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The epidemiology and control of Pythium root dieback of muck-grown carrots Wisbey, Bruce Douglas 1974

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THE EPIDEMIOLOGY AND CONTROL OF PYTHIUM ROOT DIEBACK OF MUCK-GROWN CARROTS by BRUCE DOUGLAS WISBEY B.Sc.(Agr.), U n i v e r s i t y o f B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of P l a n t S cience We accept t h i s t h e s i s as conforming to the r e q u i r e d standard. THE UNIVERSITY OF BRITISH COLUMBIA September 1974 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 degree at the U n i v e r s i t y of B r i t i s h C olumbia, I agree t h a t the 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 agree 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 purposes may be g r a n t e d by the Head o f my Department 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 . Department o f PTantt Science The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date S?eptimber 197^ ABSTRACT Olpidium b r a s s i c a e was observed i n both brown and healthy c a r r o t roots from Pythium root dieback (PRD) problem and non-problem f i e l d s . The i n c i d e n c e o f Olpidium was c o r r e l a t e d with the frequency o f p r e c i p i -t a t i o n g r e a t e r than one h a l f inch but was not c o r r e l a t e d with r o o t tem-perature, CO2 or 0^  c o n c e n t r a t i o n , s a t u r a t e d h y d r a u l i c c o n d u c t i v i t y , the h e i g h t o f the c a r r o t beds, marketable y i e l d or c u l l r a t e . Olpidium i s o l a t e s with and without TNV d i d not produce l e s i o n s on c a r r o t roots under greenhouse c o n d i t i o n s . TNV was detected i n both brown and white roots but only from problem f i e l d s . C a r r o t r o o t l e t s r u b - i n o c u l a t e d with TNV f a i l e d to produce n e c r o t i c symptoms. Olpidium and TNV were found i n onion, l e t t u c e , c e l e r y and some weed sp e c i e s common to PRD problem f i e l d s . However, no roo t t i p browning was observed i n any o f these hosts. Fast growing Pythium species were recovered e q u a l l y f r e q u e n t l y i n brown and symptomless r o o t l e t s and from problem and non-problem s o i l s . Most weeds, c e l e r y , onion and l e t t u c e a l s o had a high i n c i d e n c e of f a s t growing Pythium. The h i g h l y pathogenic, slow growing Pythium sulcatum was r e -covered o n l y from problem s o i l . The recovery r a t e from symptomless roots was very low compared to brown r o o t s . P_. sulcatum was not i s o -l a t e d from c e l e r y or any o f the weed s p e c i e s common i n problem s o i l . Lettuce and onion were found to support low l e v e l s o f i n f e c t i o n . E v i -dence suggests t h a t P_. sulcatum i s a primary i n c i t a n t . i i i i i PRD l o s s e s can be kept to a minimum and marketable y i e l d s i n -creased by using t o l e r a n t v a r i e t i e s , such as HiPak; r a i s e d beds, i f there i s a r e a d i l y a v a i l a b l e supply of i r r i g a t i o n water; p r e c i s i o n seeding a t 1 1/4 inches; and a crop r o t a t i o n o f onions preceding c a r r o t s . M a t r i c p o t e n t i a l was c o n t r o l l e d i n small c o n t a i n e r s separated from osmotic s o l u t i o n s o f p o l y e t h y l e n e g l y c o l (PEG) 6000 by P e l l i c o n u l t r a f i l t r a t i o n membranes (nominal molecular weight c u t o f f : 5 0 0 , M i l l i p o r e Corp.). M a t r i c p o t e n t i a l s could be maintained f o r periods of 3-5 weeks before m i c r o b i a l breakdown o f membranes oc c u r r e d . Flow r a t e f o r the 3 -2 -1 membranes was 1.0 cm cm day f o r a water p o t e n t i a l d i f f e r e n c e across the membrane o f 0.2 bar. Water p o t e n t i a l measured with tensiometers or thermocouple psychrometers i n a c y l i n d r i c a l c o n t a i n e r (4.3 cm diam. x 10 cm) with a membrane acrosss the bottom, remained r e l a t i v e l y constant under c o n d i t i o n s of s o i l s u r f a c e evaporation but decreased r a p i d l y when young p l a n t s were grown i n the system. S o i l c e l l s (5.5 x 2.0 x 10 cm with one 43 mm diameter membrane i n each s i d e ) , c o n t a i n i n g two young c a r r o t s , and emersed i n a -0.2 and -2.0 bar PEG s o l u t i o n had an average matric p o t e n t i a l o f -0.4 and -2.5 bars r e s p e c t i v e l y over a three week p e r i o d . The c a r r o t s t r a n s p i r e d 7.8 and 3.9 ml/day at osmotic p o t e n t i a l s of -0.2 and -2.0 bars r e s p e c t i v e l y which suggests t h a t s u f f i c i e n t water was passing through the membrane to meet the needs of a growing c a r r o t . TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES . . . . v i i i LIST OF FIGURES . X ACKNOWLEDGEMENT XI1 INTRODUCTION . . . . . . . . 1 CHAPTER I - THE ETIOLOGY, EPIDEMIOLOGY AND CONTROL OF PYTHIUM ROOT DIEBACK . 3 INTRODUCTION 3 History and Importance 3 Symptoms 4 Etiology 4 Abiotic . . . 4 Biot ic 5 Epidemiology • 7 Control of PRD • . 9 METHODS AND MATERIALS 10 Field Studies 10 Microenvironment plot 10 Grower's raised bed t r i a l 13 Precision seeding t r i a l 14 Laboratory Studies : 15 iv V Page Olpidium 15 Tobacco n e c r o s i s v i r u s . 16 Geenhouse Crop R o t a t i o n i . . . . 17 RESULTS 19 E t i o l o g y . . 19 I n d i r e c t d e t e r m i n a t i o n o f organisms r e s p o n s i b l e f o r PRD . . 19 PRD symptom p r o d u c t i o n by Olpidium and TNV . . . . . . . . 21 Epidemiology . . . . . . . 23 Olpidium p o p u l a t i o n and the microenvironment . . . . . . . 23 Grower's r a i s e d bed t r i a l 34 P r e c i s i o n seeding t r i a l 39 Weeds as a l t e r n a t e hosts f o r PRD i n c i t a n t s . . . . . . . . 44 Crop r o t a t i o n 44 DISCUSSION . . . . . . . . . . . . . . . 51 E t i o l o g y and Epidemiology . . . . . . . . . . . . . . . . . . 51 Ev a l u a t i o n o f C u l t u r a l P r a c t i c e s f o r the Control o f PRD ... 59 Raised beds ... . . . . . . . . . . . . . . 59 P r e c i s i o n seeding and t o l e r a n t v a r i e t i e s . . . . . . . . . 62 Crop r o t a t i o n 64 CONCLUSIONS 66 LITERATURE CITED . . . . . . . . . 67 APPENDIX . . . . . . . . 71 v i Page CHAPTER II - CONTROLLED WATER RELATIONS AND PYTHIUM ROOT DIEBACK DEVELOPMENT 72 INTRODUCTION . . . . . . 72 Control o f S o i l Water . 72 E f f e c t s o f S o i l Water on Pythium 75 METHODS AND MATERIALS 77 P e r i o d i c S a t u r a t i o n Experiment ^ E f f e c t s o f Osmotic P o t e n t i a l on Pythium 79 Development o f a Technique to Cont r o l M a t r i c Water P o t e n t i a l . 81 C e l l u l o s e d i a l y s i s membrane . 81 Flow r a t e 81 Moisture c o n t r o l l i n g prototypes . . . . . . . . . . . . 81 A n t i b i o t i c s f o r the PEG s o l u t i o n s . . . . . . . . . . . 84 P e l l i c o n membranes 86 Flow r a t e . . ... . . . . . . . 86 Moisture c o n t r o l l i n g prototypes . .... 86 Constant water p o t e n t i a l s as determined g r a v i m e t r i c a l l y . 91 RESULTS 9 2 P e r i o d i c S o i l S a t u r a t i o n . . . 92 E f f e c t s o f Osmotic P o t e n t i a l on Pythium 96 Control o f S o i l Moisture using C e l l u l o s e D i a l y s i s Membranes . 1°1 Control o f S o i l Moisture using P e l l i c o n Membranes ^ 5 S o i l moisture c o n t r o l l i n g prototypes ^ 5 S o i l moisture c o n t r o l as measured g r a v i m e t r i c a l l y ^ v i i Page DISCUSSION 116 P e r i o d i c S o i l S a t u r a t i o n 116 E f f e c t s o f Osmotic P o t e n t i a l on Pythium 119 C o n t r o l l e d M a t r i c P o t e n t i a l 121 CONCLUSIONS 128 LITERATURE CITED 130 LIST OF TABLES Table Page Chapter I I. Sequence o f the crops u t i l i z e d i n the r o t a t i o n experiment . 18 I I . Incidence o f Olpidium, f a s t and slow growing Pythium spp. and TNV i n brown and white r o o t s o f a non-problem and two PRD problem f i e l d s a t two sampling times i n 1973 20 I I I . Number o f c a r r o t r o o t systems d e v e l o p i n g n e c r o t i c symptoms and i n d e x i n g TNV p o s i t i v e a f t e r rub or d i p i n o c u l a t i o n i n carborundum and .05 M phosphate b u f f e r with and without TNV 23 IV. C a r r o t y i e l d i n " r a i s e d " and " c o n v e n t i o n a l " beds and a t f o u r p l a n t i n g dates i n 1972 28 V. Number o f c u l l s from r a i s e d and c o n v e n t i o n a l beds a t f o u r p l a n t i n g dates . . . . . . 30 VI. C o r r e l a t i o n between the average number o f Olpidium spores per microscope f i e l d a t v a r i o u s sampling times and the frequence o f i n f e c t i o n p e r i o d s o f p r e c i p i t a t i o n g r e a t e r than 1/2 inch i n 1972 33 VII. Average c a r r o t emergence and percentage o f doubles i n p r e c i s i o n - s e e d e d and s c a t t e r shoe-seeded beds . 35 V I I I . Organism survey i n r a i s e d and c o n v e n t i o n a l beds on PRD problem and non-problem s o i l a t two sample times 36 IX. C a r r o t y i e l d i n PRD problem and non-problem s o i l .... . 38 X. C a r r o t emergence and the percent doubles (two c a r r o t s growing i n the same l o c a t i o n ) o f two v a r i e t i e s p r e c i s i o n seeded a t a s p a c i n g o f 1 1/4, 1 1/2 and 2 inches 39 XI. Organism survey o f brown r o o t s a t two sampling times o f HiPak and GoldPak and p r e c i s i o n seed spacings o f 1 1/4, 1 1/2, and 2 inches 41 vi i i i x Table Page XII. C a r r o t y i e l d o f GoldPak andHiPak a t p r e c i s i o n seed sp a c i n g o f 1 1/4, 1 1/2 and 2 inches 43 XII I . Organism i n c i d e n c e i n some common weeds growing i n s o i l s with a h i s t o r y o f severe PRD 45 XIV. Average number o f Olpidium spores per microscope f i e l d i n r o o t s o f crops from f i v e r o t a t i o n s 46 XV. Percent s l o w growing Pythium recovered from r o o t s o f crops from f i v e r o t a t i o n s 46 XVI. Percent f a s t growing Pythium recovered from r o o t s o f crops from f i v e r o t a t i o n s 47 XVII. P e r c e n t d e t e c t i o n o f TNV i n r o o t s o f crops from f i v e r o t a t i o n s . . . . . . . . 47 XVIII. Root weight o f crops from f i v e r o t a t i o n s .... 50 2 XIX. Pythium propagule number x 10 per gram o f oven dry s o i l from f i v e r o t a t i o n s . . 50 Chapter II I. The c o n c e n t r a t i o n o f P o l y e t h y l e n e g l y c o l 6000 a t v a r i o u s osmotic p o t e n t i a l s . . . . . . . . . . 83 I I . Radial growth r a t e o f Pythium sulcatum on KC1 osmo-t i c a l l y amended basal media a t 34, 57 and 83 hours a f t e r i n o c u l a t i o n . Each measurement i s the mean o f f o u r o b s e r v a t i o n s 100 I I I . The percent water content o f s o i l i n the plumbing adaptor chamber emersed i n PEG s o l u t i o n s o f v a r i o u s osmotic p o t e n t i a l . . . . . . . . 107 IV. Percent water content o f s o i l a t v a r i o u s h e i g h t s above the membrane i n the plumbing adaptor chambers . . . . . . 109 V. S o i l water content and p o t e n t i a l o f s o i l c e l l s c o n t a i n i n g c a r r o t s over a three week i n t e r v a l maintained a t f o u r osmotic p o t e n t i a l s H4 VI. S o i l water c o n t e n t a t f o u r l o c a t i o n s w i t h i n s o i l c e l l s a f t e r two weeks of o p e r a t i o n 115 VII. Average t r a n s p i r a t i o n o f two c a r r o t s grown i n s o i l c e l l s a t f o u r osmotic p o t e n t i a l s 115 LIST OF FIGURES Figure Page Chapter I 1. Average number of 01pidium spores per microscope f i e l d (560 y) at four planting dates. '1 = May 11, 2 = May 23, 3 = May 30, 4 = June 13. Each point is the mean of 4000 counts 25 2. Average number of 01pidium spores per microscope f i e l d (560 y) at f ive carrot ages. Each point represents the mean of 3200 counts 26 3. Average number of 01pidium spores at f ive sampling ages and four planting dates. Each point represents the mean of 800 counts 27 4. Soil water content of raised and conventional beds at 8-15 cm depth in 1972 31 5. Average number of 01pidium spores per microscope f i e l d in rootlets of HiPak and GoldPak at three precision seed spacings. Each point represents the mean of 600 obser-vations 42 Chapter II 1. Apparatus used to measure the flow rate of water through test membranes 82 2. Prototypes using Pell icon membranes for controll ing so i l matric potential . (A) plumbing adaptor chamber (B) cubical chamber (C) narrow soi l ce l l (D) osmotic solution chamber . 88 3. Cross section of ceramic bulb with PVC plug holding thermocouple psychrometer in place . . . 89 4. Mean total fresh weight y ie ld of carrots at 8 weeks of age in naturally- infested f i e l d s o i l , Pythium sylvaticum-inoculated autoclaved s o i l , and non-inoculated autoclaved s o i l . Bars within a so i l treatment with the same letter do not d i f fe r s igni f icant ly (P = .05). 94 x Average number o f Olpidium and Pythium spores per micro-scope f i e l d , i n c a r r o t r o o t l e t s under f o u r watering regimes (4 c a r r o t ages combined). Bars with the same l e t t e r do not d i f f e r s i g n i f i c a n t l y (P = .05) Radial growth r a t e o f Pythium sulcatum on basal medium amended o s m o t i c a l l y w i t h NaCl, KCl, 5NaCl:3KC1:2Na 2S04 and sucrose. Each p o i n t r e p r e s e n t s the mean o f f o u r o b s e r v a t i o n s Radial growth r a t e o f Pythium s y l v a t i c u m on CMA amended o s m o t i c a l l y with NaCl, K C l , CaCl, and s u c r o s e . Each p o i n t r e p r e s e n t s a mean o f f o u r o b s e r v a t i o n s Radial growth r a t e of Pythium sulcatum on CMA, PDA, and basal medium amended with KCl. Each p o i n t r e p r e s e n t s the mean o f f o u r o b s e r v a t i o n s Radial growth r a t e o f P. ultimum, two i s o l a t e s o f P. s y l v a t i c u m and two i s o l a t e s o f P. sulcatum on basaT medium amended with KCl. Each p o i n t i s the mean o f f o u r o b s e r v a t i o n s Flow r a t e o f water acro s s a 0.001 i n c h t h i c k c e l l u l o s e d i a l y s i s membrane and a 500 NMWL P e l l i c o n u l t r a f i l -t r a t i o n membrane a t v a r i o u s p o t e n t i a l d i f f e r e n c e s .... S o i l water p o t e n t i a l as measured wi t h two tensiometers i n the plumbing adaptor apparatus emersed i n a -0.1 bar PEG s o l u t i o n . . . . . . S o i l water p o t e n t i a l o f plumbing adaptor chamber, 5 membrane c u b i c a l chamber, and 2 membrane s o i l c e l l w ith a r a d i s h s e e d l i n g . Chambers were emersed i n -0.2 bar PEG s o l u t i o n S o i l water p o t e n t i a l of the f i v e membrane chamber over a f o u r day p e r i o d S o i l water p o t e n t i a l measured with two t r i p l e j u n c t i o n two loop thermocouple psychrometers i n the plumbing adaptor apparatus emersed i n -1.2 bar PEG s o l u t i o n .... S o i l water r e t e n t i o n curve f o r UBC greenhouse s o i l ... . ACKNOWLEDGEMENT I wish to express my s i n c e r e thanks to Dr. R.J. Copeman, who suggested the t o p i c and under whose guidance the p a t h o l o g i c a l aspects of the t h e s i s were done. I am a l s o indebted to Dr. T.A. Black f o r h i s h e l p f u l d i s c u s s i o n and encouragement i n the design of a s o i l water c o n t r o l l i n g apparatus. Thanks to members of my committee, Dr. T.A. Black, Dr. R.J. Copeman, Dr. V.C. Runeckles and Dr. R. Stace-Smith f o r h e l p f u l sugges-t i o n s i n the w r i t i n g of the t h e s i s . A s s i s t a n c e o f Hedi T r a b e l s i and Frank Schneider i n c o l l e c t i n g the 1972 f i e l d data i s g r a t e f u l l y acknowledged. The author would a l s o l i k e to thank L e s l e y Tannen f o r her a s s i s t a n c e during the summer-of 1973. S p e c i a l thanks go to Ron Howard f o r c o n f i r m i n g the i d e n t i t y o f s e v eral F\ sulcatum i s o l a t e s . The w r i t e r would a l s o l i k e to express h i s a p p r e c i a t i o n to Dr. L. Chow f o r guidance i n c o n s t r u c t i n g the thermocouple psychrometers and Dr. N. Nagpal f o r advice i n determining the water r e t e n t i o n curve of the greenhouse s o i l . The c l o s e cooperation of three C l o v e r d a l e c a r r o t producers i s g r a t e f u l l y acknowledged. And f i n a l l y , I would l i k e to thank Debbie f o r her patience and moral support throughout my Master's program. F i n a n c i a l a i d i n the form o f an NRC S c h o l a r s h i p to the author and a F a c u l t y of A g r i c u l t u r a l Sciences Research and Development Fund grant to Drs. Copeman and Black i s acknowledged. x i i INTRODUCTION A r o o t d i s o r d e r o f muck-grown c a r r o t s , Daucus c a r o t a L., ob-served i n s e v e r a l l o c a t i o n s o f North America has r e c e i v e d s e v e r a l des-c r i p t i v e names. In O n t a r i o , Fushtey and Filman (1968) d e s c r i b e d the di s e a s e as " r u s t y r o o t . " Workers i n B r i t i s h Columbia c a l l e d a s i m i l a r d i s e a s e " l a t e r a l r o o t dieback" o f c a r r o t (Ormrod, 1969). Pythium  debaryanum Hesse, has been shown to be a s s o c i a t e d with the d i s e a s e i n B r i t i s h Columbia and McElroy e t al_. (1971) f e l t t h a t the name "Pythium Root Dieback" of c a r r o t was more d e s c r i p t i v e . Researchers i n Wisconsin, who have a l s o a s s o c i a t e d s e v e r a l Pythium s p e c i e s w i t h the d i s o r d e r , named the problem "brown r o o t " ( M i l d e n h a l l e t a l _ . , 1971). Since t h e r e i s now evidence t h a t : 1) the d i s e a s e s a re the same; and 2) Pythium i s the primary organism r e s p o n s i b l e f o r the d i s e a s e , Pythium Root Dieback (PRD), the name f i r s t a s s o c i a t i n g Pythium with the problem, w i l l be used throughout t h i s t h e s i s . In B r i t i s h Columbia, three organisms a r e a s s o c i a t e d with the d i s o r d e r . High l e v e l s o f Olpidium spores are observed i n d i s e a s e d t i s s u e , and tobacco n e c r o s i s v i r u s can a l s o be d e t e c t e d i n t h i s t i s s u e . Pythium s p e c i e s h i g h l y pathogenic to c a r r o t have been recovered from n e c r o t i c r o o t t i s s u e and from s o i l . The e t i o l o g y and epidemiology o f these three organisms i n PRD i n c i d e n c e and development w i l l be co n s i d e r e d i n Chapter I. 1 2 Since chemical c o n t r o l of PRD has been shown to be e i t h e r i n -e f f e c t i v e or uneconomical, other methods of c o n t r o l had to be d e v i s e d . An i n t e g r a t e d method o f c o n t r o l using f o u r c u l t u r a l p r a c t i c e s i s d i s -cussed i n Chapter I. Because s o i l moisture appears to be the most impor-t a n t parameter i n the epidemiology of PRD, Chapter II i s devoted to the r o l e o f s o i l water i n d i s e a s e development. CHAPTER I ETIOLOGY, EPIDEMIOLOGY AND CONTROL OF PYTHIUM ROOT DIEBACK INTRODUCTION H i s t o r y and Importance Pythium Root Dieback (PRD) of c a r r o t was f i r s t observed on two muck s o i l farms i n the Bradford Marsh area of O n t a r i o i n 1962, and the problem reoccurred i n 1965 and 1968 on s e v e r a l more farms i n t h a t area (Fushtey and Filman, 1968). A s i m i l a r problem had been observed f o r s e v e r a l years i n muck s o i l s i n the F r a s e r V a l l e y of B r i t i s h Columbia, but i t r e c e i v e d l i t t l e a t t e n t i o n u n t i l severe l o s s e s i n 1968. Surveys of c a r r o t f i e l d s around C l o v e r d a l e and Marine D r i v e , suggested t h a t i t was present i n many f i e l d s and caused average crop l o s s e s of 35% i n 1969 (McElroy et ah, 1971) and 10-15% i n 1970 (D.J. Ormrod et a l _ . , unpublished  d a t a ) . One small f i e l d was disked under as a r e s u l t of PRD. In the Fraser V a l l e y , there were moderate Tosses to PRD i n 1971 and 1972 and l i t t l e l o s s i n 1973. P r e s e n t l y PRD i s considered to be the most important d i s e a s e problem of c a r r o t i n o r g a n i c muck s o i l s of F l o r i d a ( P r a t t and M i t c h e l l , 1973), Wisconsin ( M i l d e n h a l l e t aj_., 1971), O n t a r i o (Fushtey and Filman, 1968), and B r i t i s h Columbia (McElroy et a l _ . , 1971). 3 4 Symptoms Above ground symptoms were f i r s t observed as a w i l t when the p l a n t s were 4 to 6 inches t a l l . The tops w i l t e d during the day and recovered a t n i g h t . A f t e r s e v e r a l days o f w i l t i n g , lower leaves began to show n e c r o s i s o f the margins and a f f e c t e d areas became q u i t e con-spicuous because o f y e l l o w i n g f o l i a g e and reduced grow'th. Below ground symptoms appeared as a r o o t t i p n e c r o s i s . R o o t l e t s became a d i s t i n c t i v e r u s t y red c o l o u r i n the v i c i n i t y o f the n e c r o t i c area (Fushtey and Filman, 1968). The decay o f the t a p r o o t o f s e e d l i n g s to w i t h i n s e v e r a l c e n t i -meters o f the s o i l s u r f a c e was another phase of the d i s e a s e ( M i l d e n h a l l ejt a l _ . , 1971). E x t e n s i v e p r o l i f e r a t i o n and branching j u s t above the p o i n t o f n e c r o s i s then o c c u r r e d (McElroy e_t a l _ . , 1971). Depending upon the weather, the p l a n t s were e i t h e r k i l l e d o r they continued producing new r o o t l e t s to r e p l a c e those l o s t . At h a r v e s t , f o l i a g e gave the appearance o f a healthy stand, but below ground the roots were s h o r t , stubby and f o r k e d . Many c a r r o t s were rough and had an e x c e s s i v e number o f f e e d e r roots which held the s o i l a t h a r v e s t . E t i o l o g y A b i o t i c . A number of attempts were i n i t i a t e d to determine i f a b i o t i c agents might be r e s p o n s i b l e f o r PRD. S a l i n i t y was measured i n c a r r o t growing muck-soils o f B r i t i s h Columbia, but there was no c o r r e l a t i o n between s o i l s a l t content and PRD (M. Driehuyzen, unpublished d a t a ) . Leaching o f PRD s o i l s i n a pot experiment had no e f f e c t on reducing r o o t symptoms (Filman and Fushtey, 1972). I t was hypothesized t h a t p e s t i c i d e residues might have some e f f e c t on PRD but O l o f f s e_t al_. (1971) found 5 lower l e v e l s o f c h l o r i n a t e d hydrocarbons i n muck-grown c a r r o t s than mineral-grown c a r r o t s . Linuron came i n t o use a t about the time PRD was f i r s t r e c o g n i z e d , but a p p l i c a t i o n s of Linuron at r a t e s 2 to 4 times the recommended r a t e had no e f f e c t on r o o t dieback symptoms (Filman, 1972a). A p p l i c a t i o n s of lime (Filman, 1972d) and d i f f e r e n t r a t e s and forms o f n i t r o g e n ( F i l m a n , 1972c) a l s o had no e f f e c t on d i s e a s e i n c i -dence. New seed reduced d i s e a s e s e v e r i t y compared with two-year-old seed i n some v a r i e t i e s but t h i s o b s e r v a t i o n has been i n c o n s i s t e n t be-tween experiments (Filman and Andersen, 1972; Filman and Fushtey, 1970). P l a n t d e n s i t y had an e f f e c t on d i s e a s e i n c i d e n c e . In a t r i a l with d i f f e r e n t numbers o f c a r r o t s per f o o t o f row, a seeding r a t e o f 12 c a r r o t s per f o o t o f row (recommended r a t e ) had such a high PRD l o s s , that i t would have been uneconomical to h a r v e s t . Seeding at h a l f the r a t e reduced d i s e a s e i n c i d e n c e by 10% but PRD was s t i l l too severe f o r economical h a r v e s t i n g (Filman, 1972b). Less f o r k i n g and fewer c u l l s were observed w i t h p r e c i s i o n seeding than P l a n e t J u n i o r seeding (A. R. Maurer, personal communication). B i o t i c . A number o f fungi have been a s s o c i a t e d with PRD. McElroy e_t al_. (1971) c o n s i s t e n t l y i s o l a t e d P_. debaryanum from brown r o o t l e t s and s o i l from d i s e a s e problem a r e a s . Since t h e i r p u b l i c a t i o n , t h i s i s o l a t e has been r e i d e n t i f i e d as a c l o s e l y r e l a t e d s p e c i e s , P_. s y l v a t i c u m Campbell and Hendrix. T h e r e f o r e , throughout the r e s t o f t h i s t h e s i s McElroy's i s o l a t e w i l l be r e f e r r e d to as s y l v a t i c u m . In p a t h o g e n i c i t y t r i a l s , P_. s y l v a t i c u m was capable o f c a u s i n g brown roots (McElroy e t a l . 1971) and c a r r o t s i n f e c t e d with P_. s y l v a t i c u m had o n l y one t h i r d the amount o f r o o t growth as n o n - i n o c u l a t e d p l a n t s (Blok, 1970). 6 A l a r g e number o f Pythium s p e c i e s have been i s o l a t e d from muck s o i l i n c a r r o t s from B r i t i s h Columbia by Wisconsin r e s e a r c h e r s . These i n c l u d e P_. i r r e g u l a r e Buisman, P_. paroecandrum " c l a s s i c a l form," P_. paroecandrum "P_. ultimum form," P_. debaryanum, P_. coloratum V a a r t a j a , £. s y l v a t i c u m , P_. sulcatum P r a t t and M i t c h e l l and s e v e r a l u n c l a s s i f i e d Pythium s p e c i e s (R.'G. P r a t t and R. J . Howard, personal communication). Four s p e c i e s of Pythium have been i s o l a t e d by b a i t i n g PRD problem s o i l s from Wisconsin ( M i l d e n h a l l et_ a l _ . , 1971). P_. i r r e g u l a r e , P_. paroecan- drum D r e c h s l e r , F\ s y l vaticum, and P_. sulcatum a l l induced r o o t browning, but only F\ i r r e g u l a r e , F\ paroecandrum and sulcatum reduced ger-mination. Root t i p n e c r o s i s was most severe w i t h P_. sulcatum and P. i r r e g u l a r e . IP. sulcatum was e a s i l y d i s t i n g u i s h e d from the o t h e r t h r e e pathogens by i t s much slower growth h a b i t ( P r a t t and M i t c h e l l , 1973). A wide range o f genera o f fungi has been i s o l a t e d from c a r r o t s i n O n t a r i o but none was pathogenic ( S u t t o n , 1973). The fungi most f r e -q uently recovered from.PRD roots were A l t e r n a r i a , C y l i n d r o c a r p o n , Fusarium, GIiocladium, Mucor, and P e n i c i l l u r n . S i m i l a r fungi were o f t e n found i n roots from non-problem a r e a s , but they were u s u a l l y fewer i n number. Sutton concluded t h a t filamentous fungi do not i n i t i a t e PRD but were probably important as secondary organisms i n d i s e a s e development. He a l s o concluded t h a t Pythium was unimportant i n the i n i t i a t i o n and develop-ment o f PRD because he i s o l a t e d very low l e v e l s from r o o t s . However, he d i d not use a s e l e c t i v e medium which i s necessary when attempting to recover Pythium from p l a n t t i s s u e . Numerous c h y t r i d spores have been observed i n r o o t s o f O n t a r i o c a r r o t s ( Sutton, 1973). Olpidium b r a s s i c a e (Woron.) Dang, was found 7 in 18 o f 30 f i e l d s i n Ontario (Anon., 1972). Tobacco n e c r o s i s v i r u s (TNV), c a r r i e d by zoospores o f 0_. b r a s s i c a e , was d e t e c t e d i n 3 o f 30 f i e l d s by i n d e x i n g c a r r o t r o o t s (Anon., 1972). The c h y t r i d and v i r u s have been d e t e c t e d i n c a r r o t r o o t s j u s t a f t e r p l a n t emergence (Kemp and Filman, 1972). 