h i c h sometimes c o n t i n -ued t o t h e end o f A u g u s t . M i n o r peaks o c c u r r e d t h r o u g h o u t t h e spor e p r o -d u c t i o n season, a f t e r t h e i n i t i a l h i g h peak, due t o t h e r u p t u r e o f new a e c i a b u t t h e s e o n l y l a s t e d a few d a y s . I n some seasons o r p e r i o d s t h e - 310 -s p o r e r e l e a s e p a t t e r n was more a f f e c t e d by weather c o n d i t i o n s . L i t t l e o r no d i s p e r s a l o c c u r r e d d u r i n g a l o n g wet p e r i o d , b u t a s e r i e s o f d r y sunny days r e s u l t e d i n a t y p i c a l d i u r n a l r e l e a s e o f s p o r e s . G e n e r a l l y t h e d i u r n a l s p o r e p e r i o d i c i t y p a t t e r n f e l l i n t o a f o r e n o o n o r a f t e r n o o n maximum w i t h a minimum a t n i g h t w h i c h i s t y p i c a l o f t h e m a j o r i t y o f t h e dayt i m e d r y spore f o r m s . T h i s s t u d y was t h e f i r s t g i v i n g d i u r n a l p e r i -o d i c i t y d a t a f o r t h e a e c i o s p o r e r u s t s t a t e and showed a peak s p o r e con-c e n t r a t i o n between 1000 and 1600 h o u r s , w i t h some e v i d e n c e o f a d o u b l e o r s e c o n d a r y peak w i t h a minor midday minimum. There was c o n s i d e r a b l e v a r i a t i o n between c a n k e r s i n t h e h o u r l y and d a i l y s p o r e c o n c e n t r a t i o n s r e l e a s e d . Peak h o u r l y c o n c e n t r a t i o n f r o m one canker was 128,000 s p o r e s / m8 and t h e maximum d a i l y c o n c e n t r a t i o n was 17,217 spores/m^, b u t on 5^ t o 63$ o f t h e days i n I966 and 1967 fewer t h a n 100 spores/m^ were c o l -l e c t e d . There was an ap p a r e n t c l o s e r e l a t i o n s h i p between s p o r e d i s p e r s a l p e r i o d i c i t y and t e m p e r a t u r e and r e l a t i v e h u m i d i t y . T u r b u l e n c e appeared more i m p o r t a n t f o r d a i l y s p o r e d i s p e r s a l , t h a n e i t h e r t e m p e r a t u r e o r h u m i d i t y i n d i v i d u a l l y , a l t h o u g h b o t h o f t h e s e do i n f l u e n c e t u r b u l e n c e . T u r b u l e n t a t m o s p h e r i c c o n d i t i o n s were a s s o c i a t e d w i t h a l l peak a e c i o s p o r e c o n c e n t r a t i o n s whether t h e y o c c u r r e d i n t h e f o r e n o o n o r a f t e r n o o n , on d r y sunny days, o r a s s o c i a t e d w i t h t h e o n s e t o f heavy r a i n showers. L i t -t l e o r no d i s p e r s a l o c c u r r e d a t t e m p e r a t u r e s b e l o w 7°C, b u t peak d i s p e r -s a l o c c u r r e d when t e m p e r a t u r e s were above 20°C, when a i r h u m i d i t y l e v e l s were a t t h e i r l o w e s t . G u s t y heavy r a i n s t o r m s i n i t i a l l y i n c r e a s e d s p o r e c o n c e n t r a t i o n s b y j a r r i n g t h e i n f e c t e d t i s s u e s w h i c h r e l e a s e d t h e s p o r e s , o r t h r o u g h r a i n s p l a s h . One heavy ^ i n c h r a i n f a l l o c c u r r i n g a f t e r t h e norm a l d a i l y d i s p e r s a l p e r i o d b r o u g h t a 25 t i m e s i n c r e a s e i n t h e h o u r l y - 311 -s p o r e c o n c e n t r a t i o n and a c o n c e n t r a t i o n 5 t i m e s l a r g e r t h a n t h e " n o r m a l " peak f o r t h a t day. L i g h t r a i n s d i d not have t h e same e f f e c t , p r o b a b l y b ecause t h e r e was l e s s t u r b u l e n c e and t h e r a i n d r o p s t e n d e d t o wash s p o r e s out o f the atmosphere and g r a d u a l l y wet t h e i n f e c t e d s u r f a c e s p r e v e n t i n g any r e m o v a l o f s p o r e s i n t o t h e a i r . Dew had l i t t l e e f f e c t , e x c e p t t h a t i t t e n d e d t o d e l a y t h e d a i l y i n i t i a t i o n o f a e c i o s p o r e d i s p e r s a l on some days. Wind, was an i m p o r t a n t f a c t o r f o r spore r e l e a s e , as i t i s n e c e s s a r y t o move t h e p a s s i v e l y l i b e r a t e d s p o r e s a c r o s s t h e l a m i n a r boun-d a r y l a y e r i n t o t h e g e n e r a l c i r c u l a t i o n o f t u r b u l e n t a i r . The h i g h e r t h e w i n d speed t h e more e a s i l y s p o r e s were p i c k e d up f r o m t h e s p o r u l a t i n g s u r f a c e b y t u r b u l e n t e d d i e s . G e n e r a l l y a w i n d speed o f 1,1 mph was r e -q u i r e d f o r c o n s i d e r a b l e i n i t i a l a e c i o s p o r e d i s p e r s a l t o t a k e p l a c e f r o m t h e c a n k e r , b u t s p o r e s c o n t i n u e d t o be d i s p e r s e d a t l o w e r w i n d speeds. There was no c l o s e c o r r e l a t i o n between h i g h w i n d speeds and h i g h s p o r e c o n c e n t r a t i o n s r e l e a s e d f r o m t h e c a n k e r s , as e q u a l l y h i g h c o n c e n t r a t i o n s o c c u r r e d a t much l o w e r w i n d speeds. I n t h e p r e s e n t s t u d y , no s p o r e s were c o l l e c t e d a t d i s t a n c e s g r e a t e r t h a n kOO f e e t f r o m a s o u r c e . T h i s may have been a case o f an i n -adequate s a m p l i n g network a t g r e a t e r d i s t a n c e s , s i n c e i t i s e x p e c t e d t h a t s p o r e s c o u l d be d i s p e r s e d t o such d i s t a n c e s b y eddy d i f f u s i o n , a l t h o u g h d i s p e r s a l d i s t a n c e would be r e d u c e d i n a f o r e s t . The a r e a was n o t s u i t -a b l e f o r l o n g d i s t a n c e s t u d i e s due t o t h e p o s s i b l e p r e s e n c e o f b a c k g r o u n d