Science, Faculty of
Botany, Department of
DSpace
UBCV
Pentland, Gertrude Draper
Doctor of Philosophy - PhD
University of British Columbia
In the first of two parts of the investigation, the stimulation of Armiil aria mellea (Fr.) Quél., a common root-rotting fungus, by Aureobastdium pullulans (DeBary) Arnaud was studied. A. pullulans is an ubiquitous imperfect fungus, inhabiting soil and other substrates. It was shown to produce in culture a volatile, heat stable, neutral substance which stimulated the mycelial development, rhizomorph initiation and rhizomorph elongation of A. mellea. A. pullulans is the only microorganism reported so far to stimulate the growth of A. mellea. The effect of ethanol on the growth of A. mellea was similar to the effect of a cell-free filtrate from a liquid culture of A. pullulans. The cell-free filtrate was shown by gas chromatography to contain ethanol. Ethanol supplied daily as 50 ppm in a glucose-asparagine medium resulted in a ten-fold increase in the number of rhizomorphs produced by A. mellea. The effects of 3-indolylacetic acid, ɣ-(indole-3)-n-butyric acid, the sodium salt of 2,4-dichlorophenoxyacetic acid and tryptophane on the growth of A. mellea were tested, but the stimulatory effect of A. pullulans was not reproduced by them. One rhizomorph tip of A. mellea could develop an extensive rhizomorph system in autoclaved soil if the stimulatory substance produced by A. pullulans was available. In the second part of the investigation the effect of soil moisture and the related effect of some soil-inhabiting Fungi imperfecti on the spread through soil of the root-rotting Basidiomycete Coniopnora puteana (Schum. ex Fr.) Karst.were studied. A soil moisture level of 20 - 25% saturation was satisfactory for the growth of C. puteana in non-sterile soil. At this moisture level C. puteana was able to grow out from a small alder disc inoculum in the centre of a petri dish and invade alder discs at the periphery of the dish. In wetter conditions (50% saturation and higher) it was unable to grow out into the soil. In autoclaved soil the cptimum moisture level for the growth of C. puteana was between 75% and 100% saturation. Small amounts of non-sterile soil were added to autoclaved soil at different moisture levels, with an effect on the growth of C. puteanas similar to that of completely non-sterile soil. Trichoderma viride Pers. ex Fr., a known antibiotic producer, was inoculated in autoclaved soil and produced a greater inhibition of C. puteana in the wetter treatments than in drier ones. The inhibitory effect of Acti-dione (cycloheximide), an antifungal antibiotic active in the pH range 3-5, was examined in non-sterile soil end in autoclaved soil. The same concentration of antibiotic resulted in a greater inhibition of the growth of C. puteana at the higher moisture levels than at the lower ones.
The University of British Columbia FACULTY COT GEADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY . of GERTRUDE.DRAPER PENTLAND B.A., The University of British Columbia, 1956 M.A., The University of British Columbia, 1959 THURSDAY, NOVEMBER 10, 1966 AT 3:30 P.M.-IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: I. McT. Cowan R. J . Bandoni T. M. C. Taylor J . E. Bier G. H. N. Towers C. A. Rowles E. B. Tregunna J . A. F. Gardner External Examiner: L . F. Roth Department of Botany and Plant Pathology Oregon State University . Corvallis, Oregon Research Supervisor: J . E. Bier SOME IWTERRELA,HONS OF ROOT~ROTTING BASIDIOMYCETES AND SOIL INHABITING FUNGI IMPERPECTI ABSTRACT In the f irs t of two parts of the investigation,, the stimulation of Armillaria mellea (Fr.) Quelog.a common root-rotting funguss by Aureobasidium. pullulans (DeBary) Arnaud was studied* A. pullulans is an ubiquitous imperfect fungus, inhabiting soil and other substrates„ It was shown to produce in culture a volatile^ heat stable,, neutral substance which stimulated the mycelial development., rhizomorph initiation and rhizomorph elongation of A<= mellea, h° PuHaLans is the only microorganism reported so far to stimulate the growth of A* mellea. The effect of ethanol on the growth of A„ mellea was similar to the effect of a cell=free fi ltrate from a l iquid culture of A, pullulans, The ce l l~£ree f i ltrate was shown by gas chromatography to contain ethanolo Ethanol supplied daily as 50 ppm in a glucose~asparagine medium resulted in a ten~fold increase in the number of rhizomorphs produced by Ao mellea. The effects of 3=indolylacetic acid s Tf-(indole-3) =n~butyric acid,, the sodium salt of 2?4=dichlorophen~ oxyacetic acid and tryptophane on the growth of A„ mellea were tested, but the stimulatory effect of A. pullulans was not reproduced by them. One^rhizomorph tip of A. mellea could develop an extensive rhizomorph system in autoclaved soil i f the stimulatory substance produced by A. pullulans was available. In the second part of the investigation the effect of soil moisture and the related effect of some soil-inhabiting Fungi Imperfecti on the spread through soil of the root-rotting Basidiomycete Coniophora puteana (Schum. ex Fr . ) Karst. were studied. A s o i l moisture l e v e l of 20 =• 25$ saturation was .satis-f a c t o r y f o r the growth of Co puteana i n non=sterile s o i l . A t t h i s moisture l e v e l Co_ puteana was able to grow out from a small alder d i s c inoculum i n the centre of a p e t r i d i s h and invade alder discs at the periphery of the d i s h . In wetter conditions (50$ saturation and higher) i t was unable to grow, out i n t o .the .soil o In autoclaved s o i l the optimum moisture l e v e l f o r the growth of C„ puteana was between 75$ and 100$ saturation. Small amounts of non=sterile s o i l were added to auto~ claved s o i l a t d i f f e r e n t moisture l e v e l s , with an e f f e c t on the growth of puteana s i m i l a r to that of completely n o n ~ s t e r i l e s o i l o Tichoderma v i r i d e Pers„ ex F r . , a known a n t i b i o t i c producer, was inoculated i n autoclaved s o i l and produced a greater i n h i b i t i o n of Co puteana i n the wetter treatments. than i n d r i e r ones. The i n h i b i t o r y e f f e c t of Acti=dione (cycloheximide), an antifungal anti= b i o t i c a c t i v e i n the pH range 3 = 5 ? was examined i n non-s t e r i l e s o i l and i n autoclaved s o i l o The same concentration of a n t i b i o t i c r e s u l t e d i n a greater i n h i b i t i o n of the growth °£ £° puteana a t the higher moisture l e v e l s than a t the lower ones,, GRADUATE STUDIES Field of Study: • Advanced Forest Pathology Advanced Mycology Related Studies: Plant Biochemistry Soil - Plant Relationships Physiology of Crop Plants ; . - ^ ^ ^ j ^ • PUBLICATIONS Bier, J.. E. and G„ D„ Pentland, Agar Plug Inocula Affect Accuracy of Cultural Tests of Inhibi-tion of Fungi by Chemicals. Forest Products Journal 14 ( 6 ) : 254-255 , 196k. Pentland, Gertrude D„, Stimulation of Rhizomorph Development of Armillaria mellea by Aureobasidium pullulans in Art i f i c ia l Culture. Canadian Journal of Microbiology 11: 345-350 , 1965. J . E. Bier R. J . Bandoni G. H. N. Towers C. A. Rowles D. P. Ormrod SOME INTERRELATIONS OF ROOT-ROTTING BASIDIOMYCETES AND SOIL-INHABITING FUNGI IMPERFECTI fay GERTRUDE DRAPER PENTLAND B.A., U n i v e r s i t y of B r i t i s h Columbia, 1956 M.A., U n i v e r s i t y of B r i t i s h Columbia, 1959 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Botany We accept t h i s t h e s i s as conforming t o the requ i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1966 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 t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t 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 n o t 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 . The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D e p a r t m e n t o f D a t e ^P^r^^^u /t>. /9S^. ft ABSTRACT In the f i r s t o f two p a r t s of the i n v e s t i g a t i o n , the s t i m u l a t i o n o f Armi11 a r i a m e l l e a ( F r . ) Q u e l . , a common r o o t - r o t t i n g fungus, by Aureobas td -ium p u l l u l a n s (DeBary) Arnaud was s t u d i e d . A. p u l l u l a n s i s an ub iqu i tous imperfect fungus , i n h a b i t i n g s o i l and o ther s u b s t r a t e s . It was shown t o produce in c u l t u r e a v o l a t i l e , heat s t a b l e , n e u t r a l substance which s t i m -u l a t e d the m y c e l i a l development, rhizomorph i n i t i a t i o n and rhizomorph e l o n g a t i o n of A. m e l l e a . A . p u l l u l a n s i s the on ly microorganism repor ted so f a r t o s t i m u l a t e the growth of A. m e l l e a . The e f f e c t o f ethanol on the growth of A . m e l l e a was s i m i l a r t o the e f f e c t of a c e l l - f r e e f i l t r a t e from a l i q u i d c u l t u r e o f A. p u l l u l a n s . The c e l l - f r e e f i l t r a t e was shown by gas chromatography t o c o n t a i n e t h a n o l . Ethanol s u p p l i e d d a i l y as 50 ppm in a g l u c o s e - a s p a r a g i n e medium r e s u l t e d i n a t e n - f o l d i n c r e a s e i n the number o f rhizomorphs produced by A . m e l l e a . The e f f e c t s o f 3 - i n d o l y l a c e t i c a c i d , y - ( i n d o l e - 3 ) - n - b u t y r i c a c i d , the sodium s a l t o f 2 , 4 - d f c h l o r o p h e n o x y a c e t i c a c i d and t ryptophane on the growth of A. m e l l e a were t e s t e d , but the s t i m u l a t o r y e f f e c t o f A. p u l l u l a n s was not reproduced by them. One rhizomorph t i p o f A. m e l l e a c o u l d develop an e x t e n s i v e rhizomorph system i n autoc laved s o i l i f t he s t i m u l a t o r y substance produced by A. p u l - l u l a n s was a v a i l a b l e . In the second par t o f the i n v e s t i g a t i o n the e f f e c t o f s o i l mo is tu re and the r e l a t e d e f f e c t of some s o i l - i n h a b i t i n g Fungi Imperfect ! on the spread through s o i l o f the r o o t - r o t t i n g Bas id iomycete Coniopnora puteana (Schum. ex F r . ) K a r s t . w e r e s t u d i e d . A s o i l mo is tu re l e v e l o f 20 - 25% s a t u r a t i o n was s a t i s f a c t o r y f o r t h e growth o f C. puteana in n o n - s t e r i l e in s o i l . At t h i s mo is tu re l e v e l C. puteana was a b l e t o grow out from a smal l e l d s r d i s c inoculum in the c e n t r e of a p e t r i d i s h and invade a l d e r d i s c s at the per iphery of the d i s h . In wet ter c o n d i t i o n s (50% s a t u r a t i o n and h igher ) i t was unable t o grow out i n t o the s o i l . In autoc laved s o i l the cptimum mois tu re l e v e l f o r the growth of C. puteana was between 75% and 100% s a t u r a t i o n . Small amounts o f n o n - s t e r i l e s o i l were added t o autoc laved s o i l at d i f f e r e n t mo is tu re l e v e l s , w i t h an e f f e c t on the growth of C. puteana s im?lar t o tha t o f comple te ly n o n - s t e r i l e s o i l . Tr ichoderma v i r i d e P e r s . ex F r . , a knoun .antibiot ic p roducer , was i n o c u l a t e d in autoc laved s o i l and produced a g rea te r i n h i b i t i o n o f puteana in the wet te r t reatments than in d r i e r ones. The i n h i b i t o r y e f f e c t o f A c t i - d i o n e (eye lohex imide ) , an a n t i f u n g a l a n t i b i o t i c a c t i v e in the pH range 3-5, was examined in n o n - s t e r i l e s o i l end in autoc laved s o i l . The same c o n c e n t r a t i o n of a n t i b i o t i c r e s u l t e d in a g r e a t e r i n h i b i t i o n o f the growth of C. puteana at the h igher mo is tu re l e v e l s than at the lower ones. i v TABLE OF CONTENTS Psge Par t I. S t i m u l a t i o n o f Rhizomorph Development o f A r m ! 1 l a r i a mellea by Aureobasidiurn p u l l u l a n s INTRODUCTION . . 1 LITERATURE REVIEW . 3 Factors A f f e c t i n g the Growth o f A. mel l e a in C u l t u r e . . . . . . . . . 3 Ni t rogen Source 3 Carbon Source k Carbon /N i t rogen R a t i o L' V i tamins 4 Hydrogen Ion Concent ra t i on 5 Temperature 5 L i g h t . . 5 S h o r t - C h a i n A l c o h o l s 5 Other Factors 6 M e t a b o l i t e s Produced by A, p u l l u l V i t a m i n Requirements o f A. pul l u l a n s 7 I n te rac t ions o f A. m e l l e a and A. p u l l u l a n s . . . . . . . . . . . . . . . . . . . G MATERIALS AND METHODS 3 EXPERIMENTS AND RESULTS ........ O r i g i n o f Fungus I s o l a t e s Media and C u l t u r e Techniques S Determinat ion of the General E f f e c t of the S t i m u l a t o r y Sub-s tance . . . . . . • • . • • • • . . • • . • • • . • • • * • • . . . . • . • . . . . . . . . . . . . . * . « 9 . 11 P h y s i c a l and Chemical P r o p e r t i e s o f the S t i m u l a t o r y Substance. 2 3 Experiments w i t h Ethanol i n L i q u i d C u l t u r e s 37 Tests f o r P lan t Growth Factors as the S t i m u l a t o r y Substsnce . . k-3 E f f e c t on I n h i b i t i o n by Triehoderma v i r i d e ^5 St imulat ion o f A. m e l l e a on Autoc1aved Soi1 . . . . . . . . . . . . . . . . . . k$ Experiments w i t h Natura l Subst ra tes DISCUSSION . . . 5 2 CONCLUSIONS 57 BIBLIOGRAPHY £3 V TABLE OF CONTENTS, c o n t ' d . Page Par t I I . The E f f e c t of S o l i M o i s t u r e on the Growth and Spread of Coniophora puteena (Schum. ex F r . ) K a r s t . Under L a b o r a t -ory Cond i t i ons INTRODUCTION 62 LITERATURE REVIEW 63 S o i l M o i s t u r e and S o i l Fungi 63 S o i l F u n g i s t s s i s 65 Spread of Wood - ro t t ing Basid iomycetes in S o i l 66 MATERIALS AND METHODS 67 EXPERIMENTS AND RESULTS 68 S o i l pH 68 N o n - s t e r i l e and Autoc laved S o i l 68 Wood Traps in N o n - s t e r i l e S o i l . . ' . . " 71 N o n - s t e r i l e S o i l Added t o Autoc laved S o i l 78 Tr ichodsrma v i r l r i e Inoculated in Autoc laved S o i l 80 A c t i - d i o n e in Autoc laved S o i l 83 DISCUSS5CN 87 BIBLIOGRAPHY 91 v i LIST OF TABLES Page Pa r t I Tab le I. E f f e c t of A. p u l l u l a n s , ethanol and increased n i t r o -gen on the development o f rhizomorphs by A. m e l l e a a f t e r three, weeks (mean and range for. f i v e p e t r i p l a t e s ) 2i: Tab le I I . E f f e c t of amount o f ethanol on dry weight o f A. mel l e a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . « 39 Tab le I I I . E f f e c t o f a low c o n c e n t r a t i o n o f ethanol s u p p l i e d d a i l y on the growth o f A. m e l l e a {\U days o l d ) k\ Tab le IV. E f f e c t o f IAA on the development o f rhizomorphs o f A. m e l l e a a f t e r t h r e e weeks (mean and range f o r f i v e p e t r i p l a t e s ) 44 Tab le V . E f f e c t of A. p u l l u l a n s and IAA together on the development o f rhizomorphs o f A . m e l l e a a f t e r two weeks (mean and range o f four p e t r i p l a t e s ) U$ Tab le V I . E f f e c t o f A. p u l l u l a n s on rhizomorph development o f A. mel l e a in autoc laved s o i l k7 Tab le V l l . Weight l o s s (%) o f D o u g l a s - f i r sapwood i n o c u l a t e d w i t h A. m e l l e a and A. p u l l u l a n s ( a f t e r k 1/2 months ) . . 50 Tab le VIII . Weight l o s s (%) o f v i n e maple sawdust by A . m e l l e a and A . pul l u l a n s ••• 51 Par t II Tab le I. Growth o f G. puteana in f o r e s t s o i l a f t e r t h r e e < . W6&|{S « i e « o « « « « 0 « 6 0 » * « « e * o « o o o e » o e e * « « » e « « « o * « « e * o « « o * 7^ v i i LIST OF FIGURES Page Pa r t I F i g u r e I, Growth o f A. m e l l e a on 5% malt agar in p e t r i p l a t e s a f t e r 10 days (viewed from be low) . L e f t , a lone ; r i g h t , w i t h A. p u l l u l a n s on same p l a t e . . . . . . . . . . . . . . 12 F i g u r e 2. Growth o f A . m e l l e a on 5% malt agar in K o l l e c u l t u r e f l a s k s a f t e r 26 days . L e f t , a l o n e ; r i g h t , w i t h A. pul l u l a n s in same f l a s k 13 F i g u r e 3. Growth o f A. m e l l e a on pota to dext rose agar in K o l l e c u l t u r e f l a s k s a f t e r 26 days . L e f t , a l o n e ; r i g h t , w i t h A. p u l l u l a n s in same f l a s k . . . . . . . . . . . . . . 