0. b r a s s i c a e zoospores, l i b e r a t e d from a c h y t r i d - v i r u s -i n f e c t e d c a r r o t , and added to c a r r o t s grown i n sand under s t e r i l e con-d i t i o n s , t r a n s m i t t e d TNV. Some brown r o o t l e t s developed but they were not as d i s c o l o u r e d , as those a s s o c i a t e d with PRD under f i e l d c o n d i t i o n s (Anon., 1973a). CL b r a s s i c a e and TNV have been d e t e c t e d i n PRD s o i l s from B r i t i s h Columbia (W. G. Kemp, personal communication). C a r r o t r o o t l e t s i n two f i e l d s o f O n t a r i o were 50-100% m y c o r r h i -zal but most f i e l d s were 1-12% myc o r r h i z a l ( S u t t o n , 1973). A nematode survey o f PRD problem and non-problem f i e l d s i n B r i t i s h Columbia i n d i c a t e d the presence o f s e v e r a l s p e c i e s o f s t y l e t b e a r i n g nematodes, but there was no c o r r e l a t i o n between a p a r t i c u l a r s p e c i e s of nematode and the dieback problem (McElroy e_t a l _ . , 1971). Epidemiology C a r r o t s , r e p l a n t e d i n f i e l d s d i s k e d under because o f severe PRD l o s s e s , grew w e l l , s u g g e s t i n g t h a t s o i l temperature or moisture may be important (Fushtey and Filman, 1968). In B r i t i s h Columbia, P. s y l v a t i c u m was a c t i v e throughout the y e a r , but i t was l e s s a c t i v e d u r i n g the dry summer months (McElroy e t a l _ . , 1971). A number o f watering regimes, i n a pot experiment, had no e f f e c t on PRD i f P_. s y l v a t i c u m was a l r e a d y present. I f the s o i l was f r e e o f the i n c i t a n t , c a r r o t s responded to the watering regimes (Maurer e_t a l _ . , 1971). 8 I n o c u l a t i o n of c a r r o t s with P_. ultimum at cool temperatures (7-18 C), l e d to a g r e a t e r PRD i n f e c t i o n than high temperatures (18-30 C) (A. R. Maurer e t a l _ . , unpublished d a t a ) . Cranston and Copeman (1972) attempted,to separate the e f f e c t s o f temperature and water i n a greenhouse experiment. They found t h a t i n f e c -t i o n by P_. s y l vaticum was g r e a t e s t at 18.5 C and was higher i n pots maintained a t a high s o i l moisture l e v e l . S o i l temperature and moisture were measured under f i e l d con-d i t i o n s i n an attempt to r e l a t e PRD to c r i t i c a l c o n d i t i o n s i n the micro-environment (Copeman and Black, 1972). A g r e a t e r d i s e a s e i n c i d e n c e was found i n p l o t s maintained a t a high s o i l moisture l e v e l by i r r i g a t i o n than d r i e r p l o t s t h a t r e c e i v e d only r a i n f a l l . R a i s i n g the moisture l e v e l l a t e r i n the season to the s p r i n g l e v e l by i r r i g a t i o n i n c r e a s e d d i s e a s e i n c i d e n c e , as measured by the number o f spores i n brown r o o t l e t s , w hile i n c i d e n c e decreased i n the n o n - i r r i g a t e d p l o t . Moisture was con-s i d e r e d more c r i t i c a l than s o i l temperature. The e f f e c t o f s o i l moisture content on PRD was a l s o s t u d i e d by growing c a r r o t s under d i f f e r e n t moisture regimes i n i n f e s t e d f i e l d s o i l i n p l a s t i c l i n e d bins (Filman and Andersen, 1972). Water treatments had l i t t l e e f f e c t on the i n c i d e n c e o f PRD as measured by c a r r o t c u l l a g e , but they s i g n i f i c a n t l y a f f e c t e d t o t a l c a r r o t y i e l d s . F l o o d i n g the s o i l f o r 7 days before p l a n t i n g reduced symptoms from 73% to 57% but t h i s r e d u c t i o n was not enough to be o f p r a c t i c a l v a l u e . 9 Control of Pythium Root Dieback A number o f f u n g i c i d e t r i a l s have been attempted but no e f f e c -t i v e c o n t r o l has been found. McElroy et_ al_. (1971) found t h a t a Dexon drench under greenhouse c o n d i t i o n s reduced c u l l s from 76.5 to 5.5%. How-ever, t h i s chemical was i n e f f e c t i v e under f i e l d c o n d i t i o n s (Ormrod e t a l . , 1970). A f t e r four y e a r s of t r i a l s i n B r i t i s h Columbia, Ormrod and Cast-l e y (1973) concluded t h a t fumigants such as Brom-0-gas were uneconomical; T e r r a c h l o r , Dexon and Demosan were not l i k e l y to be r e g i s t e r e d ; and T e r r a z o l e , though p r o m i s i n g , needed more study. F i e l d t r i a l s a p p l y i n g Dexon a g a i n s t Pythiaceous f u n g i , Benlate a g a i n s t ascomycetes and some basidiomycetes, T e r r a c h l o r a g a i n s t basidiomycetes and Nemagon a g a i n s t nematodes, had no e f f e c t i n Ontario (Anon., 1973b). The most s u c c e s s f u l r e s u l t s i n r educing l o s s e s to PRD have been obtained by s e l e c t i o n o f more r e s i s t a n t v a r i e t i e s . Hybrids from Michigan S t a t e with 5988 or Spartan parentage are more t o l e r a n t or r e s i s t a n t to PRD than any other v a r i e t i e s or h y b r i d s on the market today (Baker e t a l . , 1972). Spartan Sweet, Spartan Fancy, HiPak and Grenadier i n B r i t i s h Columbia had s i g n i f i c a n t l y l e s s f o r k i n g and c u l l a g e due to PRD than other v a r i e t i e s t e s t e d (Copeman and B l a c k , 1973). HiPak's acceptance by B r i t i s h Columbia growers has been one important f a c t o r i n reducing l o s s e s i n the past few y e a r s . To date Olpidium spp., Pythium spp., and TNV have been a s s o c i a t e d with c a r r o t roots e x h i b i t i n g PRD symptoms i n B r i t i s h Columbia. S i n c e P_. sulcatum, a very pathogenic s p e c i e s capable o f causing r o o t l e t dieback i n Wisconsin and F l o r i d a ( P r a t t and M i t c h e l l , 1973), has been found i n 10 B r i t i s h Columbia s o i l s , i t was c o n s i d e r e d important to determine i t s r o l e i n PRD. Olpidium i s o f t e n regarded as a non-pathogenic p a r a s i t e causing no macroscopic symptoms (Temmink and Campbell, 1968). However, i t i s hard to imagine t h a t the l a r g e number o f spores observed i n r o o t l e t s from B r i t i s h Columbia grown c a r r o t s are not reducing y i e l d s . I f s o i l moisture i s the most important parameter o f the microenvironment a f f e c t i n g the i n c i d e n c e o f PRD (Copeman and B l a c k , 1972), can PRD s e v e r i t y be mo d i f i e d by c u l t u r a l techniques which a l t e r s o i l moisture? The unknown r o l e o f a l t e r n a t e weed or crop hosts on pathogen p o p u l a t i o n s deserves a t t e n t i o n . T h e r e f o r e , the o b j e c t i v e s o f t h i s study were: 1) to determine the importance o f P_. sulcatum i n PRD s o i l s o f B r i t i s h Columbia; 2) to d u p l i c a t e f i e l d i n f e c t i o n o f Olpidium i n the l a b o r a t o r y and determine i f Olpidium was capable o f causing PRD symptoms; 3) to determine i f PRD l o s s e s and pathogen p o p u l a t i o n s were reduced on r a i s e d beds where the microenvironment had been m o d i f i e d ; 4) to determine i f p r e c i s i o n seeding and seed s p a c i n g a f f e c t PRD s e v e r i t y , and; 5) to observe the e f f e c t s o f weed hosts and a l t e r n a t e crops on the pop u l a t i o n s o f the organisms a s s o c i a t e d with PRD. METHODS AND MATERIALS F i e l d S t u d i e s Microenvironment p l o t . In 1972, a f i e l d experiment was conducted by Drs. R. J . Copeman and T. A. Black to determined i f " r a i s e d " c a r r o t 11 beds would reduce PRD l o s s e s by modifying the microenvironment. Mr. Hedi T r a b e l s i maintained the p l o t and c o l l e c t e d s o i l and m i c r o e n v i r o n -mental d a t a , w h i l e Mr. Frank Schneider c o l l e c t e d and prepared c a r r o t r o o t samples f o r l a t e r examination. The author observed the c a r r o t r o o t l e t s and analyzed the s o i l , y i e l d , and d i s e a s e r a t i n g d a t a . The f i e l d p l o t was s i t u a t e d on a Lumbrum Muck near C l o v e r d a l e , B. C. on the farm o f C l o v e r d a l e Produce L t d . C a r r o t s had been grown on t h i s l a n d f o r the two preceding y e a r s , and the preceding y e a r ' s crop had s u f f e r e d heavy l o s s e s from PRD. The c o o p e r a t i n g grower c u l t i v a t e d and f e r t i l i z e d the la n d as he would have done f o r a commercial c a r r o t c r o p , and then prepared beds o f maximum hei g h t with h i s equipment. The top 2 or 3 inches o f s o i l were removed from h a l f o f the beds to form "conven-t i o n a l " h e i g h t beds, and placed on the adjacent beds to form " r a i s e d " beds. T h i s r e s u l t e d i n beds having a h e i g h t d i f f e r e n c e o f 4 to 5 in c h e s . The removal o f s o i l from " c o n v e n t i o n a l " beds may have reduced inoculum p o t e n t i a l and p l a n t n u t r i e n t s , but i t was c o n s i d e r e d more d e s i r a b l e than i n t r o d u c i n g top s o i l from the ad j a c e n t f i e l d , had t h i s been p o s s i b l e . A s i d e d r e s s i n g o f n i t r o g e n was a p p l i e d a f t e r a heavy r a i n i n e a r l y J u l y . S p r i n k l e r i r r i g a t i o n was a p p l i e d to both p l o t s as was necessary to maintain a high s o i l moisture i n the " c o n v e n t i o n a l " bed. The p l o t design was a 4 x 4 L a t i n square s p l i t p l o t , w i t h p l a n t i n g dates the major f a c t o r , being s p l i t i n t o " r a i s e d " and " c o n v e n t i o n a l " bed h e i g h t s . R e p l i c a t e beds were 10 f e e t long on 72 inch c e n t e r s . C a r r o t c u l t i v a r 'GoldPak' was p l a n t e d May 11 and at three s u c c e s s i v e 10 day i n t e r v a l s , with a P l a n e t J u n i o r b e l t seeder with a 4 in c h s c a t t e r shoe. 12 Only three rows per bed could be fitted on the "raised" beds due to a reduced planting surface. The following soil and microenvironmental measurements were made every other day. 1) Soil moisture was determined gravimetrically, by oven drying at 105 C. Two soil samples were taken at 0-3, 3-8, 8-15 cm. 2) Temperature was measured with a germanium diode-bridge circuit (Sargent, 1965) at 5, 10, and 15 cm. 3) O2 and COg were measured at 5, 10, and 15 cm (Black et al. , 1965). 4) Precipitation was measured with a standard rain gauge. 5) Height of the water table in a "raised" and "conventional" bed, and level of water in the adjacent drainage ditch was determined. The saturated hydraulic conductivity, bulk density, and partial retention curve of a "raised" and "conventional" bed at 0-3, and 4-7 cm were determined at the end of the growing season (Black e_t al_., 1965). Root samples from the outside rows in two locations of each replicate were taken at successive 10 day intervals from 30 to 70 days after planting. Twenty discoloured rootlets were selected, fixed in formalin glacial actic acid (FAA) (Phillips and Hayman, 1970), cleared and stained with trypan blue (Phillips and Hayman, 1970) and micro-scopically examined under phase contrast (560 u field diameter). Counts of Pythium and Olpidium spores were made at five locations having the highest concentration of spores per root. The remaining root system was indexed for TNV by rub inoculating carborundum-dusted Chenopodium 13 quinoa W i l l d . ( T e a k l e , 1962a). The i n c i d e n c e o f Olpidium i n c a r r o t s at f o u r p l a n t i n g dates on " r a i s e d " and " c o n v e n t i o n a l " beds, and at f i v e sampling times was analyzed by a l a t i n square s p l i t - s p l i t p l o t method o f a n a l y s i s (Winer, 1971). Approximately three months a f t e r p l a n t i n g , the c e n t e r row o f c a r r o t s was h a r v e s t e d , graded i n t o A's, B's, c u l l s , and s m a l l s , and the number and weight o f each c l a s s recorded. Grower's r a i s e d bed t r i a l 1973. Two c o o p e r a t i n g C l o v e r d a l e growers, one with a PRD problem s o i l and the o t h e r with no h i s t o r y o f PRD, c u l t i -vated and f e r t i l i z e d p l o t areas as they would have done f o r a commercial c a r r o t c r o p . Then they prepared three c o n v e n t i o n a l , 300 f o o t long beds, and f o u r r a i s e d , 300 f o o t long beds, made as high as p o s s i b l e w i t h t h e i r e x i s t i n g equipment. Raised beds were c o n s t r u c t e d by throwing s o i l up i n t o beds with a wide f l a n g e d c u l t i v a t o r shoe, and then shaping w i t h a bed shaper to g i v e a f i r m p l a n t i n g s u r f a c e and f i r m s i d e s . Conventional beds were c o n s t r u c t e d o n l y with the bed shaper and were 2-3 inches lower than the r a i s e d beds. C a r r o t c u l t i v a r 'GoldPak' was p r e c i s i o n seeded using a Stan Hay seeder with a b e l t p a t t e r n o f 90:48:90. The t h r e e l i n e s were 1 1/2 inches a p a r t and the d i s t a n c e between seeds o f the out-s i d e l i n e s was 1 1/2 i n c h e s . The f o l l o w i n g row spacings were used: •0 1) three rows 14 i n c h c e n t e r s , r a i s e d beds, 2) f o u r rows 12 i n c h c e n t e r s , r a i s e d beds, 3) f o u r rows 12 i n c h c e n t e r s , c o n v e n t i o n a l beds, and 4) four rows 14 i n c h c e n t e r s , c o n v e n t i o n a l beds. An a d j a c e n t c o n v e n t i o n a l bed, seeded by the grower using a P l a n e t J u n i o r Seeder with a 4 i n c h s c a t t e r shoe, was a l s o used i n the comparison. A f t e r s e e d i n g , the grower managed the p l o t a c c o r d i n g to c u r r e n t recommended p r a c t i c e s . F i v e , 20 f o o t p l o t s evenly spaced i n the 300 f o o t row served as the sample areas. C a r r o t emergence i n a 3 f o o t s e c t i o n o f row was determined f o r each r e p l i c a t e . Root samples were taken from two l o c a -t i o n s w i t h i n a r e p l i c a t e at 50 and 80 days a f t e r p l a n t i n g and 50 d i s -c o l o u r e d and 50 white r o o t l e t s were s e l e c t e d from each sample. Twenty roots were f i x e d i n FAA, s t a i n e d with Phloxine and K0H ( T u i t e , 1969), and m i c r o s c o p i c a l l y examined with phase c o n t r a s t a t f i v e l o c a t i o n s o f hig h e s t spore c o n c e n t r a t i o n per r o o t f o r Pythium and Olpidium spores. Twenty-one r o o t l e t s were washed f o r 24 hours i n c o l d tap water and a s e c t i o n o f each was p l a t e d on a medium s e l e c t i v e f o r Pythium. The s e l e c -t i v e medium was a m o d i f i c a t i o n o f the medium o f Tsao and Ocana (1969). Ben!ate (10 ppm a c t i v e i n g r e d i e n t , 50% WP, E.I. Dupont de Nemours and Co., Wilmington) was used i n p l a c e o f P i m a r i c i n . I n g r e d i e n t s f o r the media were 10 ppm Be n l a t e , 100 ppm PCNB (75% WP, O l i n Mathieson Chem. Corp., N.Y.), 10 ppm Vancomycin HCl (Sigma Chem. Co., S t . L o u i s ) , 17 g Corn meal agar (CMA, pH 5.6, BBL), and 1 l i t e r d i s t i l l e d water. The medium was a u t o c l a v e d f o r 15 minutes a t 15 p s i . P l a t e s were incubated i n the dark a t 25 +_ 1 C and were examined every 12 hours f o r Pythium growth. The remaining r o o t l e t s and the whole ro o t system were s e p a r a t e l y indexed to carborundum-dusted C. quinoa. Ca r r o t s were harvested 100 to 110 days a f t e r p l a n t i n g , graded i n t o A's, B's, c u l l s , and s m a l l s , and the number and weight o f each c l a s s recorded. P r e c i s i o n seeding t r i a l . The p l o t was s i t u a t e d on the land pre-v i o u s l y used f o r t h i s p r o j e c t i n 1971 and 1972. The lan d and r a i s e d 15 beds were prepared by the grower i n the same f a s h i o n as 1972. A r e p l i -cated 3 x 3 l a t i n square s p l i t p l o t design was used, with p l a n t spacing the major f a c t o r being s p l i t by two c a r r o t c u l t i v a r s . GoldPak, a PRD s u s c e p t i b l e v a r i e t y , and HiPak, a PRD t o l e r a n t v a r i e t y , were p r e c i s i o n seeded with a Stan Hay Seeder a t seed spacings of 1 1/4, 1 1/2, and 2 inches with f o u r rows per bed. P l o t s were 20 f e e t i n l e n g t h . I r r i -g a t i o n was a p p l i e d to maintain a high s o i l water content to f a v o r d i s e a s e i n c i d e n c e . Weeds were c o n t r o l l e d with three a p p l i c a t i o n s o f Stoddard Solvent a t 60-80 g a l l o n s per a c r e . C a r r o t emergence and sampling f o r d i s e a s e i n c i d e n c e were per-formed i n the same manner as p r e v i o u s l y d i s c u s s e d . Samples f o r the d i s e a s e survey were taken a t 40 and 55 days a f t e r s eeding. Weed samples were c o l l e c t e d from the p l o t i n the second sampling and r o o t s were pro-cessed i n the same manner as the c a r r o t r o o t s . H a r v e s t i n g was done as i n the grower's r a i s e d bed t r i a l except t h a t the c u l l s were broken i n t o two c a t e g o r i e s : 1) " c u l l s " which i n -cluded s p l i t , bent and green shouldered c a r r o t s , and 2) " f o r k s " which i n c l u d e d f o r k e d , rough, and h a i r y c a r r o t s . Laboratory S t u d i e s Olpidium. C u l t u r e s o f Olpidium were ob t a i n e d by b a i t i n g s o i l samples with c a r r o t c u l t i v a r 'GoldPak E l i t e ' i n the greenhouse. F i e l d s o i l h eld a t greenhouse temperatures f o r one y e a r , and f r e s h f i e l d s o i l t h a t had TNV-infected c a r r o t s were used. S e e d l i n g c a r r o t s were indexed on fJ. quinoa to c o n f i r m presence or absence of TNV. 16 Attempts were made to culture Olpidium from the seedling carrots. Fifteen ml plastic beakers with no drainage, 2 x 2 inch and 4 x 4 inch plastic pots with drainage, and 3 inch clay pots with drainage were f i l l e d with washed Fraser River sand, and 14-21 day old carrot or lettuce (Lactuca sativa L. 'Grande Rapids') seedlings were transplanted in the pots. Incoulation of the seedlings was done by: 1) pouring actively swimming zoospore suspensions over the roots of seedlings (Campbell and Grogan, 1964), 2) soaking seedlings in zoospore suspensions for 15-60 minutes before transplanting (Teakle, 1962a), 3) transplanting an infected plant in with the seedlings, or 4) mixing infected roots in the sand before transplanting (D.J.S. Barr, personal communication). Zoospore suspensions were obtained by removing infected plants from soi l or sand, quickly washing roots in cool tap water, and soaking roots in either d i s t i l l e d water, tap water, 1:3 or 1:20 Hoagland's nutrient solution, or pond water (Kassanis and Macfarlane, 1964; Teakle, 1962a). The presence of Olpidium was confirmed by examining several rootlets under the microscope for actively swimming zoospores or zoosporangia. Cultures were maintained in a growth cabinet at the optimum temperature of 18 + 2 C (Fry and Campbell, 1966) with a photoperiod of 12 hours and a l ight intensity of 4300 lux. Cultures were watered daily with either tap water supplemented with nutrient solution once a week, or fu l l strength nutrient solution. Transfer of Olpidium from inoculated to healthy plants was attempted at 1 to 8 weeks after inoculation. Tobacco necrosis virus. 'GoldPak E l i t e ' carrot seed was germinated in the dark at 25 + 1 C on moist f i l t e r paper unti l the roots were 17 approximately 3 cm i n l e n g t h a t which time they were i n o c u l a t e d w i t h TNV. The v i r u s source was obtained by s i n g l e l e s i o n i s o l a t i o n from C. quinoa t h a t had been i n o c u l a t e d with PRD symptomed r o o t s . I n o c u l a t e d C. quinoa leaves having almost c o n f l u e n t l e s i o n s , were ground i n .05 M phosphate b u f f e r , pH 8.5, carborundum was added and 50 c a r r o t roots were e i t h e r dipped i n t o the v i r u s s o l u t i o n , or brushed 10 times with a p a i n t brush dipped i n the v i r u s s o l u t i o n . Control s e e d l i n g s were t r e a t e d s i m i l a r l y except t h a t only carborundum and phosphate b u f f e r were used. A f t e r f o u r days, roots were examined f o r brown l e s i o n s , dipped i n 10% Concentrate R.B.S. 25* ( F i s h e r S c i e n t i f i c Co. Ltd.) f o r 30 seconds to i n a c t i v a t e s u r f a c e v i r u s contamination and indexed on C. quinoa. Greenhouse Crop R o t a t i o n A greenhouse crop r o t a t i o n experiment was conducted from October 1973 to June 1974. Lumbrum muck, from two farms w i t h a known PRD prob-lem, was thoroughly mixed, potted i n 19.5 x 25 cm p l a s t i c pots and arranged on the greenhouse bench i n a randomized block d e s i g n . F i v e crop r o t a t i o n s (Table I) with s i x r e p l i c a t e s per sequence were used. A known number o f seeds of c a r r o t , c u l t i v a r 'GoldPak E l i t e , ' onion ( A l l i u m cepa L. 'Autumn S p i c e ' ) , o r l e t t u c e c u l t i v a r 'Pennlake' were p l a n t e d . Emergence was determined a f t e r 2 to 3 weeks. The percent germination f o r each c r o p p i n g was estimated by i n c u b a t i n g s i x l o t s o f 30 or 50 seed on moist f i l t e r paper i n p e t r i d ishes i n the dark a t 25 +_ 1 C. The f i r s t cropping was grown at 16-20 C and subsequent croppings were at 10-16 C. P l a n t s r e c e i v e d supplement l i g h t i n g o f 16 hours per day a t an i n t e n s i t y of 18 3600 l u x . Pots were f r e q u e n t l y s p r i n k l e watered so as to maintain a high s o i l moisture content. At 3 weeks, pots were thinned to e i t h e r 12 c a r r o t , 20 onion or 20 l e t t u c e p l a n t s . Four weeks a f t e r p l a n t i n g , 20-20-20 f e r t i -l i z e r (500 l b / a c r e ) was a p p l i e d as a drench. Table I. Sequence o f the crops u t i l i z e d i n the r o t a t i o n experiment R o t a t i o n 1 Cropping sequence 2 3 4 A C 1 C C C B C L 0 C C c 0 L C D c L L C E c 0 0 C C = c a r r o t ; L = l e t t u c e ; 0 = onion. S i x t y days a f t e r p l a n t i n g , p l a n t s were removed from the s o i l , r oots and tops were weighed and 41 d i s c o l o u r e d roots were removed and t r e a t e d as p r e v i o u s l y d e s c r i b e d . Within a day o f h a r v e s t i n g , the next crop was p l a n t e d . A composite s o i l sample, made up o f small q u a n t i t i e s o f s o i l from pots w i t h i n a treatment, was analyzed f o r Pythium propagule number. The s o i l was thoroughly mixed and s o i l d i l u t i o n s o f 1:25, 1:50, 1:100, 1:200 were made with 100 ml o f 0.25% water agar ( D i f c o ) plus 200 ppm Vancomycin. While the s o i l agar mixture was being s t i r r e d , 1 ml o f s o l u t i o n was removed and p i p e t t e d onto each o f f i v e p e t r i p l a t e s o f 19 Pythium s e l e c t i v e medium. The p l a t e s were incubated i n the dark at 25 +_ 1 C for 18-24 hours. The s o i l water agar suspension was washed o f f , p l a t e s were s t a i n e d with KOH and P h l o x i n e , and c o l o n i e s counted. Propagule number was c a l c u l a t e d r e l a t i v e to oven dry s o i l (Ocana and Tsao, 1966). RESULTS E t i o l o g y I n d i r e c t d e t e r m i n a t i o n o f organism r e s p o n s i b l e f o r PRD. The in-c i d e n c e o f Pythium, Olpidium and TNV were compared between a non-problem and two PRD problem f i e l d s .and between brown and white roots (Table I I ) . Fast and slow growing Pythium spp. grew out of roots p l a t e d on the s e l e c -t i v e medium. The roo t systems o f c a r r o t s in PRD s o i l showed t y p i c a l r o o t t i p browning, whereas c a r r o t r o o t s from non-problem s o i l were o n l y g r e y i s h brown in c o l o u r . These g r e y i s h brown roots were analyzed as brown r o o t s . M i c r o s c o p i c examination o f t y p i c a l d i s e a s e d r o o t l e t s r e v e a l e d t h a t the epidermal c e l l s were o f t e n sloughed o f f . Epidermal c e l l s and roo t h a i r s were i n t a c t on r o o t l e t s from non-problem JJH^ Olpidium, TNV and both slow and fas™growing Pythium spp. were found in r o o t s from PRD s o i l , but on l y 01 p i diurn and f a s t growing Pythium spp. were found in r o o t s from non-problem areas (Table I I ) . Olpidium spores were observed much more f r e q u e n t l y in problem than non-problem f i e l d s . In PRD f i e l d s , all f o u r organisms were found in brown and white Table I I . Incidence o f Olpidium, f a s t and slow growing Pythium spp. and TNV i n brown and white r o o t s o f a non-problem and two PRD problem f i e l d s a t two sampling times i n 1973 Olpidium Fast Pythium Slow Pythium TNV Av. s p o r e s / f i e l d % roots i n f e c t e d % roots i n f e c t e d % recovery Sample 1 Sample 2 Sample 1 Sample 2 Sample 1 Sample 2 Sample 1 Sample 2 Brown Roots P r e c i s i o n seeded problem s o i l 12.9 7.7 11.0 9.5 15.0 14.5 55 53 Grower's t r i a l problem s o i l 21.5 16.4 12.5 2.7 8.6 8.8 80 76 Grower's t r i a l non-problem s o i l 2.2 - 22.1 - 0.0 - 0 -White Roots P r e c i s i o n seeded problem s o i l 2.1 7.3 3.0 12.5 0.5 2.5 19 42 Grower's t r i a l problem s o i l 5.0 9.0 6.5 1.9 1.3 2.5 85 60 Grower's t r i a l non-problem s o i l 0.2 - 7.2 >;- 0.0 - 0 -21 roots, but only the slow growing Pythium isolate was consistently re-covered at much lower levels in white roots (Table II) . Olpidium spore levels decreased in brown roots and increased in white roots in the second sampling. The incidence of slow growing Pythium remained constant in brown roots and increased in white roots, but the recovery of slow Pythium in the second sampling was s t i l l 6 times less in white roots than brown roots. The selection of white roots at the second sampling was much more d i f f i c u l t as the roots were not a true white but rather an off-white or grey colour. PRD symptom production by Olpidium and TNV. Cultures of Olpidium, free of TNV, were successfully obtained by baiting one-year-old stored f ie ld soi l with carrots, while Olpidium-TNV cultures were obtained by baiting freshly collected f i e ld s o i l . Carrot and lettuce roots inoculated with Olpidium were white or grey-white in colour. TNV in the presence of Olpidium had no effect on root t ip browning. No orange-brown lateral roots were observed through-out the study even i f high levels of spores were present in the roots. Microscopic examination of the grey coloured roots sometimes revealed Olpidium zoosporangia and resting spores. Mycelium was sometimes ob-served in the grey rootlets and was most common in roots from plants in