sp o r e c o n t a m i n a t i o n . The l i t e r a t u r e g i v e s e v i d e n c e o f g r e a t e r d i s t a n c e d i s p e r s a l f o r s m a l l e r s i z e d a e c i o s p o r e s and o t h e r s p o r e s t a t e s . The p a t -t e r n o f d i s p e r s a l and d e p o s i t i o n on t h e ground n e a r t o a s o u r c e showed a - 312 -pronounced g r a d i e n t , w i t h a h o l l o w c u r v e , t h a t i s , t h e g r a d i e n t o f de-p o s i t i o n near t h e s o u r c e was v e r y s t e e p . S t u d i e s o f d e p o s i t i o n around a n a t u r a l s o u r c e one t o t h r e e f e e t above t h e ground i n d i c a t e d t h a t a t d i s -t a n c e s o v e r 6 f e e t , l e s s t h a n 1$ o f t h e c o n c e n t r a t i o n a t t h e f i r s t sam-p l i n g p o i n t (3 i n c h e s ) was d e p o s i t e d , and t h a t t h e r e was a 90$ r e d u c t i o n w i t h i n 2 f e e t . S i m i l a r r e d u c t i o n s i n s p o r e c o n c e n t r a t i o n w i t h d i s t a n c e were r e c o r d e d i n e x p e r i m e n t a l r e l e a s e s from p o i n t s o u r c e s 1 o r 5 f e e t above ground. W i t h a r e l e a s e f r o m 5 f e e t , t h e maximum c o n c e n t r a t i o n a t 1 f o o t above ground was n o t f o u n d a t t h e f i r s t s a m p l i n g p o i n t a t a d i s -t a n c e o f 5 f e e t , as o c c u r r e d w i t h a 1 f o o t r e l e a s e , b u t a t 10 o r 20 f e e t f r o m t h e s o u r c e . Spore c o n c e n t r a t i o n s a t g r e a t e r s a m p l i n g d i s t a n c e s were l o w e r a t low w i n d speeds t h a n a t h i g h w i n d speeds. These s t e e p g r a d i e n t s o f s p o r e c l o u d c o n c e n t r a t i o n and s p o r e d e p o s i t i o n agree w i t h eddy d i f -f u s i o n t h e o r y , w h i c h p r e d i c t s f a i r l y r a p i d d e p l e t i o n near t h e s o u r c e , and w i t h e x p e r i m e n t a l s t u d i e s o f s p o r e s , p a r t i c u l a t e and gaseous d e p o s i t i o n , and o b s e r v e d d i s e a s e g r a d i e n t s . Even i f no d e p o s i t i o n o c c u r r e d i t w o u l d be e x p e c t e d t h a t t h e spo r e c o n c e n t r a t i o n would- v a r y i n p r o p o r t i o n t o t h e d i s t a n c e f r o m t h e s o u r c e . As s p o r e s a r e r e l e a s e d f r o m a p o i n t s o u r c e near t h e ground i n l i g h t w i n d s , the. s p o r e c o n c e n t r a t i o n d i f f u s e s h o r i z o n t a l l y f a s t e r t h a n / v e r t i c a l l y t o f o r m a n e e i l i p s e , t h e base o f w h i c h l o s e s s p o r e s s t e a d i l y b y v a r i o u s t y p e s o f d e p o s i t i o n as i t p r o c e e d s . The r a t e o f f a l l o f a spo r e o r s e d i m e n t a t i o n i s an i m p o r t a n t f a c t o r h e l p i n g t o d e t e r m i n e t h e d i s t a n c e o f d i s p e r s a l , f o r i f t h e r a t e o f f a l l i s g r e a t e r t h a n t h e average upward a i r speed t h e n s p o r e s w i l l r e t u r n t o t h e s u r f a c e w i t h i n a d i s t a n c e not many t i m e s t h e h e i g h t a t w h i c h t h e y a r e r e l e a s e d . C. coman-drae a e c i o s p o r e s had an average v e l o c i t y o f 3.23 cm/sec i n calm a i r , - 313 -w h i c h was s l i g h t l y f a s t e r t h a n e x p e c t e d f o r t h e s i z e o f s p o r e . I f t h e spo r e s were wet t h e speed o f f a l l was two t o t h r e e t i m e s f a s t e r . Thus d r y mature s p o r e s r e l e a s e d f r o m a canker one f o o t above.ground wou l d t a k e about 10 seconds t o f a l l t h i s d i s t a n c e i n calm a i r , and t h e l i g h t 1 t o 2.5 mph winds a t t h i s l e v e l i n t h e f o r e s t w o u l d n o t be e x p e c t e d t o c a r r y t h e s p o r e s f a r from t h e s o u r c e . T h e r e f o r e , under normal c o n d i t i o n s a s u b s t a n t i a l p e r c e n t a g e o f t h e s p o r e s l i b e r a t e d n ear t h e ground w i l l be d e p o s i t e d w i t h i n a r e l a t i v e l y s h o r t d i s t a n c e o f t h e s o u r c e . However, a f r a c t i o n o f t h e s p o r e s w i l l escape d e p o s i t i o n near t h e s o u r c e and w i l l be c a r r i e d t o g r e a t e r h e i g h t s i n t h e atmosphere by m e c h a n i c a l and t h e r m a l t u r b u l e n c e . E v i d e n c e from t h e s p o r e r e l e a s e e x p e r i m e n t s i n d i c a t e d t h a t some C. comandrae s p o r e s were e s c a p i n g beyond t h e s a m p l i n g network. The chances f o r l o n g d i s t a n c e d i s p e r s a l w o u l d be h i g h e s t on warm d r y sunny days when t u r b u l e n c e was a t a maximum, and a s p o r e c l o u d w o u l d t e n d t o move i n a s e r i e s o f l o o p s . The p r i n c i p l e mechanisms i n v o l v e d i n d e p o s i t i o n o f s p o r e s a r e s e d i m e n t a t i o n under t h e i n f l u e n c e o f g r a v i t y , i m p a c t i o n i n c l u d i n g t u r b u -l e n t d o w n d r a f t d e p o s i t i o n , and wash o u t o f t h e atmosphere by r a i n . Of t h e s e , i m p a c t i o n g e n e r a l l y p l a y s t h e g r e a t e s t r o l e i n t h e open, e s p e c i a l l y a t h i g h w i n d speeds, and t h e l a r g e r t h e s p o r e t h e g r e a t e r t h e chance o f i m p a c t i o n . However, i n t h e f o r e s t where w i n d speeds and t u r b u l e n c e a r e g r e a t l y r e d u c e d a s p o r e c l o u d w i l l undergo r a p i d d e p l e t i o n b y s e d i m e n t a -t i o n as i t t r a v e l s