14 F i g u r e 4 . Growth o f A. m e l l e a on 1% malt agar in K o l l e c u l t u r e f l a s k s a f t e r 26 days . L e f t , a l o n e ; r i g h t , w i t h A . pul l u l a n s in same f l a s k 15 F i g u r e 5. Growth o f A. me l l ea on W medium i n p e t r i p l a t e s a f t e r 22 days . L e f t , a l o n e ; r i g h t , w i t h A . p u l l u l a n s on same p l a t e 16 F i g u r e 6 . Growth o f A. me l l ea on 2W medium in p e t r i p l a t e s a f t e r 22 days . L e f t , a l o n e ; r i g h t , w i t h A . p u l l u l a n s on same p l a t e 16 F i g u r e 7. The r e l a t i o n s h i p between the number o f rhizomorphs produced by A. me l l ea and the amount o f c e l l - f r e e f i l t r a t e in the medium ( a f t e r V'. days growth) 19 F i g u r e 3. The r e l a t i o n s h i p between the length o f rhizomorphs produced by A . m e l l e a and the amount o f c e l l - f r e e f i l t r a t e in the medium ( a f t e r \k days growth) 20 F i g u r e 9. The growth o f A. mel l e a a f t e r 1** days on media c o n t a i n i n g increased amounts o f c e l l - f r e e f i l t r a t e (viewed from be low) . C = 2W c o n t r o l . Numbers show per cent of c e l l - f r e e f i l t r a t e in medium 21 F i g u r e 10. Growth o f A . m e l l e a from rhizomorph t i p inoculum a f t e r \k days . L e f t , on 2V/ aga r ; r i g h t , on 2W agar w i t h 50% c e l l - f r e e f i l t r a t e 22 F i g u r e 11. Growth o f m e l l e a on W agar a f t e r 22 days in p e t r i p l a t e s . Top l e f t , a l o n e ; top r i g h t , w i t h 500 ppm o f ethanol in medium; bottom l e f t , w i t h A . p u l l u l a n s ; bottom r i g h t , w i t h A . p u l l u l a n s and 500 ppm o f ethanol In medium 25 v i i i LIST OF FIGURES, c o n t ' d . Page Pa r t I. c o n t ' d . F i g u r e 12. Growth o f A . me l l ea on 2W agar a f t e r 22 days In p e t r i p l a t e s . Top l e f t , a l o n e ; top r i g h t , w i t h 500 ppm of ethanol i n medium; bottom l e f t , w i t h A . p u l l u l a n s ; bottom r i g h t j w i t h A . p u l l u l a n s and 500 ppm o f ethanol in medium 26 F i g u r e 13. Growth o f A . m e l l e a on 2W+N agar a f t e r 22 days in p e t r i p l a t e s . Top l e f t , a l o n e ; top r i g h t , w i t h 500 ppm o f ethanol in medium; bottom l e f t , w i t h A . p u l l u l a n s ; bottom r i g h t , w i t h A. p u l l u l r n s and 500 ppm o f ethanol in medium . . . . . . . . . . . . . . . . . . . . . . . . 27 F i g u r e 14. Growth o f A. m e l l e a on 2W agar in t w o - s e c t i o n p e t r i p l a t e s a f t e r 16 days . L e f t , a l o n e ; r i g h t , w i t h A. pul l u l a n s in second s e c t i o n 29 F i g u r e 15. Growth o f A. m e l l e a on 2W agar in p e t r i p l a t e s a f t e r 12 days (viewed from be low) . L e f t , a l o n e ; r i g h t , A . m e l l e a separated from A. p u l l u l a n s by i s o l a t i o n t rench 7 • 30 F i g u r e 16. The e f f e c t of ion exchange r e s i n s on the dry weight produced by A. m e l l e a in 2W medium, 2W medium w i t h e t h a n o l , and a c e l l - f r e e f i l t r a t e o f A . p u l l u l a n s l i q u i d c u l t u r e . . . . . . . . . . . . 0 . . . . * . 33 F i g u r e 17. Growth o f A. m e l l e a on 2W medium a f t e r 14 days . L e f t , c o n t r o l ; c e n t r e , medium passed through anion exchange r e s i n ; r i g h t , medium passed through c a t i o n exchange r e s i n 34 F i g u r e 18. Growth o f A. m e l l e a a f t e r 14 days on 2W medium c o n -t a i n i n g e t h a n o l . L e f t , c o n t r o l ( c o n t a i n i n g 1160 ppm o f e t h a n o l ) ; c e n t r e , medium passed through anion exchange r e s i n ( c o n t a i n i n g 1100 ppm of e t h a n o l ) ; r i g h t , medium passed through c a t i o n e x -change r e s i n ( c o n t a i n i n g 800 ppm of e thano l ) 35 F i g u r e 19. Growth o f A. m e l l e a a f t e r 14 days on a c e l l - f r e e f i l t r a t e o f A. p u l l u l a n s l i q u i d c u l t u r e . L e f t , c o n t r o l ( c o n t a i n i n g 1200 ppm of e t h a n o l ) ; c e n t r e , medium passed through anion exchange r e s i n (con -t a i n i n g 1350 ppm of e t h a n o l ) ; r i g h t , medium passed through c a t i o n exchange r e s i n ( c o n t a i n i n g 850 ppm of e thano l ) 36 i x LIST OF FIGURES, c o n t ' d . Page Pa r t I. c o n t ' d . F i g u r e 20. Growth of A. me l lea a f t e r 14 days on media t r e a t e d as f o l l o w s ( l e f t t o r i g h t ) : 2W c o n t r o l , 2W medium changed d a i l y , 2W medium w i t h 50 ppm of e t h a n o l , 2W medium w i t h 50 ppm of ethanol changed d a i l y , 2W medium w i t h 700 ppm of ethanol 42 F i g u r e 21, Growth of A. me l l ea from rhizomorph t i p inoculum on autoc laved s o i l in two -sect ion p e t r i p l a t e s . L e f t , w i t h s t e r i l e 2W agar in second s e c t i o n ; r i g h t , w i t h A. p u l l u l a n s growing on 2W agar in second s e c t i o n 48 Pa r t 81 F i g u r e I. Growth of C_. puteana a f t e r th ree weeks in non -s t e r i l e s o i l w i t h a mo is tu re content o f 20% o f s a t u r a t i o n 69 F i g u r e 2. Growth o f C_. puteana a f t e r th ree weeks in non -s t e r i l e s o i l w i t h a mo is tu re content of 40% of s a t u r a t i o n 70 F i g u r e 3. Growth of C_. puteana a f t e r t h r e e weeks in non -s t e r i l e s o i l w i t h a mo is tu re content of 70% o f s a t u r a t i o n . Note i nvas ion of inoculum by T. v i r i d e 73 F i g u r e 4. Growth of C_. puteana a f t e r t h r e e weeks in non -s t e r i l e s o i l w i t h a mo is tu re content of 100% o f s a t u r a t i o n . Note i nvas ion of inoculum by T. v i r i d e 74 F i g u r e 5. Growth of C. puteana a f t e r two weeks in autoc laved s o i l . The f i g u r e s show the s o i l mo is tu re content as a per cent o f s a t u r a t i o n 75 F i g u r e 6. The i nvas ion of a l d e r d i s c t raps by C. puteana a f t e r 26 days in n o n - s t e r i l e s o i l w i t h a mo is tu re content of 25% o f s a t u r a t i o n 76 X LIST OF FIGURES, c o n t ' d . Par t I I , c o n t ' d . Page F i g u r e 7. The Invasion of a l d e r d i s c t raps by C. puteana a f t e r f i v e weeks in n o n - s t e r i l e s o i l w i t h a mo is tu re content of 25% o f s a t u r a t i o n . Note m y c e l i a l s t rands of C. puteana 77 F i g u r e 8 . Recovery on malt agar c o n t a i n i n g 5% agar of C. puteana from a l d e r d i s c t r a p s . Mycelium from d i s c in upper l e f t s e c t i o n of p l a t e is C. puteana 79 F i g u r e 9 . Growth of C. puteana a f t e r two weeks In p e t r i p l a t e s w i t h one gram of n o n - s t e r i l e s o i l added t o 24 grams pf autoc laved s o i l . The f i g u r e s show t h e s o i l mo is tu re content as a per cent of s a t u r a t i o n . Note the f r u i t i n g s t r u c t u r e s of T. v i r i d e in the wet te r p l a t e s 81 F i g u r e 10. Growth of C. puteana a f t e r two weeks in autoc laved s o i l i nocu la ted w i t h T. v i r i d e . The s o i l mo is tu re content i s : top l e f t , 25%; top r i g h t , 50%; bottom l e f t , 75% and bottom r i g h t , 100% of s a t u r a t i o n 82 F i g u r e 11. The mycelium of C_. puteana invading area c o l o n i z e d by !• v i r i d e on malt agar c o n t a i n i n g 2% agar and 3% malt e x t r a c t . (C. puteana has been growing f o r 6 1/2 weeks, T. v i r ide f o r f i v e weeks) 84 F i g u r e 12. Growth o f C. puteana a f t e r two weeks in n o n - s t e r i l e s o i l c o n t a i n i n g 100 jig A c t l - d i o n e per gram o f s o i l . The f i g u r e s show the s o i l mo is tu re content as a per cent of s a t u r a t i o n 85 F igu re 13. Growth o f C. puteana a f t e r two weeks in autoc laved s o i l c o n t a i n i n g 100 ug A c t i - d i o n e per gram o f s o i l . The f i g u r e s show the s o i l mo is tu re content as a per cent of s a t u r a t i o n 86 xt ACKNOWLEDGEMENTS T h i s i n v e s t i g a t i o n was c a r r i e d out w i t h the support o f a Nat iona l Research Counc i l S tudentsh ip in 1964-65 and 1965-66. The author expresses her a p p r e c i a t i o n t o Dr. J . E . B i e r , P r o f e s s o r o f Forest Patho logy , f o r f r i e n d l y guidance and encouragement throughout the s t u d y . Thanks a re a l s o extended t o Dr. E .B . Tregunna, A s s i s t a n t P r o f e s s o r o f Botany, f o r generous a s s i s t a n c e w i t h the p h y s i o l o g i c a l a s -p e c t s ; t o Dr. R . J . Bandoni , A s s o c i a t e P r o f e s s o r o f Botany, and Dr. E .H . Gardner, A s s o c i a t e P r o f e s s o r of S o i l S c i e n c e , f o r h e l p f u l a d v i c e ; and t o Miss M. Byrne, Research A s s o c i a t e i n Forest Pa tho logy , f o r her a s s i s t a n c e , p a r t i c u l a r l y in the p r e p a r a t i o n o f the manuscr ip t . 1 P a r t I. S t i m u l a t i o n of Rhizomorph Development of A r m t l l a r l a mellea by Aureobasidium p u l l u l a n s INTRODUCTION The o b s e r v a t i o n that a c u l t u r e of A r m ! l i a r i a mellea (Fr.) Quel, con-taminated by Aureobasidium p u l l u l a n s (deBary) Arnaud ( c P u l l u l a r i a p u l l u l a n s (deflary) Berkhout) grew unusually w e l l , i n i t i a t e d the i n v e s t i g a t i o n des-c r i b e d here. The p o s s i b i l i t y that A. p u l l u l a n s i n some way favourably i n -fluenced the growth of A. mellea seemed s i g n i f i c a n t because of the import-ance of A. mellea and the widespread d i s t r i b u t i o n of both f u n g i concerned. Both A. mellea and A. p u l l u l a n s are common fungi which occur through-out the world (8,34). A. melleg Is a r o o t - r o t t i n g Basidfmycete which at t a c k s woody p l a n t s i n both temperate and t r o p i c a l r e g i o n s . Raabe (34) has compiled a host l i s t of about 600 species Infected by t t . Because host spe c i e s and i n d i v i d u a l s w i t h i n a species vary i n t h e i r s u s c e p t i b i l i t y t o a t t a c k (13,32,35) there has been c o n s i d e r a b l e controversy over the r o l e of the fungus as a primary pathogen. Patton and R i k e r (31) reported that It attacked and k i l l e d apparently h e a l t h y , v i g o r o u s l y growing red p i n e s , w i t h no i n d i c a t i o n that other agencies weakened or predisposed the trees t o i n f e c t i o n . On the other hand Day (14) says " a l l the evidence goes t o show that i t [A. m e l l e a l i s always secondary t o some other f a c t o r a c t i n g as the primary cause of d i s e a s e " . Baranyay and Stevenson (3) suggest that i t was the suppressed lodgepole pine t r e e s which were attacked i n the area they s t u d i e d , and conclude that stand d e n s i t y i s an important f a c t o r p r e d i s p o s i n g trees t o s e r i o u s a t t a c k . 2 A. m e l l e a spreads by means o f rh izomorphs, which a re o rgan ised v e g e t a t i v e s t r u c t u r e s growing as a u n i t from a m e r l s t e m - l I k e apex (20). G a r r e t t (19) repor ted that on agar rhizomorphs can grow 5"6 t imes as r a p i d l y as unorganized mycel ium. On root segments under f a v o u r a b l e c o n d i t i o n s they can grow approx imate ly 50 cm per month, accord ing t o the c a l c u l a t i o n s o f Sokolov (38). Rhizomorphs can p e n e t r a t e t h e i n t a c t bark o f r oo ts (13,41) and develop in the cambial r e g i o n . A t r e e may be k i l l e d q u i c k l y w i t h g i r d l i n g in t h e cambium by rhizomorphs and m y c e l i a l f a n s . The fungus c a n , however, develop s l o w l y as a root r o t , in which case the t r e e i s k i l l e d s l o w l y but becomes s u s c e p t i b l e t o wind - th row and a t t a c k by o ther o rgan isms. Because o f the widespread occur rence of the o rgan ism, the c o n t r o v e r s y over i t s p a t h o g e n i c i t y , and i t s occur rence as an important pathogen in p l a n t a t i o n s (15,25,31) knowledge of f a c t o r s a f f e c t i n g i t s o c c u r r e n c e , spread and development a re important in f o r e s t pa tho logy . Because r h i z o -morphs a re r e l a t i v e l y s imp le o rgan i zed s t r u c t u r e s , f a c t o r s a f f e c t i n g t h e i r g rowth , d i f f e r e n t i a t i o n and o r g a n i z a t i o n may opera te at a r e l a t i v e l y s i m p l e l e v e l and a re o f i n t e r e s t at a b a s i c b i o l o g i c a l l e v e l . Thus in fo rmat ion on f a c t o r s a f f e c t i n g the growth o f A. m e l l e a rhizomorphs i s important from both a p p l i e d and b a s i c p o i n t s o f v i ew . Cooke, in a recent comprehensive rev iew o f the l i t e r a t u r e on A. p u l l u l a n s (9), has c a l l e d i t " a common u b i q u i t o u s , omnivorous, and h i g h l y po lymorphic s p e c i e s o f f u n g u s " . He d i s c u s s e s the h a b i t a t s in which It o c c u r s , i n c l u d i n g s o i l and p a r t s of l i v i n g host p l a n t s . Thus i t may occur in a s s o c i a t i o n w i t h A . m e l l e a . A means o f spread Is by wind-borne spores (9), one of which i n i t i a t e d the i n v e s t i g a t i o n repor ted here when It c o n -taminated a c u l t u r e o f A. m e l l e a . 3 Because A . m e l l e a and A . p u l l u l a n s are both common fung i which may occur together in the same h a b i t a t s , i t seemed important to i n v e s t i g a t e the means by which A. p u l l u l a n s s t i m u l a t e d the growth o f the pathogen, A. m e l l e a . LITERATURE REVIEW F a c t o r s A f f e c t i n g the Growth of A. m e l l e a in C u l t u r e I II !!• !•! I II Mil Ill Will 11 I !• f • • I •! I I II 111 •! I II i l l |1T7 I " P "" " I I H _ » l l l l mBM I • III • l» • III Ni t rogen Source A number of authors have s t u d i e d the e f f e c t o f n i t r o g e n source on the growth o f A. m e l l e a in c u l t u r e . G a r r e t t (17) and Azevedo (2) concluded that n i t r a t e s a re the poorest s o u r c e s , a l though some i s o l a t e s are a b l e t o make l i m i t e d use o f them. Reitsma (36) agreed , l i s t i n g the n i t r o g e n sources he s t u d i e d in d e c r e a s i n g o rder of a v a i l a b i l i t y as f o l l o w s : peptone, asparag!n<* g l y c o c o l l , ammonium t a r t r a t e , ammonium s u l f a t e , ammonium c h l o r i d e and p o t a s -s ium n i t r a t e . In a recent study of amino a c i d s and o ther n i t rogenous compounds as n i t r o g e n sources f o r A. m e l l e a . Weinhold and Garroway (47) repor ted that no rhizomorphs were produced on any of the substances t e s t e d in the absence of sma l l q u a n t i t i e s o f e t h a n o l . When ethanol was s u p p l i e d the r e s u l t s were as f o l l o w s : 1. very s a t i s f a c t o r y sources of n i t r o g e n - c a s e i n h y d r o l y s a t e , L - a l a n i n e , L - a s p a r a g t n e , DL -g lu tamine , L - a s p a r t i c a c i d and L - g l u t a m i c a c i d ; 2. moderately s a t i s f a c t o r y sources - L - s e r i n e , D L - l e u c l n e , ammonium phosphate and L - a r g i n l n e ; 3. u n s a t i s f a c t o r y sources - g l y c i n e , D L - p h e n y l a l a n i n e , DL -meth lon ine , D L - l y s i n e , DL - t ryp tophan , D L - v a l l n e , D L - I s o l e u c i n e , DL-t h r e o n i n e , L - h l s t l d i n e , L - p r o l i n e , L -hyd roxypro l Ine and potass ium n i t r a t e . 4 Carbon Source Rettsma (36) l i s t e d the f o l l o w i n g substances in d e c r e a s i n g o rder of the s u i t a b i l i t y as a carbon source f o r A. m e l l e a : g l u c o s e , saccharose ( s u c r o s e ) , m a l t o s e , amylum, l a c t o s e , g a l a c t o s e and c e l l u l o s e . Weinhold (45) and Weinhold and Garroway (47) reported that ethanol was an e x c e l l e n t source of carbon f o r A . m e l l e a , w i t h a c e t a t e be ing much l e s s s a t i s f a c t o r y . There was e s s e n t i a l l y no growth w i t h g l u c o s e , f r u c t o s e o r suc rose as the s o l e carbon s o u r c e . However, A. m e l l e a grew on a i l t h r e e sugars when ethanol was added as a supplement, but growth was poor on f r u c t o s e and s u c r o s e . Carbon /N i t rogen R a t i o Hamad a (24) s t u d i e d the r e a c t i o n s of n i ne s t r a i n s o f A,, mel l e a , i n -c l u d i n g luminescence, browning o f the s u b s t r a t e , g u t t a t ion from a e r i a l mycel ium, c o l o u r of the f l u i d of g u t t a t i o n , and fo rmat ion o f c a l c i u m o x a l a t e c r y s t a l s . He found t h a t these r e a c t i o n s were dependent on the C/N r a t i o o f the medium (except browning of the s u b s t r a t e , which r e s u l t e d from the o x i d a t i o n of peptone and v a r i e d on l y w i t h n i t r o g e n c o n c e n t r a t i o n ) . G a r r e t t (17) repor ted a very h i g h l y s i g n i f i c a n t i n t e r a c t i o n between carbohydrate and n i t r o g e n in t h e i r e f f e c t on the number of rhizomorph i n i t i a l s and t h e i r subsequent growth. V i tamins A. m e l l e a appears t o r e q u i r e th iamine s u p p l i e d in the medium but i s a u t o t r o p h i c f o r the o the r v i t a m i n s . G a r r e t t (17) s t a t e s that th iamine is e s s e n t i a l f o r growth on a s y n t h e t i c medium, but b i o t i n i s not e s s e n t i a l , o r even b e n e f i c i a l , f o r growth. Jennison e t a l . . (26) repor ted s i m i l a r l y t ha t A . m e l l e a c o u l d not s u b s t i t u t e b i o t i n f o r t h i a m i n e . Azevedo (2) found that the a d d i t i o n of th iamine g r e a t l y increased the growth o f the i s o l a t e s she 5 s t u d i e d . Hydrogen ion C o n c e n t r a t i o n Numerous authors (4,26,?6,42) have found the optimum pH f o r growth o f A. m e l l e a under c u l t u r a l c o n d i t i o n s t o be between pH 5-6. Rhizomorphs were formed over the range pH 3.6-8.0 and m y c e l i a l growth occur red over a s l i g h t l y wider range (42). Temperature Growth o f A . m e l l e a occurs at temperatures from 5*C t o 35*C, but Is s low at both extremes (5,22,36,38), An optimum temperature between 20°C and 25*C was repor ted In a l l the i n v e s t i g a t i o n s . L i g h t Townsend (42) repor ted that t h e r e was no d i f f e r e n c e in the number o f rhizomorphs produced in c o l o n i e s o f A. me l l ea incubated in the l i g h t and in the d a r k , but l i g h t slows down t h e i r e l o n g a t i o n . S h o r t - C h a i n A l c o h o l s Weinhold (44) found that ethanol and o the r s h o r t - c h a i n a l c o h o l s s t i m -u l a t e d the p r o d u c t i o n o f rhizomorphs ! y A. m e l l e a . The s t i m u l a t o r y e f f e c t of ethanol was d e t e c t a b l e at a c o n c e n t r a t i o n of 25 ppm but was opt imal at 500 ppm. There was l i t t l e i nc rease in s t i m u l a t i o n at 1000 ppm and 2000 ppm, w i t h no i n h i b i t i o n at c o n c e n t r a t i o n s up to 1% ethanol (10,000 ppm). R h l z o -morph p r o d u c t i o n was a l s o s t i m u l a t e d by 1 -propanol , i s o p r o p a n o l , and 1-b u t a n o l . Aceta ldehyde was l e s s e f f e c t i v e . Potassium a c e t a t e was very s l i g h t l y s t i m u l a t i n g , and methanol was complete ly i n e f f e c t i v e . Weinhold and Garroway (46) concluded that a smal l q u a n t i t y o f ethanol appeared t o be necessary u n t i l rhizomorph i n i t i a t i o n was complete and e l o n g a t i o n had begun. 