poorly drained pots. If zoosporangia were observed, zoospores could usually be seen swimming around the root within 5 minutes of soaking the roots in water. Zoospores were most active in tap water and 1:20 nutrient solut ion, and remained active for about 20 minutes. Zoospores in d i s t i l l e d water 22 and n u t r i e n t s o l u t i o n more co n c e n t r a t e d than 1:20 were s l u g g i s h and became immobile w i t h i n 5 minutes o f r e l e a s e . Several sources o f pond and stream water v/ere used to soak r o o t s , but none was s u p e r i o r to tap water i n m a i n t a i n i n g zoospore a c t i v i t y . Olpidium c u l t u r e s t r a n s f e r r e d on c a r r o t were very d i f f i c u l t to maintain from one t r a n s f e r to the next. Olpidium i n f e c t i o n o f c a r r o t s could r a r e l y be detected by m i c r o s c o p i c examination a f t e r 2 or 3 t r a n s f e r s . However, c u l t u r e s o f Olpidium on l e t t u c e , obtained from the same source as the c a r r o t i s o l a t e s , produced abundant zoospores a t each t r a n s f e r and were maintained f o r a t l e a s t s i x t r a n s f e r s . Olpidium c u l t u r e s main-t a i n e d on l e t t u c e and t r a n s f e r r e d to c a r r o t a l s o d i e d out a f t e r s e v e r a l t r a n s f e r s , but i f zoospores from c a r r o t s were t r a n s f e r r e d to l e t t u c e , Olpidium reproduced w e l l on the l e t t u c e . The d i f f e r e n t types o f pots and i n o c u l a t i o n techniques had l i t t l e e f f e c t on the a b i l i t y to c u l t u r e Olpidium on l e t t u c e , but the same procedures with c a r r o t , r e s u l t e d i n i n c o n s i s t e n t s u r v i v a l o f Olpidium. When care was taken to keep TNV-infected and TNV-free Olpidium c u l t u r e s s e p a r a t e , TNV was never d e t e c t e d i n c u l t u r e s o r i g i n a t i n g from s t o r e d , f i e l d s o i l . Both l e t t u c e and c a r r o t became i n f e c t e d w i t h TNV when i n c o u l a t e d with Olpidium-TNV c u l t u r e s . Sometimes c a r r o t s i n o c u l a t e d with Olpidium-TNV c u l t u r e s , w h i l e not having Olpidium v i s i b l e i n the r o o t s , were i n f e c t e d with the v i r u s . N e c r o t i c spots developed on c a r r o t roots r u b - i n o c u l a t e d with TNV (Table I I I ) . However, c o n t r o l r u b - i n o c u l a t e d and d i p - i n o c u l a t e d roots a l s o developed l e s i o n s . Lesions developed a t p o i n t s where the 23 roots were damaged and were more prevalent on long than short roots. The long roots were more easily damaged in handling. TNV was detected only from carrots that had been rub or dip-inoculated in the TNV solution. Symptomless rub-inoculated and dip-inoculated roots in TNV extracts were positive for TNV (Table III) . Table III . Number of carrot root systems developing necrotic symptoms and indexing TNV positive after rub or dip-inoculation in carborundum and .05 M phosphate buffer with and without TNV . Treatment Roots with necrotic symptoms No. No. TNV Symptomless roots No. ' No. TNV TNV rub TNV dip Control rub Control dip 27 6 13 10 14 2 0 0 23 44 37 40 6 8 0 0 Epidemiology Olpidium population and the microenvironment. The f i e l d experiment in 1972 was designed to study Olpidium incidence of carrot on "raised" and "conventional" beds at different planting dates. Olpidium incidence was s ignif icant ly influenced by planting date. The May 23 planting had a s ignif icant ly greater (P = .05) average number of spores per 24 microscope f i e l d than the l a t e p l a n t i n g of June 13 ( F i g u r e 1 ) . C a r r o t age a t sampling was a l s o a s i g n i f i c a n t f a c t o r i n the i n c i d e n c e o f O l p i - dium ( F i g u r e 2 ) . There was a s i g n i f i c a n t i n t e r a c t i o n between p l a n t i n g date and sampling time as can be seen i n F i g u r e 3. L a t e - p l a n t e d c a r r o t s always had a low Olpidium p o p u l a t i o n throughout the growing season. By c o n t r a s t , c a r r o t s t h a t were p l a n t e d e a r l y , had a low i n f e c t i o n when the c a r r o t s were young, but a high i n f e c t i o n l e v e l as they grew o l d e r . Bed h e i g h t and any i n t e r a c t i o n s o f p l a n t i n g date or age o f c a r r o t s a t sampling with bed h e i g h t had no s i g n i f i c a n t e f f e c t on Olpidium p o p u l a t i o n (Appendix I ) . C a r r o t r o o t l e t s i n the e a r l i e s t p l a n t e d " r a i s e d " beds had 34.8% o f the microscope f i e l d s with Olpidium. T h i s was s i g n i f i c a n t l y fewer (P = .05) than " c o n v e n t i o n a l " beds which had 50.3% o f the micro-scope f i e l d s with Olpidium. However, i n l a t e r p l a n t i n g s t h e r e was no s i g n i f i c a n t d i f f e r e n c e o f Olpidium i n c i d e n c e i n " r a i s e d " and "conven-t i o n a l " beds. In the f o u r t h p l a n t i n g , " c o n v e n t i o n a l " beds had a s l i g h t l y lower average number o f spores per microscope f i e l d than " r a i s e d " beds; 1.8 and 2.2 s p o r e s / f i e l d r e s p e c t i v e l y . The data on the number o f Olpidium spores per microscope f i e l d was transformed i n t o the number of microscope f i e l d s w i t h : one and g r e a t e r than one, f i v e and g r e a t e r than f i v e , and 10 and g r e a t e r than 10 spores. An a n a l y s i s o f v a r i a n c e o f the transformed data r e v e a l e d no changes i n s i g n i f i c a n t f a c t o r s except t h a t c a r r o t age at sampling was no lo n g e r s i g n i f i c a n t (P = .05). There was a s i g n i f i c a n t i n c r e a s e i n the marketable y i e l d on " r a i s e d " beds over " c o n v e n t i o n a l " beds (Table IV). Both bed types had 25 10 L J i i i i i 1 2 3 4 Planting date F igure 1 . Average number o f O lp id ium spores per microscope f i e l d (560 y) a t f o u r p l a n t i n g d a t e s . 1 = May 1 1 , 2 = May 2 3 , 3 = May 30 , 4 = June 13 . Each p o i n t i s the mean o f 4000 c o u n t s . ! 1 — i , i i i 30 40 50 60 70 Carrot age (days) Figure 2. Average number of Olpidium spores per microscope field (560 y) at five carrot ages. Each point represents the mean of 3200 counts. 27 T 1 — r 30 k0 50 60 70 Carrot age (days) F i g u r e 3. Average number o f Olpidium spores a t f i v e sampling ages and four p l a n t i n g d a t e s . Each p o i n t r e p r e s e n t s the mean of 800 counts. Table IV. C a r r o t y i e l d i n " r a i s e d " and " c o n v e n t i o n a l " beds and at f o u r p l a n t i n g dates i n 1972 P l a n t i n g Date No. A wt No. B wt Marketable No. wt C u l l s No. wt Smalls No. wt May 11 49.8 a 7.1 a 0.0 b 0.0 b 49.8 a 7.1 a 72.0 a 5.1 a 82.4 a 2.6 b May 23 41.5 a 4.9 a 2.9 a 1.1 a 44.4 a 6.1 a 36.0 b 3.6 a . 39.5 b 1.8 ab May 30 43.1 a 6.3 a 0.1 b 0.1 b 43.4 a 6.4 a 35.0 b 3.3 a 39.4 b 1.5 a June 11 38.8 a 6.0 a 0.0 b 0.0 b 38.8 a 6.1 a 37.0 b 4.0 a 25.6 b 1.1 a Bed Height Raised 55.3 a 7.9 a 1.2 a 0.5 a 56.5 a 8.4 a 42.6 a 4.5 a 34.8 a 1.3 a Conventional 31.3 b 4.3 b 0.3 a 0.1 a 31.6 b 4.4 fa- 47.4 a 3.5 b 58.6 b 2.1 b Means w i t h i n a column with the same l e t t e r do not d i f f e r s i g n i f i c a n t l y (P = .05). 29 the same t o t a l number o f c a r r o t s but t h e r e was a s h i f t from a l a r g e number o f small c a r r o t s i n " c o n v e n t i o n a l " beds to a g r e a t e r number o f A c a r r o t s i n " r a i s e d " beds (Table IV). The c a r r o t s i n " r a i s e d " beds were l a r g e r i n s i z e (.105 l b / c a r r o t versus .071 l b / c a r r o t ) , and as a consequence there was a s i g n i f i c a n t l y g r e a t e r weight of c u l l s from " r a i s e d " beds than " c o n v e n t i o n a l " beds even though " c o n v e n t i o n a l " beds had a g r e a t e r number o f c u l l s . However, on a percent b a s i s (wt c u l l s / t o t a l wt), " r a i s e d " beds had a lower, but not s i g n i f i c a n t l y lower, percent weight c u l l s than " c o n v e n t i o n a l " beds (31.8% versus 36.4%). Marketable y i e l d and weight o f c u l l s were not a f f e c t e d by p l a n t i n g dates. The number o f small and c u l l c a r r o t s i n the e a r l i e s t p l a n t i n g was s i g n i f i c a n t l y g r e a t e r (P = .05) than the l a t e r p l a n t i n g s . The e a r l y p l a n t i n g had a d e n s i t y o f 20 c a r r o t s per f o o t o f row compared to an average o f 11.3 c a r r o t s per f o o t o f row i n l a t e r p l a n t i n g s . There was a s i g n i f i c a n t i n t e r a c t i o n o f bed h e i g h t and p l a n t i n g date with r e s p e c t to the number o f c u l l s . "Raised" beds a t the e a r l i e s t p l a n t i n g date had s i g n i f i c a n t l y fewer c u l l s than " c o n v e n t i o n a l " beds a t the same date, but i n the t h i r d p l a n t i n g , " c o n v e n t i o n a l " beds had s i g n i f i c a n t l y fewer number o f c u l l s than r a i s e d beds (Table V). Attempts were made to c o r r e l a t e changes i n Olpidium p o p u l a t i o n with the s o i l microenvironmental measurements c o l l e c t e d by Hedi T r a b e l s i . Temperature d i d not d i f f e r g r e a t l y i n " r a i s e d " and " c o n v e n t i o n a l " beds. At the 15 cm depth, s o i l temperature s l o w l y i n c r e a s e d from 12 C to 16-18 C, the l a t t e r l e v e l being optimal f o r Olpidium r e p r o d u c t i o n . There was g e n e r a l l y h i g h e r Olpidium p o p u l a t i o n l a t e r i n the growing season, but 30 the late planting of carrots had very low levels of Olpidium during the period of most favorable temperature. Table V. Number of cul ls from raised and conventional beds at four planting dates Number of cul ls Planting date Raised Conventional May 11 58.3 b1 85.8 a May 23 36.0 cd 36.0 cd May 30 45.3 be 24.8 d June 13 30.8 cd 43.3 bed ^Means with the same letter do not d i f fer s ign i f icant ly . A three times greater saturated hydraulic conductivity in "raised" beds than in "conventional" beds had no signif icant effect on Olpidium incidence. Measurements of 0^ and C0£ in the so i l atmosphere showed sl ight changes. The concentration of C0£ s l ight ly increased after a period of rain and was at a higher concentration as the depth increased. After a ra in , the oxygen concentration in the soi l decreased but the lowest level measured was 19%. This small change in concentration was con-sidered unlikely to have had any affect on Olpidium. Soil water content, measured gravimetrically every other day at three depths was higher in "conventional" beds than "raised" beds through-out the growing season (Figure 4). The water content at the 8-15 cm depth remained r e l a t i v e l y constant between 100 and 120% water content u n t i l J u l y 11, when a heavy r a i n f a l l i n c r e a s e d s o i l water content f o r 10 days ( F i g u r e 4 ) . 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 Olpidium p o p u l a t i o n i n " r a i s e d " and " c o n v e n t i o n a l " beds, even though "conven-t i o n a l " beds had about a 10% g r e a t e r water content. The water t a b l e was measured i n one " r a i s e d " and one "conven-t i o n a l " bed a t o p p o s i t e edges o f the p l o t . The p i e z o m e t r i c l e v e l o f the " r a i s e d " bed was about 20 cm higher than the " c o n v e n t i o n a l " bed. The frequency and q u a n t i t y o f r a i n f a l l was c o r r e l a t e d with O l p i - dium p o p u l a t i o n changes. R a i n f a l l had been very low du r i n g May and the e a r l i e s t p l a n t e d c a r r o t s had low l e v e l s o f Olpidium (Table V I ) . The second p l a n t i n g o f c a r r o t s had high l e v e l o f Olpidium when f i r s t sampled on June 21. T h i s p l a n t i n g had r e c e i v e d three good waterings at n e a r l y weekly i n t e r v a l s from p l a n t i n g (Table V I ) . The t h i r d p l a n t i n g a l s o had high l e v e l s o f i n f e c t i o n a t the f i r s t sampling on J u l y 1 (Table VI). I t had r e c e i v e d two good r a i n f a l l s 14 days a p a r t which would a l s o f a v o r Olpidium spread. The mid June p l a n t i n g o f c a r r o t s had a very low l e v e l o f i n f e c t i o n even though i t had r e c e i v e d f o u r heavy p e r i o d s o f water (Table V I ) . On J u l y 20, high l e v e l s o f Olpidium spores were found i n r o ots o f the f i r s t three p l a n t i n g s . The second and t h i r d p l a n t i n g s had higher i n f e c t i o n s o f Olpidium on J u l y 20 than f o u r days e a r l i e r (Table V I ) . The heavy r a i n f a l l o f J u l y 10th to 13th fav o r e d spread and i n f e c t i o n r e s u l t i n g i n the Olpidium b u i l d up e v i d e n t i n the l a t e r sample. A f t e r the deluge o f mid-July, there was only one p o t e n t i a l i n f e c t i o n p e r i o d as the r e s t o f the summer was dry. Olpidium i n f e c t i o n i n the Table VI. C o r r e l a t i o n between the average number o f Olpidium spores per microscope f i e l d a t v a r i o u s sampling times and the frequence o f i n f e c t i o n p e r i o d s o f p r e c i p i t a t i o n g r e a t e r than 1/2 inch i n 1972 Sampling date June 13 June 21 J u l y 1 J u l y 16 J u l y 20 J u l y 28 Aug. 9 Aug. 19 P l a n t i n g date 1.3 1 May 11 1.6 8.5 10.5 9.6 May 23 7.4 9.1 6.7 • 11.4 14.5 May 30 6.5 3.2 10.5 6.9 4.9 June 13 2.1 3.4 2.2 1.9 0.4 No. i n f e c t i o n p e riods 7-21 days p r i o r to sampling 1 3 2 4 4 3 1 1 Number spores per microscope f i e l d . 34 t h i r d p l a n t i n g decreased i n l a t e r samplings but was s t i l l moderately high, w h i l e i n the f o u r t h p l a n t i n g , Olpidium i n c i d e n c e decreased a f t e r the heavy r a i n f a l l and was a t a very low l e v e l as the summer progressed (Table V I ) . In the June and e a r l y J u l y samplings, a g r e a t e r percentage o f the Olpidium spores were zoosporangia. However, l a t e r i n the summer, a g r e a t e r p r o p o r t i o n o f the spores were r e s t i n g spores. Many o f the r o o t s a f t e r J u l y 28 had v e s i c u l a r a r b u s c u l a r mycorrhizae. Grower's r a i s e d bed t r i a l . During 1973, the performance of r a i s e d beds was e v a l u a t e d under d i f f e r e n t commercial c u l t u r a l management regimes. One farm, using 68 inch wheel c e n t e r s , made con v e n t i o n a l beds with a 51 inch p l a n t i n g s u r f a c e and r a i s e d beds with a 43 inch s u r f a c e . The second farm used 72 i n c h c e n t e r s and formed beds with p l a n t i n g s u r f a c e widths o f approximately 46 and 50 inches f o r r a i s e d and c o n v e n t i o n a l beds r e s p e c t i v e l y . The emergence i n the problem and non-problem s o i l was 57% and 46% r e s p e c t i v e l y . The number o f c a r r o t s per f o o t o f row v a r i e d f o r p r e c i s i o n and s c a t t e r shoe seeded p l o t s ( T a b l e V I I ) . The c a r r o t s were growing i n the same l o c a t i o n an average o f 10.3% o f the time i n the p r e c i s i o n seeded p l o t . A comparison o f the d i s e a s e organisms found i n problem and non-problem f i e l d s , and brown and white roots has been d i s c u s s e d i n the s e c t i o n o f " I n d i r e c t d e t e r m i n a t i o n o f organism r e s p o n s i b l e f o r PRD." Only the d i s e a s e r a t i n g o f the v a r i o u s treatments w i l l be d i s c u s s e d f o r each f i e l d s e p a r a t e l y . 35 Table VII. Average c a r r o t emergence and percentage of doubles i n p r e c i s i o n - s e e d e d and s c a t t e r shoe-seeded beds No. c a r r o t s / f t row Non-problem PRD f i e l d f i e l d % doubles PRD f i e l d Non-problem f i e l d P r e c i s i o n seeded 9.2 11.4 8.0 12.5 S c a t t e r shoe seeded 8.7 7.0 On the non-problem s o i l , t h e r e was no s i g n i f i c a n t d i f f e r e n c e (P = .05) i n the i n c i d e n c e of Olpidium and fas t - g r o w i n g Pythium i n con-v e n t i o n a l and r a i s e d beds (Table V I I I ) . The f i r s t sampling o f c a r r o t r o o t l e t s from conventional beds on problem s o i l had a hig h e r number o f Olpidium spores than those from r a i s e d beds (28 versus 16 spores/micro-scope f i e l d ) , but i n the l a t e r sample t h e r e was l i t t l e d i f f e r e n c e i n spore l e v e l s (19.4 versus 18.1 s p o r e s / f i e l d ) (Table V I I I ) . The grower's s c a t t e r shoe-seeded bed had fewer spores than the p r e c i s i o n - s e e d e d beds (7.1 versus 19.7 s p o r e s / f i e l d ) i n the second sample. The percentage of r o o t s i n f e c t e d with slow-growing Pythium was n e a r l y the same i n r a i s e d and c o n v e n t i o n a l beds and between the f i r s t and second samples. There was no s i g n i f i c a n t d i f f e r e n c e (P = .05.) i n fas t - g r o w i n g Pythium and TNV recovery i n r a i s e d and conventional beds (Table V I I I ) . F a s t growing Pythium was recovered l e s s f r e q u e n t l y from c a r r o t s i n the second sample than the f i r s t (Table V I I I ) . 12 inch and 14 inch row centers appeared to have l i t t l e e f f e c t on d i s e a s e i n c i d e n c e . Table V I I I . Organism survey i n r a i s e d and conventional beds on PRD problem and non-problem s o i l a t two sample times Olpidium Fast Pythium Slow Pythium TNV Av. s p o r e s / f i e l d % roots i n f e c t e d % roots i n f e c t e d % recovery Treatment Sample 1 Sample 2 Sample 1 Sample 2 Sample 1 Sample 2 Sample 1 Sample 2 Problem S o i l a 1 S c a t t e r shoe 19.9 7.1 a 12.4 a 0.9 a 9.5 a 12.4 a 100 a 20 a 3 row 14" r a i s e d 15.8 a 15.3 ab 10.5 a 2.9 a 6.7 a 4.8 a 100 a 40 a 4 row 12" r a i s e d 16.3 a 23.5 b 15.2 a 1.9 a 9.5 a 11.0 a 100 a 20 a 4 row 12" conven-t i o n a l 25.1 a 15.0 ab 12.4 a 3.8 a 9.5 a 2.9 a 100 a 0 a 4 row 14" conven-t i o n a l 30.6 a 21.1 b 12.4 a 0.9 a 7.6 a 13.3 a 100 a 40 a Non-problem S o i l S c a t t e r shoe 1.4 a - 13.4 a - 0.0 a - 0 a -3 row 14" r a i s e d 3.0 a - 17.1 a - 0.0 a - 0 a -4 row 12" r a i s e d 2.5 a - 24.8 a - 0.0 a - 0 a -4 row 12" conven-t i o n a l 1.7 a - 27.6 a - 0.0 a - 0 a -4 row 14" conven-t i o n a l 2.6 a - 27.6 a - 0.0 a - 0 a -Means w i t h i n the same column with the same l e t t e r do not d i f f e r s i g n i f i c a n t l y (P = .05). 37 Marketable y i e l d o f c a r r o t s , from r a i s e d and c o n v e n t i o n a l beds with f o u r rows per bed on non-problem s o i l were not s i g n i f i c a n t l y d i f f e r e n t (P = .05). The r a i s e d bed with f o u r rows on 12 inch c e n t e r s y i e l d e d e q u a l l y w e l l as f o u r row 12 i n c h spaced c o n v e n t i o n a l beds (Table IX). More c u l l s were harvested from the s c a t t e r shoe-seeded bed than the p r e c i s i o n seeded beds (Table IX). There was no advantage o f using 14 inch c e n t e r s over 12 inch spaced c o n v e n t i o n a l beds. On the problem s o i l , c a r r o t y i e l d on c onventional beds was c o n s i s t e n t l y b e t t e r than on r a i s e d beds (Table IX). The s c a t t e r shoe seeded bed and the f o u r row, 12 inch c e n t e r conventional bed produced a s i g n i f i c a n t l y g r e a t e r (P = .05) weight o f marketable c a r r o t s than the r a i s e d beds. There was a g r e a t e r weight o f marketable c a r r o t s from the grower-planted s c a t t e r shoe-seeded bed because they were p l a n t e d e a r l i e r and were more mature. In a d d i t i o n , the p l a n t d e n s i t y was lower (Table V I I ) , r e s u l t i n g i n more c a r r o t s being i n the B c l a s s (Table IX). Fewer c u l l s (P = .05) were found i n the f o u r row, 14 inch c o n v e n t i o n a l bed than i n the s c a t t e r shoe-seeded and the f o u r row 12 inch c e n t e r r a i s e d bed. The three row 14 inch c e n t e r r a i s e d bed had n e a r l y the same marketable y i e l d , and s i g n i f i c a n t l y fewer small c a r r o t s (P = .05) than the f o u r row 12 i n c h c e n t e r , r a i s e d bed. The p l o t on the problem s o i l c o n t a i n e d two d i s t i n c t s o i l t y p e s : an o r g a n i c , and an i n o r g a n i c c l a y . Marketable y i e l d was b e t t e r i n con-v e n t i o n a l beds compared to the r a i s e d beds on both s o i l types. However, the c u l l r a t e was twice as great on r a i s e d compared to c o n v e n t i o n a l beds i n o r g a n i c - c l a y s o i l , but there was no d i f f e r e n c e between bed types Table IX. Carrot y ie ld in PRD problem and non-problem soi l Treatment A lb . B lb . Culls lb . Smalls lb . Problem soi l 96.5 b1 Scatter shoe 45.0 a 24.4 a 0.5 c 3 row 14 inch raised 95.8 b 7.0 b 20.2 ab 5.5 b 4 row 12 inch raised 97.6 b. 8.6 b 26.6 a .8.5 a 4 row 12 inch conventional 114.9 a 14.3 b 21.0 ab 4.1 b 4 row 14 inch 112.2 ab 9.6 b 17.6 b 6.9 ab Non-problem soi l Scatter shoe 89.0 be 15.6 a 21.4 a 4.8 a 3 row 14 inch rai sed 78.6 c 5.5 b 9.9 b 3.0 a 4 row 12 inch raised 96.4 ab 5.7 b 13.6 b 5.7 a 4 row 12 inch conventional 103.3 a 8.7 b 11.6 b 3.8 a 4 row 14 inch conventional 88.0 c 13.9 a 13.7 b 3.1 a ^Means within a f icantly (P = .05). column with the same letter do not d i f fer s ign i -39 on the or g a n i c s o i l . Due to d i s t r i b u t i o n o f the c l a y area i n the p l o t , only two r e p l i c a t e s were used to make these comparisons. C a r r o t s i n the problem s o i l s u f f e r e d a water s t r e s s . I r r i g a t i o n could not be a p p l i e d as the water i n the drainage d i t c h e s was too s a l i n e . The drought was so severe t h a t some of the c a r r o t s i n the r a i s e d beds f e l t rubbery a t harvest. P r e c i s i o n seeding t r i a l . A f i e l d t r i a l was conducted i n 1973 to determine i f p r e c i s i o n seed spacing had any e f f e c t on the i n c i d e n c e o f PRD i n s u s c e p t i b l e and t o l e r a n t c a r r o t c u l t i v a r s . HiPak (HP), a PRD t o l e r a n t v a r i e t y , had 59% emergence which was s i g n i f i c a n t l y g r e a t e r (P = .05) than the 52% emergence recorded f o r GoldPak (GP), a s u s c e p t i b l e v a r i e t y . The number o f c a r r o t s per f o o t o f row a t the th r e e seed spacings i s given i n Table X. There was a higher percentage o f two c a r r o t s growing at the same l o c a t i o n f o r HP than GP, and f o r the 1 1/4 i n c h seed s p a c i n g than o t h e r spacings (Table X). HP was much more vigorous than GP. E a r l y i n the season the d i f f e r e n c e s i n growth were e s p e c i a l l y n o t i c a b l e . The f o l i a g e growth of GP e v e n t u a l l y caught up to t h a t o f HP. Table X. C a r r o t emergence and the percent doubles (two c a r r o t s growing i n the same l o c a t i o n ) o f two v a r i e t i e s pre-c i s i o n seeded a t a spaci n g o f 1 1/4, 1 1/2, and 2 inches Emergence Doubles No. c a r r o t s / f t o f row % V a r i e t y 1 1/4" 1 1/2" 2" 1 1/4" 1 1/2" 2" GoldPak 11.7 10.9 8.1 10.3 9.2 6.2 HiPak 14.5 11.0 9.2 15.2 12.7 8.7 T h e o r e t i c a l 24 20 15 0 0 0 The three spacings had no s i g n i f i c a n t e f f e c t on the i n c i d e n c e o f Olpidiurn, TNV, and f a s t and slow growing Pythium spp. HP u s u a l l y had g r e a t e r l e v e l s of the f o u r organisms than GP (Table XI).- A s i g n i -f i c a n t (P = .05) v a r i e t y x s p a c i n g i n t e r a c t i o n o c c u r r e d with Olpidium and f a s t growing Pythiurn. In the f i r s t sample the number o f Olpidium spores decreased i n GP but i n c r e a s e d i n HP with i n c r e a s i n g p l a n t spacing ( F i g u r e 5 ) . The i n c i d e n c e o f f a s t growing Pythi urn i n the second sample a t 1 1/4, 1 1/2, and 2 inch was 10.3, 11.9, 4.8, and 7.1, 9.5, 11.9% f o r GP and HP r e s p e c t i v e l y . F a s t growing Pythium i n c i d e n c e a t the 2 inch s p a c i n g compared to other spacings s i g n i f i c a n t l y decreased (P = .05) i n GP but s i g n i f i c a n t l y i n c r e a s e d i n HP. At h a r v e s t , the c u l l s were s u b d i v i d e d i n t o two c l a s s e s , " f o r k s " and c u l l s . S i n ce PRD i s capable o f causing f o r k e d , h a i r y , rough, and stubby c a r r o t s , the " f o r k " c l a s s p r o v i d e d an estimate o f the l o s s i n y i e l d due to PRD. As the s p a c i n g d i s t a n c e i n c r e a s e d from 1 1/4 to 2 i n c h e s , the weight o f the c l a s s e s o f A's, marketables (A + B), and f o r k s , s i g n i f i c a n t l y i n c r e a s e d (P = .05) and the weight o f the c l a s s e s o f B's, c u l l s , and smalls s i g n i f i c a n t l y decreased (P = .05) (Table X I I ) . I f y i e l d o f the c l a s s e s i s analyzed on a percentage b a s i s (weight o f c l a s s / t o t a l weight x 100), 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 the percen-tage weight o f A's, B's, marketables (A + B), c u l l s and s m a l l s at the three p l a n t s p a c i n g s . However, the percentage weight o f f o r k s s i g n i -f i c a n t l y decreased (P = .05) as the p l a n t i n g d i s t a n c e i n c r e a s e d . Table XI. Organism survey o f brown roots a t two sampling times o f HiPak and GoldPak and p r e c i s i o n seed spacings of 1 1/4, 1 1/2, 2 inches Olpidium F a s t Pythium Slow Pythium TNV Av. s p o r e s / f i e l d % roots i n f e c t e d % roots i n f e c t e d % recovery Sample 1 Sample 2 Sample 1 Sample 2 Sample 1 Sample 2 Sample 1 Sample 2 Spacing 1 1 1/4" 12.7 a 1 8.4 a 11.1 a 8.7 a 10.3 a 11.1 a 59 a 50 a 1 1/2" 12.2 a 6.9 a 9.1 a 10.7 a 16.7 a 12.3 a 75 a 59 a 2" 13.8 a 7.7 a 10.7 a 8.3 a 16.2 a 17.9 a 33 a 50 a V a r i e t y GoldPak 11.7 a 6.7 a 10.8 a 9.0 a 10.8 a 13.0 a 61 a 45 a HiPak 14.1 b 8.6 a 9.7 a 9.5 a 17.9 b 14.5 a 50 a 61 a Means w i t h i n a column with the same l e t t e r do not d i f f e r e n t s i g n i f i c a n t l y (P = .05). 42 Plant spacing (inches) F i g u r e 5. Average number o f Olpidium spores per microscope f i e l d i n r o o t l e t s of HiPak and GoldPak at three p r e c i s i o n seed s p a c i n g s . Each p o i n t r e p r e s e n t s the mean o f 600 obser-v a t i o n s . 