f r o m t h e s o u r c e , w h i c h h e l p s e x p l a i n t h e v e r y s t e e p g r a d i e n t ' o f d e p o s i t i o n . The spore c l o u d w i l l f u r t h e r be d e p l e t e d b y im-p a c t i o n , as t h e a e c i o s p o r e s impact on t h e v e g e t a t i o n , and i n t h i s r e s p e c t t h e p i n e n e e d l e s w i l l a c t as b e t t e r i m p a c t o r s o f l a r g e s p o r e s t h a n b r o a d - 314 -l e a f v e g e t a t i o n . I n a s t a n d w i t h more open t r u n k space and a h i g h canopy, th e s p o r e s w i l l e n c o u n t e r l e s s f o l i a g e and stems, and d e p l e t i o n o f t h e spore c o n c e n t r a t i o n w i l l be s l o w e r . I f s p o r e s "escape" f r o m a f o r e s t s t a n d t h e n t h e y w i l l be c a r r i e d o v e r g r e a t e r d i s t a n c e s o r d e p l e t e d l e s s r a p i d l y because o f the i n c r e a s e o f wi n d v e l o c i t y and t u r b u l e n c e , c a u s i n g d e p o s i t i o n b y i m p a c t i o n t o become more i m p o r t a n t t h a n s e d i m e n t a t i o n . E n v i r o n m e n t a l f a c t o r s were a s s e s s e d f o r t h e i r i n d i v i d u a l e f f e c t on a e c i o s p o r e g e r m i n a t i o n . A e c i o s p o r e s g e r m i n a t e d o v e r the t e m p e r a t u r e range 1 t o 30°C, b u t g e r m i n a t i o n a t t h e extremes was low and on most o c -c a s i o n s t h e r e was no g e r m i n a t i o n a t 30°C. Optimum g e r m i n a t i o n o c c u r r e d c l o s e t o 15°C, w i t h good g e r m i n a t i o n o c c u r r i n g o v e r t h e range 5 t o 20°C. One t o s i x germ t u b e s were p r o d u c e d p e r s p o r e , w i t h an average o f two. The maximum number o f germ t u b e s o c c u r r e d a t t h e optimum t e m p e r a t u r e , w i t h fewer b e i n g p r o d u c e d a t t h e h i g h e r t e m p e r a t u r e s . G e n e r a l l y o n l y one germ tube d e v e l o p e d t o any l e n g t h ( a v e r a g e 400u a f t e r 24 h o u r s ) , and was l o n g e s t a t t h e optimum t e m p e r a t u r e , and s h o r t e s t a t t h e extremes. The degree o f germ tube b r a n c h i n g v a r i e d l i t t l e w i t h t e m p e r a t u r e s , b r a n c h e s were s h o r t , r a r e l y more t h a n 50uJ a r i d t h e germ t u b e r e m a i n e d n o n - s e p t a t e and b i n u c l e a t e . G e r m i n a t i o n o c c u r r e d w i t h i n 1^ hours and most s p o r e s had g e r m i n a t e d b y 4 t o 5 h o u r s . Most r a p i d g e r m i n a t i o n o c c u r r e d a t 20°C, and was s l o w e s t a t 2 and 25°C. I n i t i a l germ tube growth was a l s o f a s t e s t a t 20°C and s l o w e s t a t 2 t o 5°C. The gr o w t h c u r v e s a t most t e m p e r a t u r e s t e n d e d t o l e v e l o f f a f t e r about 6 h o u r s , a l t h o u g h growth a t 25°C l e v e l l e d o f f e a r l i e r . There was l i t t l e i n c r e a s e i n germ tube l e n g t h a f t e r 24 h o u r s , and on o c c a s i o n s , an a p p r e s s o r i u m w i t h an i n f e c t i o n peg was formed i f c o n d i t i o n s were s u i t a b l e . I f t h e t i p o f t h e germ t u b e came i n c o n t a c t - 315 -w i t h t h e media, p l a s m o p t y s i s o f t e n o c c u r r e d , and was more common a t t h e optimum and s u p r a o p t i m a l t e m p e r a t u r e s . F r e s h s p o r e s g e n e r a l l y g e r m i n a -t e d f a s t e r t h a n s t o r e d s p o r e s , and t h e t e m p e r a t u r e range and optimum f o r g e r m i n a t i o n were sometimes d i f f e r e n t , t h u s s p o r e h i s t o r y i s i m p o r t a n t i n such s t u d i e s . F r e e w a t e r was n e c e s s a r y f o r g e r m i n a t i o n as no g e r m i n a t i o n was o b t a i n e d i n s a t u r a t e d a i r o r a t a r e l a t i v e h u m i d i t y b e l o w 100%. S w e l l i n g o f d r y s p o r e s t o o k p l a c e i m m e d i a t e l y when s p o r e s were p l a c e d on a l i q u i d medium, and was a p r e r e q u i s i t e f o r i n i t i a t i o n o f a germ t u b e . The p r o -p o r t i o n a l s i z e i n c r e a s e was g r e a t e r i n t h e w i d t h d i m e n s i o n . • R o n - v i a b l e s p o r e s a l s o s w e l l b u t o n l y about h a l f as much as v i a b l e s p o r e s . V i a b l e s p o r e s began t o s w e l l when p l a c e d i n a s a t u r a t e d h u m i d i t y and t h e g e r m i n -a t i o n p e r c e n t a g e was s l i g h t l y improved by t h i s t r e a t m e n t . A e c i o s p o r e s g e r m i n a t e d and t h e i r germ t u b e s grew e q u a l l y w e l l i n t h e d a r k and i n d i f -f u s e d n a t u r a l and a r t i f i c i a l l i g h t . Ro t e s t s were made i n d i r e c t s u n l i g h t . The v a r i o u s c o l o r e d l i g h t w a v e l e n g t h s had no s i g n i f i c a n t i n d i v i d u a l e f f e c t s , a l t h o u g h i n one s e r i e s b l u e l i g h t was s l i g h t l y b e t t e r . A e c i o s p o r e s g e r -m i n a t e d o v e r t h e pH range 4.5 t o 8, w i t h optimum between pH 6.0 and 6.5 a t 15°C; a l t h o u g h optimum f o r s t o r e d s p o r e s was o f t e n c l o s e r t o pH 7.0. G e r m i n a t i o n a t t h e extreme v a l u e s was v e r y low, and o n l y o c c u r r e d a t tem-p e r a t u r e s c l o s e t o t h e optimum. There was a l s o a 50% o r more r e d u c t i o n i n t h e l e n g t h o f germ t u b e s a t t h e pH extremes. The a d d i t i o n o f Comandra l e a f m a t e r i a l t o t h e b a s i