6 I t appeared to f u n c t i o n at some p o i n t a f t e r the breakdown o f g lucose in the metabo l ic p rocess (21). Other Facto rs The growth r a t e , l u m i n o s i t y and pigment p r o d u c t i o n of a l l t he i s o l a t e s o f A . m e l l e a t e s t e d increased w i t h i n c r e a s i n g c o n c e n t r a t i o n s of malt e x t r a c t , acco rd ing t o Gibson (22). G a r r e t t (17) concluded that " a c e r t a i n t h r e s h o l d n u t r i e n t s t a t u s of the s u b s t r a t e i s e s s e n t i a l f o r rhizomorph i n i t i a t i o n " . There i s maximal p r o d u c t i o n of rhizomorph i n i t i a l s "when an inoculum d i s c of minimal n u t r i e n t content is l a i d on a f r e s h agar s u b s t r a t e c o n t a i n i n g not l ess than 1 per cent d e x t r o s e . Thus the maximum product ion of i n i t i a l s Is g iven by Inoculum d i s c s taken from c o l o n i e s growing on p l a i n water a g a r . " From a survey o f the l i t e r a t u r e the optimum c o n d i t i o n s f o r the growth of A. m e l l e a in c u l t u r e appear t o be as f o l l o w s : i t shou ld be grown in the dark at 20 -25 C on a medium w i t h pH 5-6. The medium shou ld c o n t a i n an o rgan ic source of n i t r o g e n such as peptone, asparag ine or c a s e i n h y d r o l y s a t e , w i t h at l e a s t 1% g lucose as the carbon source supplemented by a sma l l amount of e t h a n o l . The n i t r o g e n and carbon sources shou ld be present in a s u i t a b l e r a t i o and th iamine shou ld be p r o v i d e d . M e t a b o l i t e s Produced by A. p u l l u l a n s There are c o n f l i c t i n g repo r ts about the a b i l i t y o f A. p u l l u l a n s t o produce e t h a n o l . Cooke (9) has w r i t t e n a comprehensive rev iew o f in fo rma-t i o n known about the p h y s i o l o g y of A, p u l l u l a n s . Rosa, Fred and Peterson (37) found tha t the p r i n c i p l e products of x y l o s e metabol ism were C 0 2 , e t h y l a l c o h o l and c e l l s . S u m i k i , acco rd ing t o Cooke (9), i d e n t i f i e d the f o l l o w -7 ing products of A. p u l l u l a n s on a g lucose -peptone medium: e t h y l a l c o h o l , a c e t i c a c i d , s u c c i n i c a c i d and O L - l a c t i c a c i d . Q u a n t i t i e s were smal l and most of the g l u c o s e was not consumed. Under proper c o n d i t i o n s , A. p u l l u l a n s fermented g lucose to a l c o h o l , accord ing t o Ste inhaus (39). On the o ther hand Chrzaszcz and S c h i l l a k (7) repor ted that the by -p roducts of growth inc luded fo rmic a c i d , a c e t i c a c i d , p r o p i o n i c a c i d , c a l c i u m carbonate and t r a c e s of aceta ldehyde and acetone. They found no e t h y l a l c o h o l , g l y c o l a c i d , s u c c i n i c a c i d or b u t y r i c a c i d . C i f e r r i et a l _ . , as repor ted by Cooke (9), concluded that no s t r a i n s of A. p u l l u l a n s a re known in which t h e r e i s f e rmenta t ion w i t h gas on any sugar . A c i d p r o d u c t i o n occur red r e g u l a r l y when the fungus was grown on g lucose and mannose. Cooke and Matsuura (10), i n a study of four c l o s e l y - r e l a t e d spec ies of b l a c k y e a s t s i n c l u d i n g A . p u l l u l a n s . noted that "none produced s i g n i f i c a n t c a p a b i l i t i e s f o r fe rmenta -t i o n " (as determined by the p r o d u c t i o n of gas bubbles) in the sugars and at the pH l e v e l s t e s t e d . When C l a r k and Wal lace (11,12) i n v e s t i g a t e d the carbohydrate metabol ism of A. p u l l u l a n s . they concluded that i t has enzymes t o c a t a b o l i z e g lucose by some of the r e a c t i o n s of the Embden-Meyerhof system as w e l l as those of the pentose phosphate system. Thus i t may be ab le t o r e s p i r e a n a e r o b i c a l l y t o produce e t h a n o l . They a l s o found that i t s m e t a b o l -ism c o u l d take p l a c e by means of the r e a c t i o n s of the Krebs c y c l e . Jump (27) found by means of the l u p i n e hypocoty l e l o n g a t i o n techn ique that A . p u l l u l a n s conta ined a growth-promoting subs tance . He d i d not I d e n t i f y the subs tance . V i tamin Requirements of A. g u l l u l a n s Growth of A. p u l l u l a n s was shown by Ward t o be dependent on a 8 supply o f t h i a m i n e . The th iamine c o u l d be rep laced by a combinat ion o f equ iva len t amounts of t h i a z o l e and p y r i m i d i n e , but not by e i t h e r a l o n e . Cooke (8) repor ted that A. p u l l u l a n s grew in the absence o f b i o t i n when th iamine was p r e s e n t . I n t e r a c t i o n s o f A. m e l l e a and A. p u l l u l a n s Wiken (51) examined e x t r a c t s from sporophores of Swedish Hymeno-mycetes f o r a n t i b i o t i c a c t i v i t y aga ins t A . p u l l u l a n s . He found that the e x t r a c t o f A . m e l l e a produced measurable zones o f i n h i b i t i o n of the growth ° f P " 1 l u l a n s f o r a p e r i o d of 36 hou rs , but the i n h i b i t i o n d isappeared w i t h longer i n c u b a t i o n . In a study of a number of f ung i present in f o r e s t nursery s o i l s and s e e d l i n g s V a a r t a j a and S a l i s b u r y (43) p a i r e d the i s o l a t e s on cornmeal agar . A. p u l l u l a n s had no e f f e c t on A. m e l l e a , but A. m e l l e a had an a n t a g o n i s t i c e f f e c t on the growth of A. p u l l u l a n s from a d i s t a n c e of 2 - 4 mm. They a l s o noted that A. m e l l e a u s u a l l y e x h i b i t e d s t r o n g antagonism to the o the r fung i t e s t e d . MATERIALS AND METHODS O r i g i n o f Fungus I s o l a t e s The i s o l a t e of A. m e l l e a used in a l l experiments was c u l t u r e d from decayed wood of Ab ies l a s i o c a r p a (Hook.) N u t t . in August , 1962. It produced rhizomorphs r e a d i l y on s u i t a b l e media. The i s o l a t e of A. p u l l u l a n s was an a e r i a l contaminant ob ta ined in November, 1962. E igh t o ther i s o l a t e s of A. m e l l e a and two o f A . p u l l u l a n s were a l s o t e s t e d t o determine i f the s t l m u l -9 atory e f f e c t was present in o ther s t r a i n s o f the same organisms. The sources of the A. m e l l e a i s o l a t e s were as f o l l o w s : one was c u l t u r e d from a sporophore in October , 1963. and the o ther seven i s o l a t e d from m y c e l i a l fans under the bark of t h r e e t r e e s of Pseudotsuga menz ies i i (M i rb . ) Franco in May, 1965. The i s o l a t e s of A. p u l l u l a n s were obta ined as f o l l o w s : one from the heartwood of P. menzies i i in October , 1963> and the other from the bark of Populus t r i c h o c a r p a Torrev and Gray in 1962. The i d e n t i f i c a t i o n of the i s o l a t e s of A . p u l l u l a n s was conf i rmed by Dr. A. Funk, M y c o l o g i s t , Canada Department o f F o r e s t r y , Forest Research L a b o ra to ry , V i c t o r i a , B .C. A l l the i s o l a t e s were from B r i t i s h Columbia . Media and C u l t u r e Techniques I n i t i a l experiments were c a r r i e d out on malt agar prepared as 1% or 5% Mal t e x t r a c t w i t h 2% Bacto agar (designated 1% MA and 5% MA r e s p e c t i v e l y ) and on D i f c o po ta to d e x t r o s e agar (PDA). A s y n t h e t i c medium became n e c e s -sary when attempts were made to determine the nature of the s t i m u l a t o r y s u b -s tance produced by A. p u l l u l a n s . The medium used by Weinhold (W?), des ign nated W and c o n t a i n i n g 5 9 o f g l u c o s e , 2 g of L - a s p a r a g i n e , 0.75 9 o f MgS0i } .7H20, 1.75 g o f Kh^POxj, 1 mg of th iamine h y d r o c h l o r i d e and 20 g of agar in 1000 ml of d i s t i l l e d w a t e r , was mod i f i ed t o c o n t a i n 20 g (s2%) of g lucose (designated 2W). The a d d i t i o n a l g lucose was used in an attempt t o supp ly s u f f i c i e n t sugar t o prevent a carbon d e f i c i e n c y f o r A. me l l ea and was found t o be very s a t i s f a c t o r y , in l i q u i d c u l t u r e experiments agar was omi t ted from the 2W medium. Tests on s o l i d media were c a r r i e d out in 9 cm p e t r i p l a t e s c o n t a i n i n g approx imate ly 25 ml o f medium. Tests on l i q u i d media were c a r r i e d out in 10 5 1 / 2 " x 1 3/4" x 1 3/4" narrow-necked b o t t l e s w i t h a t o t a l c a p a c i t y o f 1 5 0 m l . The amount o f medium used In them v a r i e d and Is g iven In the d e s c r i p t i o n of- the experiments below. They were incubated l y i n g on t h e i r s i d e s . A l l c u l t u r e s were i n o c u l a t e d w i t h 5 mm agar d i s c s taken from the advancing zone of the fungus on water agar . The inoculum in l i q u i d c u l t u r e was f l o a t e d on the s u r f a c e of the medium (supported by s u r f a c e t e n s i o n ) . C u l t u r e s were incubated at room temperature (approximately 22°C) in the da rk . Each t e s t s e r i e s c o n s i s t e d of at l e a s t 5 r e p l i c a t e s on s o l i d media and 8 r e p l i c a t e s in l i q u i d media. On s o l i d media the number of rhizomorphs in each p l a t e was counted and the t o t a l length in each p l a t e determined w i t h a H o f f r i t z map-measuring instrument ( 4 2 , 4 4 ) . A v i s u a l obse rva t ion of the m y c e l i a l growth was made of p l a t e s in which no rhizomorphs were produced. In t e s t s us ing l i q u i d media the dry weight of fungus t h a l l u s was determined and v i s u a l e s t i m a t i o n s were made of rhizomorph growth. In experiments r e q u i r i n g A. p u l l u l a n s in l i q u i d c u l t u r e or a c e l l -f r e e f i l t r a t e of the c u l t u r e , the fungus was grown in 1 0 0 ml o f 2W l i q u i d medium in 2 5 0 ml Erlenmeyer f l a s k s f o r four days on a s low r e c i p r o c a t i n g s h a k e r . The c e l l - f r e e f i l t r a t e was prepared by f i l t e r i n g the c u l t u r e medium through an 0 . 4 5 (J p o r e - s i z e M i l l i p o r e f i l t e r . Thus the f i l t r a t e conta ined 2W medium minus the n u t r i e n t s used by A. p u l l u l a n s in four days and p l u s any m e t a b o l i t e s l i b e r a t e d i n t o the medium by the fungus. D e t a i l s o f s p e c i f i c methods employed in experiments w i l l be g iven in t h e i r context below. 11 EXPERIMENTS ANO RESULTS Determination of the General Effect of the Stimulatory Substance Preliminary experiments were carried out by inoculating 1% MA, 5% MA, PDA, W and 2W agar plates with A. mellea, with and without A. pullulans on the same plate, to determine if the stimulatory effect was present on all the media. The 1% MA, 5% MA and PDA cultures were set up in Kolle culture flasks containing 60 ml of medium. The W and 2W cultures were in petri plates with 25 ml of medium. All three isolates of A. pullulans produced a stimulatory effect on rhizomorph development and/or mycelial growth on all isolates of A. mellea in the different media tested. This effect began to be apparent after about 5 days. Figure 1 shows cultures on 5% MA after 10 days. The greatest contrast between control plates and those inoculated with A. pullulans was evident after 2-3 weeks when rhizomorphs were approach-ing the margin of those plates containing A. pullulans. On 5% MA and PDA A. mellea produced rhizomorphs which soon stopped growing and began to curl back. However, when A. pullulans was present on the plates the rhizomorphs elongated rapidly and continued to grow until they reached the margin of the plates (Figures 2 and 3 ) . In the 1% MA (Figure k) and in the W synthetic medium (Figure 5) no rhizomorphs were produced but the mycelial growth was much more dense when A. pullulans was present. By increasing the glucose concentration from 0.5% (in the W agar) to 2% (in the 2W agar) a very satisfactory medium was obtained in which A. mellea produced abundant mycelium but no rhizomorphs when inoculated alone, but rhizomorphs developed well when A. pullulans was present (Figure 6 ) . On all media there was a zone of mutual antagonism between the A. mellea 12 F i g u r e 1. Growth of A. me l l ea on 5% malt agar in p e t r i p l a t e s a f t e r 10 days (viewed from be low) . L e f t , a lone ; r i g h t , w i t h A . p u l l u l a n s on same p l a t e . 13 F igure 2 . Growth of A. me11ea on 5% malt agar in K o l l e c u l t u r e f l a s k s a f t e r 26 days . L e f t , a l o n e ; r i g h t , w i t h A. pul1u1ans in same f l a s k . 14 F i g u r e 3. G r o w t h o f A . m e l l e a o n p o t a t o d e x t r o s e a g a r i n K o l l e c u l t u r e f l a s k s a f t e r 26 d a y s . L e f t , a l o n e ; r i g h t , w i t h A . p u l l u l a n s i n s a m e f l a s k . 15 F i g u r e h. G r o w t h o f A. m e l l e a o n 1% m a l t a g a r i n K o l l e c u l t u r e f l a s k s a f t e r 26 d a y s . L e f t a l o n e ; r i g h t , w i t h A. p u l l u l a n s i n s a m e f l a s k . IS Figure 6. Growth of A. mellea on 2W medium in petri plates after 22 days. Left, alone; right, with A. pullulans on same plate. 17 mycelium and the A. p u l l u l a n s c o l o n y . Th is zone i s shown c l e a r l y in F i g -ure 3 . Subsequent t e s t s u t i l i z e d the i n i t i a l I s o l a t e of A. m e l l e a and of A. p u l l u l a n s . The g l u c o s e - p e p t o n e - t h i a m i n e medium desc r i bed by Azevedo (2) to g i v e maximum growth of her i s o l a t e s was used to determine i f the substance p r o -duced by A. p u l l u l a n s s t i m u l a t e d the growth of A. me l lea when peptone was the n i t r o g e n s o u r c e . A c e l l - f r e e f i l t r a t e of A. p u l l u l a n s l i q u i d c u l t u r e in t h i s medium was prepared as d e s c r i b e d f o r the 21/ medium. A. mel l e a was i nocu la ted in b o t t l e s c o n t a i n i n g 25 ml o f the c e l l - f r e e f i l t r a t e , and in unt reated l i q u i d medium. A f t e r two weeks the mean dry weight of A. me l l ea was 61.6 mg on the c e l l - f r e e f i l t r a t e of A. p u l l u l a n s l i q u i d c u l t u r e and 16.0 mg on the c o n t r o l . The d i f f e r e n c e was h i g h l y s i g n i f i c a n t , as d e t e r -mined by a " t " t e s t . There fo re A. p u l l u l a n s produced the s t i m u l a t o r y s u b -s tance in Azevedo's medium w i t h peptone as the n i t r o g e n s o u r c e , and i t r e s u l t e d in increased growth of A. m e l l e a in the medium. The s t i m u l a t i o n occur red on a medium found by Azevedo t o be a l ready very s u i t a b l e f o r the growth of A. m e l l e a . An experiment was c a r r i e d out t o determine i f the c e l l - f r e e f i l t r a t e of A . p u l l u l a n s l i q u i d c u l t u r e conta ined a s t i m u l a t o r y substance and i f i n -c reased c o n c e n t r a t i o n s of the f i l t r a t e added t o 2W agar r e s u l t e d in increased growth of A. m e l l e a . The c e l l - f r e e f i l t r a t e (prepared as desc r i bed in M a t e r i a l s and Methods) was added as 0 , 5 , 10, 15, 20 , 25, 30, 40 , 50 and 75% of the l i q u i d volume of 2W agar a f t e r the remaining l i q u i d and p r o p o r -t i o n a t e amounts of n u t r i e n t s had been autoc laved w i t h the agar . A. me l l ea was i nocu la ted on the p l a t e s and growth was measured a f t e r 14 days . There was a h i g h l y s i g n i f i c a n t c o r r e l a t i o n (p = <0.01, us ing a c u r v i -l i n e a r r e g r e s s i o n a n a l y s i s and the m u l t i p l e r e g r e s s i o n c o e f f i c i e n t ) between 18 the amount of c e l l - f r e e f i l t r a t e in the medium and the number and t o t a l length o f rhizomorphs per p l a t e . F igures 7 and 8 show the mean number and t o t a l length of rhizomorphs per p l a t e a f t e r 14 days growth. F igu re 9 shows the growth p a t t e r n of A. me l l ea in the d i f f e r e n t t rea tments . No rhizomorphs were present in the p l a t e s c o n t a i n i n g l e s s than 25% f i l t r a t e in the medium, but the number and t o t a l length increased s t e a d i l y from 25% t o 75%. Thus the c e l l - f r e e f i l t r a t e promoted rhizomorph development. The e f f e c t increased w i t h the amount of f i l t r a t e in the medium. In a second experiment p e t r i p l a t e s c o n t a i n i n g 50% c e l l - f r e e f i l t r a t e and 50% 2W medium were i nocu la ted w i t h rhizomorph t i p s cut o f f where they prot ruded above the medium. That i s , the inoculum c o n s i s t e d o f a e r i a l rhizomorphs approximate ly 1 cm long w i t h no agar a t tached to them. A f t e r 14 days the growth on the c e l l - f r e e f i l t r a t e p lus 2W agar was compared t o that on 2W agar c o n t r o l s t o determine i f the s t i m u l a t o r y e f f e c t was apparent a f t e r rhizomorphs had been d i f f e r e n t i a t e d . F igu re 10 shows the growth on the two t rea tments . The growth of rhizomorphs on the c e l l - f r e e f i l t r a t e agar was s i g n i f i c -a n t l y increased (p = <0.01) both in t o t a l length and in number per p l a t e as compared w i t h the growth on 2W agar . The mean t o t a l length of rhizomorphs per p l a t e on the f i l t r a t e agar was 214 mm and on the 2W agar 61 mm. There was an average of 8 rhizomorphs per p l a t e on the f i l t r a t e agar compared w i t h 3 .5 on 2w agar . Because Weinhold (44) repor ted that ethanol s t i m u l a t e d the growth of m e l l e a rhizomorphs at an optimum c o n c e n t r a t i o n of 500 ppm (weight /vo lume) , experiments were set up t o t e s t the e f f e c t of ethanol in comparison t o the e f f e c t o f A. p u l l u l a n s on the growth of A. m e l l e a . The f i r s t experiment was c a r r i e d out in p e t r i p l a t e s , us ing W agar , 2W agar , and 2W agar w i t h hi I-< 0. or m a. CO x a. or o z o N X or u. o 100 % F I L T R A T E I N M E D I U M F igu re 7. The r e l a t i o n s h i p between the number of rhizomorphs produced by A. m e l l e a and the amount of e e l I - f r e e f i l t r a t e in the medium ( a f t e r \k days growth) . 