43 Table XII. C a r r o t y i e l d o f GoldPak and HiPak a t p r e c i s i o n seed spacing o f 1 1/4, 1 1/2, and 2 inches A B C u l l s Forks Smalls l b . l b . l b . l b . l b . Spacing 1 1/4" 129.5 a 1 3.5 a 11.4 a 18.5 a 5.0 a 1 1/2" 119.9 b 5.1 b 10.9 a 21.2 b 3.7 b 2" 107.3 c 5.4 b 8.5 b 20.7 b 2.2 c V a r i e t y GoldPak 103.0 a 2.5 a 8.3 a 19.8 a 3.7 a HiPak 134.8 b 6.8 b 12.2 b 20.5 a 3.6 a Means w i t h i n a column wi t h the same l e t t e r do not d i f f e r s i g n i f i -c a n t l y (P = .05). HP had a s i g n i f i c a n t l y g r e a t e r (P = .05) weight o f A's, B's, marketables, and c u l l s than GP (Table X I I ) . The marketable y i e l d a t spacings o f 1 1/4, 1 1/2, and 2 inches was 28.1, 25.5, and 23.5 tons/ a c r e , and 20.1, 19.9, and 17.4 to n s / a c r e f o r HP and GP r e s p e c t i v e l y . GP had a g r e a t e r number (P = .05) of f o r k e d c a r r o t s , and a s i g n i f i c a n t l y g r e a t e r (P = .05) percentage weight of small s than HP. There was no s i g n i f i c a n t space x v a r i e t y i n t e r a c t i o n i n the weight o f the c l a s s e s o f A's, B's, marketables, and s m a l l s . HP had a s i g n i f i c a n t decrease i n the weight o f c u l l s a t three spacings compared to c l o s e r spacings w h i l e GP had no s i g n i f i c a n t change i n the weight o f c u l l s a t the three s p a c i n g s . S i m i l a r l y , GP had l i t t l e change i n the 44 weight o f f o r k s a t the three spacings whereas HP had a s i g n i f i c a n t i n -crease (P = .05) i n the weight of f o r k s a t spacings g r e a t e r than 1 1/4 inches. Weeds as a l t e r n a t e hosts f o r PRD i n c i t a n t s . A number o f weeds growing i n the p r e c i s i o n - s e e d e d p l o t were indexed f o r f a s t and slow growing Pythium spp., TNV, and Olpidium (Table X I I I ) . Yellowed, stunted c e l e r y p l a n t s s u f f e r i n g from an unknown r o o t d i s o r d e r , and l e t t u c e p l a n t s s u f f e r i n g from hollow r o o t were a l s o surveyed. None o f the weed s p e c i e s e x h i b i t e d r o o t t i p n e c r o s i s . The e p i -dermis and ro o t h a i r s showed no s i g n s o f breakdown and decay as i s com-monly observed i n c a r r o t . C e l e r y roots from both s t u n t e d and h e a l t h y p l a n t s c o n t a i n e d high l e v e l s o f 01pidium. Mycelium was f r e q u e n t l y ob-served i n the c o r t e x o f roots from only the stunted c e l e r y p l a n t s . Crop r o t a t i o n . The e f f e c t s o f a c a r r o t , l e t t u c e and onion r o t a t i o n on the p o p u l a t i o n s o f PRD organisms was s t u d i e d i n the greenhouse. A f t e r the f i r s t crop o f c a r r o t s , high l e v e l s o f Olpidium, f a s t and slow growing Pythium spp. and TNV were recovered from a l l pots (Tables XIV-XVII). However, i n the second and t h i r d c r o p p i n g s , low l e v e l s o f Olpidium and slow growing Pythium were recovered only from r o t a t i o n s c o n t a i n i n g l e t t u c e and onion. In c a r r o t s by c o n t r a s t , the i n c i d e n c e o f these organisms remained a t the o r i g i n a l recovery r a t e . In the second c r o p p i n g , t h e r e was no s i g n i f i c a n t d i f f e r e n c e (P = .05) i n the l e v e l o f f a s t growing Pythium but c a r r o t roots had the h i g h e s t l e v e l (Table XVI). A f t e r the t h i r d c r o p p i n g , there was s i g n i f i c a n t l y (P = .05) more f a s t growing Pythium recovered from c a r r o t than from l e t t u c e or onion r o o t s . 45 Table X I I I . Organism i n c i d e n c e i n some common weeds growing i n s o i l s with a h i s t o r y of severe PRD Fast Slow Host Pythium Pythium Olpidium TNV Lamb's q u a r t e r s -, Chenopodium album L. +++ +' Common chickweed S t e l l a r i a media (L.) V i l l . + - + -Sheperd's purse C a p s e l l a b u r s a - p a s t o r i s (L.) Medic. + - + Barnyard grass E c h i n o c h l o a c r u s g a l l i (L.) Beauv. ++++ - + + Redroot pigweed Amaranthus r e t r o f l e x u s L. ++++ - - -Pineapple weed M a t r i c a r i a m a t r i c a r i o i d e s (Less.) P o r t e r + - ++ + Hop c l o v e r (?) T r i f o l i u m sp. -Ce l e r y Apium graveolens L. ++++ - ++++ Lettu c e Lactuca s a t i v a L. ++ + ++ - = 0; + = 1-3; ++ = 4-7; +++ = 8-12; ++++ = 12; roo t s i n f e c t e d with f a s t and slow growing Pythium spp. o r Olpidium. 2 + = p o s i t i v e ; - = negative TNV on Chenopodium quinoa. 46 T a b l e XIV. Average number o f 01 pi d i u r n s p o r e s p e r m i c r o -scope f i e l d i n r o o t s o f c r o p s from f i v e r o t a -t i o n s R o t a t i o n 1 C r o p p i n g s 2 3 4 A (C-C-C-C) 10.3 a 1 7.4 a 7.0 a 3.1 a B (C-L-O-C) 11.0 a 1.1 b 0.1 b 2.5 a C (C-O-L-C) 12.6 a 0.4 b 0.8 b 2.3 a D (C-L-L-C) 7.8 a • 0.5 b 0.4 b 2.7 c E (C-O-O-C), 8.0 a 0.4 b 0.2 b 3.8 a ^ Means f i c a n t l y (P w i t h the same l e t t e r w i t h i n a = .05). c o l u n n do n o t d i f f e r s i g n i C = c a r r o t ; L = l e t t u c e ; 0 = o n i o n . T a b l e XV. P e r c e n t slow growing P y t h f r o m r o o t s o f c r o p s from ium r e c o v e r e d f i v e r o t a t i o n s R o t a t i o n 1 C r o p p i n g s 2 3 4 A (C-C-C-C) 17.5 a b c 1 33.5 a 41.5 a 34.0 a B (C-L-O-C) 15.0 be 0.0 b 0.9 b 36.5 a C (C-O-L-C) 26.5 a 0.9 b 1.8 b 24.0 a D (C-L-L-C) 22.5 ab 0.9 b 0.9 b 39.0 a E (C-O-O-C) 11.0 c 0.9 b 0.9 b 21.5 a Means w i t h the same l e t t e r w i t h i n a column do n o t d i f f e r s i g n i -f i c a n t l y (P = .05). Table XVI. Percent f a s t growing Pythium recovered from roots o f crops from f i v e r o t a t i o n s R o t a t i o n 1 Croppings 2 3 4 A (C-C-C-C) 18.5 1 a 14.0 a 17.5 a 12.5 a B (C-L-O-C) 21.0 a 2.0 a 0.9 b 16.0 a C (C-O-L-C) 20.0 a 0.9 a 4.2 b 10.0 a D (C-L-L-C) 11.5 a 9.0 a 7.5 b 11.0 a E (C-O-O-C) 29.0 a 6.5 a 5.0 b 13.5 a Means w i t h i n a column with the same l e t t e r do not d i f f e r s i g n i -f i c a n t l y (P = .05). Table XVII. Percent d e t e c t i o n o f TNV i n r o o t s o f crops from f i v e r o t a t i o n s R o t a t i o n 1 2 Croppings 3 4 A (C-C-C-C) 100 a 1 67 a 100 a 33 a B (C-L-O-C) 100 a 17 b 33 b 0 a C (C-O-L-C) 100 a 0 b 100 a 33 a D (C-L-L-C) 100 a 33 ab 100 a 66 a E (C-O-O-C) 100 a 0 b 66 ab 33 a ^ Means f i c a n t l y (P w i t h i n a column with = .05). the same l e t t e r do not di f f e r s i g n i -48 TNV was not recovered from onion i n the second cropping and was recovered l e s s f r e q u e n t l y from onion than from c a r r o t and l e t t u c e i n the t h i r d c r o p p i n g (Table XVII). In the f o u r t h c r o p p i n g , when a l l pots were p l a n t e d back to c a r r o t , no s i g n i f i c a n t d i f f e r e n c e (P = .05) was de t e c t e d i n the i n c i d e n c e o f a l l f o u r organisms (Tables XIV-XVII). The change i n recovery r a t e o f f a s t growing Pythium between the f i r s t and f o u r t h croppings was s i g n i f i c a n t only f o r crop r o t a t i o n B. The number o f roots i n f e c t e d with both f a s t and slow growing Pythium spp. were s i g n i f i c a n t l y h i g h e r i n the l a s t crop o f c a r r o t s than the f i r s t crop. In the f i r s t h a r v e s t , there were some brown c a r r o t r o o t l e t s , but very few c a r r o t s e x h i b i t e d f o r k i n g . In the second and t h i r d c r o p p i n g s , only c a r r o t s had brown r o o t l e t s . The l e t t u c e r o o t l e t s were an o f f - w h i t e c o l o u r and the onion roots were p e a r l y white. No breakdown o f the e p i -dermis and c o r t e x and l i t t l e mycelium i n the r o o t l e t s o f l e t t u c e and onion were observed by m i c r o s c o p i c examination. By c o n t r a s t c a r r o t r o o t l e t s showed breakdown o f the epidermis and had mycelium permeating the c o r t e x . In the f i n a l h a r v e s t , the crop sequence o f f o u r s u c c e s s i v e c a r r o t crops c o n t a i n e d more t y p i c a l PRD symptoms than c a r r o t grown a f t e r other crop sequences. Between 10-20% o f the c a r r o t s from the "only c a r r o t s " r o t a t i o n d i s p l a y e d f o r k i n g and stubby r o o t symptoms. C a r r o t s from the o t h e r crop sequences, had few red brown r o o t l e t s and o n l y a few were f o r k e d . G e n e r a l l y , t h e i r r o o t l e t s were grey o r grey-brown i n c o l o u r . 49 Throughout the experiment, the c a r r o t s had an emergence o f 55 to 62%; onions, 84 to 93%; and l e t t u c e , 2.3 to 4.5% o f maximum germin a t i o n . Germination was determined by the p e t r i d i s h germination t e s t , and these r e s u l t s were used to c o r r e c t the percent emergence. L e t t u c e r e q u i r e d reseeding to get a s u f f i c i e n t l y good stand, so t h a t the s o i l would be well permeated with r o o t s . Supplementary i l l u m i n a t i o n was i n s u f f i c i e n t to compensate f o r changes i n day l i g h t between c r o p p i n g s , which prevented meaningful s t a t i s -t i c a l a n a l y s i s o f y i e l d s between r o t a t i o n s . The f i r s t t h r e e croppings were conducted through the w i n t e r months and had much s h o r t e r p e r i o d s o f d a y l i g h t than the f i n a l c ropping which was conducted during the l a t e s p r i n g . The e f f e c t o f d a y l i g h t was q u i t e e v i d e n t i n a comparison o f the r o o t y i e l d s i n crop sequence A (Table X V I I I ) . In the f i r s t t h r e e h a r v e s t s , c a r r o t r o o t weight was one t h i r d to one q u a r t e r the top weight but i n the f i n a l h a r v e s t , r o o t weight was 60% g r e a t e r than top weight. Carrots grown i n s o i l t h a t had p r e v i o u s l y been i n onions had a s i g n i -f i c a n t l y g r e a t e r (P = .05) r o o t weight than c a r r o t s grown i n s o i l t h a t had p r e v i o u s l y been i n l e t t u c e or c a r r o t s (Table XVIII). C a r r o t s grown a f t e r two crops o f onions had a s i g n i f i c a n t l y g r e a t e r r o o t weight than a f t e r a l l o t h e r r o t a t i o n s . The p o p u l a t i o n o f Pythium spp. i n the s o i l as measured by d i l u -t i o n p l a t i n g appeared to f o l l o w a c y c l i c p a t t e r n . No crop sequence s i g n i f i c a n t l y changed the l e v e l o f Pythium i n the s o i l (Table XIX). A decrease from 3600 to 1600 propagules o f Pythium per gram oven dry s o i l was found a f t e r the f i r s t cropping o f c a r r o t s . A f t e r the second Tablve XVIII. Root weight o f crops from f i v e r o t a t i o n s Harvest Rotation 1 2 3 4 A (C-C-C-C) 10.6 a 1 14.1 15.1 45.3 c B (C-L-O-C) 9.9 a 8.4 16.6 61.5 b C (C-O-L-C) 8.5 a 2.3 14.6 45.6 c D (C-L-L-C) 7.5 a 6.4 17.8 40.2 c E (C-O-O-C) 8.8 a 1.3 19.9 80.0 a Means w i t h i n a column with the same l e t t e r do not s i g n i f i c a n t l y d i f f e r (P = .05). Table XIX. Pythium propagule number x 10 per gram of over dry s o i l from f i v e r o t a t i o n s Rotation P r e - p l a n t 1 Croppings 2 3 4 A (C-C-C-C) 36 13 a 1 22 a 11 be 21 a B (C-L-O-C) 36 17 a 25 a 11 be 28 a C (C-O-L-C) 36 14 a 13 b 8 c 26 a D (C-L-L-C) 36 17 a 13 b 18 a 31 a E (C-0-0-C) 36 21 a 8 b 16 ab 25 a Means w i t h i n a column with the same l e t t e r do not s i g n i f i c a n t l y d i f f e r (P = .05). 51 h a r v e s t , Pythiurn propagule number i n s o i l t h a t had been i n onions was s i g n i f i c a n t l y l e s s (P = .05) than s o i l i n c a r r o t s . The propagule l e v e l i n s o i l i n which l e t t u c e had grown, showed an i n c o n s i s t e n t change. In r o t a t i o n B, the l e v e l o f Pythium i n the s o i l i n c r e a s e d and i n r o t a t i o n D, the l e v e l decreased, even though both sequences had been i n the same crops up to t h i s stage of the experiment. There were o f t e n l a r g e v a r i -a t i o n s i n the number o f Pythium propagules recovered a t the f o u r s o i l d i l u t i o n s . U s u a l l y the 1:200 d i l u t i o n had only one h a l f to one t h i r d the propagule number per gram oven dry s o i l , as the 1:100 and 1:50 d i l u -t i o n s . DISCUSSION E t i o l o g y and Epidemiology o f PRD Koch's f i r s t p o s t u l a t e r e q u i r e s the pathogen always to be asso-c i a t e d with the d i s e a s e . An i n d i r e c t method of proving t h a t a g i v e n i n c i t a n t i s the cause o f a d i s e a s e when there i s more than one organism present i s to show t h a t the suspected i n c i t a n t i s not present w h i l e the others are present i n symptomless t i s s u e . To t h i s end, p o t e n t i a l patho-gens i n brown c a r r o t r o ots were compared with those i n symptomless r o o t s from both PRD problem and non-problem s o i l s . The number o f 01pidium spores, the presence o f TNV, and the recovery r a t e o f f a s t growing Pythium were s i m i l a r i n brown and white roots from PRD problem s o i l s . Very low l e v e l s o f slow growing Pythium were recovered from symptomless r o o t s , while high l e v e l s were recovered from brown r o o t s . Slow growing Pythium 52 spp. could be recovered from r o o t l e t s of p l a n t s grown only i n problem f i e l d s and were not recovered from c a r r o t s grown i n non-problem s o i l s . In a second sampling o f the same p l o t s , there was an i n c r e a s e i n the i n c i d e n c e i n the r a t e of recovery o f slow growing Pythium from white r o o t s . S i nce i t was very d i f f i c u l t to f i n d white r o o t l e t s i n the second sampling some o f the o f f - w h i t e or grey r o o t l e t s s e l e c t e d may have been i n the e a r l y stages o f developing PRD. By c o n t r a s t none o f the weed spe c i e s i n v e s t i g a t e d was found to harbor the slow growing Pythium. Based on f i e l d i s o l a t i o n s and greenhouse r o t a t i o n s i n PRD problem s o i l , both l e t t u c e and onion supported only very low l e v e l s o f t h i s organism. However, the i n c i d e n c e i n greenhouse grown c a r r o t s f o l l o w i n g e i t h e r l e t t u c e or onion crops was not s i g n i f i c a n t l y reduced, even though the two preceding crops supported very low l e v e l s . T h i s slow growing Pythium has been i d e n t i f i e d on the b a s i s o f growth r a t e and d i a g n o s t i c a n t h e r i d a l morphology as the very pathogenic Pythium sulcatum. These r e s u l t s c e r -t a i n l y i m p l i c a t e P_. sulcatum as a causal agent o f PRD i n B r i t i s h Columbia s o i l s and suggest t h a t the organism has a r a t h e r narrow host range. A causal r o l e f o r the f a s t growing Pythium s p e c i e s i n PRD has not been r u l e d out, but t h e r e was a high recovery r a t e from both white and brown c a r r o t r o o t s . L e t t u c e , o n i o n , c e l e r y , and a number o f weed species (Table XIII) were good hosts f o r the f a s t growing Pythium s p e c i e s . P.. i r r e g u l a r , P_. paroecandrum " c l a s s i c a l form," JP. paroecandrum "P. ui t i -mum" form, P_. debaryanum, P_. s y l v a t i c u m , and P_. coloratum and s e v e r a l u n i d e n t i f i e d f a s t growing Pythium sp. were i s o l a t e d from B r i t i s h Columbia s o i l s (R. J . Howard and R. G. P r a t t , personal communication). Probably 53 not a l l o f these s p e c i e s are pathogenic to c a r r o t , but at l e a s t two s p e c i e s , P_. debaryanum and P_. i r r e g u l a r e have a l s o been i s o l a t e d from l e t t u c e and onion (MacFarlane, 1968). In F l o r i d a and Wisconsin, Pythium spp. are r e s p o n s i b l e f o r 50% pre-emergence damping o f f (R. J . Howard and J . 0. Strandburg, personal  communication). S i m i l a r l y , 40-50% seed f a i l u r e has been observed i n f i e l d t r i a l s and greenhouse experiments using PRD i n f e s t e d s o i l . Pre-l i m i n a r y experiments suggest t h a t some i s o l a t e s o f P_. s y l v a t i c u m and P_. sulcatum caused seed f a i l u r e . In p a t h o g e n i c i t y t r i a l s , F. D. McElroy (unpublished r e s u l t s ) found t h a t i t was necessary to germinate seeds on f i l t e r paper before i n o c u l a t i n g with P_. s y l v a t i c u m , because i f seeding and i n o c u l a t i n g were done s i m u l t a n e o u s l y , a higher r a t e o f seed f a i l u r e o c c u r r e d . Pythium s p e c i e s were probably r e s p o n s i b l e f o r the poor emer-gence r a t e o f l e t t u c e i n PRD s o i l . Farmers on muck s o i l i n the F r a s e r V a l l e y sow l e t t u c e a t very heavy r a t e s to compensate f o r poor emergence. The importance and r o l e o f Pythium i n pre-emergence damping o f f o f c a r r o t s and l e t t u c e r e q u i r e s f u r t h e r study. The presence o f 80-100 Olpidium zoosporangia and r e s t i n g spores per 560 y s e c t i o n of r o o t l e t and the s p o r a d i c recovery of TNV from symp-tomed r o o t s were e a r l y o b s e r v a t i o n s . As a consequence, c o n s i d e r a b l e time was spent i n determining what r o l e TNV, Olpidium b r a s s i c a e , or both, were p l a y i n g i n the d i s e a s e complex. TNV was recovered o n l y from c a r r o t r o o t s i n PRD problem s o i l which suggests a causal r o l e . Smith (1937) concluded t h a t TNV o c c u r r e d i n symptomless r o o t s , but Teakle (1962b) has shown t h a t s t e r i l e cowpea 54 roots produce n e c r o t i c l e s i o n s when r u b - i n o c u l a t e d with TNV. Attempts to determine i f TNV alone c o u l d reproduce f i e l d symptoms, were i n c o n -c l u s i v e . Lesions were only s p o r a d i c a l l y observed on TNV r u b - i n o c u l a t e d c a r r o t r o o t s , and these were s i m i l a r to l e s i o n s which developed on phos-phate b u f f e r i n o c u l a t e d c o n t r o l p l a n t s . Because c a r r o t roots are much more f r a g i l e and more e a s i l y i n j u r e d than cowpea or mung bean r o o t s used by Teakle (1962b), the l e s i o n s observed were probably the r e s u l t of i n j u r y during i n o c u l a t i o n r a t h e r than TNV i n f e c t i o n . Roots dipped i n TNV e x t r a c t s a l s o developed symptoms and indexed p o s i t i v e f o r v i r u s . Handling the s e e d l i n g s d u r i n g d i p - i n o c u l a t i o n , must have s u f f i c i e n t l y i n j u r e d the r o o t s to enable v i r u s p e n e t r a t i o n and i n f e c t i o n to occur. TNV c o u l d a l s o have been recovered from symptomless and d i p - i n o c u l a t e d roots i f i n f e c t i v e TNV p a r t i c l e s on the r o o t s u r f a c e were not i n a c t i v a t e d by s u r f a c e d i s i n f e c t i n g the roots i n Concentrate R.B.S. *. Attempts to e s t a b l i s h a r o l e f o r Olpidium alone as the c a u s a t i v e agent of PRD were u n s u c c e s s f u l . The i n c i d e n c e o f spores was not h i g h l y c o r r e l a t e d with d i s e a s e s e v e r i t y as measured by marketable y i e l d and c u l l r a t e . T y p i c a l n e c r o t i c r o o t symptoms were not observed wi t h O l p i - dium alone or i n mixed i n f e c t i o n s w i t h TNV. No symptoms were produced on l e t t u c e even when i n f e c t i o n l e v e l s were g r e a t e r than 50 sporangia per 560 y diameter f i e l d . Furthermore, the presence o f Olpidium was observed i n both PRD problem and non-problem f i e l d s but TNV, t r a n s m i t t e d by Olpidium was only observed i n PRD problem f i e l d s . The c o r r e l a t i o n o f the v i r u s but not the v e c t o r to PRD problem f i e l d s may p o s s i b l y be e x p l a i n e d by Teakle's (1962b) o b s e r v a t i o n t h a t r o o t n e c r o s i s o f l e t t u c e 55 occurred only under c o n d i t i o n s o f high inoculum p o t e n t i a l s o f both 01 pi - dium and TNV. Perhaps the v i r u s t i t e r i n the non-problem s o i l was too low to i n i t i a t e symptom development. Olpidium was a l s o observed i n roots o f a number o f weeds, c e l e r y , l e t t u c e and onion and TNV was detected i n a l l but a few weed s p e c i e s . In the crop r o t a t i o n experiments, onion was the poorest host, and a s i g n i f i c a n t l y lower l e v e l o f Olpidium was a l s o observed i n l e t t u c e than i n c a r r o t . T h i s l a t e r o b s e r v a t i o n i s c o n t r a d i c t o r y to c u l t u r e s t u d i e s of Olpidium where on c a r r o t , Olpidium i s o l a t e s d i e d a f t e r s e v e r a l t r a n s -f e r s but on l e t t u c e , the i s o l a t e s reproduced w e l l . D i f f e r e n t l e t t u c e v a r i e t i e s were used i n two experiments. Host s p e c i f i c i t y may a l s o be i n v o l v e d (D.J.S. Barr, personal communication). Garnet and Tomlinson (1967) found t h a t l e t t u c e i s o l a t e s f a i l e d to i n f e c t b r u s s e l s p r o u t , cabbage and t u r n i p and c o n v e r s e l y cabbage i s o l a t e s i n f e c t e d these crops but not l e t t u c e . However, i t i s strange t h a t the Olpidium i s o l a t e s recovered from c a r r o t were not more s p e c i f i c to c a r r o t c o n s i d e r i n g the problem s o i l used f o r b a i t i n g had been p l a n t e d to c a r r o t s the t h r e e preceding y e a r s . Another c o m p l i c a t i n g f a c t o r i n t h i s i s s u e , and the f i e l d surveys as w e l l , i s t h a t Pythium s p e c i e s are capable o f reducing Olpidium i n f e c t i o n (D.J.S. B a r r , personal communication). The high l e v e l s o f Pythium i n the r o t a t i o n experiment may have i n h i b i t e d Olpidium i n f e c t i o n o f l e t t u c e . This mechanism c o u l d a l s o be important i n pre-venting Olpidium i n f e c t i o n s from becoming so numerous as to cause a measurable y i e l d l o s s . E x p e r i m e n t a l l y determining a r o l e f o r Olpidium a t high p o p u l a t i o n s must await the development o f s u i t a b l e techniques f o r the maintenance o f these a p p a r e n t l y s t r a i n s p e c i f i c i s o l a t e s . 56 Previous d i s e a s e r a t i n g had been based on o b s e r v a t i o n s of un-s t a i n e d i n f e c t e d r o o t s which d i d not permit d i s t i n g u i s h i n g Pythium and Olpidium s p o r a n g i a . With the i n t r o d u c t i o n o f the s t a i n i n g technique i n 1972 f i e l d s t u d i e s , i t was apparent t h a t only a few of the sporangia were Pythiaceous w h i l e the m a j o r i t y were 01pidiaceous. T h i s r e s u l t e d i n the switch to r o o t i s o l a t i o n s on s e l e c t i v e media i n subsequent attempts to monitor Pythium. Attempts to e v a l u a t e microenvironmental f a c t o r s with Olpidium i n c i d e n c e were cont i n u e d , s i n c e c o n c l u s i v e proof o f the non-involvement o f Olpidium had not been shown. The only environmental f a c t o r t h a t appears to be r e l a t e d to Olpidium p o p u l a t i o n f l u c t u a t i o n s i n 1972 f i e l d s t u d i e s was r a i n f a l l . S o i l temperature, s o i l CO- and 0^, s a t u r a t e d h y d r a u l i c c o n d u c t i v i t y , and s o i l moisture d i f f e r e n c e between " r a i s e d " and " c o n v e n t i o n a l " beds, appa r e n t l y had no d i r e c t e f f e c t s on Olpidium. R a i n f a l l o r i r r i g a t i o n s i n s u f f i c i e n t q u a n t i t y to s a t u r a t e the s o i l f o r a s h o r t p e r i o d appeared to i n f l u e n c e Olpidium spread and b u i l d up i n c a r r o t r o o t s . T h i s was not unexpected because Olpidium r e q u i r e s f r e e water f o r zoospore r e l e a s e and spread. Heavy r a i n f a l l s or i r r i g a t i o n may i n c r e a s e the f r e e water i n the r o o t zone f o r a s h o r t p e r i o d and thus allow f o r zoospore r e l e a s e and i n f e c t i o n o f r o o t s . Under optimum c o n d i t i o n s o f temperature and moisture, 01 pidiurn's l i f e c y c l e r e q u i r e s 5 to 7 days but under l e s s i d e a l c o n d i t i o n s i t i s 7-14 days (Fry and Campbell, 1966). T h e r e f o r e , the amount and frequency o f r a i n f a l l and i r r i g a t i o n 7 to 21 days p r i o r to sampling are important i n determining the spread and b u i l d up o f Olpidium i n young c a r r o t r o o t l e t s . One h a l f inch o f p r e c i p i t a t i o n i s g e n e r a l l y c o n s i d e r e d to be the minimum b e n e f i c i a l q u a n t i t y of water 57 required to wet a s o i l , and amounts less than this only dampen the soi l surface. Rainfall of less than 1/2 inch was thought not to influence Olpidium. A very dry May resulted in a low i n i t i a l infection of early planted carrots. Carrots planted in late May were subjected to 2 or 3 periods of soi l saturation and thus had high levels of Olpidium infection. The mid-June planting of carrots appeared to be more resistant to Olpi - dium despite being subjected to at least four potential infection periods. It appears that later plantings of carrots escaped infection even though conditions were favorable for spread of Olpidium. Olpidium levels de-creased during August, a month with only one infection period. It appeared that once there was a high inoculum level in the roots, the population of Olpidium was high in new rootlets even i f infection periods were less favorable. Even though there was no precipitation after the July cloudburst, soi l moisture was maintained at an optimum level for carrot growth by adjusting the level of water in the drainage ditches to maintain a constant water table. Soil moisture during this period was less favorable for zoospore spread but was optimum for carrot growth. Zoospore movement is dependent upon the water f i l l e d pore s ize. Flagella propel zoospores relat ively short distances in water f i l l e d pores. The maximum movement of zoospores becomes possible when the matric potential allows for the greatest frequency of water f i l l e d pores large enough to accommodate the zoospore (Cook and Papendick, 1972). The maximum pore diameter that remains f i l l e d with water as a so i l drains can be determined from the capil lary r ise equation Ymd = 2.94, where 58 ^m = m a t r i c p o t e n t i a l i n bars, and d = pore diameter i n p. S t o l z y e t a l . (1965) suggested (without any s u p p o r t i n g data) t h a t a water f i l l e d pore diameter o f 40 to 60 u i s necessary f o r Phytophthora zoospore movement. Phytophthora zoospores are s l i g h t l y l a r g e r than Olpidium zoospores, but i f r e l a t i v e l y the same pore diameter i s necessary f o r movement of Olpidium zoospores, t h i s would correspond to a m a t r i c p o t e n t i a l o f -.05 to -.07 bar. These p o t e n t i a l s are equal to a s o i l water content o f 110 to 115% ( c a l c u l a t e d from the r e t e n t i o n curve F i g u r e 3, R u s s e l , 1972). At the 15 cm depth, the s o i l moisture i n " c o n v e n t i o n a l " beds was g r e a t e r than 110% u n t i l August. S o i l moisture i n the " r a i s e d " bed was below 110% d u r i n g l a t e May and mid-June, i t then i n c r e a s e d and d i d not become l e s s than 110% u n t i l l a t e J u l y ( F i g u r e 4 ) . The i n c r e a s e i n water content above 110% i n " r a i s e d " beds corresponded to the p e r i o d o f h i g h e s t O l p i - dium i n f e c t i o n . The type o f spore formed by Olpidium w i l l i n f l u e n c e p o p u l a t i o n b u i l d up. Under f a v o r a b l e c o n d i t i o n s , zoosporangia are formed i n r o o t s and there i s a s h o r t p e r i o d between zoospore i n f e c t i o n and r e l e a s e . Under l e s s f a v o r a b l e c o n d i t i o n s , r e s t i n g spores develop. The r e s t i n g spores are r e s i s t a n t to adverse c o n d i t i o n s and r e q u i r e the presence of c e r t a i n f a c t o r s to t r i g g e r the breaking o f dormancy and p r o d u c t i o n o f zoospores to i n i t i a t e new i n f e c t i o n s . T h e r e f o r e , roots with a high p r o p o r t i o n o f zoosporangia have a higher i n f e c t i o n c a p a b i l i t y than r o o t s with a high p r o p o r t i o n o f r e s t i n g spores. As the season progressed, a h i g h e r p r o p o r t i o n o f the spores were r e s t i n g s p o r e s , so t h a t these roots were l e s s capable o f spreading Olpidium even i f weather c o n d i t i o n s had been f a v o r a b l e f o r spread. 