c media d i d n o t i n c r e a s e g e r m i n a t i o n , b u t t h e r e was e v i d e n c e ltho>ugh i n c o n s i s t e n t , t h a t G e o c a u l o n l e a f m a t e r i a l i n c r e a s e d g e r m i n a t i o n . Why t h e r e was no r e s -ponse w i t h Comandra e x t r a c t , b u t some w i t h G e o c a u l o n was unknown as i n - 316 -i n o c u l a t i o n e x p e r i m e n t s , i n f e c t i o n was always o b t a i n e d on Comandra l e a v e s , b u t not on t h e l o c a l G e o c a u l o n l e a v e s . The pH o f t h e l e a v e s o f b o t h p l a n t s was on t h e a c i d s i d e o f t h e optimum pH f o r g e r m i n a t i o n . Czapek-Dox and d e x t r o s e media p r o v e d b e t t e r g e r m i n a t i o n s u b s t r a t e s t h a n a b a s i c w a t e r a g a r , b u t y e a s t and m a l t e x t r a c t s gave r e l a t i v e l y poor g e r m i n a t i o n . .The m a l t e x t r a c t s had a pH a t t h e extreme a c i d end o f t h e range f o r g e r m i n a -t i o n , b u t t h a t o f t h e y e a s t was c l o s e t o t h e optimum pH. G e r m i n a t i o n r e -q u i r e m e n t s appear t o i n v o l v e m a i n l y an u p t a k e o f w a t e r , as t h e mature sp o r e s a l r e a d y c o n t a i n e d t h e e s s e n t i a l c o n s t i t u e n t s f o r g e r m i n a t i o n and growth o f a s h o r t germ t u b e ; added compounds were, t h e r e f o r e , not r e q u i r e d . Samples o f a e c i o s p o r e s c o l l e c t e d d a i l y f r o m s e l e c t e d c a n k e r s showed c o n s i d e r a b l e v a r i a t i o n i n d a i l y g e r m i n a t i o n p e r c e n t a g e between can-k e r s and between y e a r s . G e n e r a l l y a r e l a t i v e l y h i g h g e r m i n a t i o n p e r c e n t -age was o b t a i n e d d u r i n g t h e f i r s t two t o f o u r weeks o f s p o r u l a t i o n , b u t g e r m i n a t i o n was much l o w e r d u r i n g t h e second h a l f o f t h e p r o d u c t i o n p e r i o d . I n most y e a r s t h e r e was a good r e l a t i o n s h i p between t h e c a n k e r s i n t h e t r e n d o f t h e day t o day g e r m i n a t i o n , and a s i m i l a r c o r r e l a t i o n was f o u n d f o r t h e g e r m i n a t i o n t r e n d o f i n d i v i d u a l p u s t u l e s on t h e same c a n k e r , a l -though t h e r e c o u l d be c o n s i d e r a b l e v a r i a t i o n i n t h e a c t u a l g e r m i n a t i o n p e r c e n t a g e s . There was no c o n s i s t e n t r e l a t i o n s h i p between spo r e g e r m i n a -t i o n and accompanying weather c o n d i t i o n s , e x c e p t t h a t i f t h e a e c i o s p o r e s were wet f r o m r a i n t h e r e was o f t e n a marked r e d u c t i o n i n g e r m i n a t i o n p e r -centage t h a t day o r t h e f o l l o w i n g day. E a r l y i n t h e s t u d y , f l u c t u a t i o n s i n t h e d a i l y g e r m i n a t i o n p e r c e n t a g e s were o f t e n a r e s u l t o f t a k i n g sam-p l e s f r o m newly opened p u s t u l e s on a c a n k e r , b u t when s a m p l i n g was s t a n -dardized i n l a t e r y e a r s , o v e r a l l g e r m i n a t i o n f r o m i n d i v i d u a l p u s t u l e s - 317 -showed a g r a d u a l d e c l i n e i n p e r c e n t g e r m i n a t i o n f o l l o w i n g t h e i n i t i a l h i g h p e r i o d . Spores t a k e n f r o m p r o t e c t e d a e c i a gave b e t t e r g e r m i n a t i o n t h a n s p o r e s f r o m an aecium w h i c h was exposed t o t h e elements and t o con-t a m i n a t i o n f r o m m i c r o o r g a n i s m s . A t t i m e s m i c r o f l o r a l c o n t a m i n a n t s r e -duced t h e d a i l y g e r m i n a t i o n p e r c e n t a g e m a r k e d l y . These c o n t a m i n a n t s c o n t r i b u t e d g r e a t l y t o t h e s e a s o n a l d e c l i n e o f sp o r e v i a b i l i t y . There was a b u i l d u p o f t h e s e m i c r o o r g a n i s m s i n a s s o c i a t i o n w i t h t h e s p o r e s as th e s p o r u l a t i o n s e a s o n p r o g r e s s e d . C l a d o s p o r i u m spp., P e n i c i l l i u m spp. and v a r i o u s b a c t e r i a , were p r o m i n e n t c o n t a m i n a n t s , many o f w h i c h were washed i n t o t h e exposed p u s t u l e s f r o m t h e b a r k d u r i n g heavy r a i n . The e f f e c t o f t h e v a r i o u s e n v i r o n m e n t a l f a c t o r s on a e c i o s p o r e v i a b i l i t y a l l u d e d t o above was i n v e s t i g a t e d s e p a r a t e l y . A e c i o s p o r e s l o s t v i a b i l i t y v e r y r a p i d l y when exposed t o t e m p e r a t u r e s o f 25°C and above. Spores exposed t o 4o°C, became b l e a c h e d and l o s t t h e i r v i a b i l i t y i n l e s s t h a n 30 m i n u t e s . Few s p o r e s s t o r e d a t 25 and 30°C re m a i n e d v i a -b l e much l o n g e r t h a n a day. A t t h e i n t e r m e d i a t e t e m p e r a t u r e s o f 10 t o 15°C, v i a b i l i t y was r e d u c e d t o 1% i n 10 t o 30 days. V i a b i l i t y a t -k and 2°C was r e t a i n e d by some samples f o r o v e r a y e a r a l t h o u g h t h e p e r -centage was v e r y low. Spores s t o r e d a t -20'°C, f o l l o w i n g r a p i d c o o l i n g i n l i q u i d n i t r o g e n , r e t a i n e d some v i a b i l i t y f o r 17 months, b u t r e s u l t s were not c o n s i s t e n t . H y d r a t i o n o f s p o r e s f o l l o w i n g s h o r t p e r i o d s o f s t o r a g e improved g e r m i n a t i o n , b u t a f t e r l o n g s t o r a g e g e r m i n