500 " Z z Ul < _J a. or ui a. co i 0. or o Z o N X or o z U l 400 -300 -2 0 0 " 100 • I0O % F I L T R A T E I N M E D I U M F i g u r e 8 . The r e l a t i o n s h i p between the length of rhizomorphs produced by A . me l l ea and the amount of c e l l - f r e e f i l t r a t e in medium ( a f t e r 14 days growth) . 21 F igu re 9. The growth of A. m e l l e a a f t e r 14 days on media c o n t a i n i n g increased amounts of c e l l - f r e e f i l t r a t e (v iew-ed from be low) . C = 2W c o n t r o l . Numbers show per cent of c e l l - f r e e f i l t r a t e in medium. 22 F i g u r e 10. Growth o f A. me l l ea from rhizomorph t i p i n o c -ulum a f t e r \k days. L e f t , on 2W agar ; r i g h t , on 2W agar w i th 50% c e l l - f r e e f i l t r a t e . 23 enough L -asparag lne (6g) added to b r i n g i t t o the same p r o p o r t i o n as in the W medium (designated 2W+N). The 2W+N medium was used t o check the p o s s i b i l -i t y tha t A. p u l l u l a n s was a f f e c t i n g the C/N r a t i o . Each medium was used w i t h and wi thout the a d d i t i o n of ethanol as 500 ppm (volume/volume) and w i t h and w i thout A. p u l l u l a n s growing on the same p l a t e . The r e s u l t s o f the a d d i t i o n of 500 ppm ethanol t o W, 2W and 2W-1-N agar media are g iven in Tab le I and i l l u s t r a t e d in F igures 11, 12 and 13. The ethanol had a s t i m u l a t o r y e f f e c t on rhizomorph p r o d u c t i o n in the 2W and 2W*N media , but A. p u l l u l a n s w i t h or w i thout t h e a d d i t i o n of ethanol had a much g reate r e f f e c t . Weinhold (kk) reported that 500 ppm ethanol was o p -timum f o r rhizomorph development and a d d i t i o n a l amounts had l i t t l e f u r t h e r e f f e c t . Thus i t appeared that A. p u l l u l a n s produced something in a d d i t i o n to e t h a n o l . Rhizomorph growth was g reate r in the p l a t e s w i t h a d d i t i o n a l n i t r o g e n (2W+N) than in the 2W p l a t e s . However, there was a marked s t i m -u l a t i o n in the presence of A. p u l l u l a n s . i n d i c a t i n g that the e f f e c t was not a s imp le i n f l u e n c e on the C/N r a t i o . The r e s u l t s of t h i s experiment led t o attempts t o determine the p h y s i c a l and chemica l p r o p e r t i e s of the s t i m u l a t o r y subs tance . When i t became apparent that ethanol was the substance produced by A. p u l l u l a n s . the use of an agar s u b s t r a t e was re -examined . Perhaps agar i n t e r f e r e d w i t h d i f f u s i o n of ethanol through the s u b s t r a t e . T h e r e f o r e , f u r t h e r work w i t h ethanol was c a r r i e d out us ing l i q u i d s u b s t r a t e s r a t h e r than agar media. P h y s i c a l and Chemical P r o p e r t i e s of the S t i m u l a t o r y Substance Two experiments were c a r r i e d out to determine i f the s t i m u l a t o r y s u b -s tance produced by A. p u l l u l a n s was v o l a t i l e . The f i r s t c o n s i s t e d of grow-TABLE I. E f f e c t of A. p u l l u l a n s , ethanol and increased n i t r o g e n on the development o f rhizomorphs by A. me l lea a f t e r th ree weeks (mean and range f o r f i v e p e t r i p l a t e s ) . D e s c r i p t i o n s o f the media are g iven in the t e x t . T o t a l rhizomorph length per p l a t e (mm) No. o f rhizomorphs per p l a t e per Mean length rhizomorph (mm) Med i urn Alone + A. p u l l u l a n s A lone + A. p u l l u l a n s A lone + A . p u l l u l a n s W 0 0 0 0 0 0 W + EtOH 0 * 0 * 0 * 2W 0 509 (65-925) 0 19.6 (3-25) 0 26 .0 2W + EtOH 56 (0-125) 525 (390-890) 2.2 (0-5) 16.2 (12-28) 25.5 32.4 2W + N 0 918 (460-1120) 0 31.8 (16-43) 0 28 .8 2W + N + EtOH 136 (0-500) 534.6 (405-600) 4.8 (0-17) 20 (J8-22) 28 .3 26 .7 One p l a t e had one rhizomorph 16 mm long . 25 F i g u r e 11. Growth of A. m e l l e a on W agar a f t e r 22 days in p e t r i p l a t e s . Top l e f t , a l o n e ; top r i g h t , w i t h 500 ppm of ethanol in medium; bottom l e f t , w i t h A. p u l l u l a n s ; bottom r i g h t , w i t h A. p u l l u l a n s and 500 ppm of ethanol in medium. F i g u r e 12. Growth of A. me11ea on 2W agar a f t e r 22 days in p e t r i p l a t e s . Top Teft, a l one ; top r i g h t , w i t h 500 ppm o f ethanol in medium; bottom l e f t , w i t h A. p u l J u l a n s ; bottom r i g h t , w i t h A. p u l l u l a n s and 500 ppm o f ethanol in medium. 27 F igu re 13. Growth of A. me l l ea on 2W+N agar a f t e r 22 days in p e t r i p l a t e s . Top l e f t , a lone ; top r i g h t , w i t h 500 ppm of ethanol in medium; bottom l e f t , w i t h A. p u l - l u l a n s ; bottom r i g h t , w i t h A. p u l l u l a n s and 500 ppm of ethanol in medium. 2 3 i n g A . m e l l e a a n d A . p u l l u l a n s i n t w o - s e c t i o n p e t r i p l a t e s c o n t a i n i n g 2W a g a r . T h e r e was n o c o n t a c t b e t w e e n t h e a g a r o n w h i c h A . p u l l u l a n s was g r o w -i n g a n d t h a t o n w h i c h A . m e l l e a was g r o w i n g . H o w e v e r , t h e a i r s p a c e o v e r t h e t w o f u n g i w a s c o n t i n u o u s . In a s e c o n d e x p e r i m e n t t h e t w o f u n g i w e r e s e p a r a t e d i n s t a n d a r d p e t r i p l a t e s b y r e m o v i n g a s t r i p o f a g a r a c r o s s t h e d i a m e t e r o f t h e p l a t e b e f o r e i n o c u l a t i o n ( G a r r e t t ' s i s o l a t i o n t r e n c h m e t h o d , 15). When t h e m e d i u m o n w h i c h A . p u l l u l a n s w a s g r o w i n g was s e p a r a t e d b y a p h y s i c a l b a r r i e r f r o m t h e m e d i u m o n w h i c h A . m e l l e a was g r o w i n g , b u t t h e a i r s p a c e was c o n t i n u o u s o v e r t h e t w o f u n g i , t h e g r o w t h o f A . m e l l e a was s t i m u l a t e d . F i g u r e s )k a n d 15 i l l u s t r a t e t h e s e r e s u l t s , w h i c h i n d i c a t e t h a t a v o l a t i l e s u b s t a n c e p r o d u c e d by A . p u l l u l a n s w a s an e f f e c t i v e g r o w t h s t i m u l a n t f o r A . m e l l e a . N e x t , t h e h e a t s t a b i l i t y a n d v o l a t i l i t y o f t h e s t i m u l a t o r y s u b s t a n c e w e r e e x a m i n e d . A l i q u i d c u l t u r e o f A. p u l l u l a n s was f i l t e r e d t h r o u g h f i v e l a y e r s o f f i l t e r p a p e r , t h e n t r e a t e d zs f o l l o w s : o n e - t h i r d w a s b o i l e d i n a n o p e n c o n t a i n e r f o r t h r e e h o u r s , o n e - t h i r d was r e f l u x e d f o r t h r e e h o u r s a n d o n e - t h i r d was M i 1 1 i p o r e - f i l t e r e d a s a c o n t r o l . E a c h t r e a t e d p o r t i o n was a d d e d es 5 0 % o f t h e l i q u i d i n 2W agar a n d t h e p l a t e s i n o c u l a t e d w i t h A . m e l l e a . A c u l t u r e o f A . p u l l u l a n s s i m i l a r l y p a p e r - f i l t e r e d was a u t o c l a v e d a n d a d d e d t o 2W a g a r a n d c o m p a r e d t o i t s M i 1 1 i p o r e - f i l t e r e d c o n t r o l . B o t h s e t s o f p l a t e s w e r e c o m p a r e d t o 2W a g a r c o n t r o l p l a t e s . When t h e f i l t e r e d l i q u i d c u l t u r e was b o i l e d i n an o p e n f l a s k f o r t h r e e h o u r s m o s t o f t h e s t i m u l a t o r y e f f e c t was l o s t . When i t w a s r e f l u x e d f o r t h r e e h o u r s o r a u t o c l a v e d f o r 2 0 m i n u t e s i t wa s j u s t a s e f f e c t i v e a s t h e u n t r e a t e d l i q u i d . T h e r e f o r e t h e s t i m u l a t o r y s u b s t a n c e w a s v o l a t i l e a n d h e a t s t a b l e . F igu re \ht Growth of A. m e l l e a on 2W agar in t w o - s e c t i o n p e t r i p l a t e s a f t e r 16 days. L e f t , a l o n e ; r i g h t , w i t h A. p u l l u l a n s in second s e c t i o n . 30 Figure 15. Growth of A. mellea on 2W agar in pe t r i plates after 12 days (viewed from below). Left, alone; r i g h t , A. mellea separated from A. pullulans by iso l a t i o n trench? 31 Attempts to determine the v o l a t i l e products in the c e l l - f r e e f i l t r a t e of A. pullulans l i q u i d culture were made by analysing i t in a Perkin-Elmer 810 Gas Chromatograph and comparing i t with known substances. The details of the packed column used are as follows: coating (Liq. Phase): "K" Carbo-wax K 1500; Support Material: Teflon #6; Mesh s i z e : 35; Length: 6 1; Out-side Diameter: 1/8"; Temperature Range: 40 -150 C. This column is recom-mended for use in separating the components of alcoholic beverages. The a l i p h a t i c compounds are separated and the position of ethanol is different from those of formaldehyde, acetone, short-chain acids and other alcohols. The chromatograph was operated with the thermal conductivity detector at a current of 245 milliamps and a gas flow rate of 23 ml/min. The temperatures used were: hot wire 170°C, injector 150°C and oven 50°C. Gas chromatographic analysis of the c e l l - f r e e f i l t r a t e showed a peak for only one v o l a t i l e compound in addition to the water. There appeared to be no other v o l a t i l e compounds present. The substance came through the column before the water and corresponded to the peak produced when ethanol was analysed. To determine whether the stimulatory substance was a c i d i c , basic or neutral, portions of a 2W l i q u i d culture of A. pullulans were passed through ion exchange resin columns. An untreated portion was retained as a control. The amount of ethanol present in each portion was measured with the gas chromatograph after i t was put through the ion exchange resin. This amount of ethanol was added to 2W medium, which was then put through the ion ex-change resin. A f i n a l adjustment was made so that the amount of ethanol in the 2W medium was approximately equal to that in the equivalent A. pullulans medium. Portions of 2W medium containing no ethanol were also put through the resins. 32 The ion exchange r e s i n s used were Dowex 50W-X2, i o n i c form H , a s t r o n g l y a c i d i c c a t i o n exchange r e s i n and Dowex 1-X8, i o n i c form C l " , a s t r o n g l y b a s i c anion exchange r e s i n . To r e p l a c e the ions removed from the media by the r e s i n s , the inorgan ic s a l t s Kh^PO^ and MgSO^^h^O were rep laced in a l l t reatments a f t e r exchange and the L -asparag ine was rep laced in a l l t reatments through the c a t i o n exchange column. The pH was ad justed t o approx imate ly 5.0 w i t h KOH. A l l t reatments were s t e r i l i z e d by M i l l i p o r e -f i I t e r I n g and the amount of ethanol present in each was measured w i t h the gas chromatograph. L i q u i d c u l t u r e s of each treatment were se t up in b o t t l e s c o n t a i n i n g 30 ml of medium and were i nocu la ted w i t h A. m e l l e a . The dry weight of A. me11ea was measured a f t e r ]k days . The mean dry weights of A. m e l l e a and the amount of ethanol present in the d i f f e r e n t t reatments are shown in F i g u r e 16. F igures 17, 18 and 19 show the growth p a t t e r n s of A. m e l l e a in the d i f f e r e n t t rea tments . The u n -t r e a t e d 2W medium c o n t a i n i n g ethanol produced a s t i m u l a t o r y e f f e c t which was s t a t i s t i c a l l y the same as that produced by the unt reated A. p u l l u l a n s ee l 1-f r e e f i l t r a t e . A l l o ther t reatments were s i g n i f i c a n t l y d i f f e r e n t from each other as determined by " t " t e s t s , except the 2W anion exchange treatment compared w i t h the 2W c a t i o n exchange t reatment . The dry weight of A. m e l l e a in a l l t reatments c o n t a i n i n g ethanol was very s i g n i f i c a n t l y h igher than that in the 2W medium treatments wi thout e t h a n o l . When the A. p u l l u l a n s l i q u i d c u l t u r e was passed through the ion exchange r e s i n s , the s t i m u l a t o r y s u b -s tance (as i n d i c a t e d by the b ioassay w i t h A. me l l ea ) came through both the anion and c a t i o n exchange r e s i n s . However, there was l e s s s t imu la t i on of growth a f t e r the c a t i o n exchange treatment than a f t e r the anion exchange treatment or the c o n t r o l . A n a l y s i s w i t h the gas chromatograph of the A. p u l l u l a n s l i q u i d c u l t u r e treatments showed that t h e r e was l e s s ethanol in 32 The ion exchange r e s i n s used were Dowex 50W-X2, i o n i c form H + , a S t r o n g l y a c i d i c c a t i o n exchange r e s i n and Oowex 1-X8, i o n i c form C l " , a s t r o n g l y b a s i c anion exchange r e s i n . To r e p l a c e the ions removed from the media by the r e s i n s , the ino rgan ic s a l t s Kh^PO^ and MgSO^.yHjO were rep laced in a l l t reatments a f t e r exchange and the L -asparag ine was rep laced in a l l t reatments through the c a t i o n exchange column. The pH was ad justed t o approx imate ly 5.0 w i t h KOH. A l l t reatments were s t e r i l i z e d by M i l l i p o r e -f i I t e r i n g and the amount of ethanol present in each was measured w i t h the gas chromatograph. L i q u i d c u l t u r e s of each treatment were se t up in b o t t l e s c o n t a i n i n g 30 ml of medium and were i nocu la ted w i t h A. m e l l e a . The dry weight of A. m e l l e a was measured a f t e r \h days . The mean dry weights of A. m e l l e a and the amount of ethanol present in the d i f f e r e n t t reatments are shown in F igu re 16. F igures 17, 18 and 19 show the growth p a t t e r n s of A. me l l ea in the d i f f e r e n t t rea tments . The u n -t r e a t e d 2W medium c o n t a i n i n g ethanol produced a s t i m u l a t o r y e f f e c t which was s t a t i s t i c a l l y the same as that produced by the unt reated A. p u l l u l a n s ee l 1-f r e e f i l t r a t e . A l l o ther t reatments were s i g n i f i c a n t l y d i f f e r e n t from each o ther as determined by M t " t e s t s , except the 2W anion exchange treatment compared w i t h the 2W c a t i o n exchange t reatment . The dry weight of A. m e l l e a in a l l t reatments c o n t a i n i n g ethanol was very s i g n i f i c a n t l y h igher than that in t h e 2W medium treatments wi thout e t h a n o l . When the A. p u l l u l a n s l i q u i d c u l t u r e was passed through the ion exchange r e s i n s , the s t i m u l a t o r y s u b -s tance (as i n d i c a t e d by the b ioassay w i t h A. me l lea ) came through both the anion and c a t i o n exchange r e s i n s . However, t he re was l e s s s t imu la t i on of growth a f t e r t h e c a t i o n exchange treatment than a f t e r the anion exchange treatment o r the c o n t r o l . A n a l y s i s w i t h the gas chromatograph o f the A. p u l l u l a n s l i q u i d c u l t u r e t reatments showed that t h e r e was l e s s ethanol in 33 125 100 E X © 75 UJ $ > cc o 50 25 • CONTROL 0 CATION RESIN 0 A N I O N RE8IN EtOH-ppm 1 i 0 0 2W 1 23 V/i 160 8 0 0 100 1200 8 5 0 1350 EtOH A.pullulans filtrate F i g u r e 16. The e f f e c t o f Ion exchange r e s i n s on the dry weight produced by A . me l l ea in 2W medium,2W medium w i t h e t h a n o l , and a c e l l - f r e e f i l t r a t e o f A, p u l l u l a n s l i q u i d c u l t u r e . 