59 M o d i f i c a t i o n o f the microenvironment with " r a i s e d " beds, i n 1972, i n f l u e n c e d Olpidium i n c i d e n c e only i n the e a r l i e s t p l a n t e d c a r r o t s . In the f i r s t sampling o f 1973, Olpidium spore l e v e l s were l e s s i n r a i s e d beds made on a commercial s c a l e than conventional beds. However i t was concluded t h a t r a i s e d beds had l i t t l e o v e r a l l e f f e c t i n reducing Olpidium i n f e c t i o n . Olpidium appears to have no d i r e c t e f f e c t on y i e l d , as y i e l d was s i g n i f i c a n t l y a f f e c t e d by bed h e i g h t , whereas the i n c i d e n c e of Olpidium was not. The use of p r e c i s i o n seeding to modify the micro-environment o f i n d i v i d u a l p l a n t s a l s o had no e f f e c t on Olpidium i n c i -dence. E v a l u a t i o n o f C u l t u r a l P r a c t i c e s to Control PRD Raised beds . Y i e l d o f marketable c a r r o t s from " r a i s e d " beds was g r e a t e r than t h a t from " c o n v e n t i o n a l " beds i n 1972 but not 1973. In 1972, " r a i s e d " beds were made hig h e r by removing s o i l from a d j a c e n t beds, whereas i n 1973, r a i s e d beds were made only as high as was p o s s i b l e with the grower's equipment. T h e r e f o r e , the comparison between bed heights o f r a i s e d and conventional beds was much more extreme i n 1972 than i n 1973. In 1972 there was a s h i f t i n the p o p u l a t i o n from a l a r g e number o f small c a r r o t s i n " c o n v e n t i o n a l " beds to fewer small c a r r o t s and more marketable c a r r o t s i n " r a i s e d " beds. I f the l a t e r a l r o o t d i e -back phase o f the disease i s important t h i s would prevent many c a r r o t s from r e a c h i n g a l a r g e r s i z e . I t would seem t h a t one of the major benefits o f " r a i s e d " beds was to i n c r e a s e marketable y i e l d by de c r e a s i n g the time from p l a n t i n g to m a t u r i t y . In C a l i f o r n i a , narrow beds, 20 inches from 60 center to c e n t e r with an 8 inch wide top are being used because narrow beds have a g r e a t e r s u r f a c e area exposed to heat u n i t s so crop m a t u r i t y i s e a r l i e r (Anon., 1973c). "Raised" beds a l s o i n c r e a s e the exposed s u r f a c e area, and t h i s may e x p l a i n why c a r r o t s i n " r a i s e d " beds appear to mature e a r l i e r . On non-problem s o i l , where there was no v i s i b l e water s t r e s s , bed h e i g h t had no e f f e c t on marketable y i e l d . The average marketable y i e l d from beds wi t h f o u r rows was 18.5 T/acre f o r r a i s e d beds and 19.4 T/acre f o r c o n v e n t i o n a l beds. On the problem s o i l , where there was a s e r i o u s water s t r e s s , f o u r row c o n v e n t i o n a l beds o u t - y i e l d e d the f o u r row r a i s e d beds, 22.8 to 19.3 T/acre r e s p e c t i v e l y . Under c o n d i t i o n s where moisture was l i m i t i n g and i r r i g a t i o n was u n a v a i l a b l e , c a r r o t s on r a i s e d beds s u f f e r e d a g r e a t e r water s t r e s s than c a r r o t s on c o n v e n t i o n a l beds. The c a r r o t s from the r a i s e d beds were s m a l l e r than those from conventional beds: .156 l b / c a r r o t and .174 l b / c a r r o t r e s p e c t i v e l y . Under severe water s t r e s s , the marketable y i e l d from r a i s e d beds with three rows per bed n e a r l y e q u a l l e d t h a t from the f o u r rows r a i s e d bed. Plants on the f o u r row r a i s e d beds d i d not have enough water to " s i z e up" as was i n d i c a t e d a t h a r v e s t by g r e a t e r percent weight o f small c a r r o t s from the f o u r row r a i s e d beds than from the three row r a i s e d bed. Raised beds with f o u r rows per bed w i l l y i e l d as well as c o n v e n t i o n a l f o u r row beds i f s o i l moisture i s adequate, and t h e r e f o r e should o n l y be used i f i r r i g a t i o n water i s a v a i l a b l e . Bed h e i g h t had no e f f e c t on the c u l l r a t e of c a r r o t s on non-problem s o i l . The 1973 growing season was very dry and as a r e s u l t 61 there was only a minor i n c i d e n c e o f PRD. There was no o p p o r t u n i t y to evaluate the r a i s e d beds' a b i l i t y to reduce d i s e a s e i n c i d e n c e and c u l l r a t e due to PRD. The grower with the problem s o i l used much higher c o n v e n t i o n a l beds than employed by most other growers. As a consequence, the width o f the p l a n t i n g s u r f a c e o f h i s c o n v e n t i o n a l beds was l e s s . Problems were encountered i n keeping the seeder on the bed when seeding f o u r rows with 14 i n c h c e n t e r s . T h i s may have been one cause f o r the poor performance o f the f o u r row 14 in c h c e n t e r conventional beds. Several f a c t o r s o t h e r than the q u a l i t y and a v a i l a b i l i t y o f i r r i -g a t i o n water should be c o n s i d e r e d when using r a i s e d beds. Equipment must be capable o f throwing the s o i l up i n t o a bed, r a t h e r than j u s t making furrows. A bed shaper i s necessary to f i r m the s o i l on the s i d e s o f the bed and make a smooth packed s u r f a c e f o r p r e c i s i o n s e e d i n g . Raised beds should be 45 inches wide i f f o u r row 12 in c h spacing i s used, otherwise i t i s d i f f i c u l t to keep the seeder on the bed, and the s o i l may f a l l away from the edges o f the bed l a t e r i n the season and expose the r o o t s . Mechanical weeders must be a d j u s t e d f o r the narrower between row s p a c i n g . The between row spacing o f 14 i n c h c e n t e r to c e n t e r p r e c i s i o n seeded c a r r o t s seemed s l i g h t l y l e s s than the between row spaci n g of the 14 in c h c e n t e r to c e n t e r s c a t t e r shoe seeded c a r r o t s and t h e r e f o r e , the p r e c i s i o n seeded c a r r o t s were damaged by the mechanical weeder s e t f o r the s c a t t e r shoe between row s p a c i n g . Y i e l d was i n c r e a s e d by 10-25% with f o u r rows o f c a r r o t s per bed over 3 rows per bed. The y i e l d i n c r e a s e was most n o t i c e a b l e i n 62 c a r r o t s not s u f f e r i n g from drought. I f moisture i s a v a i l a b l e , f i v e rows per bed may f u r t h e r i n c r e a s e y i e l d . The between row space does not have to be as g r e a t as i n the past because more e f f e c t i v e h e r b i c i d e s are now used so l e s s c u l t i v a t i o n i s r e q u i r e d . Filman (1972e) has found t h a t y i e l d s were s i g n i f i c a n t l y i n c r e a s e d , the nearer the p l a n t s were evenly spaced at about 12 cm per p l a n t ( 4 x 3 i n c h g r i d ) compared to the standard 16 i n c h row s p a c i n g . However, B r i t i s h Columbia growers have expressed doubt t h a t present h a r v e s t i n g combines co u l d handle the i n c r e a s e d c a r r o t d e n s i t y o f a s o l i d l y sown bed or f i v e row bed. P r e c i s i o n seeding and t o l e r a n t v a r i e t i e s . HiPak (HP), a PRD t o l e r a n t v a r i e t y , had a g r e a t e r marketable y i e l d and fewer number o f f o r k e d c a r r o t s than GoldPak (GP), a PRD s u s c e p t i b l e v a r i e t y d e s p i t e having a h i g h e r i n c i d e n c e o f Olpidium and slow growing Pythium. F o r k i n g o f c a r r o t s was c o n s i d e r e d one measure o f PRD i n c i d e n c e . On t h i s b a s i s GP was more a d v e r s e l y a f f e c t e d by PRD than HP. The l o s s o f y i e l d due to PRD was 13.5-15.8% and 9.2-13.3% f o r GP and HP r e s p e c t i v e l y a t the d i f f e r e n t spacings. HP's a b i l i t y to t o l e r a t e PRD has been p r e v i o u s l y observed (McElroy e t al-» 1 9 7 1 ) • In the grower's r a i s e d bed t r i a l , p r e c i s i o n seeding s i g n i f i c a n t l y reduced c u l l r a t e over s c a t t e r shoe-seeding, c o n f i r m i n g A. R. Maurer's unpublished r e s u l t s . However 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 the i n c i d e n c e o f the PRD a s s o c i a t e d organisms. Marketable y i e l d was g r e a t e r as the p r e c i s i o n p l a n t spacing decreased. At a s p a c i n g o f 1 1/4 i n c h e s , marketable y i e l d was the g r e a t e s t but on a percentage weight b a s i s , p l a n t s p a c i n g had no s i g n i f i c a n t e f f e c t on y i e l d . Y i e l d i n c r e a s e d at the c l o s e r spacing only because p l a n t p o p u l a t i o n was g r e a t e r . The i n c i d e n c e o f Olpidium, f a s t and slow growing Pythium spp., and TNV were not a f f e c t e d by p l a n t s p a c i n g , but the percent weight o f f o r k s s i g n i f i c a n t l y decreased as p l a n t s p a c i n g i n c r e a s e d . The i n c r e a s e i n marketable y i e l d f a r exceeded the small i n c r e a s e i n c u l l s a t the c l o s e r s p a c i n g s . Filman (1972b) found t h a t by reducing the seeding r a t e by h a l f from the recommended r a t e o f 15 to 20 seed per f o o t , PRD was reduced by 10%, but the d i s e a s e i n c i d e n c e was s t i l l too high f o r economic h a r v e s t i n g . In the p r e s e n t t r i a l , PRD, as measured by f o r k i n g , was l e a s t a t the c l o s e s t s p a c i n g . A high d i s e a s e i n c i d e n c e would have been p r e d i c t e d because the p l o t had been i n c a r r o t s the 3 previous y e a r s , the area had a h i s t o r y o f severe PRD i n c i d e n c e , and the s o i l was maintained a t a high s o i l water content throughout the growing season. P r e c i s i o n seeding and r a i s e d beds appear to have s u f f i c i e n t l y m o d i f i e d the s o i l microenvironment to l i m i t b u i l d up and spread o f the PRD a s s o c i a t e d organisms and as a consequence, PRD l o s s e s were kept to a minimum. Nonnecke (1973) has a l s o noted the PRD was kept to a minimum by p r e c i s i o n s e e d i n g , because " s t r e s s " between i n d i v i d u a l p l a n t s was removed. The high frequency o f two c a r r o t s growing i n the same l o c a t i o n i n p r e c i s i o n seeded beds c o u l d be caused by a number o f f a c t o r s . The seed may have been po o r l y coated, and crumbled o f f i n the p l a n t e r , so t h a t two seeds c o u l d then f a l l through the same hole i n the b e l t . T h i s e x p l a n a t i o n seems u n l i k e l y as the two v a r i e t i e s had been coated by d i f -f e r e n t companies. The stop and s t a r t p l a n t i n g o f s h o r t p l o t s may have caused some seeding i r r e g u l a r i t i e s , but n e a r l y the same percentage of 64 doubles were observed i n the grower's t r i a l . Two c a r r o t s growing i n the same l o c a t i o n may become c u l l s by twi n i n g around each o t h e r , but i n t h i s t r i a l , they helped to f i l l i n the row because o f the poor emer-gence. The cause o f two c a r r o t s growing i n the same l o c a t i o n r e q u i r e s f u r t h e r study. Crop r o t a t i o n . L e t t u c e and o n i o n , two crops commonly grown i n r o t a t i o n w i t h c a r r o t s on muck s o i l i n the F r a s e r V a l l e y , were ev a l u a t e d as to t h e i r e f f e c t i v e n e s s i n reducing PRD. There was no simple c o r r e -l a t i o n between crop y i e l d and the i n c i d e n c e o f the f o u r PRD organisms. C a r r o t s , grown a f t e r onions had a s i g n i f i c a n t l y g r e a t e r y i e l d , but the same l e v e l o f i n f e c t i o n o f Olpidium, f a s t and slow growing Pythium and TNV as c a r r o t s grown a f t e r l e t t u c e o r c a r r o t s . A higher percentage o f the c a r r o t s were f o r k e d and e x h i b i t e d PRD symptoms i n s o i l t h a t was c o n t i n u o u s l y cropped i n c a r r o t s than s o i l t h a t had been i n onions or l e t t u c e . T h i s was i n c o n f l i c t with Sutton's (1973) q u e s t i o n a b l e obser-v a t i o n c o r r e l a t i n g PRD i n c i d e n c e and previous crops o f . o n i o n s . Olpidium and slow growing Pythium were a t low l e v e l s i n l e t t u c e and onion f o r two crop p e r i o d s , but were at high l e v e l s when the s o i l was r e t u r n e d to c a r r o t s . These organisms may maintai n t h e i r p o p u l a t i o n i n the s o i l by a r e s t i n g spore, which can s u r v i v e adverse c o n d i t i o n s such as poor h o s t s , or by i n f e c t i n g the poor host j u s t enough to maintain i t s p o p u l a t i o n , but not enough to cause damage. A greenhouse crop r o t a -t i o n experiment i s unable to d u p l i c a t e the e f f e c t s o f w i n t e r . Periods o f s o i l f r e e z i n g and f l o o d i n g , and the long p e r i o d the s o i l i s i d l e without a crop would probably reduce the hold-over o f an organism from 65 one crop to the next. In the greenhouse experiment another crop was planted w i t h i n a day a f t e r h a r v e s t i n g the previous crop. Filman and Andersen (1972) have shown t h a t PRD i s reduced 16% by 10 days of s o i l f l o o d i n g before p l a n t i n g . Many of the muck sou l s around C l o v e r d a l e are f l o o d e d f o r a c o n s i d e r a b l e p a r t of the w i n t e r but PRD i s s t i l l a problem. F l o o d i n g d u r i n g the w i n t e r may reduce the PRD a s s o c i a t e d organisms enough to permit economic growing o f c a r r o t s i n B r i t i s h Columbia. Under f i e l d c o n d i t i o n s , l e t t u c e and onions i n a r o t a t i o n with c a r r o t s , would probably have g r e a t e r e f f e c t s on Pythium and Olpidium than was observed i n the greenhouse. Losses due to PRD can be kept to a minimum by f o l l o w i n g an i n t e -grated method o f c o n t r o l . F i r s t l y , Copeman and Black (1973) showed t h a t marketable y i e l d c o u l d be i n c r e a s e d and c u l l r a t e decreased by using PRD t o l e r a n t v a r i e t i e s . Raised beds s i g n i f i c a n t l y i n c r e a s e d marketable y i e l d over low conventional h e i g h t beds i f there i s a s u f f i c i e n t s o i l moisture throughout the season. P r e c i s i o n seeding reduces the c u l l r a t e over s c a t t e r shoe-seeding. The c a r r o t s are more evenly spaced so t h e r e i s probably l e s s spread o f the d i s e a s e from one c a r r o t to the next. And f i n a l l y , PRD i s reduced i f other crops such as l e t t u c e and onion are i n c l u d e d i n the r o t a t i o n . Onions appear to be the best crop to precede c a r r o t s . PRD l o s s e s can probably be kept to a minimum by using crop r o t a t i o n , t o l e r a n t v a r i e t i e s , r a i s e d beds, and p r e c i s i o n seeding. 66 CONCLUSIONS 1. Negative evidence from p a t h o g e n i c i t y t e s t s and the l a c k o f c o r r e -l a t i o n between the i n c i d e n c e o f Olpidium and c a r r o t marketable y i e l d or f o r k i n g suggest that Olpidium and TNV play a minor r o l e i n PRD. 2. I n d i r e c t f i e l d evidence suggests t h a t Pythium sulcatum i s a primary causal agent. Other f a s t growing Pythium spp. may a l s o be important. 3. Olpidium i n c i d e n c e appears to be d i r e c t l y c o r r e l a t e d with frequency o f p r e c i p i t a t i o n g r e a t e r than 1/2 i n c h . 4. PRD l o s s e s can be kept to a minimum and marketable y i e l d s i n c r e a s e d by f o l l o w i n g an i n t e g r a t e d method o f c o n t r o l using a) t o l e r a n t v a r i e t i e s , b) r a i s e d beds, c) p r e c i s i o n seeding, d) crop r o t a t i o n . LITERATURE CITED Anonymous. 1972. Vir u s e s o f u m b e l l i f e r o u s p l a n t s . Res. Rep. 1971. Can. Agr. Res. Branch, Information Canada, Ottawa, p. 114. . 1973a. A c h y t r i d - t r a n s m i t t e d v i r u s o f c a r r o t s . Res. Rep. Can. Agr. Res. Branch, Information Canada,Ottawa, p. 115. . 1973b. S o i l borne d i s e a s e s and i n t e g r a t e d c o n t r o l . Ann. Rep. Res. I n s t , o f O n t a r i o . A p r i l 1, 1972 to March 31, 1973. p. 159. . 1973c. C a r r o t s take to the s t r a i g h t and narrow. Amer. Veg. Grower 21:15-16. Baker, L. R., C. C. Filman and T. F. Lowndes. 1972. 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Bot. 46:951-956. Tsao, P. H. and G. Ocana. 1969. S e l e c t i v e i s o l a t i o n o f s p e c i e s o f Phytophthora from n a t u r a l s o i l s on an improved a n t i b i o t i c medium. Nature 223:636-638. T u i t e , J . 1969. P l a n t p a t h o l o g i c a l methods, fun g i and b a c t e r i a . Burgess Pub. Co., Minn. 239 pp. Winer, B. J . 1971. S t a t i s t i c a l p r i n c i p l e s i n experimental d e s i g n . 2nd ed. McGraw-Hill Book Co., N.Y. pp. 375-378. 71 APPENDIX I Analysis of Variance of Olpidium spores per micro-scope f ie ld from the 1972 microenvironment plot le No. Source df MS EMS F 1 Row 3 4680.6 Error A 2.5 2 Column 3 845.1 Error A 0.5 3 Date 3 41355.0 I 3.8559' 4 Error A 6 1854.4 Error B 0.9 5 Water 1 2488.5 II 1.067 6 W x D 3 2518.0 III 1.0485 7 Error B 12 1965.4 Error C 1.0 8 Age 4 8973.4 IV 3.1104 9 A x W 4 1779.8 AxWxD 0.9 10 A x W 12 7406.7 AxWxD 3.53* 11 A x W x D 12 2098.5 Error C 1.11 ** 12 Error C 96 1889.2 Root 3.47 ** 13 Roots 3040 543.8 L/Rt 6.95 14 L/Rt 12800 78.2 * Significance at the 5% level (P = .05). ** Significance at the 1% level (P = .01). Quasi F" test and degrees of freedom for numerator/denominator. I M S 3 + 7 + 1 1 / M S 4 + 6 + 1 0 df 3.6/19.0 II M S 5 + 1 1 / M S 6 + g df 3.2/19.0 III M S 6 + 1 2 / M S 7 + n df 9.0/24.0 IV M S 8 + 1 1 / M S g + 1 0 df 6.0/15.7 71 CHAPTER II CONTROLLED WATER RELATIONS AND PYTHIUM ROOT DIEBACK DEVELOPMENT INTRODUCTION F i e l d work conducted by Copeman and Black (1972) had suggested t h a t s o i l moisture was the most important s o i l parameter i n the i n c i -dence o f Pythium r o o t dieback (PRD) o f c a r r o t . Roots from p l o t s main-t a i n e d a t a high s o i l water content c o n t a i n e d a g r e a t e r number o f Pythium and Olpidium spores than r o o t s from d r i e r p l o t s . Greater marketable y i e l d s were a l s o harvested from these d r i e r p l o t s , s u g g e s t i n g t h a t s o i l m oisture, the presence o f spores i n the ro o t s and y i e l d s were c o r r e l a t e d . Control o f S o i l Water S o i l water i s best d e s c r i b e d i n terms o f p o t e n t i a l energy. Water ( l i k e heat) flows from areas o f high to low p o t e n t i a l energy, i n a c c o r -dance with the Second Law of Thermodynamics, u n t i l the two areas a re i n e q u i l i b r i u m . S o i l water p o t e n t i a l i s the sum o f p r e s s u r e , m a t r i c , osmotic, g r a v i t a t i o n a l , and s e v e r a l n e g l i g i b l e p o t e n t i a l s . The pres s u r e p o t e n t i a l r e s u l t s when h y d r o s t a t i c pressures occur i n the s o i l , f o r example when the s o i l s u r f a c e i s f l o o d e d . The mat r i c p o t e n t i a l r e s u l t s from the p h y s i c a l a f f i n i t y o f water to the s o i l p a r t i c l e s u r f a c e s and 72 73 c a p i l l a r y pores. Osmotic p o t e n t i a l i s i n f l u e n c e d by the presence o f s o l u t e s i n the s o i l water. The g r a v i t a t i o n a l p o t e n t i a l i s the r e s u l t o f . g r a v i t a t i o n a l f o r c e s and i s important only between s a t u r a t i o n c a p a c i t y (SC) and f i e l d c a p a c i t y (FC). Under most f i e l d c o n d i t i o n s , except those of high water and high s a l t content, m a t r i c p o t e n t i a l i s the most impor-t a n t component o f the t o t a l s o i l water p o t e n t i a l . Osmotic p o t e n t i a l may dominate i n the r h i z o s p h e r e and r o o t s i n c e roots are known to exude s a l t s , amino a c i d s and sugars. E a r l i e r work on the e f f e c t s o f osmotic p o t e n t i a l on fungal germination and growth have been reviewed by S c o t t (1957) and G r i f f i n (1963a). Sommers e t a l (1970) have r e c e n t l y shown t h a t growth o f Phytophthora s p e c i e s i s stimu-l a t e d as p o t e n t i a l energy decreases from -1.2 bars to -8.0 bars on agar media o s m o t i c a l l y amended with s a l t s or sucrose. Phytophthora p a r a s i t i c a was i n a c t i v a t e d a t a p o t e n t i a l o f -40 to -50 bars (Sommers e t a l _ . , 1970) and Fusarium oxysporum f . sp. vasinfectum a t -120 bars (Manandhar and B r u e h l , 1973). Experimental methods f o r c o n t r o l l i n g s o i l moisture o f growing p l a n t s w i t h i n narrow l i m i t are s u b j e c t to a g r e a t deal of e r r o r . High s o i l water p o t e n t i a l s can be maintained by the Haines apparatus; a s i n -t e red g l a s s funnel connected to a hanging water column (Haines, 1930). The Haines technique i s g e n e r a l l y l i m i t e d to p o t e n t i a l s o f g r e a t e r than -0.15 bars because the flow r a t e of water through the funnel i s too slow at lower p o t e n t i a l s . This technique i s i d e a l f o r spore germination s t u d i e s ( B a i n b r i d g e , 1970) and seed exudate s t u d i e s ( K e r r , 1964). Weighing pots and adding enough water to maintain the p l a n t s a t the d e s i r e d l e v e l of s o i l moisture works reasonably well i n the range 74 between SC and FC but i s not s a t i s f a c t o r y i n the range of FC to permanent w i l t i n g p o i n t (PWP) (Couch e t a]_., 1967). The whole s o i l mass cannot be u n i f o r m l y wetted a t s t r e s s e s below FC because when water i s added to the top o f a pot, i t tends to move down along the s i d e s . As a r e s u l t some areas o f s o i l are s a t u r a t e d and others are near the PWP (Hendickson and Veihmeyer, 1941). To avoi d the problem o f only wetting p a r t o f the s o i l , the recommended procedure i s to a l l o w the pots to dry out to a predetermined moisture l e v e l and then add enough water to b r i n g the s o i l back up to FC (Couch e_t a l _ . , 1967). E s s e n t i a l l y , t h i s technique only p r o v i d e s i n f o r m a t i o n on the e f f e c t s of adding d i f f e r e n t amounts o f water to pots. For example, pea r o o t r o t , caused by Pythium ultimum was most severe when water f l u c t u a t e d between -1/3 bar and -1 bar, and g r e a t e r d r y i n g p r i o r to rewatering to FC, reduced d i s e a s e s e v e r i t y ( K r a f t and Roberts, 1967). Roth and Riker (1943) s t u d i e d the e f f e c t s o f moisture on damping o f f o f red pine s e e d l i n g s by Pythium and R h i z o c t o n i a by using d i f f e r e n t heights o f s o i l c y l i n d e r s above a c o n s t a n t water t a b l e . Bateman (1961) improved the technique by r e s t r i c t i n g the r o o t zone i n small c l a y pots b u r i e d i n v a r y i n g depths of sand above the water t a b l e . The h e i g h t o f sand column necessary f o r a c e r t a i n water p o t e n t i a l i s o b t a i n e d by t r i a l and e r r o r and i s d i f f i c u l t to d u p l i c a t e . C a p i l l a r y movement o f water through the sand, the w a l l s o f the pot, and the s o i l w i t h i n the pot probably w i l l not keep up with the demands o f a growing p l a n t , and as a r e s u l t , water p o t e n t i a l decreases. Double-walled porous pots have a s i m i l a r s h o r t coming--the flow r a t e o f water through them i s very slow at water p o t e n t i a l s l e s s than FC. 75 An osmotic system has been developed to c o n t r o l s o i l water m a t r i c p o t e n t i a l (Zur, 1966). A narrow s l i c e o f s o i l i s separated from an os-motic s o l u t i o n by a semi-permeable membrane. Water moves from the os-motic s o l u t i o n to the s o i l when the p o t e n t i a l of the s o i l i s l e s s than t h a t o f the s o l u t i o n . By m a i n t a i n i n g the volume o f the osmotic s o l u t i o n c o nstant, water l o s t by s o i l s u r f a c e e v a p o r a t i o n and t r a n s p i r a t i o n i s c o n t i n u o u s l y r e p l a c e d . A membrane with a high water c o n d u c t i v i t y and r e s i s t a n c e to m i c r o b i a l breakdown i s necessary i f t h i s technique i s to be used to c o n t r o l s o i l moisture f o r s e v e r a l weeks. E f f e c t s o f S o i l Water on Pythium Pythium d i s e a s e s are u s u a l l y a s s o c i a t e d with s o i l s o f high s o i l water content. A number o f r o l e s f o r s o i l water i n d i s e a s e development and Pythium p o p u l a t i o n dynamics have been suggested. Oogonia o f f_. ultimum were more p r e v a l e n t at high s o i l p o t e n t i a l s than sporangia ( B a i n -b r i d g e , 1970). Oogonia p r o d u c t i o n took p l a c e i n water f i l l e d pores, whereas spo r a n g i a ! production only took p l a c e i n a i r f i l l e d pores. G r i f f i n (1963b) found t h a t P_. ultimum produced oospores i n wet s o i l s o n l y when s o i l pores were o f s u f f i c i e n t s i z e to accommodate the oogonia. Sexual r e p r o d u c t i o n d i d not occur i n a r t i f i c i a l s o i l s with a pore space l e s s than 15 u diameter, though m y c e l i a l growth was u n a f f e c t e d . The s i z e o f w a t e r - f i l l e d pores or pore necks and pore s i z e d i s -t r i b u t i o n may l i m i t the passage o f c e r t a i n Phycomycetes such as Olpidium which r e l y on zoospores f o r spread. As the m a t r i c p o t e n t i a l decreases, the diameter of w a t e r - f i l l e d pores decreases. S t o l z y et_ al_. (1965) suggested t h a t zoospores o f Phytophthora s p e c i e s r e q u i r e d w a t e r - f i l l e d 76 pores at least 40-60 u in diameter. At saturation with d i s t i l l e d water, no zoospores were produced by oospores or sporangia of P. aphanidermatum but at three times soi l saturation, 30% of the oospores and 90% of the sporangia formed zoospores. If nutrients were in the water, the propagules germinated directly (Stanghellini and Burr, 1973). They concluded that zoospores are only produced in surface water of saturated soi ls and are not an important form of inoculum spread. Kerr (1964)concluded that so i l moisture did not affect P_. ultimum  per se but influenced the amount of sugar exuded from pea seeds. Under high so i l moisture, bean exudates permeated further from the seed so that a greater number of Pythium spores were stimulated to germinate and infect the seedlings (Stanghellini and Burr, 1973). Germination and growth was restricted at higher water potentials because of a reduced ava i lab i l i t y of nutrients. When a soi l becomes saturated, the concentration of 0 2 decreases and CO- increases. Gr i f f in (1963b) has suggested that the ab i l i t y of Pythium to withstand saturated conditions is related to the a b i l i t y of the fungus to withstand either low concentrations of 0- or high concen-trations of CO,,. P_. ul timum growth was reduced at 1.3% 0 2 but not 4% 0 2 and P_. irregulare and P_. vexans were relat ively unaffected by high CO- and low 0 2 concentrations (Gardner and Hendrix, 1973). Periodic or continuous saturation may have different effects on Pythium species. P_. irregulare was unaffected by periodic or con-tinuous saturation because i t infected holly roots only by direct ger-mination of the sporangia, whereas P_. vexans was most severe under con-tinuous saturation because i t produced zoospores (Biesbrock and Hendrix, 1970a). 