a t i o n was o f -t e n r e d u c e d when h y d r a t e d . Spores s t o r e d o v e r c a l c i u m c h l o r i d e l o s t v i a b i l i t y r a p i d l y . S t o r a g e o f d r y s p o r e s a t h i g h h u m i d i t i e s appeared t o f a v o u r r e t e n t i o n o f v i a b i l i t y o v e r s h o r t s t o r a g e p e r i o d s , b u t co n t a m i n -a n t s soon became a pr o b l e m . I f s p o r e s were s t o r e d wet v i a b i l i t y was - 318 -soon l o s t , s i m i l a r l y , i f wet s p o r e s were d r i e d some i r r e v e r s i b l e p r o c e s s appeared t o have t a k e n p l a c e as v i a b i l i t y was never r e g a i n e d . D i r e c t s u n l i g h t r e d u c e d g e r m i n a t i o n o f d r y f r e s h s p o r e s c o l l e c t e d f r o m p r o t e c t e d a e c i a t o z e r o i n 5 t o 6 hours and t o l e s s t h a n 50$ o f i n i t i a l g e r m i n a t i o n i n 1 t o 3 h o u r s . E v e n on c l o u d y days d i f f u s e l i g h t a d v e r s e l y a f f e c t e d v i a b i l i t y , a l t h o u g h t h e r a t e o f l o s s was r e t a r d e d . The e x p e r i m e n t a l r e s u l t s h e l p e x p l a i n t h e g r e a t v a r i a t i o n i n d a i l y a e c i o s p o r e g e r m i n a t i o n . E x p o s u r e o f s p o r e s en masse t o h i g h l i g h t i n t e n s i t i e s w o u l d r e d u c e t h e v i a b i l i t y o f s p o r e s v e r y r a p i d l y , as does w e t t i n g o f spores.. D r y i n g does n o t a d v e r s e l y a f f e c t t h e s p o r e s as t h e y a r e c h a r a c t e r i s t i c a l l y low i n m o i s t u r e c o n t e n t a t m a t u r i t y . H i g h tem-p e r a t u r e s , r e d u c e d v i a b i l i t y r a p i d l y , but' d a i l y t e m p e r a t u r e s o f 10 t o 15°C and b e l o w do not r e d u c e v i a b i l i t y v e r y q u i c k l y . F r e e z i n g does n o t ad-v e r s e l y a f f e c t v i a b i l i t y , a l t h o u g h t h e e f f e c t o f r e p e a t e d f r e e z i n g and t h a w i n g i s not known. I t can be c o n c l u d e d t h a t i f s p o r e s a r e exposed on wet r a i n y days and on c l e a r warm sunny days v i a b i l i t y w i l l be m a r k e d l y r e d u c e d , o n l y on c l o u d y c o o l days w i l l v i a b i l i t y l o s s be r e t a r d e d . V i a -b i l i t y w o u l d be r e d u c e d a l m o s t t o z e r o a f t e r o n l y two o r t h r e e days o f e x p o s u r e t o u n f a v o u r a b l e w e a t h e r . C o n s i d e r i n g t h e e f f e c t s o f t h e i n d i v i d u a l e l e m e n t s o f t h e en-v i r o n m e n t , i t was not s u r p r i s i n g t h a t c o n d i t i o n s were not f a v o u r a b l e f o r t h e d i s p e r s a l and g e r m i n a t i o n o f a l l a e c i o s p o r e s p r o d u c e d . A e c i o s p o r e s t h a t were not d e s t r o y e d b y t h e v a r i o u s b i o l o g i c a l a g e n t s were exposed t o t h e e f f e c t s o f m e t e o r o l o g i c a l f a c t o r s . I f d r y a e c i o s p o r e s became wet t h e y o f t e n c l u n g t o g e t h e r i n l a r g e a g g r e g a t e s w h i c h were rot e a s i l y d i s -p e r s e d f r o m t h e a e c i a , most clumps e v e n t u a l l y f e l l d i r e c t l y t o t h e ground. - 319 -Even i f such wet sp o r e s were d i s p e r s e d on d r y i n g , t h e y l o s t t h e i r c o l o r and v i a b i l i t y . D r y a e c i o s p o r e s r e m a i n i n g u n d i s p e r s e d i n t h e a e c i a were s u b j e c t e d t o h i g h d a y t i m e t e m p e r a t u r e s and h i g h l i g h t i n t e n s i t i e s , w h i c h t e n d e d t o re d u c e s p o r e v i a b i l i t y . The n o r m a l d i u r n a l p a t t e r n o f a e c i o -spore d i s p e r s a l was f o r s p o r e s t o be d i s p e r s e d on d r y , warm sunny t u r -b u l e n t days between 0800 and 1900 h o u r s . Those s p o r e s w h i c h were d i s p e r -sed d u r i n g t h i s p e r i o d , and d e p o s i t e d on s u i t a b l e a l t e r n a t e h o s t p l a n t s were d e p o s i t e d i n c o n d i t i o n s w h i c h were g e n e r a l l y u n f a v o u r a b l e f o r g e r -mination, as f r e e w a t e r was n e c e s s a r y f o r t h e i n i t i a t i o n o f g e r m i n a t i o n . Such s p o r e s were t h e r e f o r e f u r t h e r exposed t o h e a t and l i g h t f o r s e v e r a l h o u r s , and n o t ; . u n t i l d a r k n e s s were more f a v o u r a b l e c o n d i t i o n s f o r r e t a i n -i n g v i a b i l i t y e x p e r i e n c e d . D u r i n g p e r i o d s o f d r y warm we a t h e r , m o i s t u r e c o n d i t i o n s s u i t a b l e f o r g e r m i n a t i o n may n o t be e x p e r i e n c e d . A t m o s p h e r i c h u m i d i t y a t n i g h t w o u l d o n l y a p p r o a c h 100% on some n i g h t s and t h e n p e r -haps f o r o n l y an hour, w h i c h was i n s u f f i c i e n t t i m e f o r t h e average s p o r e t o i n i t i a t e g e r m i n a t i o n . Any c o n d e n s a t i o n i n t h e fo r m o f dew t h a t o c c u r -r e d on p l a n t l e a v e s a l s o d i s a p p e a r e d r a p i d l y . T h e r e f o r e , t h e main p e r i o d s when s p o r e s were g e n e r a l l y d i s p e r s e d were u n f a v o u r a b l e f o r r e t a i n i n g v i a -b i l i t y and f o r g e r m i n a t i o n , w h i c h means t h a t most o f t h e s e s p o r e s were l o s t . I f s p o r e s were d i s p e r s e d a t t h e o n s e t o f heavy r a i n , , o r i n d r y c o n d i t i o n s j u s t