34 Figure 1 7 . Growth of A. mellea on 2W medium after 14 days. Left, control; centre, medium passed through anion exchange resin; right, medium passed through cation ex-change resin. F igu re 18. Growth of A . me11ea a f t e r 14 days on 2W med-ium c o n t a i n i n g e t h a n o l . L e f t , c o n t r o l ( c o n t a i n i n g 1160 ppm of e t h a n o l ) ; c e n t r e , medium passed through anion e x -change r e s i n ( c o n t a i n i n g 1100 ppm of e t h a n o l ) ; r i g h t , medium passed through c a t i o n exchange r e s i n ( c o n t a i n i n g 800 ppm of e t h a n o l ) . F igu re 19. Growth of A. m e l l e a a f t e r 14 days on a c e l l -f r e e f i l t r a t e of A. p u l l u l a n s l i q u i d c u l t u r e . L e f t , c o n t r o l ( c o n t a i n i n g 1200 ppm of e t h a n o l ) ; c e n t r e , medium passed through anion exchange r e s i n ( c o n t a i n i n g 1350 ppm of e t h a n o l ) ; r i g h t , medium passed through c a t i o n exchange r e s i n ( c o n t a i n i n g 850 ppm of e t h a n o l ) . 37 the cation exchange treatment and slightly more ethanol in the anion ex-change treatment than in the control. The treatments with A. pullulans liquid culture showed a pattern similar to those with ethanol in 2W medium; i.e., there was an increase in the dry weight of A. mellea when they were passed through the anion ex-change resin and a decrease when they were passed through the cation ex-change resin, as compared with the controls. When there was no ethanol present, the dry weight of A. mellea was greater in the 2W control than in the 2W medium passed through either the anion or cation exchange resins. Although passing the A. pullulans liquid culture through the anion exchange resin resulted in increased stimulation of A. mellea and passing it through the cation exchange resin resulted in decreased stimulation, it is not necessary to postulate the presence of a substance other than ethanol as the effective one. 2W Medium containing ethanol was affected in the same way by the ion exchange resins. Thus there was no evidence from this ex-periment to indicate that the stimulatory substance produced by A. pullulans was not ethanol, and no need to postulate a stimulatory substance in addi-tion to ethanol. Experiments with Ethanol in Liquid Cultures An experiment was set up to determine if an increased supply of the same concentration of ethanol would result in increased growth of A. mellea. The treatments were 15. 30 and 60 ml per bottle of 2W liquid medium c o n -taining ethanol in a concentration of 500 ppm and 15, 30 and 60 ml per bottle of 2W medium without ethanol. The dry weight of A. mellea was measured after \k days and the concentration of ethanol remaining in the medium was measured 33 with the gas chromatograph. The dry weight of A. mellea increased with an increased volume of 2W medium containing 500 ppm ethanol. Table II shows the effect of the amount of ethanol in the medium on the dry weight of A. mellea, when the concentra-tion of ethanol remained constant. In the control cultures (2W) there was no significant difference in the dry weight of A. mellea when the volume of medium was increased; hence the weight per ml of medium decreased consider-ably as the volume increased. The increase in the amount of nutrients available did not result in increased growth of A. mellea. When ethanol was supplied as 500 ppm in the medium the dry weight of A. mellea Increased with an increased volume of medium, but the weight per ml of medium and the weight per ul of ethanol remained essentially constant. Analysis with the gas chromatograph showed that there was less than 50 ppm ethanol remaining in any of the media. Thus it appears that the growth of A. mellea, as measured by the dry weight produced, varies with the amount of ethanol in the medium when the concentration is held at 500 ppm. The increase in dry weight with the addition of ethanol could not be accounted for by postulating that the ethanol was used solely as an addi-tional source of carbon. In 60 ml of 2W medium, containing 8 g of carbon, the dry weight of A. mellea was 9.1 mg. In 60 ml of 2W medium with 30 ul of ethanol there was an additional 19 lJg of carbon, but the dry weight was increased by 54.9 mg to 65.0 mg (Table II). Thus the ethanol had a sig-nificant effect in addition to supplying carbon. An experiment was carried out to determine if ethanol added at regular intervals as lower concentrations in the medium would stimulate the growth of A. mellea as much as one higher initial concentration of ethanol. Liq-uid cultures containing 25 ml of medium per bottle were grown for 21 days. TABLE I I . E f f e c t of amount of ethanol on dry weight o f A. me l l ea Vo l medium Conc 'n EtOH Am't EtOH Dry wt A . m e l l e a Wt/ml medium Wt /u l EtOH Treatment (ml) (ppm) (u l ) a f t e r 14 days (mg) (mg) (mg) 2W 15 0 0 6.7 0.45 30 0 0 7.6 0.25 60 0 0 9.1 0 .15 EtOH 15 500 7.5 16.5 1.10 2.20 30 500 15 34.7 1.16 2.31 60 500 30 67.3 1.12 2 .24 EtOH 15 500 7.5 23.9 1.59 3.19 30 500 15 53.9 1.80 3.59 60 500 30 65.0 1.08 2.17 40 The treatments were as f o l l o w s : (1) an i n i t i a l c o n c e n t r a t i o n of ethanol as 1000 ppm in 2W medium w i t h no ethanol added du r ing the exper iment , (2) an i n i t i a l c o n c e n t r a t i o n of ethanol as 333 ppm in 2W medium, w i t h an a d d i t i o n a l 333 ppm a f t e r seven days and again a f t e r 14 days to g i v e a t o t a l amount of ethanol the same as in (1 ) , (3) an i n i t i a l c o n c e n t r a t i o n of ethanol as 143 ppm in 2W medium, w i t h an a d d i t i o n a l 143 ppm every t h i r d day (seven t imes) t o g i v e a t o t a l amount of ethanol the same as in (1 ) , (4) 2W medium without e t h a n o l . Ethanol added at r e g u l a r i n t e r v a l s as lower c o n c e n t r a t i o n s in the 2W medium s t i m u l a t e d the growth of A. me l l ea at l e a s t as much as one h igher i n i t i a l c o n c e n t r a t i o n . The mean dry weight produced w i t h a s i n g l e i n i t i a l c o n c e n t r a t i o n of ethanol as 1000 ppm (treatment 1) was 82 .9 mg, w i t h 333 ppm added t h r e e t imes (treatment 2) was 91.9 mg, and w i t h 143 ppm added seven t imes (treatment 3) was 83.2 mg. The dry weight produced w i t h the a d d i t i o n of 333 ppm ethanol t h r e e times (treatment 2) was s i g n i f i c a n t l y h igher than that w i t h the o ther two t rea tments , which were not s i g n i f i c a n t -l y d i f f e r e n t from each o t h e r . A l l t reatments were s i g n i f i c a n t l y h igher than the 2W c o n t r o l (treatment 4) in which the mean dry weight was 21.5 mg. In a s i m i l a r experiment to determine i f ethanol as 50 ppm s u p p l i e d d a i l y in 2W l i q u i d medium was as e f f e c t i v e in s t i m u l a t i n g the growth of A. m e l l e a as 700 ppm (=14 days x 50 ppm) s u p p l i e d i n i t i a l l y , the f o l l o w i n g t reatments were set up: 1. Ethanol s u p p l i e d as 50 ppm in l i q u i d medium. The medium was withdrawn and rep laced d a i l y . 2 . 2W L i q u i d medium, withdrawn and rep laced d a i l y . 3. Ethanol s u p p l i e d as 700 ppm in 2W l i q u i d medium. The medium was not changed du r ing the exper iment . 4 . 2W L i q u i d medium, not changed. 41 5 . Ethanol s u p p l i e d as 50 ppm in the medium. The medium was not changed du r i ng the exper iment . Each c u l t u r e b o t t l e conta ined 25 ml of medium. It was i nocu la ted w i t h A. me l l ea growing on water agar , the d i s c s being 8 mm in diameter and approx -imate ly 10 mm deep. The d i s c was l a r g e enough t o r e s t on the lower s u r f a c e of the b o t t l e and p ro t rude above the s u r f a c e of the medium. A f t e r 14 days the c u l t u r e s were photographed, the number of rhizomorphs counted and the dry weights determined. The r e s u l t s o f the experiments d e s c r i b e d above a re g iven in Tab le III and i l l u s t r a t e d in F i g u r e 20. There was a great i nc rease in the number of rhizomorphs when 50 ppm ethanol was changed d a i l y (treatment 1 ) , as com-pared w i t h a l l the o ther t rea tments . However, the dry weight produced was not s i g n i f i c a n t l y d i f f e r e n t from that in which 50 ppm ethanol or 700 ppm TABLE I I I . E f f e c t of a low c o n c e n t r a t i o n of ethanol s u p p l i e d d a i l y on the growth of A. me l l ea (14 days o l d ) . Treatment Mean Dry Weight (mg) Mean No, o f Rhizomorphs 2W 30.5 0 2W changed d a i l y 22 .5 0.2 50 ppm ethano l 45 .9 0 50 ppm ethanol changed d a i l y 46 .8 10.4 700 ppm ethanol 51 .5 0 F igu re 20. Growth of A . me l lea a f t e r \k days on media t r e a t e d as f o l l o w s ( l e f t t o r i g h t ) : 2W c o n t r o l , 2W medium changed d a i l y , 2W medium w i t h 50 ppm of e t h a n o l , 2W medium w i t h 50 ppm of ethanol changed d a i l y , 2W med-ium w i t h 700 ppm of e t h a n o l . 43 ethanol was s u p p l i e d i n i t i a l l y . In a l l t h r e e t reatments c o n t a i n i n g ethanol the dry weight was s i g n i f i c a n t l y h igher than in the treatments wi thout e t h a n o l . There was a s i g n i f i c a n t decrease in dry weight when the 2W medium was changed d a i l y , as compared w i t h 2W medium not changed. A. m e l l e a , i n o c u l a t e d as water agar p l u g s , was grown in 60 ml of 2W l i q u i d medium f o r s i x weeks t o determine i f i t c o u l d a t t a i n in t ime the amount of growth i t ob ta ined w i t h ethanol present in the medium. The mean dry weight a f t e r s i x weeks was 44 .0 mg w i t h no rhizomorphs p r e s e n t . Th is was l e s s than the 65 .0 mg a t t a i n e d by A. m e l l e a water agar p lugs in two weeks w i t h 500 ppm ethanol in 60 ml of 2W medium. If the A. m e l l e a inoculum was 2W agar p lugs w i t h no rhizomorph i n i t i a l s p r e s e n t , instead o f water agar p l u g s , the mean dry weight a f t e r s i x weeks was 146.5 mg, w i t h r h i z o -morphs present in most samples. Th is growth was a t t a i n e d w i t h no e x t e r n a l source of ethanol in the 2W medium. Thus i t appears that A. me11ea on water agar p lugs cannot a t t a i n i n s i x weeks the growth on 2W medium that i t a t t a i n s in two weeks w i t h ethanol in the medium. However, growth was much more e x t e n s i v e when the inoculum was 2W agar p lugs on a medium w i t h no e t h a n o l . Tests f o r P l a n t Growth Facto rs as the S t i m u l a t o r y Substance Because Weinhold , Hendr ix and Raabe (48) repor ted that 5 ppm of i ndo le - 3 - a c e t i c a c i d s t i m u l a t e d the growth of A. m e l l e a rhizomorphs an experiment was c a r r i e d out t o determine i f the e f f e c t o f A. p u l l u l a n s c o u l d be r e p r o -duced by d i f f e r e n t p l a n t growth subs tances . The substances used were 3~ i n d o l y l a c e t i c a c i d ( IAA) , Y- ( i n d o l e - 3 ) - n - b u t y r i c a c i d ( IBA) , the sodium s a l t o f 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d (2 ,4 -0 ) and t ryptophane. Each one kk was d i s s o l v e d in s t e r i l e d i s t i l l e d water and added t o 2W agar t o g i v e c o n -c e n t r a t i o n s ranging from 0.1 ppm t o 100 ppm, A. m e l l e a was i n o c u l a t e d a lone and w i t h A. p u l l u l a n s on each t reatment . P a r t i c u l a r a t t e n t i o n was p a i d t o the e f f e c t of IAA. The growth of A. m e l l e a rhizomorphs was s l i g h t l y s t i m u l a t e d by 0.1 ppm and 5 ppm of IAA (Table IV ) . The s t i m u l a t o r y e f f e c t of A. p u l l u l a n s on the growth of A. m e l l e a was somewhat i n h i b i t e d by 10 ppm IAA. A. m e l l e a was s t r o n g l y i n h i b i t e d by 50 ppm of IAA even when A. p u l l u l a n s was present (Table V ) . The o ther p l a n t growth substances had l i t t l e e f f e c t on the growth of A. m e l l e a . TABLE IV. E f f e c t of IAA on the development of rhizomorphs of A. m e l l e a a f t e r t h r e e weeks (mean and range f o r f i v e p e t r i p l a t e s ) . Medium T o t a l rhizomorph length per p l a t e (mm) No. o f rhizomorphs per p l a t e Mean length per rhizomorph (mm) 2W 0 0 0 2W + 0.1 ppm \k 1.4 10 IAA (0-30) (0-3) 2W + 5 ppm 117 11 10.7 IAA (0-225) (0-18) A . p u l l u l a n s was grown in 2W l i q u i d medium f o r e igh t days . The medium was then t e s t e d f o r IAA u s i n g the c o l o r l m e t r i c t e s t o u t l i n e d by Gordon and Weber (21) . T h i s t e s t i s s e n s i t i v e t o a c o n c e n t r a t i o n of IAA as low as 0 .2 ppm. The medium gave a nega t i ve t e s t . 45 TABLE V . E f f e c t of A. p u l l u l a n s and IAA together on the development of rhizomorphs of A. m e l l e a a f t e r two weeks (mean and range of four p e t r i p l a t e s ) . T o t a l rhizomorph length No. o f rhizomorphs Mean length Medium per p l a t e per p l a t e per rhizomorph (mm) (mm) 2W 470 19 24 .7 (315-835) (13-30) 2W + 0.1 ppm 637 28 22 .4 IAA (500-795) (21-36) 2W + 5 ppm 485 30 15.9 IAA (310-815) (19-47) 2W + 10 ppm 150 23 6 .4 IAA (85-260) (12-45) 2W + 50 ppm 0 0 0 IAA Thus i t appeared that the s t i m u l a t o r y substance produced by A. p u l - l u l a n s was not 3 - i n d o l y l a c e t i c a c i d . The p l a n t growth substances t e s t e d c o u l d not reproduce the s t i m u l a t o r y e f f e c t of A. p u l l u l a n s . E f f e c t on I n h i b i t i o n by TrJchodenna v i r i d e Tr ichoderma v i r i d e P e r s . ex F r . has been repor ted by B l i s s (6) and G a r r e t t (19) t o i n h i b i t the growth o f A. me l l ea in s o i l and In woody i n o c u l a . It seemed p o s s i b l e tha t A, p u l l u l a n s c o u l d s t i m u l a t e the growth of A. m e l l e a t o the extent tha t i t would no longer be i n h i b i t e d by T. v i r i d e . To t e s t t h i s hypothes is in c u l t u r e A. m e l l e a and A. p u l l u l a n s were i nocu la ted on 5% malt agar in p e t r i p l a t e s . They were grown f o r e igh t days t o a l l o w them t o 46 become w e l l e s t a b l i s h e d , then T. v i r i d e was i nocu la ted on the p l a t e s . It q u i c k l y overgrew the e n t i r e s u r f a c e of the agar and i n h i b i t e d f u r t h e r growth of A. m e l l e a . Under the c u l t u r e c o n d i t i o n s used here , the s t i m u l a t i o n of growth of A. m e l l e a by A. p u l l u l a n s was not s u f f i c i e n t t o prevent i n h i b i t i o n by T. v i r i d e . S t i m u l a t i o n o f A. me l l ea on Autoc laved S o i l An experiment was set up to determine if. the substance produced by A. p u l l u l a n s s t i m u l a t e d the growth of A. me l l ea in s t e r i l e s o i l . A loamy g a r -den s o i l was autoc laved in p e t r i p l a t e s f o r one hour before use. Two-s e c t i o n p e t r i p l a t e s were used to keep the media and the fung i s e p a r a t e . In a l l the p l a t e s one s e c t i o n conta ined autoc laved s o i l i nocu la ted w i t h A. m e l l e a . The o ther s e c t i o n conta ined e i t h e r autoc laved s o i l o r 2W agar . Inoculum of A. m e l l e a was e i t h e r 2W agar p lugs or rhizomorph t i p s . The 2W agar p lugs were cut from the margin of mycelium growing on 2W agar . The rhizomorph t i p s were approximate ly 1 cm l o n g , cut o f f where they p r o j e c t e d through the medium ( i . e . , they were a e r i a l rhizomorph t i p s c o n t a i n i n g no e x t e r n a l source of n u t r i e n t ) . In one se t of p l a t e s the second s e c t i o n was i nocu la ted w i t h A. p u l l u l a n s and in the other i t was l e f t as an un inocu la ted c o n t r o l . The e igh t t reatments a re shown in Tab le V I . The r e s u l t s of the experiment us ing autoc laved s o i l in t w o - s e c t i o n p e t r i p l a t e s a re shown in Tab le VI and i l l u s t r a t e d in F igu re 21 . A. m e l l e a produced rhizomorphs in the autoc laved s o i l on l y when A. p u l l u l a n s was grown on 2W agar in the second s e c t i o n of the p l a t e . The development o f rhizomorphs d i d not appear t o be i n f l uenced by the type of inoculum used. TABLE V I . E f f e c t of A. p u l l u l a n s on rhizomorph development o f A. me l l ea in autoc laved s o i l . Treatments Type of inoculum of A. m e l l e a (on autoc laved s o i l in f i r s t s e c t i o n o f p l a t e ) Medium in second s e c t i o n of p l a t e Rhi zomorph development 1. 2W agar p l u g autoc laved s o i l 2. " autoc laved s o i l i nocu la ted w i t h A. p u l l u l a n s 3 . " 2W agar 4. " 2V/ agar i nocu la ted w i t h A. p u l l u l a n s ++ 5 . Rhizomorph t i p autoc laved s o i l 6. " autoc laved s o i l Inoculated w i t h A. p u l l u l a n s 7. " 2W agar 8. " 2W agar i nocu la ted w i t h A. p u l l u l a n s ++ 48 F igu re 2 1 . Growth of A. me l l ea from rhizomorph t i p i n o c -ulum on autoc laved s o i l i n t w o - s e c t i o n p e t r i p l a t e s , L e f t , w i t h s t e r i l e 2W agar in second s e c t i o n ; r i g h t , w i t h A. p u l - 1u1ans growing on 2W agar in second s e c t i o n . 