77 Since s o i l moisture appeared to be the c r i t i c a l f a c t o r i n PRD, i t s r o l e i n d i s e a s e development was s t u d i e d . The o b j e c t i v e s o f the study were 1) to determine the e f f e c t s o f p e r i o d i c s o i l s a t u r a t i o n on Pythium and Olpidium p o p u l a t i o n s , 2) to observe the e f f e c t o f os-motic p o t e n t i a l on Pythium growth, and 3) to develop a technique f o r c o n t r o l l i n g s o i l moisture of a growing c a r r o t . METHODS AND MATERIALS P e r i o d i c S a t u r a t i o n Experiment A greenhouse experiment was done to determine the e f f e c t s o f p e r i o d i c s o i l s a t u r a t i o n on PRD i n c i d e n c e . The s o i l f o r the experiment was a Lumbrum Muck from the farm o f C l o v e r d a l e Produce Farms L t d . , which had produced a c a r r o t crop with symptoms of PRD the past summer. A p o r t i o n o f the s o i l was a u t o c l a v e d a t 15 p s i f o r 90 minutes. One week a f t e r a u t o c l a v i n g , the a u t o c l a v e d and unautoclaved s o i l was potted i n square 4 inch p l a s t i c pots. C a r r o t , Daucas c a r o t a L. 'GoldPak E l i t e ' , was seeded and l i g h t l y covered w i t h s o i l . A f t e r s e e d l i n g s had emerged, they were thinned to two uniform s e e d l i n g s per pot. The c a r r o t s were grown at 16-20 C, under a 14 hour photoperiod (3600 lux) provided by "Cool White" f l u o r e s c e n t lamps and i n c a d e s c e n t b u l b s . Pythium s y l v a t i c u m Campbell and Hendrix was grown on D i f c o Corn meal agar (CMA, 17 g/1) p l a t e s a t 25 + 1 C f o r e i g h t days. The c u l t u r e s were examined f o r sporangia and then the agar and the organism were 78 homogenized with an O s t e r i z e r blender a t low speed f o r 10 seconds i n 250 ml o f d i s t i l l e d water. Subsequent to t h i n n i n g , f o u r 1 ml a l i q u a n t s of s o l u t i o n were i n j e c t e d with a s y r i n g e i n t o h a l f of the pots c o n t a i n i n g a u t o c l a v e d s o i l . N a t u r a l l y - i n f e s t e d , f i e l d s o i l served as a t h i r d s o i l treatment. Pots were placed i n p l a s t i c l i n e d cedar f l a t s (12 x 18 inches) with a d r a i n hole a t one end. Four watering regimes were used: 1) s p r i n k l i n g the pots from above when they appeared to be nearing the p o i n t o f w i l t i n g ; 2) s a t u r a t i n g pots by f i l l i n g the f l a t s with water f o r 10 minutes d a i l y ; 3) s a t u r a t i n g pots 10 minutes every f o u r t h day; and, 4) s a t u r a t i n g the pots 8 hours every f o u r t h day. Pots o f d i f f e r e n t inoculum treatments were kept i n separate f l a t s . Every two weeks, f o r 8 weeks, t h r e e pots i n each water and s o i l treatment were harvested. The t o t a l f r e s h weight o f the c a r r o t s was measured and 20 brown or d i s c o l o u r e d r o o t l e t s from p l a n t s i n each pot were p i c k e d o f f and preserved i n f o r m a l i n a c e t i c a c i d (FAA) ( P h i l l i p s and Hayman, 1970). Roots were s t a i n e d w i t h Phloxine and KOH ( T u i t e , 1969), and examined under phase c o n t r a s t . The number o f spores a t f i v e l o c a t i o n s o f estimated h i g h e s t d e n s i t y were recorded f o r each r o o t . The remainder o f the r o o t system was macerated i n a small amount o f 0.05 M phosphate b u f f e r , pH 8.5 ( T e a k l e , 1962), and r u b - i n o c u l a t e d on carborundum-dusted l e a v e s o f Chenopodium quinoa W i l l d . The data was analyzed by a 3 x 4 f a c t o r i a l a n a l y s i s . 79 E f f e c t s o f Osmotic P o t e n t i a l on Pythium To determine the a b i l i t y o f Pythium to i n f e c t and c o l o n i z e a r o o t , i t was necessary to determine Pythiurn's a b i l i t y to t o l e r a t e v a r i o u s osmotic p o t e n t i a l s . The osmotic p o t e n t i a l s o f agar media were ad j u s t e d to v a r i o u s l e v e l s by adding s o l u t e s of the a p p r o p r i a t e molal c o n c e n t r a t i o n s . For a s i n g l e s o l u t e , the molal c o n c e n t r a t i o n s were determined by i n t e r p o l a t i o n from the water a c t i v i t y ( a ) t a b l e s o f Robinson and Stokes (1955). A W s a l t mixture c o n s i s t i n g o f 5NaCl:3KC1 ^ i ^ S O ^ was a l s o used to a d j u s t the osmotic p o t e n t i a l s . The a w values o f ^ SO^. were c a l c u l a t e d from the formula: l o g a w = -O.OO7824vm0 where v = no. o f ions per molecule ( 3 ) , m = m o l a l i t y and 0 = osmotic c o e f f i c i e n t . Robinson and Stokes (1955) l i s t the osmotic c o e f f i c i e n t s . The a, values o f the s a l t mixture i s the sum o f the 3 s a l t a,, values w w at the 5:3:2 molal r a t i o . Water p o t e n t i a l (Y) i s d e r i v e d from the a w values by the formula: RT * = \ T l o g 1 0 aw = 1 0- 6 T 1 0 9 aw where R = i d e a l gas c o n s t a n t , T = a b s o l u t e temperature and V = volume o f a mole o f water (Manandhar and B r u e h l , 1973). The general procedure used i n a l l experiments was to d i s s o l v e the s a l t s i n d i s t i l l e d water before the agar medium was added. Calcium c h l o r i d e was d i s s o l v e d i n d i s t i l l e d water and a u t o c l a v e d , b e f o r e adding the agar to prevent p r e c i p i t a t i o n . The p o t e n t i a l s o f the s o l u t e amended . 80 media were c a l c u l a t e d as the sum of the medium plus s o l u t e osmotic poten-t i a l s . Media were au t o c l a v e d a t 15 p s i f o r 15 minutes. A f t e r the media had c o o l e d to about 46 C, 20-25 mis were poured i n t o 8.5 cm ID p l a s t i c p e t r i d i s h e s . Dishes were l e f t a j a r i n a laminar a i r flow bench to prevent condensation on the l i d s . A m y c e l i a l agar plug taken with a #1 cork bore (4 mm diameter) from the l e a d i n g edge of an a c t i v e l y growing Pythium c u l t u r e (grown on the same base medium) served as inoculum. Each treatment was r e p l i c a t e d f o u r times. P e t r i d i s h e s were s e a l e d i n p l a s t i c bags and incubated a t 25 + 1 C. Colony diameter was measured at r e g u l a r i n t e r v a l s u n t i l the f a s t e s t growing treatment had covered the p l a t e . The e f f e c t o f the s o l u t e s NaCl, KCl, CaCl-, 5NaCl:3KC1:2NaS0 4 and sucrose on growth o f P_. s y l v a t i c u m on CMA ( D i f c o 17 g/1) and P_. s u l - catum P r a t t and M i t c h e l l on basal medium (Sommers ejt a l _ . , 1970) were t e s t e d . To determine i f n u t r i t i o n might have an e f f e c t on osmotic poten-t i a l t o l e r a n c e , CMA, potato dextrose agar (PDA, D i f c o 39 g/1) and basal medium were a d j u s t e d o s m o t i c a l l y with KCl and i n o c u l a t e d w i t h £_. sulcatum. The osmotic p o t e n t i a l o f nonamended basal medium, CMA and PDA were -1.2, -1.2 and -3.2 bars r e s p e c t i v e l y (Sommers et_ a l _ . , 1970). P_. ul timum Trow, and two i s o l a t e s each o f P_. s y l v a t i c u m and P_. sulcatum were i n o c u l a t e d on KCl o s m o t i c a l l y a d j u s t e d basal medium to determine i f d i f f e r e n t s p e c i e s and i s o l a t e s o f Pythium a c t s i m i l a r l y to osmotic p o t e n t i a l s . 81 Development of a Technique to Control Matric Water Potential After a comparison of the techniques used to control soi l mois-ture, I decided that Zur's osmotic system had the greatest potential in controll ing soi l moisture within narrow l im i t s . Cellulose dialysis membrane. Flow rate. The flow rate of water through the membrane was determined by measuring the quantity of water passing through the mem-brane from a reservoir of d i s t i l l e d water to a reservoir of Polyethylene glycol (PEG) solution. A glass mil l ipore f i l t e r holder was attached to a funnel by means of rubber tubing (Figure 1). A single thickness of a 1 5/16 inch f l a t diameter, 0.001" wall thickness cellulose dialysis membrane (Arthur H. Thomas Co., Philadelphia) was placed on the screen of the mill ipore f i l t e r holder. The funnel was f i l l e d with d i s t i l l e d water and a l l the a i r bubbles were removed from between the screen and dialys is membrane. A 2.5 bar PEG 6000 solution (J . T. Baker Chemical Co., New Jersey) was placed in the f i l t e r holder. The concentration of PEG at various osmotic potentials was determined by interpolation from graphs of Zur (1966) and Williams and Shaykewich (1970) (Table I) . The levels of the two solutions were adjusted to the same i n i t i a l height. The change in volume of the PEG solution was measured and removed dai ly . Moisture controll ing prototypes using dialysis membrane. Soil cel ls were constructed by f i t t i n g a cellulose dialysis membrane over a Polyvinylchloride (PVC) frame. Three sixteenth inch PVC was used to make a frame 12.5 x 1.0 x 30.0 cm. Dialysis tubing, 5 3/4 inch f l a t 82 F i g u r e 1. Apparatus used to measure the flow r a t e o f water through t e s t membranes. 83 Table I. The c o n c e n t r a t i o n o f P o l y e t h y l e n e g l y c o l 6000 a t various osmotic p o t e n t i a l s Osmotic p o t e n t i a l C o n c e n t r a t i o n PEG 6000 (-bars) (g/U 0.0 0 0.1 20 0.2 30 0.4 42 0.5 55 0.6 60 1.0 88 1.2 90 2.0 125 2.5 132 5.0 175 84 diameter, 0.0043 inch wall t h i c k n e s s (Arthur H. Thomas Co., P h i l a d e l p h i a ) was soaked i n water, f o r 15 minutes, p u l l e d over the frame, and s e a l e d at the base by clamping the end o f the tubing between a p l a s t i c book binder. The c e l l was placed between two sheets o f plywood and then c a r e -f u l l y f i l l e d and packed with f r e s h l y a u t o c l a v e d greenhouse s o i l . The c e l l s were then placed i n -0.4 bar and -2.5 bar PEG s o l u t i o n s and the plywood s i d e s were removed. S o i l tubes were c o n s t r u c t e d by k n o t t i n g one end o f a 15 cm, 1 5/16 inch f l a t diameter, 0.001 i n c h wall t h i c k n e s s d i a l y s i s tube and f i t t i n g the ot h e r end over a s i n g l e holed No. 3 1/2 cork. The tu b i n g was held on the cork with an e l a s t i c band. Tubes were f i l l e d with f r e s h l y a u t o c l a v e d s o i l and placed i n 600 ml g l a s s beakers o f PEG s o l u t i o n s o f 0, -0.2, -0.4, -0.6, -1.0, -1.5, or -2.0 bar (Table I ) . A n t i b i o t i c s f o r the PEG s o l u t i o n s . B a c t e r i a and fungi growing i n the PEG s o l u t i o n , s o i l , and on the membrane s u r f a c e were i s o l a t e d by p l a t i n g small q u a n t i t i e s o f s o i l and d i a l y s i s membrane o r by making st r e a k s o f PEG s o l u t i o n on PDA and N u t r i e n t Agar. A l l c u l t u r e s were incubated i n the dark a t 25 +_ 1 C. The i s o l a t e d organisms capable o f degrading the c e l l u l o s e membrane were determined by the f o l l o w i n g technique. E i g h t cm long s o i l tubes were placed i n 250 ml Erlenmeyer f l a s k s and held u p r i g h t by a s t r i n g attached to the cork and passed to the o u t s i d e o f the f l a s k . A p p r o x i -mately 100 ml of a Glucose Yeast E x t r a c t broth (1% Glucose, 0.2% Yeast E x t r a c t , 1 l i t e r water) was poured i n the f l a s k and i n the tube so as to h a l f fill the s o i l tube. The f l a s k was stoppered with a foam plug and 85 the whole assembly was autoclaved f o r 20 minutes a t 15 p s i . A 1 cm agar d i s k o f inoculum from each i s o l a t e was placed i n s i d e a s o i l tube, t a k i n g care not to contaminate the s o l u t i o n around the s o i l tube. F l a s k s were ge n t l y shaken a t a temperature o f 20 + 3 C. A f t e r 9 days, the s o i l tube was removed and the s t r e n g t h of the d i a l y s i s t ubing was t e s t e d by g e n t l y p u l l i n g on the membrane. Benlate (50% WP, E.I. DuPont de Nemours and Co., Wilmington), PCNB (75% WP, 01 i n Mathieson Chem. Corp., New York), and Streptomycin s u l f a t e (Sigma Chem. Co., S t . Louis) were t e s t e d f o r t h e i r e f f e c t i v e n e s s a g a i n s t the organisms capable o f degrading the c e l l u l o s e membrane, by i n c o r p o r a t i n g 1, 10, 100, and 1000 ppm a c t i v e i n g r e d i e n t o f each chemical s e p a r a t e l y i n t o a u t o c l a v e d .CMA a t 45 to 50 C. M y c e l i a l d i s k s from the l e a d i n g edge o f a c t i v e l y growing fungal c u l t u r e s and s t r e a k s o f b a c t e r i a l i s o l a t e s were used as i n o c u l a . P l a t e s were incubated a t 25 + 1 C and colony diameter o r growth was determined d a i l y . B e n l a t e , PCNB, and Streptomycin were added s e p a r a t e l y i n concen-t r a t i o n s o f 1000 ppm, 1000 ppm, and 125 ppm r e s p e c t i v e l y to a -0.4 bar PEG s o l u t i o n i n 600 ml beakers. S o i l tubes c o n t a i n i n g e i t h e r f r e s h l y a u t o c l a v e d or aged a u t o c l a v e d s o i l were placed in the PEG s o l u t i o n s . C a r r o t seeds were p l a n t e d i n the s o i l o f some o f the tubes. The s o i l water c o n t r o l l i n g apparatus was s e t up on a l a b o r a t o r y bench a t 20 + 3 C. A f t e r two weeks, the water content o f the s o i l was determined g r a v i -m e t r i c a l l y by oven d r y i n g the s o i l a t 105 C. Throughout the t h e s i s , water content w i l l be expressed on a weight b a s i s . P e l l i c o n membrane In p r e l i m i n a r y experiments, the P e l l i c o n u l t r a f i l t r a t i o n mem-branes ( M i l l i p o r e Corp., Bedford) appeared capable o f r e s t r i c t i n g l a r g e molecules such as PEG 6000, and were durable f o r a t l e a s t t h r e e weeks. Flow r a t e . The flow r a t e o f water through a P e l l i c o n PSAC, 500 nominal molecular weight l i m i t (NMWL) and a PSAL 1000 NMWL u l t r a f i l -t r a t i o n membrane were determined by the procedure p r e v i o u s l y o u t l i n e d . The c o n c e n t r a t i o n o f PEG was monitored throughout the experiment by d r y i n g and weighing the PEG s o l u t i o n removed from the M i l l i p o r e f i l t e r h o l d e r . To determine i f any PEG passed through the membrane, 100 ml o f s o l u t i o n from the d i s t i l l e d water s i d e o f the apparatus was d r i e d and weighed f o r PEG r e s i d u e . Moisture c o n t r o l l i n g p r o t o t y p e s . A p r e l i m i n a r y experiment was conducted to t e s t the membranes' a b i l i t y to c o n t r o l the moisture o f small q u a n t i t i e s o f s o i l . A c r y l o n i t r i l e Butadiene S t r y r e n e (ABS) plumbing adapters (4 cm i n s i d e diameter, ID) were used to hold 4.3 cm diameter 500 NMWL P e l l i c o n membranes. Membranes were placed between the nylon r i n g s and caps, which had been l i g h t l y coated w i t h stop cock grease. Chambers were then p l a c e d i n a d i s t i l l e d water bath f o r one hour to check f o r lea k s before f i l l i n g with s o i l . P a s t e u r i z e d greenhouse mix s o i l , screened through a 10 mesh s i e v e was used i n t h i s and a l l o t h e r s o i l moisture c o n t r o l l i n g experiments. The s o i l was packed s e v e r a l times d u r i n g the f i l l i n g o f the chambers to a height o f 3 cm. Extreme care had to be e x e r c i s e d not to ruptu r e the membrane. Chambers were 87 placed i n s o l u t i o n s o f PEG o f 0, -0.2, -0.4, -1.0 or -2.5 bars. A f t e r 6 weeks, the water content o f the bottom 2 cm o f s o i l was determined g r a v i m e t r i c a l l y . Tensiometers and thermocouple psychrometers were used to measure s o i l water p o t e n t i a l s over time i n the ranges o f 0 to -0.8 bars and l e s s than -1.0 bars r e s p e c t i v e l y . S o i l chambers were made l a r g e r by f i t t i n g a 4 cm ID ABS pipe i n the plumbing adapter to g i v e a t o t a l h e i g h t o f 9.5 cm ( F i g u r e 2a). A 2 cm x 0.6 cm o u t s i d e diameter (0D) ceramic t e n -siometer ( S o i l Moisture Equip. Corp., Santa Barbara) was glued w i t h epoxy to a 0.65 cm 0D a c r y l i c tube. Tensiometers were i n s e r t e d through the w a l l o f the s o i l chambers and cemented i n place w i t h epoxy a t 2 cm and 7.5 cm from the membrane s u r f a c e . Tensiometers were connected to mercury manometers by means o f tygon t u b i n g . Three t e r m i n a l double loop thermocouple psychrometers were b u i l t a c c o r d i n g to the s p e c i f i c a t i o n s o f Chow and De V r i e s (1973) and were c a l i b r a t e d with KCl s o l u t i o n s (Camp-b e l l e_t a l _ . , 1966). Thermocouple output was measured with a K e i t h l e y Model 155 Mic r o v o l t m e t e r and recorded on a s t r i p c h a r t r e c o r d e r . A ceramic bulb made from a 1.0 cm o u t s i d e diameter (0D) tensiometer and ground to 0.8 cm 0D was used to p r o t e c t the thermocouple psychrometer. The thermocouple psychrometer was held i n the bulb by means o f a tapered PVC plug ( F i g u r e 3 ) . The ceramic bulbs were i n s e r t e d through the wal l of s o i l chambers a t 2 cm and 7.5 cm from the membrane s u r f a c e . Rubber cement (Black P l a s t i c Rubber, D u r o - P l a s t i c , Woodhill Chem Sales Corp., Cleveland) was a p p l i e d to prevent water from l e a k i n g i n t o the thermo-couple psychrometer chamber. 88 Figure 2. Prototypes using P e l l i c o n membranes fo r c o n t r o l l i n g s o i l matric p o t e n t i a l . (A) plumbing adaptor chamber (B) cubical chamber (C) narrow s o i l c e l l (D) osmotic so lut ion chamber. 89 Thermocouple psychrometer Set screw Ceramic bulb' 1 4H PVC plug, Wall of s o i l chamber Thermocouple wire Figure 3. Cross section of ceramic bulb with PVC plug holding thermocouple psychorometer in place. 90 PEG s o l u t i o n c o n t a i n e r s were made o f 15 x 10 cm ID ABS sewer pipe glued to a 0.65 cm c l e a r p l e x i g l a s s bottom ( F i g u r e 2d). A 0.65 cm a c r y l i c tube near the base o f the c o n t a i n e r was connected to an a i r m a n i f o l d system. A small stream of a i r a g i t a t e d the PEG s o l u t i o n . The s o l u t i o n c o n t a i n e r s were covered with plywood l i d s having holes only l a r g e enough to accommodate the s o i l chambers. S o i l chambers were f i l l e d w ith s o i l and placed i n PEG s o l u t i o n s o f -0.1, -0.2, -0.4, -0.6, -1.2, -2.5 or -5.0 bars. Saran wrap was used to c over the s o i l chambers f o r s e v e r a l days w h i l e the system came to e q u i l i b r i u m . S o i l s u r f a c e e v a p o r a t i o n was then allowed to take p l a c e and the water p o t e n t i a l o f the chambers was measured 3 or 4 times d a i l y . In some cases, s u r f a c e e v a p o r a t i o n was hastened with the a i d o f a f a n . The volume o f the PEG s o l u t i o n was kept c o n s t a n t throughout the e x p e r i -ment by adding water every second day. PEG s o l u t i o n s were renewed weekly. To determine whether the system c o u l d keep up with the water demands o f a young p l a n t , r a d i s h , Raphanus s a t i v u s L., was p l a n t e d i n s o i l chambers which were then placed i n PEG s o l u t i o n s o f -0.1 and -0.2 bars. The water p o t e n t i a l was measured with t e n s i o m e t e r s . The p l a n t s were grown at 20 +_ 3 C under a 14 hour photoperiod (2800 lux) provided by "cool white" f l u o r e s c e n t lamps and incandescent bulbs. Two s o i l c e l l designs were e v a l u a t e d i n an attempt to maintai n the water p o t e n t i a l w i t h i n a narrower range than p r e v i o u s l y done. A c u b i c a l s o i l chamber, 5.6 x 5.6 x 10 cm o f 1/4 i n c h c l e a r p l e x i g l a s s , had a 3.8 cm diameter hole i n each of the f o u r s i d e s and bottom. A 4.3 cm diameter 500 NMWL P e l l i c o n membrane was cemented with rubber cement 91 (Black P l a s t i c Rubber) over the i n s i d e o f each hole ( F i g u r e 2b). The second design was 5.6 x 2.5 x 10 cm o f 1/4 i n c h c l e a r p l e x i g l a s s with a membrane i n each s i d e ( F i g u r e 2 c ) . A tensiometer, e n t e r i n g the chambers from the s o i l s u r f a c e , was p l a c e d a t a p o i n t equal d i s t a n c e from the membranes. The s o i l c e l l s were placed i n a -0.2 bar PEG s o l u t i o n and water p o t e n t i a l s were monitored during the growth o f a r a d i s h s e e d l i n g . T r a n s p i r a t i o n was approximated from the amount of water added to maintai n a c o n s t a n t l e v e l o f the PEG s o l u t i o n . Constant water p o t e n t i a l as determined g r a v i m e t r i c a l l y . The measurement o f water p o t e n t i a l by i n s t r u m e n t a t i o n f o r extended p e r i o d s of time i n the -0.4 to -2.0 bar range i s s u b j e c t to many problems. G r a v i -m e t r i c measurement o f s o i l water content i s the most r e l i a b l e i n d i r e c t method o f determining the water p o t e n t i a l and t h e r e f o r e , i t was used as a check to see i f water p o t e n t i a l s were con s t a n t f o r s e v e r a l weeks. S o i l c e l l s , 5.6 x 2.0 x 10.5 cm were c o n s t r u c t e d o f 1/8 i n c h p l e x i g l a s s ( F i g u r e 2 c ) . One P e l l i c o n (4.3 cm diameter, 500 NMWL) membrane was glued with rubber cement (Devcon.Rubber, Devcon Canada L t d . , S c a r -borough) on the i n s i d e o f each s i d e . S o i l c e l l s were f i l l e d with s o i l and p l a c e d i n osmotic s o l u t i o n s o f -0.2, -0.5, -1.0, -2.0 bars. Two chambers were p l a c e d i n each bath. The change i n volume of the PEG s o l u t i o n r e s u l t i n g from t r a n s p i r a t i o n was determined d a i l y by measuring the change i n he i g h t o f the s o l u t i o n i n a 1 ml d i s p o s a b l e p i p e t t e t h a t was bent and f i t t e d i n the base of the PEG c o n t a i n e r to f u n c t i o n as a l e v e l gauge. 92 Two uniform 15 day o l d c a r r o t s e e d l i n g s were t r a n s p l a n t e d i n t o each c e l l and were grown at 20 +_ 3 C under a 14 hour photoperiod (2800 lux) p rovided by "cool white" f l u o r e s c e n t lamps and incandescent b u l b s . S o i l water content was determined f o r twelve l o c a t i o n s i n each pot by measuring the l o s s o f water a t 105 C. Two c e l l s a t each of f o u r osmotic p o t e n t i a l s were analyzed each week f o r 3 weeks. Leaf area was measured with a planimeter a t the end o f the t h i r d week. The water r e t e n t i o n c h a r a c t e r i s t i c s o f the greenhouse s o i l was determined by a hanging column apparatus and a pre s s u r e p l a t e e x t r a c t o r ( R i c h a r d s , 1947). S o i l water contents c o u l d then be converted i n t o p o t e n t i a l s . RESULTS P e r i o d i c S o i l S a t u r a t i o n C a r r o t s grown i n n a t u r a l l y i n f e s t e d s o i l , under a l l f o u r watering regimes, had a t each h a r v e s t some n e c r o t i c r o o t t i p s . Post emergence damping o f f was observed and at h a r v e s t , some o f the c a r r o t s e x h i b i t e d t y p i c a l f i e l d symptoms o f f o r k i n g . By c o n t r a s t , c a r r o t s from a u t o c l a v e d s o i l were not f o r k e d , but some o f the r o o t l e t s from Pythium i n o c u l a t e d p l a n t s were l i g h t grey i n c o l o u r . Any roots t h a t had grown through the drainage holes i n the bottom o f the pots were brown, but these were e a s i l y d i s t i n g u i s h e d because of t h e i r t h i c k e r diameter and b r i g h t brown or orange c o l o u r . A v i s u a l comparison of c a r r o t s a t each h a r v e s t suggested t h a t growth i n autoclaved s o i l was much g r e a t e r than i n non-autoclaved s o i l . 93 Fresh weight of c a r r o t s i n autoclaved s o i l was s i g n i f i c a n t l y g r e a t e r (P = .05) a t each h a r v e s t than i n non-autoclaved s o i l ( F i g u r e 4 ) . Weight o f c a r r o t s was not s i g n i f i c a n t l y d i f f e r e n t i n Pythium i n o c u l a t e d and c o n t r o l treatments. P e r i o d i c s o i l s a t u r a t i o n i n c r e a s e d y i e l d s over s p r i n k l e watering i n au t o c l a v e d s o i l , whereas i n n a t u r a l l y - i n f e s t e d s o i l , water regimes had no s i g n i f i c a n t e f f e c t on y i e l d . Olpidium b r a s s i c a e (Woron.) Dang, was the only organism con-s i s t e n t l y observed i n roots from n a t u r a l l y - i n f e s t e d s o i l . While t h e r e was mycelium i n the r o o t s , r a r e l y were Pythium spores observed. Olpidium spore counts per microscope f i e l d were s i g n i f i c a n t l y g r e a t e r (P = .05) i n r o o t l e t s exposed to d a i l y 10 minute s a t u r a t i o n than a l l o t h e r water treatments ( F i g u r e 5 ) . R o o t l e t s s a t u r a t e d f o r 10 minutes every f o u r t h day had a s i g n i f i c a n t l y g r e a t e r (P = .05) l e v e l o f spores than those s a t u r a t e d f o r 8 hours every f o u r t h day or s p r i n k l e watered. I f data were analyzed on the b a s i s o f p o s i t i v e i n c i d e n c e o f Olpidium, counts g r e a t e r than or equal to f i v e s p o r e s , or counts g r e a t e r than o r equal to 10 spores, the same b a s i c r e s u l t s were o b t a i n e d . C a r r o t s a t s i x weeks o f age, had a s i g n i f i c a n t l y g r e a t e r (P = .05) number o f spores than a l l o t h e r ages. No Olpidium spores were observed i n r o o t l e t s from a u t o c l a v e d s o i l . Tobacco n e c r o s i s v i r u s (TNV) was recovered only from c a r r o t s grown i n n a t u r a l l y - i n f e s t e d s o i l t h a t was s u b j e c t to p e r i o d i c s a t u r a t i o n . C a r r o t s from n a t u r a l l y - i n f e s t e d s o i l t h a t was s p r i n k l e watered, w h i l e having r e l a t i v e l y high l e v e l s o f Olpidium, d i d not c o n t a i n the v i r u s at l e v e l s d e t e c t a b l e by the assay used. Y//A Sprinkle watering KS^ M Saturation 10 min./ Non-inoculated Pythium inoculated Naturally-infested autoclaved s o i l autoclaved s o i l f i e l d s o i l Figure 4. Mean t o t a l f r e s h weight y i e l d o f c a r r o t s a t 8 weeks of age i n n a t u r a l l y - i n f e s t e d s o i l , Pythium s y l v a t i c u m - i n o c u l a t e d autoclaved s o i l , and non-inoculated a u t o c l a v e d s o i l . Bars w i t h i n a s o i l treatment with the same l e t t e r do not s i g n i f i c a n t l y (P = .05) d i f f e r . 