b e f o r e r a i n , when p l a n t s u r f a c e s r e m a i n e d wet f o r s e v e r a l h o u r s , t h e n t h e s p o r e s g e r m i n a t e d under f a v o u r a b l e c o n d i t i o n s . Low tem-p e r a t u r e s , l i k e l y under such c o n d i t i o n s , were n o t l i m i t i n g f o r g e r m i n a t i o n . When such c o n d i t i o n s o c c u r r e d i n t h e f i r s t p o r t i o n o f t h e s p o r u l a t i o n p e r -i o d , t h e n l a r g e numbers o f v i a b l e s p o r e s f o u n d f a v o u r a b l e g e r m i n a t i n g con-d i t i o n s , b u t i f such c o n d i t i o n s o c c u r r e d l a t e r i n t h e s p o r e p r o d u c t i o n - 320 -p e r i o d , t h e d a i l y g e r m i n a t i o n t e s t s i n d i c a t e d much l o w e r p e r c e n t a g e s o f v i a b l e s p o r e s were a v a i l a b l e f o r d i s p e r s a l . D u r i n g p e r i o d s o f l i g h t r a i n o r g e n e r a l l y humid weather when m o i s t u r e c o n d i t i o n s were s u i t a b l e f o r spore g e r m i n a t i o n , s p o r e s were u n l i k e l y t o be d i s p e r s e d . T h e r e f o r e , on o n l y a few o c c a s i o n s i n any one s p o r u l a t i o n p e r i o d were c o n d i t i o n s f a -v o u r a b l e f o r d i s p e r s a l , r e t e n t i o n o f v i a b i l i t y and f o r g e r m i n a t i o n , and even t h e n t h e chances were h i g h t h a t d e p o s i t e d s p o r e s c o u l d be washed o f f t h e l e a f s u r f a c e s o f t h e a l t e r n a t e h o s t . The i n i t i a l s t e e p g r a d i e n t s o f spor e d e p o s i t i o n , e s p e c i a l l y w i t h i n a f o r e s t , i n d i c a t e d t h a t u n l e s s s u i t a b l e a l t e r n a t e h o s t p l a n t s were g r o w i n g w i t h i n a r a d i u s o f about 200 f e e t f r o m t h e s p o u r b a t i n g can-k e r , t h e chances o f s p o r e s r e a c h i n g s u s c e p t i b l e h o s t m a t e r i a l were p o o r . The m a j o r i t y o f c a n k e r s o c c u r r e d w i t h i n 3 f e e t o f t h e ground, t h e odd canker o c c u r r e d a t h e i g h t s above 15 f e e t and t h e chances f o r g r e a t e r d i s -p e r s a l f r o m t h e s e c a n k e r s w o u l d be r h i g b e r , b u t s t i l l o n l y i n o r d e r o f hundreds o f f e e t . I f s p o r e s d i d escape f r o m t h e immediate f o r e s t area, t h e n t h e y were once a g a i n exposed t o t h e f a c t o r s o f heat and l i g h t w h i c h r e d u c e d v i a b i l i t y . D u r i n g t h e peak p e r i o d s o f d i s p e r s a l around midday, t h e i n t e n s i t i e s o f h e a t and l i g h t w o u l d be c l o s e t o maximum due t o t h e n o r m a l d i u r n a l p e r i o d i c i t y o f t h e s e p a r a m e t e r s , e s p e c i a l l y under sunny c o n d i t i o n s w i t h a s s o c i a t e d t h e r m a l l y u n s t a b l e t u r b u l e n c e . T h e r e f o r e , t h e i r chances o f b e i n g d e p o s i t e d a t g r e a t e r d i s t a n c e s i n a v i a b l e c o n d i -t i o n w o u l d be f u r t h e r r e d u c e d . E v e n where s u i t a b l e h o s t s were p r e s e n t w i t h i n a s h o r t d i s -t a n c e o f a b u n d a n t l y s p o r u l a t i n g c a n k e r s , known t o p r o d u c e • s p o r e s h a v i n g a h i g h l e v e l o f v i a b i l i t y , t h e p e r i o d n e c e s s a r y f o r i n f e c t i o n t o o c c u r - 321 -was most v a r i a b l e between y e a r s (12 t o 39 c l a y s ) . T h e r e f o r e , t h e o c c a s i o n when a l l t h e complex m e t e o r o l o g i c a l f a c t o r s n e c e s s a r y f o r s p o r e d i s p e r s a l , d e p o s i t i o n and g e r m i n a t i o n are so i n t e g r a t e d t h a t abundant i n f e c t i o n can t a k e p l a c e i n t h i s a e r o b i o l o g i c a l phase o f t h e r u s t i s f a i r l y r a r e . Be-s i d e s t h i s complex o f f a c t o r s , t h e b i o l o g i c a l and m e t e o r o l o g i c a l f a c t o r s a c t i n g t o r e d u c e t h e amount o f v i a b l e i n o c u l u m a v a i l a b l e f o r d i s p e r s a l and g e r m i n a t i o n must be remembered. When f a c t o r s o f t h e e n v i r o n m e n t de-l a y t h e s u c c e s s f u l c o m p l e t i o n o f t h e a e c i o s p o r e a e r o b i o l o g i c a l phase o f t h e r u s t , t h e n t h e u r e d i o s p o r e phase i s a l s o d e l a y e d w h i c h r e d u c e d t h e b u i l d up o f t h i s and subsequent s t a t e s o f t h e r u s t , p r o b a b l y a d v e r s e l y a f f e c t i n g t h e chances f o r t h e r u s t t o i n t e n s i f y t h a t y e a r . However, i n -f e c t i o n b y comandra b l i s t e r r u s t i s s t i l l a c o n s i d e r a b l e ' t h r e a t , e s p e c i -a l l y i n young p i n e s t a n d s and p l a n t a t i o n s , o r i n s m a l l s t a n d o p e n i n g s where r e g e n e r a t i o n has become e s t a b l i s h e d . I n s u c h a r e a s t h e e n v i r o n -ment i s u s u a l l y more f a v o u r a b l e f o r r u s t s u r v i v a l , and t h e a l t e r n a t e h o s t s o f t e n o c c u r i n t h e same o r a d j a c e n t open a r e a s . I n t h e s e s t a n d s o r o p e n i n g s , l o w e r d a i l y maximum t e m p e r a t u r e s and l o n g e r p e r i o d s o f c o o l m o i s t c o n d i t i o n s o c c u r . They t e n d t o have l o n g e r p e r i o d s o f a v a i l a b l e m o i s t u r e on p i n e n e e d l e s , n e c e s s a r y f o r g e r m i n a t i o n , t h a n o l d e r s t a n d s where dew o n l y forms on t h e o v e r s t o r y canopy l e a v e s , o r i n s t a n d open-i n g s . A i r movement i s a l s o m i n i m a l , e s p e c i a l l y w i t h t e m p e r a t u r e i n v e r -s i o n s , w h i c h h e l p t o e s t a b l i s h and m a i n t a i n f a v o u r a b l e t e m p e r a t u r e s and m o i s t u r e c o n d i t i o n s f o r l o n g e r p e r i o d s , c a u s i n g r a p i d d e p l e t i o n o f s p o r e c l o u d s e n t e r i n g ' t h e s t a n d . There i s a l s o abundant young n e e d l e growth, s u s c e p t i b l e t o i n f e c t i o n , on stems and b r a n c h e s near t h e ground where most r u s t c a n k e r s o c c u r . - 322 -LITERATURE CITED ADAMS, J.F. 1919. R u s t s on c o n i f e r s i n P e n n s y l v a n i a . Penn. 