49 There was m y c e l i a l growth but no rhizomorph development by A, m e l l e a in a l l the o ther t r e a t m e n t s , i n c l u d i n g those in which A. p u l l u l a n s was growing on autoc laved s o i l i n the second s e c t i o n . The r e s u l t s of t h i s experiment i n d i c a t e that A. me l l ea can produce rhizomorphs in the autoc laved s o i l i f the s t i m u l a t o r y substance produced by A. p u l l u l a n s i s p r e s e n t . Apparent ly A. p u l l u l a n s was ab le to produce i t in s u f f i c i e n t q u a n t i t y when growing on 21/ agar but not when growing on a u t o -c l a v e d s o i l which has a much lower n u t r i e n t c o n t e n t . Experiments w i t h Natura l Subs t ra tes Spec ies of t r e e s d i f f e r in t h e i r s u s c e p t i b i l i t y t o a t t a c k by A. m e l l e a . Perhaps they a l s o vary as food bases f o r the spread of A. m e l l e a by r h i z o -morphs both under n a t u r a l c o n d i t i o n s and as sources of inoculum f o r l a b o r -a to ry exper iments . To t e s t the second pa r t o f t h i s hypothes is smal l b locks o f s e v e r a l d i f f e r e n t hardwood s p e c i e s were autoc laved and i nocu la ted w i t h A. m e l l e a . They were then p laced in wet n o n - s t e r i l e s o i l and autoc laved s o i l in g l a s s storage d ishes 100 mm in diameter and 80 mm h i g h . Species t e s t e d were Acer c i r c i n a t u m P u r s h , Alnus rubra Bong . , Cornus n u t t a l 1 i I Audubon, and Prunus emarqinata (Dougl . ) D, D i e t r . The inoculum b locks were p i e c e s of branches cut t o approximate ly 2 cm long x I cm in d iameter . The development of rhizomorphs showed no c o n s i s t e n t p a t t e r n in e i t h e r n o n - s t e r i l e o r autoc laved s o i l . They were most f requent from A. c i r c i n a t u m b l o c k s , w i t h A . rub ra the next best s o u r c e . However, t h e r e were some b locks o f a l l s p e c i e s from which rhizomorphs were never produced. To determine i f A , p u l l u l a n s s t i m u l a t e d the growth of A. m e l l e a on D o u g l a s - f i r wood, sapwood samples were i n o c u l a t e d w i t h the two f u n g i . Sap -50 wood p i e c e s approx imate ly 2" x 3 / 4" x 3/8" were cut from the basal p o r t i o n of a young, f r e s h l y - c u t , apparent ly hea l thy D o u g l a s - f i r t r e e . They were d ry -hea t s t e r i l i z e d at 105*C f o r s i x hours , then s a t u r a t e d in s t e r i l e water and d r i e d t o the a p p r o p r i a t e water c o n t e n t . Two water l e v e l s were used, the w e t t e r having a water content of approximate ly 52% by weight and the d r i e r approx imate ly 30%. The wood samples were p laced on s l i d e s in s to rage j a r s w i t h caps t i gh tened t o f i n g e r - t i p t i g h t n e s s . Inoculum c o n s i s t e d of A. m e l l e a or A. p u l l u l a n s growing on smal l segments o f S a l i x s p . branches. Samples at both mo is tu re l e v e l s were i n o c u l a t e d w i t h each fungus s e p a r a t e l y and w i t h the two fung i t o g e t h e r . A f t e r 4 1/2 months the per cent weight l oss o f the samples was determined. The weight l oss in the d i f f e r e n t t reatments i s shown in Tab le V l l . It was very sma l l in a l l samples. Growth of the fung i was poor whether they were a lone or t o g e t h e r . No rhizomorphs were formed by A . m e l l e a on any o f the wood samples. TABLE V l l . Weight l oss (%) of D o u g l a s - f i r sapwood inocu la ted w i t h A. m e l l e a and A. p u l l u l a n s ( a f t e r 4 1/2 months) . Inoculum Weight l oss Wet treatment Dry treatment A. m e l l e a A , p u l l u l a n s A. m e l l e a & A. p u l l u l a n s mean range mean 0.380 0.151 - 0.534 0.285 0.408 0.329 " 0.568 0.306 0.610 0.403 - 0.884 0.361 range 0.105 - 0.548 0.124 - 0.564 0.228 - 0.493 51 Sawdust from v i n e maple (Acer c i r c i n a t u m Pursh) and D o u g l a s - f i r (Pseudotsuga menz tes i i (M i rb . ) Franco) was used t o determine whether A. p u l l u l a n s c o u l d s t i m u l a t e the p r o d u c t i o n of rhizomorphs and the amount o f decay by A, m e l l e a on these s p e c i e s . The sawdust from each s p e c i e s was used in p e t r i p l a t e s e i t h e r in a n o n - s t e r i l e c o n d i t i o n or d ry -heat s t e r i l -i zed at 95*C f o r 24 hours . The dry weight of the s t e r i l i z e d sawdust was measured be fo re 20 ml o f s t e r i l e water were added to each p l a t e . The p l a t e s of each treatment were i n o c u l a t e d w i t h A. m e l l e a growing on v i n e maple branch segments and /or A. p u l l u l a n s growing on sawdust. M y c e l i a l growth and rhizomorph development of A. m e l l e a and the weight l o s s of the s t e r i l i z e d v i n e maple sawdust were recorded a f t e r S 1/2 weeks. No rhizomorphs were formed in any of the t rea tments . There was no v i s i b l e d i f f e r e n c e in the growth of A. m e l l e a w i t h i n any o f the t reatments on D o u g l a s - f i r sawdust or on v i n e maple sawdust but growth was b e t t e r on v i n e maple sawdust than on D o u g l a s - f i r sawdust. The weight l oss on s t e r i l -i zed v i n e maple sawdust is shown in Tab le V I I I . It was not great enough TABLE V I I I . Weight l o s s (%) of v i n e maple sawdust by A. m e l l e a and A. p u l l u l a n s . Treatment % Weight l o s s mean range A. m e l l e a 1.94 0.60 - 2.83 A. p u l l u l a n s 3.08 2.53 - 3.48 A. m e l l e a & 2.38 1.38-2.98 A. p u l l u l a n s Un inocu la ted c o n t r o l 0.88 0.56 - 1.14 52 i n any of the t reatments t o i n d i c a t e that A. p u l l u l a n s i n f l uenced the growth of A. m e l l e a , DISCUSSION Severa l p o i n t s are apparent in a c o n s i d e r a t i o n of the s t i m u l a t i o n of growth o f A. m e l l e a by A. p u l l u l a n s . The f i r s t is the r o l e of the n u t r i e n t l e v e l o f the s u b s t r a t e . A. p u l l u l a n s produced a s t i m u l a t o r y substance which promoted the growth of A. m e l l e a rhizomorphs in a medium c o n t a i n i n g s u f f i c i e n t n u t r i e n t s . There was a n u t r i e n t l e v e l below which rhizomorphs were not i n i t i a t e d by A . meI lea when A. p u l l u l a n s was p r e s e n t , even though m y c e l i a l growth was s t i m u l a t e d . The s t i m u l a t o r y substance produced by A. p u l l u l a n s c o u l d not t r i g g e r rhizomorph d i f f e r e n t i a t i o n when the n u t r i e n t l e v e l was too low to support rhizomorph development. Th is s i t u a t i o n i s in agreement w i t h G a r r e t t ' s o b s e r v a t i o n (17) tha t a c e r t a i n n u t r i e n t s t a t u s o f the s u b s t r a t e i s e s s e n t i a l f o r rhizomorph i n i t i a t i o n . Second ly , the s t i m u l a t i o n of A. m e l l e a by A. p u l l u l a n s appears t o be an e f f e c t on growth in general - on m y c e l i a l development as w e l l as on rhizomorph i n i t i a t i o n and e l o n g a t i o n . When a water agar p l u g inoculum c o n -t a i n i n g on ly sparse mycelium o f A. m e l l e a was p l a c e d on 2W agar , no r h i z o -morphs were formed du r i ng the 21 days of the exper iment . If A . p u l l u l a n s was a l s o growing on 2W p l a t e s , rhizomorphs were d i f f e r e n t i a t e d and e longated r a p i d l y . However, when the A. m e l l e a inoculum was a rhizomorph t i p ( i . e . , the rhizomorph was a l ready d i f f e r e n t i a t e d ) the 2W medium supported c o n t i n -ued e l o n g a t i o n . If the rhizomorph t i p was p l a c e d on 2W agar c o n t a i n i n g the c e l l - f r e e f i l t r a t e of an A . p u l l u l a n s l i q u i d c u l t u r e , i t grew more r a p i d l y and formed more new rhizomorphs than when i t was p l a c e d on 2W agar a l o n e . 53 When the n u t r i e n t l e v e l of the s u b s t r a t e was too low t o a l l o w rhizomorph i n i t i a t i o n t o take p l a c e , as on the W agar, m y c e l i a l growth was s t i m u l a t e d by the presence of A. p u l l u l a n s . One can draw two c o n c l u s i o n s from these ob s e r v a t i o n s : f i r s t , the 2W medium i s capable of supporting rhizomorph growth once i t has been d i f f e r e n t i a t e d , and second, the s t i m u l a t o r y sub-stance produced by A. p u l l u l a n s f u n c t i o n s i n m y c e l i a l development, r h i z o -morph d i f f e r e n t i a t i o n and rhizomorph e l o n g a t i o n . The p o s s i b i l i t y that A. p u l l u l a n s s u p p l i e d a v i t a m i n r e q u i r e d by A. m e l l e a was c o n s i d e r e d , but both fungi have been reported t o r e q u i r e only thiamine s u p p l i e d i n the medium (17,49). Therefore i t seemed u n l i k e l y that the substance produced by A. p u l l u l a n s was a v i t a m i n . Weinhold and Garroway (46) reported t h a t , on the glucose-ammonium phosphate medium used f o r t h e i r t e s t s , a small amount of ethanol appeared t o be necessary u n t i l rhizomorph i n i t i a t i o n was complete and e l o n g a t i o n had begun. Th e i r c o n c l u s i o n i s e s s e n t i a l l y s i m i t a r t o that reported here f o r the s t i m u l a t o r y substance produced by A. p u l l u l a n s . The experiments described i n t h i s report i n d i c a t e that the s t i m u l a t o r y substance produced by A. p u l l u l a n s i s e t h a n o l . It was the only v o l a t i l e substance recorded In an a n a l y s i s w i t h the gas chromatograph of the metabol-i t e s l i b e r a t e d i n t o the 2W l i q u i d medium by A. p u l l u l a n s . The c e l l - f r e e f i l t r a t e o f an A. p u l l u l a n s l i q u i d c u l t u r e and the 2W medium c o n t a i n i n g ethanol were a f f e c t e d the same way by the ion exchange r e s i n s . Thus there were no r e s u l t s which suggested that tnore than one s t i m u l a t o r y compound was produced and none of the evidence r e f u t e d the hypothesis that ethanol i s the e f f e c t i v e substance produced by A. p u l l u l a n s . There have been numerous reports of organisms which i n h i b i t the growth °f mellea (6,28,38*43). A. p u l l u l a n s i s the o n l y microorganism reported 54 so f a r which s t i m u l a t e s i t s growth. Experiments us ing D o u g l a s - f i r sap wood samples , D o u g l a s - f i r sawdust or v i n e maple sawdust t o t e s t the s t i m u l a t i o n o f A. m e l l e a by A. p u l l u l a n s y i e l d e d r e s u l t s which d i d not i n d i c a t e t h e i n f l u e n c e of A . p u l l u l a n s on the growth o f A . m e l l e a . A. me l l ea f a i l e d t o grow w e l l on any o f the s u b -s t r a t e s under the c o n d i t i o n s o f the exper iments . However, D o u g l a s - f i r i s a s p e c i e s f r e q u e n t l y a t tacked by A. m e l l e a under n a t u r a l c o n d i t i o n s , i n d i c -a t i n g that the l a c k o f growth here was the r e s u l t o f the l abo ra to ry c o n d i -t i o n s used . There was, t h e r e f o r e , no s a t i s f a c t o r y assessment of the i n f l u -ence o f A. p u l l u l a n s on the growth of A. m e l l e a on the s u b s t r a t e s . I n v e s t i g a t i o n o f the p h y s i c a l p r o p e r t i e s of the s t i m u l a t o r y substance produced by A. p u l l u l a n s showed that i t was a v o l a t i l e compound. An import -ant c o r a l l o r y Is t ha t A. m e l l e a can u t i l i z e i t at a s i t e removed from the source o f p r o d u c t i o n . Th is a b i l i t y may g i v e A. m e l l e a a c o m p e t i t i v e advan-tage in i t s n a t u r a l h a b i t a t . A . p u l l u l a n s o r o the r organisms produc ing the same substance may r e l e a s e i t i n t o the s o i l atmosphere where i t would d i f f u s e and become a v a i l a b l e t o A. m e l l e a at a d i s t a n c e from the s i t e o f p r o d u c t i o n . A . reel l e a would then not be in d i r e c t c o m p e t i t i o n w i t h the producers o r o the r organisms f o r the n u t r i e n t s at the p r o d u c t i o n s i t e or p o s s i b l y f o r the s u b s t a n c e . One rhizomorph t i p o f A . m e l l e a w i t h no a d d i t i o n a l n u t r i e n t s c o u l d develop an e x t e n s i v e rhizomorph system in autoc laved s o i l I f t he s t i m u l a t o r y substance produced by A. p u l l u l a n s was a v a i l a b l e . The n u t r i e n t s t a t u s o f autoc laved s o i l was s u f f i c i e n t t o support the growth o f A. meI lea r h i z o -morphs when the s t i m u l a t o r y substance was p r e s e n t , even when t h e r e was no food base incorporated i n the Inoculum. It i s dangerous t o r e l a t e the behaviour of an organism i n au toc laved s o i l in a p e t r i p l a t e t o i t s behav-55 lour under n a t u r a l c o n d i t i o n s , but the p o s s i b i l i t y e x i s t s that the a v a i l -i b i l i t y o f a s t i m u l a t o r y substance such as ethanol in the s o i l may g r e a t l y a f f e c t the development o f A. m e l l e a . Weinhold (43) repor ted that the optimum c o n c e n t r a t i o n o f ethanol appeared t o be 5--0 ppm i n c u l t u r e s of A. mel l e a which he grew f o r 14 days a f t e r adding the e t h a n o l . However, experiments c a r r i e d out here i n d i c a t e tha t t h e cont inuous presence of lower c o n c e n t r a t i o n s of ethanol can s u b -s t i t u t e f o r t h i s i n i t i a l h igher c o n c e n t r a t i o n . The same c o n c e n t r a t i o n of ethanol in increased volumes o f medium, and t h e r e f o r e w i t h increased amounts of e t h a n o l , r e s u l t e d in increased growth o f A. m e l l e a . T h i s i n d i c -ated that c o n c e n t r a t i o n i s not an abso lu te c r i t e r i o n . A lower c o n c e n t r a -t i o n o f ethanol renewed over a p e r i o d of t ime to g i v e the same f i n a l amount o f e thano l as an i n i t i a l h igher c o n c e n t r a t i o n was as e f f e c t i v e in s t i m u l a t -ing the growth o f A . m e l l e a as the i n i t i a l h igher one. There fo re It appears tha t i t i s not one optimum c o n c e n t r a t i o n o f ethanol in the medium, but a very sma l l amount c o n t i n u o u s l y p r e s e n t , which Is necessary t o s t i m u l a t e the growth o f A. m e l l e a . The s i g n i f i c a n c e of the f a c t tha t the cont inued p r e s -ence of a c o n c e n t r a t i o n o f ethanol as low as 50 ppm can s t i m u l a t e the growth o f A . m e l l e a i s tha t A. m e l l e a is more l i k e l y t o encounter a cont inuous supply of sma l l amounts o f ethanol under n a t u r a l c o n d i t i o n s . A number of s o ! I - I n h a b i t i n g microorganisms and w o o d - r o t t i n g fung i have been repor ted t o produce ethanol as a r e s u l t o f t h e i r metabol ism (29, 30,33,50). Stevenson and Katzne lson (40) s t u d i e d the o x i d a t i o n of ethanol and a c e t a t e by the s o i l microorganisms in i n t a c t s o i l . They found that ethanol was o x i d i z e d t o a c e t a t e wi thout a p r e l i m i n a r y l a g p e r i o d . The r a t e o f o x i d a t i o n remained r e l a t i v e l y constant f o r one s o i l but v a r i e d markedly w i t h s o i l s o f d i f f e r e n t f e r t i l i t y l e v e l s . Alexander (1) s a y s : 56 "S imp le substances that a re added t o s o i l a re r e a d i l y metabo l i zed but always w i t h an apparent l a g p e r i o d p r i o r t o the maximal o x i d a -t i o n r a t e . The l a g represents the t ime necessary f o r the p o p u l a -t i o n t o i nc rease t o an extent s u f f i c i e n t to cause r a p i d o rgan ic t u r n o v e r . E t h a n o l , however, i s o x i d i z e d r e a d i l y w i t h no p r e l i m i n -ary l a g . Th is anomalous c h a r a c t e r i s t i c o f ethanol decomposi t ion has been repor ted in a number o f s o i l t y p e s . . . . A c e t a t e i s a l s o o x i d i z e d wi thout a l ag p e r i o d , but a c t i v i t y q u i c k l y d e c l i n e s . S i n c e ethanol and a c e t a t e a re metabo l i zed immediately , i t seems l i k e l y tha t the autochthonous p o p u l a t i o n i s adapted t o these s u b -s t r a t e s through repeated and f requent encounters . Thus, the two compounds are probab ly c o n t i n u o u s l y formed and m e t a b o l i z e d . Should t h i s Indeed be t r u e , ethanol and a c e t a t e would be n a t u r a l i n te rmed -i a t e s i n humus d e c o m p o s i t i o n . " Thus i t appears tha t A . m e l l e a may indeed f r e q u e n t l y encounter sma l l amounts o f ethanol In t h e s o i l . One of the f a c t o r s which may i n f l u e n c e the amount of ethanol present i n the s o i l i s s o i l m o i s t u r e . A number o f authors have s t u d i e d the e f f e c t of s o i l m o i s t u r e on the development o f A. m e l l e a . Sokolov (38) concluded that Increased s o i l mo is tu re promotes the spread of rh izomorphs, but does not a f f e c t the d i s t r i b u t i o n of the mycelium under the bark of d i s e a s e d t r e e s . In N i g e r i a Fox (16) found that h igh r a i n f a l l and impeded d ra inage appeared t o enhance the i nc idence and s e v e r i t y o f a t t a c k by A. m e l l e a . Under exper imenta l c o n d i t i o n s w i t h s o i l in g l a s s tubes G a r r e t t (18) r e p o r t -ed no s i g n i f i c a n t d i f f e r e n c e In the development of A. m e l l e a between 40%, 60% and 80% o f the m o i s t u r e h o l d i n g c a p a c i t y o f the s o i l . Jenn ison et al_ . (26) found that good growth o f A. m e l l e a was obta ined on l y in a l e s s h i g h l y o x i d i z e d medium (Eh = +300 mv) than that used f o r most o f the w o o d - r o t t i n g fung i (Eh = +400 t o 500 mv). It t h e r e f o r e appears tha t A. m e l l e a grows very w e l l in wet s o i l s , i n which t h e r e may be a l a r g e r number of m i c r o -organisms produc ing ethanol under semi -anaerob ic c o n d i t i o n s than in d r i e r s o i l s . Forest s i t e s , s o i l types and host t r e e s may vary c o n s i d e r a b l y in t h e 57 amount of ethanol that is a v a i l a b l e t o a fungus such as A. mellea. Steven-son and Katznelson (40) found that the r a t e of o x i d a t i o n of ethanol v a r i e d markedly w i t h s o i l s o f d i f f e r e n t f e r t i l i t y l e v e l s . There may a l s o be seasonal v a r i a t i o n s at one l o c a t i o n , as the population of s o i l microorgan-isms develops and changes. The p o s s i b i l i t y e x i s t s that i n d i v i d u a l t r e e s or t r e e species vary i n the amount of ethanol a v a i l a b l e t o an invading fungus such as A, mellea. Perhaps a study of the amount of ethanol a v a i l a b l e in d i f f e r e n t s o i l s and i n d i f f e r e n t t r e e s would account f o r some of the unex-p l a i n e d v a r i a t i o n in p a t h o g e n i c i t y and occurrence of A. mellea and c l a r i f y i t s r o l e as a primary pathogen. CONCLUSIONS 1. Aureobasidlum p u l l u l a n s produces a v o l a t i l e , h e a t - s t a b l e substance which s t i m u l a t e s the growth of A r m i l l a r i a mellea in c u l t u r e . 