95 i-H <U <4-t (X o ° 3 o }-l o u CU (0 o M O 9 (0 IH o J-l a> 1 r S p r i n k l e w a t e r i n g KSXH S a t u r a t i o n 10 rain, p e r day I I S a t u r a t i o n 10 rain. e v ery 4th day S a t u r a t i o n 8 hours every 4th day Olpidium Pythium Figure 5. Average number of Olpidium and Pythium spores per. microscope f i e l d , in carrot rootlets under four watering regimes (4 carrot ages combined). Bars with the same letter do not d i f fer s ign i -f icantly (P = .05). 96 Low levels of Pythium infection were observed in roots from a r t i f i c i a l l y infested s o i l , and no spores were observed in non-inoculated autoclaved s o i l . Mycelium was observed in the cortex of the rootlets, and in some cases, mycelia and sporangia were observed externally around the rootlets. No oospores were observed in any of the rootlets. Watering regimes had no signif icant effect on the number of Pythium spores observed per microscope f ie ld (Figure 5). Carrots at 10 weeks of age had a s ign i -f icantly greater (P = .05) Pythium infection than four week old carrots. There was a signif icant (P = .05) sample date X water regime interaction, but this was caused by one replicate having an exceptionally high infec-tion leve l , and therefore, the interaction was not considered important. Effects of Osmotic Potential on Pythium For a l l solutes tested, growth of P_. sulcatum at decreasing osmotic potentials was nearly identical (Figure 6). The growth rate of Pythium was expressed in mm of radial growth per day, to f a c i l i t a t e direct comparisons between experiments. Radial growth decreased as the water potential decreased from -1.2 bars. sylvaticum exhibited a greater var iab i l i t y in growth to different solutes than P_. sulcatum (Figure 7). Radial growth was stimulated, approximately 25%, over non-amended medium at a potential of -3 .2 bars created by KCl or NaCl and -4.2 bars created by sucrose. The growth rate of F\ sulcatum on CMA and basal medium was nearly ident ica l ; however, on PDA, growth ceased at -15 bars as compared to -30 bars on the other media (Figure 8) . !120 £ 1 0 0 to £ 60. o u M 4 0 to 20 A A NaCl — a KCl • a 5NaCl:3KCl:2Na 2S0 4 o o Sucrose 10 15 Osmotic p o t e n t i a l (-bars) F i g u r e 6. Radial growth r a t e o f Pythium sulcatum on basal medium amended o s m o t i c a l l y with NaCl, KCl, 5NaCl:3KC1:2NagS0 4, sucrose. Each p o i n t r e p r e s e n t s the mean of four o b s e r v a t i o n s . so T 1 1 r Osmotic pote n t i a l (-bar) F i g u r e 7. Radial growth r a t e o f Pythium s y l v a t i c u m on CMA amended o s m o t i c a l l y with NaCl, KC1, CaCl2» sucrose. Each p o i n t represents a mean of f o u r o b s e r v a t i o n s . co Figure 8. Radial growth rate of Pythium sulcatum on CMA, PDA, and basal medium amended with KCl. Each point represents a mean of four observations. to U3 100 Colony diameter o f P. sulcatum was measured at three times i n t e r -v a l s to determine i f there was a l a g phase a t lower p o t e n t i a l s . Growth d i d show a s l i g h t l a g a t p o t e n t i a l s l e s s than -15 bars but there was no growth at -30 bars even a f t e r 2 to 3 weeks (Table I I ) . Table I I . Radial growth r a t e o f Pythium sulcatum on KCl o s m o t i c a l l y amended basal media a t 34, 57 and 83 hours a f t e r i n o c u l a t i o n . Each measurement i s the mean o f f o u r o b s e r v a t i o n s Osmotic p o t e n t i a l Radial growth r a t e (mm/day) (-bars) 34 hours 57 hours 83 hours 1.2 1.01 .97 1.01 2.2 .99 .97 .98 3.2 .89 .90 .94 5.2 .86 .81 .78 7.2 .69 .72 .67 9.2 .63 .60 .57 11.2 .47 .45 .44 15.2 .27 .31 .34 19.2 .09 .10 .13 25.2 Trace .02 .02 30.2 . .00 .00 .00 P. ul timum, two i s o l a t e s of P_. s y l v a t i c u m and an i s o l a t e o f P_. sulcatum responded s i m i l a r l y to osmotic p o t e n t i a l s . Each e x h i b i t e d s t i m u l a t e d growth a t osmotic p o t e n t i a l s i n the range of -1.2 to -3.2 101 bars, over non-amended media ( F i g u r e 9 ) . Only the Wisconsin P_. sulcatum (65) i s o l a t e had d e c r e a s i n g growth r a t e with d e c r e a s i n g osmotic p o t e n t i a l in. the range o f -1.2 to -3.2 bars ( F i g u r e 9). The growth r a t e was 50% l e s s than on non-amended media a t osmotic p o t e n t i a l s o f approximately -11.5, -19.0 and -20.8 bars f o r P_. sulcatum, P_. s y l v a t i c u m , and P_. ultimum r e s p e c t i v e l y . However, m y c e l i a l d e n s i t y a t low p o t e n t i a l s appeared l e s s than a t high water p o t e n t i a l s . The growth r a t e as measured by colony diameter does not r e f l e c t changes i n d e n s i t y . Control o f S o i l Moisture using C e l l u l o s e D i a l y s i s Membranes The flow r a t e o f water acro s s a membrane i s d i r e c t l y r e l a t e d to the p o t e n t i a l energy g r a d i e n t and the t h i c k n e s s o f the membrane. P r e l i m i n a r y experiments i n d i c a t e d t h a t the 5 3/4 inch f l a t diameter (FD) membrane had a much slower flow r a t e than the 1 5/16 i n c h FD mem-brane a t the same pres s u r e g r a d i e n t , because o f i t s g r e a t e r t h i c k n e s s . The flow r a t e s o f water acro s s the 1 5/16 inch FD membrane determined a t v a r i o u s p r e s s u r e g r a d i e n t s are shown i n F i g u r e 10. For a pres s u r e d i f f e r e n t i a l o f 1.0 bar, the flow r a t e o f water acro s s the c e l l u l o s e -1 -2 membrane was 0.65 ml/day cm . U s u a l l y w i t h i n 5 to 10 days, the 1 5/16 in c h FD membrane had d e t e r i o r a t e d and was a l l o w i n g u n c o n t r o l l e d flow o f water i n t o the s o i l . A white and blue-green c o l o u r e d fungus was observed growing i n the s o i l tubes and i n a bag o f week o l d a u t o c l a v e d s o i l . T h i s fungus was i d e n -t i f i e d as Trichoderma sp., a common s o i l fungus t h a t i s a c t i v e i n f r e s h l y autoclaved s o i l . Several mucor l i k e f i n g i and b a c t e r i a were i s o l a t e d from the membranes, s o i l , and PEG s o l u t i o n . 1 1 r - r 1 r Osmotic po t e n t i a l (-bars) F i g u r e 9. Radial growth r a t e o f ultimum, two i s o l a t e s o f ?. s y l v a t i c u m and two i s o l a t e s o f P_. sulcatum on basal medium amended wi t h KCl. Each p o i n t i s the mean o f f o u r o b s e r v a t i o n s . o Flow rate (mlday'^cm"^) F i g u r e 10. Flow r a t e o f water across a 0.001 in c h t h i c k c e l l u l o s e d i a l y s i s membrane and a 500 NMwl P e l l i c o n u l t r a f i l t r a t i o n membrane at various p o t e n t i a l d i f f e r e n c e s . o CO 104 S o i l tubes, i n the glucose y e a s t b r o t h , i n o c u l a t e d with Pythium, Trichoderma and b a c t e r i a were s t i l l i n t a c t a f t e r nine days. The tubes i n o c u l a t e d with the mucors, were b r i t t l e and t o r e e a s i l y . In some ca s e s , the fungi had p i e r c e d and grown through the wall o f the membrane. The f e a s i b i l i t y o f using f u n g i c i d e s and a n t i b i o t i c s o f s l i g h t t o x i c i t y to Pythium s p e c i e s to p r o t e c t the membrane from c e l l u l o s e de-grading organisms was t e s t e d . Trichoderma was completely i n h i b i t e d by 10 ppm Benlate and p a r t i a l l y by 1000 ppm PCNB. The growth o f one mucor-l i k e i s o l a t e was 70% i n h i b i t e d by 1 to 1000 ppm Be n l a t e , and t h a t o f a second mucor i s o l a t e was completely i n h i b i t e d by 1 ppm PCNB and 80% i n h i b i t e d by 1000 ppm Benlate. The b a c t e r i a were o n l y s l i g h t l y i n h i b i t e d by 100 ppm Streptomycin. Pythium growth was s l i g h t l y i n h i b i t e d by 100 ppm Streptomycin, 80% i n h i b i t e d by 1000 ppm Benlate and not i n h i b i t e d by 100 ppm PCNB. 10 ppm Benlate had no observable e f f e c t on Pythium growth. This f i n d i n g was l a t e r used i n the m o d i f i c a t i o n o f the medium of Tsao and Ocana (1969). 10 ppm Benlate r e p l a c e d P i m a r i c i n when i t became u n a v a i l a b l e . The a d d i t i o n o f B e n l a t e , PCNB and Streptomycin i n c r e a s e d mem-brane l o n g e v i t y to 14-17 days. However, any han d l i n g o f the tubes a f t e r t h i s time caused them to f a l l a p a r t as they were very b r i t t l e . The use o f aged a u t o c l a v e d s o i l i n which Trichoderma had been growing pre-sented no advantage over f r e s h l y a u t o c l a v e d s o i l by i n c r e a s i n g the l i f e of the membranes. Car r o t s germinated and grew to a he i g h t o f about 2 inches i n s o i l tubes t h a t had been placed i n fungicide-PEG s o l u t i o n s . C a r r o t s i n the s o i l tubes immersed i n PEG s o l u t i o n s c o n t a i n i n g 105 Streptomycin died shortly after emergence. Some of the soi l tubes were so fragi le that the carrot root grew through the membrane. Control of Soil Moisture using Pell icon Membranes The flow rates of water through the Pell icon 500 NMWL membrane at various pressure gradients exceeded that of the cellulose membranes (Figure 1 0 ) . At a pressure di f ferent ial of 1 bar, the flow of water - 1 - 2 across the membrane was 4 . 3 5 ml day cm and even at a low pressure - 1 - 2 dif ferent ial of 0 . 2 bar, the flow rate was nearly 1 ml day cm . No PEG residue was detected in 100 ml of solution from the d i s t i l l e d water reservoir after 7 days of operation. The 1000 NMWL Pell icon membrane had s l ight ly greater flow rates but 0.081 g of PEG per 100 ml water was detected as having passed through the membrane after 7 days of operation. Therefore, the 500 NMWL membrane was used in a l l further experiments. Soil moisture controll ing prototypes. Preliminary experiments using the Pell icon membrane in the plumbing adaptor apparatus indicated that soi l moisture could be controlled for 4 to 6 weeks. The water con-tent of small quantities of so i l in the apparatus decreased as the os-motic potential of the solution decreased (Table I I I ) . . When a larger volume of soi l was used, and there was only sur-face evaporation, the soi l in the top of the chamber was drier than soi l near the membrane (Figure 1 1 ) . Water was not able to pass through the membrane and the 9 . 5 cm column of so i l fast enough to keep up with eva-porative demand, so the potential of the top so i l decreased. However, the potential of the lower layer of so i l remained constant (Figure 1 1 ) . Removed top 4 cm of s o i l Continuity l o s t i n upper tensiometer f o r f i r s t 8 days. Lower tensiometer Upper tensiometer 10 Membrane leaking 15 Time (days) 20 25 Figure 11. Soil water potential as measured with two tensiometers in the plumbing adaptor apparatus emersed in a -0.1 bar PEG solution. 107 The top 4 cm of soi l was removed to permit drying out of the lower soi l regions because the capi l lary conductivity of the soi l was so low in the upper region. After removing the s o i l , the water potential in the lower region of the chamber decreased from - .02 to - .125 bars in 5 days (a change of .02 bars per day) and remained constant for the next 6 days (Figure 11). Table I I I . The percent water content of soi l in the plumbing adaptor chamber emersed in PEG solutions of various osmotic potential Osmotic potential (-bars) 0.0 0.2 0.4 1.0 2.5 Water content (%) 63.3 51.9 34.1 25.1 15.3 The water potential in the lower region of the plumbing adaptor chambers remained relat ively constant for seven days after planting a radish seed. Thereafter, as the plant grew, the potential decreased slowly over an 18 day period at .023 and .025 bars per day for a -0.1 and -0 .2 bar osmotic solution respectively (Figure 12). The rate of potential decrease appeared to be slowly accelerating as the plants became older. F i g u r e 12. S o i l water p o t e n t i a l o f plumbing adaptor chamber, 5 membrane c u b i c a l chamber, and 2 membrane s o i l c e l l with a r a d i s h s e e d l i n g . Chambers were emersed i n -0.2 bar PEG. o co 109 On d i s m a n t l i n g the chamber, a dense mat of f i n e roots was found i n the f i r s t 0.5 cm of s o i l from the membrane. The water content o f the s o i l at d i f f e r e n t heights above the membrane are given i n Table IV. Table IV. Percent water content o f s o i l a t v a r i o u s heights above the membrane i n the plumbing adaptor chambers Height above membrane (cm) S o i l water content (%) of ' i n osmotic s o l u t i o n s -0.1 bar chambers -0.2 bar 0.0 to 0.5 62.3 74.2 0.5 to 1.5 44.3 55.4 1.5 to 3.0 46.6 52.3 3.0 to 4.5 44.3 49.3 4.5 to 6.0 36.1 39.1 6.0 to 9.5 - -Leaf area (cm ) 42 62 The s o i l chamber wit h f i v e membrane s u r f a c e s hada slow and steady decrease i n p o t e n t i a l o f 0.02 bars per day over 17 days a f t e r s e e d l i n g emergence ( F i g u r e 12). However, the two membrane chamber had o n l y a decrease o f 0.007 bar/day over the same 17 day p e r i o d ( F i g u r e 12). The r a d i s h s e e d l i n g s were approximately t r a n s p i r i n g 8 ml/day towards the end o f the 17 day p e r i o d . The water p o t e n t i a l o f the f i v e membrane chamber decreased more ' r a p i d l y d u r i n g the l a t e hours o f the l i g h t p e r i o d than during the n i g h t p e r i o d ( F i g u r e 13). In some cases the p o t e n t i a l even i n c r e a s e d during the n i g h t p e r i o d . S o i l water p o t e n t i a l (-bars) 4. JL I l l S o i l moisture c o n t r o l as measured g r a v i m e t r i c a l l y . Due to innumer-able problems o f measuring water p o t e n t i a l with tensiometers ( F i g u r e 11) and thermocouple psychrometers ( F i g u r e 14), an experiment was designed whereby moisture c o u l d be determined g r a v i m e t r i c a l l y f o r s e v e r a l weeks. However, i t was f i r s t necessary to determine the s o i l water r e t e n t i o n curve f o r the greenhouse s o i l ( F i g u r e 15) to enable c o n v e r s i o n o f s o i l water contents i n t o p o t e n t i a l s . The greenhouse s o i l was packed i n the _3 s o i l c e l l s to g i v e an average bulk d e n s i t y of 0.7 gmcm . The water content o f the s o i l s decreased as the osmotic poten-t i a l o f the s o l u t i o n decreased (Table V). The s o i l water content o f s o i l c e l l s i n the -0.2 bar s o l u t i o n remained c o n s t a n t a t 46% over the 3 week p e r i o d . A f t e r one week, s o i l c e l l s i n the -2 bar s o l u t i o n had a p o t e n t i a l o f -1.0 bar. The system had probably not reached e q u i l i b r i u m as the c a r r o t s had been "watered i n " at t r a n s p l a n t i n g . The water d i s t r i b u t i o n w i t h i n a s o i l c e l l a t 2 weeks shows t h a t the top l a y e r of s o i l f u r t h e s t from the membrane was the d r i e s t (Table V I ) . This was more n o t i c a b l e a t lower p o t e n t i a l s . The s o i l r e g i o n d i r e c t l y between the two membranes had the h i g h e s t water content and i t s p o t e n t i a l was the c l o s e s t to t h a t o f the osmotic s o l u t i o n . T r a n s p i r a t i o n , estimated by measuring the change i n l e v e l o f the PEG s o l u t i o n and c a l c u l a t i n g the change i n volume, i s given i n Table VII. A f t e r 3 weeks, t r a n s p i r a t i o n was g r e a t e r f o r c a r r o t s i n the -0.2 bar than the -2.0 bar PEG s o l u t i o n s . At the end o f the 3 week p e r i o d , 2 p l a n t s a t -0.2 bar had three t r u e leaves and a l e a f area o f 8.5 cm w h i l e 2 p l a n t s at 2 bar had one to two t r u e leaves and a l e a f area o f 3 cm . In t h i s experiment 75% o f the c e l l s were c o n t r o l l i n g s o i l moisture a f t e r two weeks but only 37.5% were o p e r a t i n g p r o p e r l y a f t e r 3 weeks. 5 10 15~ 20 25 30 Time (days) Figure 14. Soil water potential measured with two t r ip le junction two loop thermocouple psychrometers in the plumbing adaptor apparatus emersed in -1.2 bar PEG. 113 _0~ 20 30 40 50 60~~ 70 Percent water (g water/g oven dry s o i l x 100) Figure 15. Soil water retention curve for UBC greenhouse s o i l . Table V. Soil water content and potential of soi l cel ls containing carrots over a three week interval maintained at four osmotic potentials Soil water content {%) 1 of cel ls in osmotic solutions Weeks -0 .2 bar -0 .5 bar .1 .0 bar 2.0 bars 1 46.31 ( .40) 2 42.6 (.60) 38.3 (.98) 37.8 (1.0) 46.3 (.40) 43.6 (.54) 39.0 (.88) 37.8 (1.0) 2 48.4 (.33) _3 - 35.3 (1.35) 26.8 (3.6) 46.3 (.40) - - 39.5 (.85) 33.5 (1.7) 3 46.2 (.40) - - - 33.0 (1.8) 25.3 (4.2) Each measurement is an average of 3 soi l samples taken from the region between the two membranes. p Soil water potential (-bars). 3 Chambers developed leaks. 115 Table VI. S o i l water content at f o u r l o c a t i o n s w i t h i n s o i l c e l l s a f t e r two weeks o f o p e r a t i o n S o i l water content {%) of c e l l s i n osmotic s o l u t i o n s L o c a t i o n -0.2 bar -1.0 bar -2.0 bars 16.7 19.8 37.3 39.0 48.4 46.0 51.3 47.1 1 9.4 12.7 30.3 34.0 35.4 39.5 35.4 39.5 10.4 9.5 22.8 24.6 26.6 33.5 23.8 31.6 1 Average o f 3 determinations per l o c a t i o n . Table V I I . Average t r a n s p i r a t i o n o f two c a r r o t s grown i n s o i l c e l l s a t f o u r osmotic p o t e n t i a l s T r a n s p i r a t i o n (ml/day) i n osmotic s o l u t i o n s Time -0.2 bar -0.5 bar -1.0 bar -2.0 bars 3 to 7 1.6 0.8 1.2 1.2 8 to 10 3.2 1.9 1.9 3.8 11 to 15 11.3 - 3.3 5.6 16 to 18 13.6 - 8.0 4.8 19 to 21 7.8 - - 3.9 116 DISCUSSION P e r i o d i c S o i l S a t u r a t i o n Olpidium p o p u l a t i o n was a f f e c t e d by d i f f e r e n t watering regimes. D a i l y s a t u r a t i o n and s a t u r a t i o n f o r s h o r t periods o f time, induced g r e a t e r i n f e c t i o n l e v e l s than s p r i n k l e watering or s a t u r a t i o n f o r a prolonged p e r i o d o f time. These f i n d i n g s c o n f i r m f i e l d r e s u l t s o f 1972 which suggested t h a t Olpidium i n f e c t i o n was r e l a t e d to the number o f r a i n f a l l s or i r r i g a t i o n s t h a t s a t u r a t e d the s o i l (Wisbey, 1974). E a r l y p l a n t e d c a r r o t s which r e c e i v e d o n l y 1 or 2 heavy waterings had lower l e v e l s o f Olpidium than l a t e r p l a n t e d c a r r o t s t h a t had r e c e i v e d 3 o r 4 s o i l s a t u r a -t i o n s . 01pidiurn's only form o f spread i s by swimming zoospores and under experimental c o n d i t i o n s zoospore r e l e a s e i s s t i m u l a t e d by s a t u r a t i n g roots o f n e a r l y w i l t e d p l a n t s . Frequent p e r i o d i c s o i l s a t u r a t i o n pro-vided the best growing c o n d i t i o n s f o r c a r r o t s w h i l e s t i m u l a t i n g zoospore r e l e a s e . C a r r o t s grown under "the 4 days between s a t u r a t i o n " watering regimes were n e a r l y w i l t e d b e f o r e r e s a t u r a t i n g the s o i l . Under such s t r e s s growing c o n d i t i o n s , Olpidium may have been induced to form a g r e a t e r percentage o f r e s t i n g spores than zoosporangia. T h e r e f o r e , inoculum p o t e n t i a l was reduced even though s a t u r a t i o n of n e a r l y w i l t e d p l a n t s was i d e a l f o r zoospore r e l e a s e . This experiment has a l s o confirmed, c o n c l u s i o n s t h a t Olpidium has no d i r e c t e f f e c t on y i e l d (Wisbey, 1974). Watering regimes a f f e c t e d Olpidium i n c i d e n c e but not y i e l d , w h i l e i n the f i e l d , r a i s e d and conven-t i o n a l beds had no s i g n i f i c a n t e f f e c t on Olpidium i n c i d e n c e , but marketable y i e l d from r a i s e d beds was s i g n i f i c a n t l y g r e a t e r . 117 The d i f f e r e n t s o i l s a t u r a t i o n periods o f 10 minutes o r 8 hours, were dev i s e d to simulate c o n d i t i o n s o f high and low s a t u r a t e d h y d r a u l i c c o n d u c t i v i t y (HC) r e s p e c t i v e l y . A s o i l with a high HC d r a i n s q u i c k l y , so the r o o t system i s s a t u r a t e d f o r a s h o r t l e n g t h o f time, whereas, a s o i l with a low HC remains s a t u r a t e d a lo n g e r p e r i o d o f time. Olpidium i n f e c t i o n was s i g n i f i c a n t l y g r e a t e r (P = .05) under c o n d i t i o n s o f 10 minutes s a t u r a t i o n (high HC) than under 8 hours s a t u r a t i o n (low HC, Table V), but 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 c a r r o t y i e l d a t these two simulated HC's i n n a t u r a l l y - i n f e s t e d s o i l . In the 1972 f i e l d study, the HC was determined to be th r e e times g r e a t e r i n r a i s e d than c o n v e n t i o n a l beds, but Olpidium i n c i d e n c e was not s i g n i f i c a n t l y d i f f e r e n t . These f i e l d r e s u l t s are i n d i r e c t c o n t r a d i c t i o n to greenhouse r e s u l t s . A s o i l with a high HC would be expected to have l e s s 01 pi diurn i n f e c t i o n because zoospores would have l e s s chance to i n f e c t r o o t s before s o i l pore diameter r e s t r i c t e d t h e i r movement. As a s o i l d r a i n s , the l a r g e pores are the f i r s t to become a i r f i l l e d . Prolonged s a t u r a t i o n f o r 8 hours may not si m u l a t e a s o i l with a low HC, as the s a t u r a t i o n p e r i o d may have been too lon g . C o n c e n t r a t i o n s of 0 2 and C0 2 i n the s o i l may change c o n s i d e r a b l y under prolonged s a t u r a -t i o n , and these c o n c e n t r a t i o n changes may have had unknown e f f e c t s on zoospore i n f e c t i o n . No p r o v i s i o n was made i n t h i s s i m u l a t i o n to s l o w l y d r a i n the s o i l as would happen under n a t u r a l c o n d i t i o n s o f low HC. Olpidium was present i n a l l water treatments but TNV was r e -covered only i n the p e r i o d i c s a t u r a t i o n treatments. TNV i s c l o s e l y a s s o c i a t e d with the zoospore so i t i s d i f f i c u l t to e x p l a i n the reason f o r the apparent l a c k o f v i r u s i n c a r r o t s under the s p r i n k l e water regime. 118 C a r r o t s i n autoclaved s o i l responded to the v a r i o u s water r e -gimes, whereas c a r r o t s i n non-autoclaved s o i l showed no response to water. Maurer e_t al_. (1971) found t h a t c a r r o t s grown i n s o i l which was allowed to dry to v a r i o u s moisture l e v e l s b efore r e s a t u r a t i n g , showed a y i e l d response to water only when i n f e c t i o n with Pythium debaryanum (£_. s y l vaticum ?) was not a f a c t o r . In both s t u d i e s , c a r r o t y i e l d s were g r e a t e r under f r e q u e n t watering regimes. P_. s y l v a t i c u m was a poor c h o i c e of pathogen i n t h i s experiment f o r two reasons. F i r s t l y , t h i s s p e c i e s does not produce zoospores (Camp-b e l l and Hendrix, 1967) which are dependent upon water f o r spread. In pure c u l t u r e , terminal and i n t e r c e l a r y sporangia are the o n l y i n f e c t i o n s t r u c t u r e s . I n f e c t i o n takes place by germination of the sporangia to form a germ tube which i n f e c t s r o o t s . In s i m i l a r s t u d i e s , Biesbrock and Hendrix (1970b) found t h a t r o o t damage to peach by P. i r r e g u l a r e was not a f f e c t e d by excess water because i t i n f e c t e d peach r o o t s only by d i r e c t germination o f the s p o r a n g i a . However, damage induced by F\ vexans was most severe a t c o n d i t i o n s o f excess water and was r e l a t e d to the c a p a c i t y o f P_. vexans to produce zoospores. Secondly, a low i n c i d e n c e of P_. s y l v a t i c u m was observed because i t may not be the primary pathogenic Pythium s p e c i e s i n f e c t i n g muck grown c a r r o t s . P_. sulcatum i s now con-s i d e r e d the most pathogenic s p e c i e s on c a r r o t , and i t produces some zoo-spores ( P r a t t and M i t c h e l l , 1973). Had the l a t t e r been used i n the a u t o c l a v e d s o i l , the r e s u l t s may have been q u i t e d i f f e r e n t . 