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A g r . , F o r . B i o . P i v . , V i c t o r i a , B.CJ. 117 pp. (mimeo). ZILLER, W.G. and MOLNAR, A.C. 1953. F o r e s t P i s e a s e S u r v e y , pp. 14-2-154. i n Can. P e p t . A g r . , P i v . F o r . B i o l . , A n n u a l R e p o r t o f t h e F o r e s t I n s e c t and P i s e a s e S u r v e y 1952. 154 pp. ZOBERI, M.H. 1961. T a k e - o f f o f mould s p o r e s i n r e l a t i o n t o w i n d speed and h u m i d i t y . A n n a l s Botany, 25: 5 3-64. - 353 -APPENDIX I L i s t o f t h e s p e c i e s o f f u n g i , y e a s t and b a c t e r i a c o l l e c t e d from C r o n a r t i u m comandrae on P i n u s c o n t o r t a f r o m 1964 t o 1966. The l i s t f o l -lows c l o s e l y t h e s y s t e m a t i c arrangement o f A i n s w o r t h (1961) f o r t h e f u n g i , and C l i f t o n (1950) f o r t h e b a c t e r i a . F i n a l column i n d i c a t e s whether t h e f u n g i o r b a c t e r i a were i s o l a t e d f r o m t h e s p o r u l a t i n g a r e a o f t h e canker o r f r o m a spo r e c o l l e c t i o n . CLASS HOW ISOLATED ORDER Canker S p o r e s FAMILY SPECIES PHYCOMYCETES MUCORALES MUCORACEAE Mucor sp. x ASCOMYCETES U n i d e n t i f i e d sp. x HYSTERIALES M y t i l i d i o n sp. x HELOTIALES D u r a n d i e l l a sp. x L a c h n e l l u l a a r i d a ( P h i l l . ) D e n n i s x BASIDIOMYCETES U n i d e n t i f i e d sp. x AGARICALES POLYPORACEAE P o l y p o r u s a d u s t u s W i l l d . ex F r . x DEUTEROMYCETES (FUNGI IMPERFECTl) SPRAEROPSIDALES - 354 -SPHAERIOIDACEACE C o n i o t h y r i u m o l i v a c e u m Bonord D i p l o d i e l l a sp. Phoma sp. Phomopsis sp. P l e u r o p h o m e l l a sp. ? s t a t e o f Tympanis h y p o p o d i a N y l . S c l e r o p h o m a p i t h y a p h i l a (Cda) Honn. MELANCONIALES MELANCONIACEAE S e i m a t o s p o r i u m sp. n r . S. d i s c o s i o i d e s ( E l l . & Ev.) Shoemaker MONILIALES CRYPTOCOCCACEAE T o r u l o p s i s a e r i a ( S a i t o ) Lodder R h o d o t o r u l a f l a v a ( S a i t o ) L o d der R h o d o t o r u l a spp. SPOROBOLOMYCETACEAE Sporobolomyces sp. MOWILIACEAE A s p e r g i l l u s j a n u s Raper & Thorn A s p e r g i l l u s v e r s i c o l o r ( V u i l l . ) T i r a b o s c h B o t r y t i s sp. C e p h a l o s p o r i u m sp. G o n a t o b o t r y s sp. M o n o c i l l i u m sp. nov. ? P a e c i l o m y c e s f a r i n o s u s ( D i c k s , ex F r . ) Brown & S m i t h P e n i c i l l i u m t h o m i i M a i r e s e r i e s P e n i c i l l i u m r e s t r i c t u m G i l m a n & Abbot P e n i c i l l i u m r a i s t r i c k i i S m i t h s e r i e s n r . P. r o l f s i i Thorn P e n i c i l l i u m j a n t h i n e l l u m B i o u r g e P e n i c i l l i u m o c h r o - c h l o r o n B i o u r g e P e n i c i l l i u m g o d l e w s k i i Z a l e s k i P e n i c i l l i u m c o r y l o p h i l u m D i e r c k x . P e n i c i l l i u m b r e v i - c o m p a c t u m D i e r c k x . P e n i c i l l i u m commune Thorn s e r i e s ' n r . - P . lanosum W e s t l i n g - 355 -P e n i c i l l i u m c y c l o p i u m W e s t l i n g P e n i c i l l i u m a u r a n t i o - v i r e n s B i o u r g e P e n i c i l l i u m f u n i c u l o s u m Thorn s e r i e s P e n i c i l l i u m r u g u l o s u m Thorn P e n i c i l l i u m r u g u l o s u m Thorn s e r i e s P e n i c i l l i u m tardum Thorn P e n i c i l l i u m spp. S p i c a r i a v i b l a c e a Abbot S p o r o t r i c h u m epigaeum Brun. v a r . t - e r r e s t r e Daszewska V e r t i c i l l i u m tenerum Nees DEMATIACEAE A c r e m o n i e l l a sp. ? A l t e r n a r i a t e n u i s Nees C l a d o s p o r i u m herbarum L i n k ex F r . C l a d o s p o r i u m sp. 1. (C.M.I, t a x X L I l ) C l a d o s p o r i u m sp. 2 C l a d o s p o r i u m sp. P h i a l o c e p h a l a sp. n r . f u s c a K e n d r i c k P u l l u l a r i a p u l l u l a n s (de B a r y ) B e r k h . V i r g a r i a sp. TUBERCULARIACAE E p i c o c c u m n i g r u m L i n k . F u s a r i u m sp. T u b e r c u l i n a maxima R o s t . SCHIZOMYCETES EUBACTERIALE S PSEUDOMONADACEAE Pseudomonas f l u o r e s c e n s ( F l u g g e ) M i g u l a Pseudomonas sp. Xanthomonas sp. ' CORYJEBACTERIACEAE A r t h o b a c t e r g l o b i f o r m i s Conn. & Dimmick A r t h o b a c t e r c i t r e u s Sacks A r t h o b a c t e r sp. ACHROMOBACTERIACEAE Achromobacter sp. BACILLACEAE B a c i l l u s sp. - 356 -APPENDIX I I L i s t o f the s p e c i e s o f i n s e c t s , m i t e s and s p i d e r s c o l l e c t e d f r o m C r o n a r t i u m comandrae on P i n u s c o n t o r t a and P . b a n k s i a n a f r o m 196U t o 1968. The l i s t f o l l o w s c l o s e l y t h