2. The s t i m u l a t i o n appears t o be an e f f e c t on s e v e r a l phases of the growth of A. m e l l e a , i n c l u d i n g mycelia) development, rhizomorph i n i t i a t i o n and rhizomorph e l o n g a t i o n . 3. The s t i m u l a t o r y substance produced by A. p u l l u l a n s appears t o be et h a n o l . 4. A low c o n c e n t r a t i o n of ethanol c o n t i n u o u s l y present Is s u f f i c i e n t t o s t i m u l a t e the growth of A. mellea on a glucose-asparagine medium. Ethanol as 50 ppm s u p p l i e d d a l l y r e s u l t e d in a great increase i n the number of rhizomorphs produced by A. mellea In 14 days. 58 BIBLIOGRAPHY 1 . A lexander , M. 1961 . I n t roduct ion t o S o i l M i c r o b i o l o g y . John Wi ley and Sons I n c . , New York , 472 p. 2 . 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N i t rogen and carbon n u t r i t i o n of A r m ? 1 l a r i a me l l ea in r e l a t i o n t o growth-promoting e f f e c t s of e t h a n o l . Phytopathology 56: 108-112. 4 3 . Weinhold , A . R . , F . F . Hendr ix and R.D. Raabe. 1962. S t i m u l a t i o n of rhizomorph growth of Armi11 a r i a m e l l e a by i n d o l e - 3 - a c e t i c a c i d and figwood e x t r a c t . A b s t r . in Phytopathology 52: 757. 49 . Ward, E.W.B. I960. Thiamine requirement of P u l l u l a r i a p u l l u l a n s (de Bary) Berkhout . Can. J . Botany 38: 891-894. 50 . Wh i take r , D.R. and P . E . George. 1951. S tud ies in the b iochemis t ry of c e l l u l o l y t i c microorganisms. I i . Metabo l i c products of Polyporus a b i e t i n u s . Peniophora g i g a n t e a . and Hydnum s e p t e n t r i o n a l e . Can^ J . Botany 29: 176-181". 5 1 . Wiken, T. 1946. Examinat ion of e x t r a c t s from sporophores of Swedish Hymenomycetes f o r a n t i b i o t i c a c t i v i t y aga inst P u l l u l a r i a p u l l u l a n s (De Bary et Loew) Berkhout . A r k i v f u r Botan ik 33 A: 1-12. 62 Par t I I . The E f f e c t o f S o i l M o i s t u r e on the Growth and Spread o f C o n i o -phora puteana (Schum. ex F r . ) K a r s t . under Laboratory Cond i t i ons INTRODUCTION Very l i t t l e i s known about the sap rophy t i c behaviour in s o i l o f r o o t -d i s e a s e pathogens of f o r e s t t r e e s . There i s a l a c k o f ev idence f o r m y c e l -i a l growth o f these pathogens in f o r e s t s o i l s , w i t h l i t t l e c o n s i d e r a t i o n g iven t o l e v e l of s o i l mo is tu re as an important f a c t o r a f f e c t i n g t h e i r s p r e a d . The amount of mois tu re i s one of the most v a r i a b l e f a c t o r s in the s o i l , i t a f f e c t s , t o a c o n s i d e r a b l e e x t e n t , o the r s o i l f a c t o r s such as a e r a t i o n and temperature . As Raney (27) s a y s , " . . . water dominates a l l o f the s o i l p h y s i c a l f a c t o r s . It i s g e n e r a l l y imposs ib le t o change mo is tu re wi thout s i m u l t a n e o u s l y i n f l u e n c i n g o ther p r o p e r t i e s . " There fo re a l a b o r a t -ory study was undertaken o f the e f f e c t o f d i f f e r e n t l e v e l s o f s o i l mo is tu re on the development and spread o f Coniophora puteana (Schum. ex F r . ) K a r s t . in a f o r e s t s o i l . C. puteana is a thelephoraceous fungus which causes a brown c u b i c a l root and butt ro t o f f o r e s t t r e e s (1,8,9,21). It is a l s o r e s p o n s i b l e f o r d e t e r i o r a t i o n o f s l a s h (20) and decay of wood in s e r v i c e , p a r t i c u l a r l y in Great B r i t a i n and Europe, where i t i s f r e q u e n t l y c a l l e d Coniophora c e r e b e l l a ( P e r s . ) Duby (7). C. puteana was chosen f o r t h i s study because i t has a wide geograph ica l range and because the mycelium i s compara t i ve ly easy t o recogn i ze in s o i l and in pure c u l t u r e . 63 LITERATURE REV JEW S o i l M o i s t u r e and S o i l Fungi G r i f f i n (13) has d i s c u s s e d the b a s i c concepts of s o i l mo is tu re c o n -tent and i t s i n f l u e n c e on the ecology of s o i l f u n g i . He p o i n t s out tha t the h y d r a u l i c c o n d u c t i v i t y of a s o i l i s s h a r p l y reduced when the mo is tu re content f a l l s below f i e l d c a p a c i t y and the major n o n - c a p i l l a r y pore systems are empty. Th is in i t s e l f w i l l not impede the growth of fung i because, " i t seems probab le that n e a r l y a l l fung i w i l l be ab le t o exer t the necessary f o r c e to absorb water and t o grow unimpeded by reduced h y d r a u l i c c o n d u c t i v -i t y o f the s o i l throughout the s u c t i o n range pF 0 - h.2 and even in d r i e r s o i l s . In s o i l s d r i e r than permanent w i l t i n g p o i n t , however, a b i l i t y w i l l be l i k e l y t o d i f f e r from one s p e c i e s t o another , probably l ead ing t o an e c o l o g i c a l d i v e r s i f i c a t i o n . " G r i f f i n concludes that a e r a t i o n i s the e f f e c -t i v e agent i n f l u e n c i n g fungal a c t i v i t y , and that a e r a t i o n i s a f f e c t e d by such f a c t o r s as mo is tu re c o n t e n t , t e x t u r e and s t r u c t u r e . In a d i s c u s s i o n of the e f f e c t on s o i l microorganisms of s o i l p h y s i c a l f a c t o r s , Raney (27) suggests that s o i l microorganisms can probably w i t h -stand h igher mo is tu re tens ions than p l a n t s when the s o i l mo is tu re l e v e l i s c o n s t a n t . When changes in s o i l mo is tu re are cont inuous microorganisms probably respond about the way p l a n t s do. Sewel l (23) notes that a l though s o i l mo is tu re is r e a d i l y manipulated in the f i e l d the i n t e r p r e t a t i o n of i t s a f f e c t s on d i seases and pathogens requ i res c o n s i d e r a b l e c a u t i o n . Even when s o i l mo is tu re i s known t o a f f e c t the pathogen d i r e c t l y , host responses t o mo is tu re content may s i g n i f i c a n t l y a f f e c t d i s e a s e development. Thus i t i s d i f f i c u l t t o a t t r i b u t e any d i s e a s e 64 response t o mo is tu re a l o n e . In f l ooded s o i l , where the a n t a g o n i s t i c mechanism may be in f l uenced by other f a c t o r s i n c l u d i n g oxygen a v a i l a b i l i t y , m i c r o b i a l a c t i v i t y may be d i r e c t l y or i n d i r e c t l y r e s p o n s i b l e f o r the d e s -t r u c t i o n of c e r t a i n pathogens. Stover (29), in a study of the e f f e c t of s o i l mo is tu re on s p e c i e s o f Fusarlum found that the optimum growth and s u r v i v a l o f the fung i occur red in s o i l at 15% s a t u r a t i o n . Optimum s o l i mo is tu re content f o r act inomycetes was s i m i l a r to that f o r Fusariurn, w h i l e the optimum f o r b a c t e r i a was 75% s a t u r a t i o n . He concluded that a c o m p e t i t i v e b a c t e r i a l f l o r a at h igh mo is tu re l e v e l s was a f a c t o r reduc ing Fusarium p o p u l a t i o n s . The b a c t e r i a l f l o r a may be in pa r t a n t a g o n i s t i c but i t a l s o was capab le of competing s u c c e s s f u l l y f o r a v a i l a b l e oxygen and food . In a l a t e r rev iew Stover (30) noted that the e f f e c t s of s o i l mo is tu re on r o o t - i n f e c t i n g fung i i n d i c a t e tha t the optimum f o r growth and s u r v i v a l i s broad f o r most f u n g i . However, fungus growth and s u r v i v a l c o u l d be i n f l uenced by a change of n u t r i e n t o r f u n g i t o x i n s t a t u s as a r e s u l t o f d e s i c c a t i o n . Maciejowska and W i l l i a m s (22) examined the e f f e c t of mo is tu re l e v e l and c e l l u l o s e a d d i t i o n s on the myco f lo ra of s o i l s . They concluded that a v a i l a b l e inoculum and the mo is tu re l e v e l o f s o i l s o f s i m i l a r pH va lues are major f a c t o r s in dete rmin ing the compos i t ion of the myco f l o ra of ce l lu lose -amended s o i l . In 1934 G a r r e t t (14) concluded that "o the r c o n d i t i o n s being s u i t a b l e , b i o l o g i c a l antagonism increases w i t h s o i l mo is tu re over the range 30 to 80% s a t u r a t i o n " . Th is c o n c l u s i o n came from a study of f a c t o r s a f f e c t i n g the p a t h o g e n i c i t y of c e r e a l f o o t - r o t f u n g i . In h i s more recent books Gar re t t (15,16) has not f o l l o w e d up t h i s c o n c l u s i o n . The e f f e c t i v e n e s s of t h r e e f u n g i c i d e s t e s t e d by Purdy (26) f o r c o n -65 t r o ) o f the wheat f l a g smut fungus was in f l uenced by both s o i l mo is tu re and s o i l temperature . The f u n g i c i d e s f a i l e d t o c o n t r o l the d i s e a s e at low s o i l mo is tu re c o n t e n t s , but p rov ided some p r o t e c t i o n at h igher mo is tu re l e v e l s . S o i l F u n g i s t a s i s In 1953 Oobbs and Hinson (11) desc r i bed a "widespread f u n g i s t a t i c f a c t o r " which e x i s t s in s o i l . The e x t e n s i v e research t o e l u c i d a t e the nature of s o i l f u n g i s t a s i s and the c o n d i t i o n s a f f e c t i n g i t has been summar-i zed in s e v e r a l r e v i e w s , i n c l u d i n g those by Dobbs, Hinson and Bywater (12), B r i a n (3), Jackson (19) and o t h e r s . In h i s rev iew of the mechanisms l i m i t i n g a c t i v i t y and s u r v i v a l o f fung i in s o i l , B r i a n (3) cons idered the f o l l o w i n g mechanisms: compet i t i on f o r n u t r i e n t s , c a p a c i t y f o r r a p i d hyphal e x t e n s i o n , p a r a s i t i s m , p roduct ion of a n t i b i o t i c s and r e s i s t a n c e t o chemical i n h i b i t o r s . He concluded that none of them alone c o u l d p o s s i b l y e x p l a i n a l l cases of d i f f e r e n t i a l s u r v i v a l , but a n t i b i o t i c p roduct ion seemed the most l i k e l y e x p l a n a t i o n of f u n g i s t a s i s . B r i a n d i d not d i s c u s s the p o s s i b i l i t y that the i n d i v i d u a l mechanisms may act d i f f e r e n t l y as o ther f a c t o r s change. Jackson (19), r ev iewing a n t i b i o s i s and f u n g i s t a s i s in s o i l m i c r o -o rgan isms, concluded that " a n t i b i o s i s in one form or another , p l ays a key r o l e in the ecology o f s o i l microorgan isms, be ing perhaps second in import -ance o n l y t o c o m p e t i t i o n f o r n u t r i e n t s " . There is l i t t l e doubt of the b i o l o g i c a l o r i g i n of f u n g i s t a s i s , accord ing t o Dobbs, Hinson and Bywater (12), but " i n v iew of the complex i ty o f the s o i l p o p u l a t i o n i t may not be easy t o d i s t i n g u i s h the p a r t s p layed by any p a r t i c u l a r organism which when i s o l a t e d can i n h i b i t fungal growth o r 66 g e r m i n a t i o n " . Oobbs ( iO) commented that the phenomenon of non-germ(nat ion o f spores or growth of hyphae on c e l l u l o s e f i l m or o the r m a t e r i a l s permeable t o the s o i l s o l u t i o n c o u l d on l y be e x p l a i n e d by the presence of a w a t e r -s o l u b l e chemica l i n h i b i t o r . Spread of Wood - ro t t ing Basid iomycetes in S o i l Menzies (2k) in a rev iew of the s u r v i v a l of p l a n t pathogens in s o i l , po in ted out tha t there were few c o n t r o l l e d experiments t o determine the c o m p e t i t i v e s a p r o p h y t i c a b i l i t y o f w o o d - r o t t i n g Bas id iomycetes . He s u g -gested that they probab ly do not encounter in tense c o m p e t i t i o n f o r food because o f t h e i r s p e c i a l i z e d a b i l i t i e s t o u t i l i z e c e l l u l o s e and l i g n i n . Buck land , Molnar and Wal l i s (6) found no spread of " a n n u a l " P o r i a w e i r i i Murr . from O o u g l a s - f i r inoculum b locks embedded in humus and minera l s o i l h o r i z o n s . They a t t r i b u t e d the lack of spread t o s o i l m i c r o -organisms which had an a n t a g o n i s t i c or s u p p r e s s i v e a c t i o n on P. w e i r i i and prevented or g r e a t l y impeded i t s spread through s o i l under n a t u r a l c o n d i -t i o n s . However, no exper imenta l c o n s i d e r a t i o n was g iven t o s o i l mo is tu re o r o the r v a r i a b l e s o i l f a c t o r s which may have a f f e c t e d the a b i l i t y o f P.. w e i r i i t o spread in s o i l . In a study of the growth of Fomes annosus ( F r . ) Cke . in n o n - s t e r i l e s o i l in v i t r o . Francke-Grosmann (13) found the r e s u l t s of her experiments were not un i fo rm. She concluded that the r e a l f a c t o r c o n t r o l l i n g the r e s u l t s was the presence or absence of a n t a g o n i s t i c microorgan isms. The s o i l mo is tu re and the s i z e o f s o i l p a r t i c l e s were a l s o important f a c t o r s . Oobbs, Hinson and Bywater (12) s t u d i e d the m y c e l i a l growth o f 11 Bas id iomycetes , i n c l u d i n g C. c e ^ e b e l l a . in 12 f o r e s t - s o i l samples. They 67 found an average of 48% reduct ion of growth,of C. cerebe l1 a in a l l samples, us ing an agar and c e l l u l o s e f i l m techn ique in " m o i s t " s o i l . The amount of i n h i b i t i o n of C_. ce rebe l l a was w ide l y v a r i a b l e , ranging from none t o com-p l e t e i n h i b i t i o n , as compared w i t h the c o n t r o l s . Other w o o d - r o t t i n g fung i showed average percentage reduct ions ranging from 54% t o 100%. MATERIALS AND METHODS The s o i l used in a l l experiments c o n s i s t e d of the H and A] ho r i zons from a D o u g l a s - f i r p l a n t a t i o n approximate ly 35 years o l d on the campus of the U n i v e r s i t y of B r i t i s h Columbia . Thus the s o i l c o n s i s t e d of a m ix tu re of the undecomposed and p a r t i a l l y decomposed humus m a t e r i a l s , and the upper minera l s o i l in which o rgan ic matter had become i n c o r p o r a t e d . It was a sandy s o i l w i t h an o rgan ic content of about 15% and a pH o f 5 . 