119 E f f e c t o f Osmotic P o t e n t i a l on Pythium Roots are known to exude a number o f compounds t h a t have an e f f e c t on the osmotic p o t e n t i a l o f the r h i z o s p h e r e . T h e r e f o r e , the c a p a b i l i t y o f Pythium spp. to i n f e c t and c o l o n i z e c a r r o t r o o t s may be r e l a t e d to i t s a b i l i t y to t o l e r a t e osmotic p o t e n t i a l s . Pythium growth a t d e c r e a s i n g osmotic p o t e n t i a l s was r e l a t e d more to water s t r e s s than s p e c i f i c ion t o x i c i t y . Radial growth r a t e s o f P_. s y l v a t i c u m and P_. sulcatum responded s i m i l a r l y to d i f f e r e n t s o l u t e s . Sommers ejt a]_. (1970) obtained s i m i l a r r e s u l t s with Phytophthora spp. P_. sulcatum, P_. s y l v a t i c u m , and P_. ul timum ceased growth a t -25, -31 and l e s s than -31 bars r e s p e c t i v e l y . According to the c l a s s i -f i c a t i o n o f Walter ( G r i f f i n , 1963a), Pythium would be c o n s i d e r e d a hygro-p h i l e ; minimum r e l a t i v e humidity f o r growth o f 95% or h i g h e r , and maximum growth a t about 100% R.H. On PDA, P_. sulcatum was i n a c t i v a t e d a t a much high e r p o t e n t i a l than on other media. T h i s r e l a t i v e l y r i c h media and basal medium amended with sucrose d i d not i n c r e a s e growth r a t e s as Sommers e t al_. (1970) found with Phytophthora p a r a s i t i c a . The e f f e c t o f n u t r i t i o n on the growth response curve a t v a r i o u s p o t e n t i a l s with d i f f e r e n t media suggest t h a t the e f f e c t o f water p o t e n t i a l should not be i n t e r p r e t e d s o l e l y on a water p o t e n t i a l b a s i s but t h a t the n u t r i e n t s t a t u s o f the s o i l or p l a n t may be important (Sommers e t a l _ . , 1970). Side d r e s s i n g s o f f e r t i l i z e r may a f f e c t the osmotic p o t e n t i a l and n u t r i e n t s t a t u s o f the r o o t zone, and thus a f f e c t Pythium i n f e c t i o n and c o l o n i z a t i o n o f r o o t l e t s growing i n t o the f e r t i l i z e r zone. 120 Pythium d i s e a s e s are a s s o c i a t e d with s o i l s o f high water conte n t , but the three Pythium sp e c i e s were growing we l l on o s m o t i c a l l y amended media at -15 bars, a p o t e n t i a l which i s c o n s i d e r e d to be the PWP of most p l a n t s . Gardner (1968) has hypothesized t h a t the water p o t e n t i a l i n the r h i z o s p h e r e i s c o n s i d e r a b l y l e s s than i n the s o i l mass a d i s t a n c e from r o o t s . In a s a t u r a t e d s o i l there i s probably only a small p o t e n t i a l g r a d i e n t near the r o o t . I f the s o i l i s at a p o t e n t i a l o f -1 bar, there i s probably a p o t e n t i a l g r a d i e n t o f 1 bar near the r o o t so the p o t e n t i a l at the s o i l - r o o t i n t e r f a c e would be -2.0 bar. But a t the PWP (-15 b a r s ) , the p o t e n t i a l g r a d i e n t between the s o i l and the r o o t s u r f a c e w i l l probably be 100 bars (Gardner, 1968). A p o t e n t i a l of -115 bars a t the r o o t s u r f a c e i s p r o h i b i t i v e to Pythium growth. In o r d e r to c o l o n i z e a r o o t , Pythium spp. must be capable of growing at lower water p o t e n t i a l s than are u s u a l l y a s s o c i a t e d with s o i l s because the r o o t s u r f a c e - s o i l i n t e r f a c e i s at a lower p o t e n t i a l . Average s o i l water p o t e n t i a l s determined i n f i e l d p l o t s i n 1972 i n the 0-3 cm, 4-8 cm and 9-15 cm o f s o i l o f r a i s e d and conven-t i o n a l beds were -0.4 to -2.0, -0.02 to -0.8, and -0.02 to -0.2 bars r e s p e c t i v e l y ( c a l c u l a t e d from s o i l water r e t e n t i o n curve, F i g u r e 3, R u s s e l l , 1972). M y c e l i a l growth and i n f e c t i o n o f c a r r o t r o o t l e t s was probably not r e s t r i c t e d a t these p o t e n t i a l s . The s t i m u l a t i o n e f f e c t o f osmotic p o t e n t i a l s i n the range o f -2 to -5 bars, was observed with a l l Pythium i s o l a t e s t e s t e d , except one i s o l a t e of P_. sulcatum. T h i s i n c r e a s e d growth r a t e may be due to an i n c r e a s e d energy requirement by c e l l s to r e t a i n s o l u t e s i n very d i l u t e media ( S c o t t , 1957). Another p o s s i b i l i t y i s t h a t an ion d e f i c i e n c y i n 121 d i l u t e media may r e s t r i c t the f u n c t i o n of some enzymes, r e s u l t i n g i n a slower growth r a t e (Manandhar and B r u e h l , 1973). Manandhar and Bruehl (1973) have suggested t h a t the s t i m u l a t i o n e f f e c t o f lower water poten-t i a l s i s not due to decreased water per se. Adebayo and H a r r i s (1971) compared the e f f e c t s o f m a t r i c and osmotic p o t e n t i a l on fungal growth. They found t h a t growth was not s t i m u l a t e d as the m a t r i c p o t e n t i a l decreased from -1 bar, and t h a t growth was n ot n e a r l y as g r e a t a t low m a t r i c p o t e n t i a l s . The m a t r i c p o t e n t i a l a t which growth e x t i n c t i o n occured was one h a l f to two t h i r d s o f the corresponding osmotic p o t e n t i a l . Fungal response to d e c r e a s i n g water cannot be e x p l a i n e d s o l e l y on an energy b a s i s but i n c l u d e s o t h e r changes i n water s o i l p r o p e r t i e s such as s o l u t e t r a n s p o r t . T h e i r f i n d i n g s empha-s i z e a need f o r both m a t r i c and osmotic p o t e n t i a l s t u d i e s on Pythium growth. C o n t r o l l e d M a t r i c P o t e n t i a l In d e s i g n i n g a s o i l moisture c o n t r o l l i n g apparatus f o r a growing p l a n t an important c o n s i d e r a t i o n i s the r a t i o o f the volume o f s o i l t o the membrane s u r f a c e area. For a cons t a n t membrane s u r f a c e a r e a , a design with a l a r g e volume o f s o i l (e.g. tube) w i l l be s u b j e c t to g r e a t e r water p o t e n t i a l g r a d i e n t s than a design with a s m a l l e r volume o f s o i l (e.g. narrow s o i l c e l l ) . The 1 5/16 i n c h FD d i a l y s i s tube works we l l as a tube because i t has a l a r g e s u r f a c e area to s o i l volume. The 5 3/4 inch FD diameter d i a l y s i s tube would not work we l l as a tube because the r a t i o o f s o i l volume to membrane s u r f a c e area i s very l a r g e . S o i l chambers 122 had to be c o n s t r u c t e d by f i t t i n g the membrane over a frame so as to l i m i t s o i l volume and decrease the r a t i o o f s o i l volume to s u r f a c e a r e a . However, the chambers of t h i s design were s u b j e c t to s e v e r a l problems. I t was im p o s s i b l e to get a permanent, water t i g h t seal with rubber or s i l i c o n e cement when a thermocouple psychrometer was f i t t e d through the membrane and the wall o f the frame. Pressure had to be main-t a i n e d a g a i n s t the frame and membrane s u r f a c e a t a l l times, otherwise the membrane would bulge and the s o i l would move. This was not a problem f o r B a b a l o l a e t al_. (1968) because they put the membrane around a de-veloped r o o t system t h a t h e l d the s o i l i n p l a c e . The narrow diameter s o i l tubes avoided the above problems, were easy to c o n s t r u c t , had a r e l a t i v e l y l a r g e s u r f a c e area to volume and were made o f a t h i n n e r mem-brane t h a t had a g r e a t e r flow r a t e . One o f the problems with the t h i n n e r membrane s o i l tubes was th a t the membranes were degraded by microorganisms w i t h i n two weeks. Mucor-like fungi were capable o f degrading the c e l l u l o s e membrane i n l i q u i d c u l t u r e a f t e r only nine days. T r i b e (1966) st u d y i n g the i n t e r -a c t i o n o f s o i l f i n g i on c e l l u l o s e f i l m i n the s o i l , found t h a t Trichoderma was c e l l u l o l y t i c and t h a t Pythium was not. The a v a i l a b i l i t y o f n u t r i e n t s may be an important f a c t o r i n the c e l l u l o l y t i c a b i l i t y shown by f u n g i . Under c o n d i t i o n s o f high n u t r i t i o n such as Glucose Yeast b r o t h , f u n g i may not produce c e l l u l o s e degrading enzymes. Membrane l o n g e v i t y was not i n c r e a s e d by f u n g i c i d e s o r aged auto-c l a v e d s o i l i n which Trichoderma had been growing. Trichoderma i s one of the f i r s t organisms to r e c o l o n i z e a u t o c l a v e d s o i l ; i t b u i l d s up r a p i d l y , 123 and produces an a n t i b i o t i c , i n h i b i t o r y to many other s o i l microorganisms ( A g r i o s , 1969). P r e l i m i n a r y experiments i n d i c a t e d t h a t the P e l l i c o n 500 NMWL membrane had the f o l l o w i n g d e s i r a b l e c h a r a c t e r i s t i c s : 1) i t r e s i s t e d m i c r o b i a l breakdown f o r 4 to 6 weeks, 2) i t had a flow r a t e s u f f i c i e n t to meet the water requirements o f a c a r r o t f o r the f i r s t f o u r weeks a f t e r emergence, 3) the design o f the chamber t h e r e f o r e d i d not r e q u i r e as much conducting s u r f a c e as p r e v i o u s l y needed i n c e l l u l o s e models (Cox and Boersma, 1966). The working hypothesis was t h a t a p l a n t i n such a system would send i t s roots down to the membrane and e x t r a c t most of i t s water a t the membrane s u r f a c e . As a r e s u l t , the water p o t e n t i a l o f the whole s o i l system would be r e l a t i v e l y constant throughout. However, when a p l a n t was grown i n the chamber, the water p o t e n t i a l s l o w l y decreased a f t e r germination o f the p l a n t u n t i l i t c o u l d no lon g e r be monitored with the tensiometers. There was a high c o n c e n t r a t i o n o f roots next to the membrane and the water content o f the s o i l i n the 0.5 to 4.5 cm r e g i o n v a r i e d s l i g h t l y . As the d i s t a n c e from the membrane i n c r e a s e d , the s o i l became d r i e r . T h i s f a c t s t r o n g l y suggested t h a t the flow r a t e o f water through the membrane was not the l i m i t i n g f a c t o r , but r a t h e r t h a t the c a p i l l a r y c o n d u c t i v i t y o f water through the s o i l was not gre a t enough to maintain a constant water p o t e n t i a l o f the whole s o i l mass. A change i n s o i l c e l l design confirmed t h a t the c a p i l l a r y con-d u c t i v i t y o f the s o i l was the l i m i t i n g f a c t o r , as a narrow s o i l c e l l , with two membrane conducting s u r f a c e s maintained a constant water p o t e n t i a l , 124 while a c u b i c a l chamber with f i v e membrane s u r f a c e s had a de c r e a s i n g p o t e n t i a l as s e e d l i n g s grew. The narrow s o i l c e l l design decreased the volume o f s o i l , w h i l e i n c r e a s i n g the membrane s u r f a c e area and the c u b i c a l chamber, only i n c r e a s e d the membrane s u r f a c e area, w h i l e the s o i l volume remained n e a r l y the same as with the plumbing adaptor chamber. The c e n t r e o f the narrow s o i l c e l l was o n l y 0.7 cm from the membrane compared to 2.2 cm to the c e n t r e o f the c u b i c a l chamber. S i n c e water moves from one p o i n t to another along an energy g r a d i e n t , a l a r g e r energy g r a d i e n t would be r e q u i r e d f o r water to move t h i s g r e a t e r d i s t a n c e . The g r e a t e r v a r i a b i l i t y o f water p o t e n t i a l observed i n the c u b i c a l cham-ber compared to the narrow s o i l c e l l was not unexpected from t h e o r e t i c a l c o n s i d e r a t i o n s . The measurement o f water p o t e n t i a l by i n s t r u m e n t a t i o n f o r extended periods o f time i n the 0-.5 bar to -2 bar range i s s u b j e c t to many prob-lems. Tensiometers draw a i r r e l a t i v e l y r a p i d l y a t te n s i o n s g r e a t e r than -0.4 bars. When a i r i s removed from the system, the pr e s s u r e i s r e l e a s e d and water passes from the tensiometer i n t o the s o i l because the s o i l i s a t a lower p o t e n t i a l . I t takes s e v e r a l days f o r the system to r e t u r n t o i t s o r i g i n a l p o t e n t i a l , by which time the tensiometers again r e q u i r e b l e e d i n g . On s e v e r a l o c c a s i o n s g r e a t d i f f i c u l t y was ex-perien c e d i n g e t t i n g the tensiometers to operate a f t e r b l e e d i n g . I f an a i r bubble was trapped i n the tensiometer cup i n s t e a d o f r i s i n g to the h i g h e s t p o i n t i n the tube, the tensiometer d r i e d out. I t was d i f f i -c u l t to s a t u r a t e the porous cup and r e e s t a b l i s h c o n t i n u i t y so t e n s i o n s could be again measured. In one case, where c o n t i n u i t y was l o s t and 125 r e e s t a b l i s h e d ( F i g u r e 11), s o i l p o t e n t i a l readings were s t i l l q u e s t i o n -a b l e . The t e n s i o n readings i n d i c a t e d a s o i l p o t e n t i a l o f -0.225 bars but a g r a v i m e t r i c d e t e r m i n a t i o n i n d i c a t e d a t e n s i o n of -0.6 bars. The t r i p l e j u n c t i o n two loop thermocouple psychrometer, used to measure p o t e n t i a l s l e s s than -1 bar, a l s o posed problems. Measure-ments were probably only a c c u r a t e w i t h i n 0.5 bars as the thermocouple output fluctuated q u i t e widely from one reading to the next ( F i g u r e 14). Immediately a f t e r s t a r t i n g the fan to i n c r e a s e s o i l s u r f a c e e v a p o r a t i o n , measurements o f water p o t e n t i a l i n the top r e g i o n o f the pots decreased r a p i d l y and much more than was expected i n such a s h o r t time i n t e r v a l . G r a v i m e t r i c d e t e r m i n a t i o n of s o i l water content i n d i c a t e d t h a t psychrometer readings were i n f a c t much too low. The psychrometer reading was l e s s than -15 bars but g r a v i m e t r i c d e t e r m i n a t i o n suggested a p o t e n t i a l o f -2 bars. While d i s m a n t l i n g the s o i l chambers, a white s a l t d e p o s i t was found around some o f the porous bulbs. A few of the psychrometers had v i s i b l e c o r r o s i o n a t the copper-constantan, copper-chromel j u n c t i o n s . C a l i b r a t i o n curves a f t e r the s i x week experiment were much lower than curves b e f o r e the experiment. I n t e r p r e t a t i o n o f r e s u l t s using the thermo-couple psychrometer to measure water p o t e n t i a l s o f s o i l maintained a t osmotic p o t e n t i a l s o f l e s s than -1 bar were t h e r e f o r e i m p o s s i b l e because of these unforseen problems. G r a v i m e t r i c d e t e r m i n a t i o n o f the s o i l water p o t e n t i a l e v e n t u a l l y had to be used because of the problems i n measurement with the a v a i l a b l e instruments. T his i n d i r e c t method of determining water p o t e n t i a l has the advantage o f high accuracy, but the disadvantage t h a t i t i s a 126 d e s t r u c t i v e method. The water p o t e n t i a l s o f s o i l c e l l s i n -0.2 bar osmotic s o l u t i o n were constant at -0.4 bars at each of the three weeks. The water p o t e n t i a l o f the s o i l c e l l s i n -2.0 bars was more v a r i a b l e and p o t e n t i a l s ranged from -1.7 to -4.2 bars i n the second and t h i r d weeks o f the experiment. This technique o f using osmotic s o l u t i o n s to c o n t r o l the s o i l m a t r i c p o t e n t i a l , permitted c o n t r o l a t much lower water p o t e n t i a l s than are p o s s i b l e using other techniques. To show t h a t t h i s system c o u l d keep up with the water demands of young p l a n t s , the f o l l o w i n g c a l c u l a t i o n s were done. The s o i l c e l l s 3 3 had a s o i l volume o f 57.5 cm , and a t a bulk d e n s i t y o f 0.7 g/cm , they would c o n t a i n 40 g o f oven dry s o i l . I f no water was e n t e r i n g the system, and a p l a n t was t r a n s p i r i n g 7.8 ml/day, i t would take o n l y one day f o r the s o i l water p o t e n t i a l to decrease from -0.2 bar to -2.0 bars and l e s s than a f u r t h e r h a l f day f o r the s o i l to have reached a p o t e n t i a l of the permanent w i l t i n g p o i n t . S i n c e the c a r r o t s were growing we l l and the water content o f the s o i l remained c o n s t a n t , water must have been f l o w i n g from the PEG s o l u t i o n i n t o the s o i l . T h e r e f o r e , t h i s system i s able to maintain the water requirement o f a young a c t i v e l y t r a n s -p i r i n g p l a n t . T r a n s p i r a t i o n decreased as the osmotic p o t e n t i a l o f the PEG s o l u -t i o n decreased. P l a n t s a t -2.0 bars t r a n s p i r e d about h a l f as much water as p l a n t s a t -0.2 bars. P l a n t growth a f t e r 3 weeks was l e s s a t -2.0 bars than -0.2 bars. I t appeared t h a t the lower water p o t e n t i a l was p l a c i n g a s t r e s s on the p l a n t s which r e s u l t e d i n decreased growth. Rawlins et_ al_. (1968) found t h a t t r a n s p i r a t i o n was u n a f f e c t e d by s o i l 127 water content u n t i l s o i l p o t e n t i a l s were l e s s than -6 to -8 bars. A s h o r t p e r i o d o f low s o i l p o t e n t i a l before i r r i g a t i n g the s o i l to s a t u r a -t i o n had l i t t l e e f f e c t on t r a n s p i r a t i o n (Rawlins et_ a l _ . , 1968). The l i m i t i n g f a c t o r i n t h i s technique f o r c o n t r o l l i n g s o i l mois-ture i s the c a p i l l a r y c o n d u c t i v i t y o f the s o i l . T h i s was p a r t i c u l a r l y n o t i c a b l e i n t h e upper l a y e r s o f the narrow s o i l c e l l s a t low water poten-t i a l s . In c e l l s a t -2 bars, the s o i l d i r e c t l y between the membranes was c l o s e r to the p o t e n t i a l o f the s o l u t i o n than the s o i l above or below the membranes. As the p o t e n t i a l o f a s o i l decreased, the c a p i l l a r y c o n d u c t i v i t y r a p i d l y decreased (e.g. the c o n d u c t i v i t y o f a s a t u r a t e d , -3 -5 -1 bar and -15 bar s o i l i s i n the o r d e r o f 1, 10 and 10 cm/day r e s p e c t i v e l y (Gardner, 1968)), so t h a t water movement a t lower poten-t i a l s i s very l i m i t e d . I f t h i s technique i s to be s u c c e s s f u l l y used at low p o t e n t i a l s , the d i s t a n c e between membranes should not be any g r e a t e r than 1.4 cm as used i n t h i s experiment. At high p o t e n t i a l s such as -0.2 bars, the c e l l width c o u l d be s l i g h t l y i n c r e a s e d without causing a decrease i n s o i l p o t e n t i a l . A high percentage o f s o i l c e l l s , a f t e r 3 weeks, had developed l e a k s . A d i f f e r e n t l o t o f membranes was used i n t h i s experiment than previous experiments. S i n c e these membranes were s t i l l i n the e x p e r i -mental stages o f development at the time o f use, the q u a l i t y may not have been r e p r o d u c i b l e from one l o t to the next. In a d d i t i o n , the mem-branes were cemented to the p l e x i g l a s s with Devcon rubber cement, a d i f f e r e n t brand o f rubber cement than used p r e v i o u s l y . T h i s change was n e c e s s i t a t e d by a temporary removal o f Dexon rubber cement from the 128 market, pending r e l i c e n s i n g . S o i l c e l l s t h a t were n e g l e c t e d c o n t r o l l e d s o i l moisture l o n g e r than c e l l s t h a t were weekly washed and placed i n f r e s h PEG s o l u t i o n s . Chambers were washed with a s q u i r t o f water from a p l a s t i c wash b o t t l e to remove s l i m e t h a t b u i l t up on the chamber s u r -f a c e . The f o r c e o f the spray d i d not seem e x c e s s i v e enough to damage the membrane. I f the problems observed with the 500 NMWL P e l l i c o n membrane can be overcome or i f 1000 NMWL membranes and PEG 20,000 are used i n p l a c e o f the 500 NMWL membranes and PEG 6000, t h i s technique w i l l be i d e a l f o r c o n t r o l l i n g s o i l moisture w i t h i n a narrow range f o r a growing p l a n t . The technique i s now at a stage where the organisms a s s o c i a t e d with PRD can be added to the system, and d i s e a s e development a t va r i o u s water p o t e n t i a l s can be observed. This technique i s a l s o i d e a l for. s t u d y i n g the e f f e c t o f s o i l moisture on spore germination and behavior a t poten-t i a l s much lower than i s p o s s i b l e with o t h e r t e c h n i q u e s . CONCLUSIONS 1. A greenhouse p e r i o d i c , s o i l - s a t u r a t i o n experiment confirmed f i e l d o b s e r v a t i o n s t h a t the frequency o f s o i l s a t u r a t i o n i s d i r e c t l y c o r r e -l a t e d with Olpidium i n c i d e n c e . 2. Pythium s p e c i e s ceased growth on o s m o t i c a l l y amended media a t about -30 bars, w h i l e a t -15 bars they g e n e r a l l y had a growth r a t e o f gr e a t e r than 50% t h a t on non-amended media. T h e r e f o r e s o i l water content per se may not be the l i m i t i n g f a c t o r i n Pythium i n f e c t i o n . 129 3. A method o f c o n t r o l l i n g s o i l water m a t r i c p o t e n t i a l of a growing p l a n t f o r w i t h i n narrow l i m i t s f o r a t l e a s t t hree weeks was developed. LITERATURE CITED Adebayo, A. A. and R. F. H a r r i s . 1971. Fungal growth responses to os-motic as compared to m a t r i c water p o t e n t i a l . S o i l S c i . Soc. Amer. 35:465-469. A g r i o s , G. N. 1969. P l a n t pathology. Academic P r e s s , New York. 624 pp. B a b a l o l a , 0., L. Boersma, and C. T. Youngberg. 1968. Photosynthesis and t r a n s p i r a t i o n of Monterey pine s e e d l i n g s as a f u n c t i o n of s o i l water s u c t i o n and s o i l temperature. P l a n t P h y s i o l . 43:515-521. Ba i n b r i d g e , A. 1970. S p o r u l a t i o n by Pythium ultimum a t v a r i o u s s o i l moisture t e n s i o n s . Trans. B r i t . Mycol. Soc. 55:485-488. Bateman, D. F. 1961. The e f f e c t of s o i l moisture upon development of p o i n s e t t i a r o o t r o t s . Phytopathology 51:445-451. Biesb r o c k , J . A. and F. F. Hendrix. 1970a. I n f l u e n c e o f continuous and p e r i o d i c s o i l water c o n d i t i o n s on r o o t n e c r o s i s o f h o l l y caused by Pythium spp. Can. J . Bot. 48:1641-1645. and . 1970b. I n f l u e n c e o f s o i l water and temperature on r o o t n e c r o s i s of peach caused by Pythium spp. Phytopathology 60:880-882. Campbell, W. A. and F. F. Hendrix. 1967. A new h e t e r o t h a l l i e Pythium from southern United S t a t e s . Mycologia 59:274-278. Campbell, G. S., W. D. Z o o l i n g e r and S. A. T a y l o r . 1966. Sample changer f o r thermocouple psychrometers: c o n s t r u c t i o n and some a p p l i c a t i o n s . Agronomy J . 58:315-318. Chow, T. L. and J . DeVries. 1973. Dynamic measurement o f s o i l and l e a f water p o t e n t i a l with a double loop p e l t i e r type thermocouple psychrometer. S o i l S c i . Soc. Amer. Proc. 37:181-188. Copeman, R. J . and T. A. Black. 1972. E f f e c t o f the microenvironment on l a t e r a l r o o t dieback of c a r r o t under f i e l d c o n d i t i o n s . Rep. o f the Comm. on Hort. Res. 1971, Can. Hort. C o u n c i l , p. 176. Couch, H. B., L. H. Purdy and D. W. Henderson. 1967. A p p l i c a t i o n o f s o i l moisture p r i n c i p l e s to the study of p l a n t d i s e a s e s . B u l l . 4. Res. Div. V i r g i n i a P o l y t e c h n i c I n s t i t u t e , 23 pp. 130 131 Cox, L. M. and L. Boersma. 1967. T r a n s p i r a t i o n as a f u n c t i o n o f s o i l temperature and s o i l water s t r e s s . P l a n t P h y s i o l . 42:550-556. Gardner, W. R. 1968. A v a i l a b i l i t y and measurement o f s o i l water, pp. 107-135. In T. T. Kozlowski (ed.) Water d e f i c i t s and p l a n t growth. V o l . I. Academic Press, New York. Gardner, D. E. and F. F. 1973. Carbon d i o x i d e and oxygen c o n c e n t r a t i o n s i n r e l a t i o n to s u r v i v a l and s a p r o p h y t i c growth o f Pythium i r r e g u l a r e and Pythium vexans i n s o i l . Can. J . Bot. 51:1593-1598. G r i f f i n , D. M. 1963a. S o i l moisture and the ecology of s o i l f u n g i . B i o l . Rev. 38:141-166. . 1963b. S o i l p h y s i c a l f a c t o r s and the ecology o f f u n g i . I I . Behavior o f Pythium ultimum a t small s o i l water s u c t i o n s . Trans. B r i t . Mycol. Soc. 46:368-372. Haines, W. B. 1930. Stu d i e s i n the p h y s i c a l p r o p e r t i e s of s o i l . V. The h y s t e r e s i s e f f e c t i n c a p i l l a r y p r o p e r t i e s and the mode o f mois-ture d i s t r i b u t i o n a s s o c i a t e d t h e r e w i t h . J . Agr. S c i . 20:97-117. Hendickson, A. H. and F. J . Veihmeyer. 1941. Moisture d i s t r i b u t i o n i n s o i l i n c o n t a i n e r s . P l a n t P h y s i o l . 16:821-826. Kerr, A. 1964. The i n f l u e n c e o f s o i l moisture o f i n f e c t i o n o f peas by Pythium ultimum. A u s t r a l i a n J . B i o l . S c i . 17:676-685. K r a f t , J . M. and D. D. Roberts. 1969. I n f l u e n c e s o f s o i l water and temperature on the pea r o o t r o t complex caused by Pythium ultimum and Fusarium s o l a n i f . s p . pi s i . Phytopathology 59:149-152. Manandhar, J . B. and G. W. B r u e h l . 1973. In v i t r o i n t e r a c t i o n s o f Fusarium and V e r t i c i l l i u m w i l t fungi with water, pH and temperature. Phytopathology 63:413-419. Maurer, A. R., J . F. Conroy and T. K. Watson. 1971. The response o f muck grown c a r r o t s to fumiga t i o n and s o i l water s t r e s s . H ortScience 6:42-43. P h i l l i p s , J . M. and D. S. Hayman. 1970. Improved procedures f o r c l e a r i n g r o ots and s t a i n i n g p a r a s i t i c and v e s i c u l a r a r b u s c u l a r m y c o r r h i z a l fungi f o r r a p i d assessment o f i n f e c t i o n . Trans. B r i t . Mycol. Soc. 55:158-161. P r a t t , R. G. and J . E. M i t c h e l l . 1973. A new s p e c i e s o f Pythium from Wisconsin and F l o r i d a i s o l a t e d from c a r r o t s . Can. J . Bot. 51: 333-339. 132 Rawlins, S. L., W. R. Gardner and F. N. Dalton. 1968. In s i tu measure-ment of soi l and plant leaf water potential . Soil Sc i . Soc. Amer. J . 32:468-470. Richards, L. A. 1947. Pressure membrane apparatus - construction and use. Agr. Eng. 28:451-454. Robinson, R. A. and R. H. Stokes. 1955. Electolyte solutions. Academic Press,New York. 571 pp. Roth, L. F. and A. J . Riker. 1943. Influence of temperature, moisture and soi l reactions in the damping off of red pine seedlings by Pythium and Rhizoctonia. J . Agr. Res. 67:273-293. Russell , D. W. 1972. The effects of so i l temperature and soi l water on Pythium sp. induced lateral root dieback of carrots. Bachelor thesis, University of Br i t ish Columbia, 25 pp. Scott, W. J . 1957. Water relations of food spoilage microorganisms. Advance in Food Res. 7:83-127. Sommers, L. E., R. F. Harris and F. N. Dalton. 1970. Water relations in three root- infecting Phytophthora species. Phytopathology 60: 932-934. Stanghell ini , M. E. and T. J . Burr. 1973. Effect of so i l water potential on disease incidence and oospore germination of Pythium aphani- dermatum. Phytopathology 63:1496-1498. Stolzy, L. H., J . Letey, L. J . Klotz and C. K. Labanauskas. 1965. Water and aeration as factors in root decay of Citrus sinensis. Phyto-pathology 55:270-275. Teakle, D. S. 1962. Necrotic symptoms of TNV in roots. Phytopathology 52:1037-1040. Tribe, H. T. 1966. Interactions of soi l fungi on cellulose f i lm . Trans. B r i t . Mycol. Soc. 49:457-466. Tsao, P. H. and G. Ocana. 1969. Selective isolat ion of species of Phytophthora from natural soi ls on an improved antibiot ic medium. Nature 223:636-638. Tuite, J . 1969. Plant pathological methods, fungi and bacteria. Bur-gess Pub. Co., Minn. 239 pp. Williams, J . and C. F. Shaykewich. 1969. An evaluation of Polyethylene Glycol (PEG) 6000 and PEG 20,000 in the osmotic control of so i l water matric potential. Can. J . Plant S c i . 43:397=-401. 133 Wisbey, B. D. 1974. The epidemiology and control of Pythium root die-back of muck-grown carrots. Chapter I. Master's thesis, University of Br i t ish Columbia. Zur, B. 1966. Osmotic control of the matric soi l water potential . I. Soil water system. Soil Sc i . 102:394-398. 

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