e s y s t e m a t i c arrangement o f B o r r o r and DeLong (196I+). Numbers f o l l o w i n g each s p e c i e s a r e t h e number o f i n d i v i d u a l s c o l l e c t e d . F i n a l column i n d i c a t e s whether i n s e c t s were c o l -l e c t e d f r o m a cage arou n d t h e c a n k e r , d i r e c t l y f r o m an exposed c a n k e r , o r r e a r e d f r o m a c a n k e r . ORDER • No. o f HOW COLLECTED FAMILY SPECIMENS Cage Canker R e a r e d SPECIES .INSECTA COLLEMBOLA PODURIDAE H y p o g a s t r u r a armata ( N i c o l e t ) 6 x H y p o g a s t r u r a sp. n r . matura (Folsom) 8 x H y p o g a s t r u r a s o c i a l i s U z e l 10 x H y p o g a s t r u r a sp. immature h x ENTOMOBRYIDAE Entomobrya n i v a l i s ( L . ) 2 x Entomobrya comparata F o l s o m 1 x Entomobrya sp. imm. 1 x Tomocerus f l a v e s c e n s T u l l b e r g 27 x PLECOPTERA U n i d e n t i f i e d sp. PSOCOPTERA (CORRENTIA) U n i d e n t i f i e d sp. imm. THYSANOPTERA THRIPIDAE 83+ x F r a n k l i n i e l l a sp. T a e n i o t h r i p s sp. 1 1 x x - 357 -PHLOEOTHRIPIDAE T u b i l i f e r a imm. G n o p h o t h r i p s f u s c u s (Morgan) (G. p i n i p h i l u s C r a w f o r d ) HEMIPTERA NABIDAE U n i d e n t i f i e d sp. PENTATOMIDAE C h l o r o c h r o a l i g a t a Say HOMOPTERA ACHILIDAE E p i p t e r a , n r . p a l l i d a Say APHIDIDAE NEUROPTERA HEMEROBIIDAE Hemerobius sp. ? COLEOPTERA CARABIDAE C a l a t h u s i n g r a t u s D e j . STAPHYLINIDAE A t h e t a sp. CANTHARIDAE C a n t h a r i s sp. imm. S i l i s d i f f i c i l i s L e c . 1 12 1 6 E s s i g e l l a sp. C i n a r a m e d i s p i n o s a G i l l e t t e & P a l m e r C i n a r a murrayanae G i l l e t t e & Palmer k7+ C i n a r a n r . murrayanae G i l l e t t e -.&". Palmer 10+ 60+ 1 1 x x X X X X X X X - 358 -NITIDULIDAE E p u r a e a o b l i q u u s H a t c h LATHEIDIIDAE C o r t i c a r i a sp. Enicmus sp. Melanophthalma sp. Microgramme f i l u m (Aube) C0CCI3MELLIDAE U n i d e n t i f i e d sp. TEKE BRIONIDAE T r i b o l i u m confusum J . du V a l . T r i b o l i u m sp. C o r t i c e u s sp. p r o b . p r a e t e r m i s s u s ( F a l l ) ANOBIIDAE E r n o b i u s sp. S CARABAEIDAE Ap h o d i u s f i m e t a r i u s ( L . ) CERAMBYCTDAE ? L e p t u r a sp. U n i d e n t i f i e d sp. imm. CURCULIOWIDAE S c y t h r o p u s sp. P i s s o d e s s c h w a r z i H o p k i n s M a g d a l i s sp. C y l i n d r o c o p t u r u s d e l e o n i Buchanan SCOLYTIDAE Den d r o c t o n u s murrayanae H o p k i n s Dendroctonus sp. P i t y o g e n e s k n e c h t e l i Sw. O r t h o t o m i c u s l a t i d e n s L e c . 200+ x x x Ik x 1 x 7 x 2k x 1 x 2 x k x k x 29 x x 1 x 1 x 2 x 1 X 22 x x x 8 x 68 x x x 1 x 1 x 7 x x 1 x - 359 -LEPIDOPTERA PYRALIDAE D i o r y c t r i a zimmermani Grt, D i o r y c t r i a sp. imm. OLETHREUTIDAE E p i n o t i a sp. L a s p r e y r e s i a sp. L a s p r e y r e s i a Hbn. gp. 2 imm. ( b u t not c u p r e s s a n a K f t . ) GELECHIIDAE P u l i c a l v a r i a sp. C o l e o t e c h n i t e s ( R e c u r v a r i a ) f l o r a e F r e e . C o l e o t e c h n i t e s ( R e c u r v a r i a ) s t a r k i F r e e . C o l e o t e c h n i t e s ( R e c u r v a r i a ) sp. BLASTOBASIDAE U n i d e n t i f i e d sp. imm. DIPTERA MYCE TOPHILIDAE U n i d e n t i f i e d sp. S CIARIDAE B r a d y s i a sp. n r . v a r i a n s Johannsen B r a d y s i a sp. 1 B r a d y s i a sp. 2 B r a d y s i a sp. 3 CECIDOMYIIDAE L e s t r e m i i n a e imm. L e s t r e m i i n i U n i d e n t i f i e d sp. imm. DOLICHOPODIDAE 17+ 9 1 2k+ 1 2 10 21+ 63 183+ 1 10+ 10 1 110+ x x x x x x x x x x x x x x X X X X M e d e t e r a sp. (? l ) U n i d e n t i f i e d sp. 2 1 x x X - 360 -PHORIDAE P h o r a sp. k x x M e g a s e l i a ( A p h i o c h a e t a ) sp. k x CHAMAEMYIIDAE L e u c o p i s o r b i t a l i s group 1 x PIOPHILIDAE P i o p h i l a x a nthopoda M e l a n d e r & S p u l e r 1 x DROS OPHILIDAE P a r a c a c o x e n u s g u t t a t u s Hardy & Wheeler 8 0 + x x x Chymomyza sp. ? 1 x CHLOROPIDAE O s c i n e l l i n a e 1 x HYMENOPTERA BRACONIDAE B r a c h i s t e s sp. 6 x T r i a s p i s sp. 1 x A g a t h i s b i n o m i n a t a M. & W. k x A p a n t e l e s sp. 2 x A s c o g a s t e r sp. 1 x M i c r o c h e l o n u s sp. 2 x Braeon sp. 5 x ICHNEUMONIDAE D o l i c h o m i t u s t e r e b r a n s n u b l i p e n n i s ( V i e r . ) 1 x Phrudus sp. 1 x M a s t r u s sp. 1 x Phygadeuon sp. 1 x Phaeogenes sp. 1 Coelichneumon b r u n n e r i Rohw. h x G l y p t a sp. 7 x T r a t h a l a sp,. 3 x CHALC1DC,IDEA U n i d e n t i f i e d sp. 1 x - 361 -ENCYRTTDAE Copidosoma s p. 1 PTEROMALIBAE ? Asaphes sp. ? nov. gen. o r sp. 1 x T r i t n e p t i s sp. ? 1 x CERAPHROWIDAE Ce r a p h r o n s p . 1 x FORMICIDAE Camponotus h e r c u l e a n u s ( L . ) 3+ x L a s i u s c r y p t i c u s W i l s o n 3+ x L a s i u s s i t - k a e n s i s Pergande 1 x F o r m i c i n a e 1 x ACARINA MESOSTIGMATA ASCIDAE P r o c t o l a e l a p s sp. nov. 1 1 LAELAPTIDAE A n d r o l a e l a p s ( H a e m o l a e l a p s ) c a s a l i s ( B e r l e s e ) 60+ PROSTIGMATA EUPOPIDAE U n i d e n t i f i e d sp. imm. 6 x BDELLIDAE B d e l l o d e s l o n g i r o s t r i s (Hermann) 3 x T h o r i b d e l l a s p . n r . s i m p l e x A t y e o 3 x TYDEIDAE U n i d e n t i f i e d sp. 2 x ANYSTIDAE A n y s t i s sp. 2 x - 362 -ERYTHRAEIDAE A b r o l o p h u s s p. B a l a u s t i u m s p . B o c h a r t i a k u y p e r i Oudemans TROMBIDIIDAE U n i d e n t i f i e d sp. imm. 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