3 . The s o i l was a i r - d r i e d and s i e v e d through a 2 mm p o r e - s i z e s i e v e before use. D i s t i l l e d water was added t o 25 g o f a i r - d r y s o i l in each p e t r i d i s h t o b r i n g i t to the requ i red mo is tu re l e v e l . In the p r e l i m i n a r y experiments mo is tu re l e v e l s of 20%, 40%, 70% and 100% of s a t u r a t i o n were used. The l e v e l s in l a t e r experiments were changed t o 25%, 50%, 75% and 100% of s a t u r a t i o n f o r convenience in c a l c u l a t i o n s . When s t e r i l i z e d s o i l was used the mo is tu re l e v e l was ad justed be fo re a u t o c l a v i n g f o r one hour at 15 lbs p r e s s u r e . The l e v e l was read jus ted w i t h s t e r i l e water a f t e r a u t o c l a v i n g because some water was l o s t du r ing the p r o c e s s . In a l l experiments t h e r e were at l e a s t 5 r e p l i c a t e s of each t reatment . An I s o l a t e of C. puteana. obta ined from the Forest Products L a b o r a t o r y , Canada Department o f F o r e s t r y , Vancouver , B .C . ,was used throughout the s t u d y . Inoculum f o r a l l experiments c o n s i s t e d of segments of a l d e r branches (Alnus 68 rubra Bong.) approx imate ly 10 mm high by 7 mm in diameter which had been autoc laved and i nocu la ted w i t h a m y c e l i a l suspension of C. puteana. They were incubated f o r approx imate ly two months so the fungus was w e l l e s t a b -l i s h e d on the a l d e r d i s c s be fo re use . A l l p e t r i d i shes were incubated in the dark at room temperature (approximately 23°C) under p l a s t i c cove rs to prevent e x c e s s i v e d r y i n g . V i s u a l measurements of r a d i a l growth were d i f f i c u l t because the fungus o f t e n d i d not invade the whole p l a t e at the same r a t e and because the m y c e l -i a l s t rands and hyphae were not always v i s i b l e on the s u r f a c e o f the s o i l . There fo re the r e s u l t s are presented as d e s c r i p t i o n s and i l l u s t r a t i o n s of the growth p a t t e r n s . EXPERIMENTS AND RESULTS S o i l pH The i n i t i a l pH of the s o i l was 5.3. Measurements of pH showed that the re was l i t t l e change in the a c i d i t y du r ing any of the exper iments . N o n - s t e r i l e and Autoc laved S o i l The f i r s t experiment c o n s i s t e d of i n o c u l a t i n g n o n - s t e r i l e and a u t o -c l a v e d s o i l at mo is tu re l e v e l s of 20%, 40%, 70% and 100% of s a t u r a t i o n w i t h C. puteana. The experiment was repeated us ing mo is tu re l e v e l s of 25%, 50%, 75% and 100%. In the n o n - s t e r i l e s o i l the fungus spread s l o w l y but s t e a d -i l y through the s o i l at the 20 - 25% mo is tu re l e v e l , forming m y c e l i a l s t rands r a t h e r than d i s p e r s e d hyphae (F igure 1 ) . It grew somewhat more s l o w l y at the 40 - 50% l e v e l (F igu re 2 ) . At l e v e l s of 70 - 75% and 100% 69 F i g u r e 1. T h e g r o w t h o f C . p u t e a n a a f t e r t h r e e w e e k s i n n o n - s t e r i l e s o i l w i t h a m o i s t u r e c o n t e n t o f 20% o f s a t u r a t i o n . 70 Figure 2. The growth of C. puteana after three weeks in non-sterile s o i l with a moisture content of 40% of saturat ion. 71 s a t u r a t i o n C. puteana d i d not invade the s o i l , and the inoculum was f r e q -u e n t l y invaded by Trichoderma v i r i d e P e r s , ex F r , and by nematodes (F igures 3 and k). The hyphae of C. puteana u s u a l l y d isappeared from the inoculum at the h igher mo is tu re l e v e l s i Table I shows the r e l a t i v e amount of growth of C_. puteana in the d i f f e r e n t s o i l t reatments . In autoc laved s o i l the fungus was ab le t o complete ly c o l o n i z e the s o i l at a l l mo is tu re l e v e l s , w i t h growth be ing more dense at the 70 - 75% and 100% l e v e l s than in the d r i e r ones (F igu re 5 ) . An autoc laved c o n t r o l s e r i e s was inc luded in a l l subsequent exper iments , g i v i n g s e v e r a l r e p l i c a t i o n s of t h i s t reatment . Apparent ly the mo is tu re l e v e l a lone d i d not i n h i b i t the growth o f C. puteana in n o n - s t e r i l e s o i l , but d i d i n f l u e n c e other f a c t o r s a f f e c t i n g the growth of the fungus. V/ood Traps in N o n - s t e r i l e S o i l To determine whether C, puteana was capab le of invading s o i l and c o l o n i z i n g wood which was not in d i r e c t contact w i t h the inoculum, the fungus was i nocu la ted in n o n - s t e r i l e s o i l at mo is tu re l e v e l s of 25%, 50%, 75% and 100% o f s a t u r a t i o n . Fresh n o n - s t e r i l e a l d e r d i s c s (not c o l o n i z e d by C. puteana) were p laced in a c i r c l e sur rounding the inoculum and approx-imately 25 mm from i t (F igure 6). The p e t r i p l a t e s were incubated f o r 28 days . In a s i m i l a r experiment the C. puteana inoculum was p laced at the margin of p e t r i p l a t e s c o n t a i n i n g n o n - s t e r i l e s o i l w i t h mo is tu re l e v e l s of 25% and 100% s a t u r a t i o n . A lde r d i s c t raps were p laced in two arcs approx-imate ly 12 mm and 27 mm from the inoculum. F i g u r e 7 shows the arrangement of the d i s c s and the growth of C. puteana in the s o i l at the 25% mo is tu re l e v e l . TABLE I. Growth o f C, puteana in f o r e s t s o i l a f t e r t h r e e weeks. Treatment S o i l mo is tu re l e v e l (% mois tu re of s a t u r a t i o n ) N o n - s t e r ? l e s o i I 20 - 25 +++ hO - 50 70 - 75 100 Autoc laved s o i l *+++ ++++ ++++ N o n - s t e r i l e so i 1 added t o autoc laved so i1 T. v i r i d e in autoc laved s o i 1 +++ 100 ug /g A c t i ' d ione in non -s t e r i l e so i1 -H-100 ug /g A c t i -d lone in auto -c1aved so i1 ++++ growth to margin o f p l a t e +++ 3/4 o f p l a t e c o l o n i z e d ++ 1/2 o f p l a t e c o l o n i z e d + s l i g h t growth from inoculum - no growth F i g u r e 3. The growth of C. puteana a f t e r t h r e e weeks in n o n - s t e r i l e s o i l w i t h a mois ture content of 70% of s a t u r a t i o n . Note invas ion of inoculum by T. v i r i d e . Figure 4. The growth of C. puteana a f t e r three weeks in n o n - s t e r i l e s o i l w i t h a moisture content of 100% of s a t u r a t i o n . Note invasion of inoculum by T. v i r i d e . F i g u r e 5. The growth of C_. puteana a f t e r two weeks in au toc laved s o i l . The f i g u r e s show the s o i l mo is tu re content as a per cent o f s a t u r a t i o n . 76 F i g u r e 6. The invas ion of a l d e r d i s c t raps by C. puteana a f t e r 26 days in n o n - s t e r i l e s o i l w i t h a mo is tu re content of 25% o f s a t u r a t i o n . F igu re 7. The invas ion of a lde r d i s c t raps by C. puteana a f t e r f i v e weeks in n o n - s t e r i l e s o i l w i t h a mo is tu re c o n -tent of 25% o f s a t u r a t i o n . Note m y c e l i a l s t rands o f C. puteana. 78 To recover C. puteana i f i t had invaded the a l d e r t r a p s , the t r a p s were removed from the s o i l and p laced on malt agar made up as 5% agar and 3% malt e x t r a c t , The hard agar was used because B i e r and Rowat (2) found that Hvpoxylon pruinatum ( K l o t . ) Cke . , a bark canker pathogen, was ab le t o compete s u c c e s s f u l l y w i t h bark saprophytes on a hard agar . If C_, puteana was present in the t r a p d i s c , i t was a b l e t o grow out s l o w l y i n t o the hard agar in c o m p e t i t i o n w i t h the dther s o i l microorganisms present in the d i s c . F igu re 8 shows the growth of C, puteana from one of the d i s c s a f t e r f i v e weeks on the hard malt agar . In n o n - s t e r i l e s o i l at the 25% mo is tu re l e v e l , C. puteana was a b l e t o grow out and c o l o n i z e the a l d e r d i s c s (F igures 6 and 7). None o f the t r c p s in s o i l at h igher mo is tu re l e v e l s was c o l o n i z e d . There fo re C. puteana can spread through the s o i l and c o l o n i z e a p i e c e of wood in com-p e t i t i o n w i t h the other s o i l microorganisms at a mo is tu re l e v e l o f 25% o f s a t u r a t i o n , but not at h igher s o i l mo is tu re l e v e l s . N o n - s t e r i l e S o i l Added t o Autoc laved S o i l To c l a r i f y the e f f e c t of s o i l mo is tu re on C_, puteana s e v e r a l a d d i t i o n a l experiments were c a r r i e d o u t . F i r s t , one gram of n o n - s t e r i l e s o i l was added over the s u r f a c e o f each p e t r i p l a t e c o n t a i n i n g 2 f g of autoc laved s o i l at 20%, 40%,70% and 100% mo is tu re l e v e l s . If the e f f e c t In n o n - s t e r i l e s o i l was a n u t r i t i o n a l one overcome by the r e l e a s e of n u t r i e n t s du r i ng a u t o c l a v -i n g , C. puteana ought t o grow as i t d i d on autoc laved s o i l . However, i f the e f f e c t was caused by the i n f l u e n c e of s o i l mo is tu re on the m i c r o b i a l p o p u l a -t i o n in the s o i l , one gram o f n o n - s t e r i l e s o i l would be s u f f i c i e n t t o r e -i n o c u l a t e the organisms i n t o the autoc laved s o i l . 79 F i g u r e 3. Recovery on malt agar c o n t a i n i n g 5% agar of C. puteana from a l d e r d i s c t r a p s . Mycelium from d i s c in upper l e f t s e c t i o n of p l a t e i s C, puteana. 80 The growth p a t t e r n s of C. puteana were s i m i l a r to those on non -s t e r i l e s o i l , but w i t h some growth at the 70% and 100% l e v e l s before the o ther organisms became e s t a b l i s h e d (F igure 9).. T_» v i r i d e f r u i t e d abund-a n t l y on p l a t e s at the h igher mo is tu re l e v e l s . There fo re i t appeared that the e f f e c t o f s o i l mo is tu re on the growth of C. puteana was r e l a t e d t o the e f f e c t of the s o i l m i c r o b i a l p o p u l a t i o n s . Trichpderma v i r i d e Inoculated in Autoc laved S o i l Tr ichoderma v i r i d e , an imperfect fungus known to be antagon1s t ic to many s o i l fungi (15), was present in the n o n - s t e r i l e s o i l and was p a r t i c u l a r -ly ev ident at the h igher mo is tu re l e v e l s , i t was i s o l a t e d from the s o i l and a suspension of i t s c o n i d i a in s t e r i l e water was sprayed on the s u r f a c e of autoc laved s o i l at 25%, 50%, 75% and 100% mo is ture l e v e l s . C. puteana was i nocu la ted on the s o i l immediately a f t e r . It grew s l o w l y out i n to the s o i l at the 25% and 50% l e v e l s but d id not develop at the 75% and 100% l e v e l s (F igure 10 and Tab le 1). The f r u i t i n g s t r u c t u r e s o f T. v i r i d e were more ev ident in the wet te r s o i l than in the d r i e r s o i l . These r e s u l t s were s i m i l a r to those in n o n - s t e r i l e s o i l and in autoc laved s o i l r e ^ i n o c u l a t e d w i t h n o n - s t e r i l e s o i l . It should be noted here that the growth of C_. puteana was i n h i b i t e d by T. v i r ? d e on malt agar c o n t a i n i n g 2% agar and 3% malt e x t r a c t . However, 6 1/2 weeks a f t e r C_. puteana was i nocu la ted on the agar and f i v e weeks a f t e r !• v ' r i d e was i nocu la ted (C. puteana was g iven 10 days to become e s t a b l i s h e d ) hyphae of C. puteana were ab le to invade the area c o l o n i z e d by v l r f .de (F igure 11). By that t ime the agar had d r i e d out' c o n s i d e r a b l y . P o s s i b l y ! • v i r i d e was no longer m e t a b o l i z i n g a c t i v e l y . Thus c o n d i t i o n s were such 31 F i g u r e 9 . G r o w t h o f C . p u t e a n a a f t e r t w o w e e k s i n p e t r i p l a t e s w i t h o n e g r a m o f n o n - s t e r i l e s o i l a d d e d t o 2k g r a m s o f a u t o c l a v e d s o i l . T h e f i g u r e s s h o w t h e s o i l m o i s t u r e c o n t e n t a s a p e r c e n t o f s a t u r a t i o n . N o t e t h e f r u i t i n g s t r u c t u r e s o f T . v i r i d e i n t h e w e t t e r p l a t e s . Figure 10. Growth of C. puteana after two weeks in auto-claved s o i l inoculated with T. v i r i d e . The s o i l moisture content i s : top l e f t , 25%; top r i g h t , 50%; bottom l e f t , 75% and bottom right, 100% of saturation. 83 t h a t C_. p u t e a n a was no l o n g e r i n h i b i t e d b y T . v i r i d e . A c t i - d t o n e i n A u t o c l a v e d S o i l An e x p e r i m e n t was s e t up t o d e t e r m i n e i f a n i n d i v i d u a l a n t i b i o t i c a d d e d t o a u t o c l a v e d s o i l a t d i f f e r e n t m o i s t u r e l e v e l s w o u l d a f f e c t t h e g r o w t h o f C_. p u t e a n a i n t h e s a m e way a s n o n - s t e r i l e s o i l a t d i f f e r e n t m o i s t u r e l e v e l s . A c t i - d i o n e ( c y c l o h e x i m i d e ) was c h o s e n f o r t h e e x p e r i m e n t b e c a u s e i t i s a n a n t i f u n g a l a n t i b i o t i c , s t a b l e a t pH 3 - 5 , w i t h a w a t e r s o l u b i l i t y o f 2 % ( a c c o r d i n g t o t h e s p e c i f i c a t i o n s o f t h e U p j o h n C o m p a n y , K a l a m a z o o , M i c h i g a n ) . G o t t l i e b , S i m i n o f f a n d M a r t i n ( 1 7 ) r e p o r t e d t h a t A c t i - d i o n e was n o t r e m o v e d f r o m a q u e o u s s o l u t i o n by s o i l a n d i t i n h i b i t e d t h e g r o w t h o f s e n s i t i v e m i c r o o r g a n i s m s i n s o i l . In t h i s e x p e r i m e n t A c t i -d i o n e w a s a d d e d a s 100 m i c r o g r a m s p e r g r a m o f s o i l t o b o t h n o n - s t e r i l e a n d a u t o c l a v e d s o i l a t 2 5 % , 5 0 % , 7 5 % a n d 1 0 0 % m o i s t u r e l e v e l s . T h e g r o w t h p a t t e r n s o f C_. p u t e a n a i n t h e d i f f e r e n t t r e a t m e n t s a r e s h o w n i n F i g u r e s 12 a n d 1 3 . In n o n - s t e r i l e s o i l w i t h A c t i - d i o n e , t h e g r o w t h p a t t e r n o f C . p u t e a n a was s i m i l a r t o t h a t i n n o n - s t e r i l e s o i l a t t h e d i f f e r e n t m o i s t u r e l e v e l s . In a u t o c l a v e d s o i l w i t h A c t i - d i o n e a d d e d , g r o w t h was l e s s a t a l l s o i l m o i s t u r e l e v e l s t h a n i n a u t o c l a v e d s o i l w i t h o u t A c t i - d i o n e . T h e r e was m o r e g r o w t h a t t h e 2 5 % a n d 5 0 % l e v e l s t h a n a t t h e 7 5 % . T h e r e w a s l i t t l e g r o w t h o f C_. p u t e a n a a t 1 0 0 % o f s a t u r a t i o n ( F i g u r e 1 3 ) . T h u s t h e p a t t e r n o f i n h i b i t i o n o f g r o w t h o f C_. p u t e a n a a t d i f f e r e n t s o i l m o i s t u r e l e v e l s b y A c t i - d i o n e was s i m i l a r t o t h a t i n n o n - s t e r i l e s o i l , a l t h o u g h 1 00 u g o f A c t i - d i o n e p e r g r a m o f s o i l d i d n o t p r o d u c e t h e c o m p l e t e i n h i b i t i o n a t t h e h i g h e r m o i s t u r e l e v e l s w h i c h was e v i d e n t i n n o n - s t e r i l e s o i l . F igu re 11, The mycelium of C. puteana invading area c o l -on i zed by T. v i r i d e on malt agar c o n t a i n i n g 2 % agar and 3 % malt e x t r a c t ^ fc. puteana has been growing f o r 6 1/2 weeks, T. v i r i d e f o r f i v e weeks. ) 35 F i g u r e 12. G r o w t h o f C. p u t e a n a a f t e r t w o w e e k s i n n o n -s t e r i l e s o i l c o n t a i n i n g 100 p g A c t i - d i o n e p e r g r a m o f s o i l . T h e f i g u r e s s h o w t h e s o i l m o i s t u r e c o n t e n t a s a p e r c e n t o f s a t u r a t i o n . F i g u r e 13. G r o w t h o f p u t e a n a a f t e r t w o w e e k s i n a u t o -c l a v e d s o i l c o n t a i n i n g 100 \ig A c t i - d i o n e p e r g r a m o f s o i l . T h e f i g u r e s s h o w t h e s o i l m o i s t u r e c o n t e n t a s a p e r c e n t o f s a t u r a t i o n * 87 DISCUSSION The e f f e c t o f s o i l mo is tu re l e v e l on the growth of C. puteana through n o n - s t e r i l e s o i l in p e t r i p l a t e s was very c o n s i s t e n t . C. puteana was a b l e t o spread through the s o i l and c o l o n i z e wood when the s o i l mo is tu re l e v e l was 20 - 25% o f s a t u r a t i o n . At mo is tu re l e v e l s of 50% o r h igher i t was u n -ab le t o spread in the s o i l . These r e s u l t s agree w i t h G a r r e t t ' s repor t in 1934 (14) that b i o l o g i c a l antagonisms Increase w i t h s o i l mo is tu re over the range 30 - 30% s a t u r a t i o n . When i n d i v i d u a l f a c t o r s such as smal l amounts o f n o n - s t e r i l e s o i l , an i n d i v i d u a l organism a n t a g o n i s t i c t o C_. puteana. or a p u r i f i e d a n t i b i o t i c , were added to autoc laved s o i l the growth p a t t e r n of C. puteana was s i m i l a r t o that in n o n - s t e r i l e s o i l . Some i n d i v i d u a l f a c t o r s were more i n h i b i t o r y t o the growth of C. puteana than o thers and none e x a c t l y reproduced the p a t t e r n of i n h i b i t i o n present in n o n - s t e r i l e s o i l . However the r e l a t i o n of s o i l mo is tu re t o the va r i ous f a c t o r s was c o n s i s t e n t throughout , w i t h C. puteana ab le to grow through the d r i e r s o i l t r ea tments , but not the wet te r ones. The amount o f water in the s o i l , w h i l e be ing the c o n t r o l l i n g f a c t o r in a l l exper iments , appeared t o be i n d i r e c t r a t h e r than d i r e c t in i t s e f f e c t on the growth of C_. puteana. The fungus was not i n h i b i t e d in wet autoc laved s o i l . The s o i l mo is tu re l e v e l t h e r e f o r e appeared t o a f f e c t the s o i l m i c r o b i o l o g i c a l community, which in tu rn a f f e c t e d the growth of C. puteana. There a re s e v e r a l p o s s i b l e ways of i n t e r p r e t i n g the e f f e c t of h igh s o i l mo is tu re on the m i c r o b i a l p o p u l a t i o n which produced an i n h i b i t i o n of growth o f C« puteana. It may be a c o m p e t i t i o n f o r n u t r i e n t s , w i t h more a c t i v e m y c e l i a at h igher water l e v e l s and hence g reater c o m p e t i t i o n . A n -38 other f a c t o r is a e r a t i o n , which v a r i e s at d i f f e r e n t water contents (18 ,27) and may a f f e c t the development of C_. puteana and the o ther microorganisms. Carbon d i o x i d e may accumulate t o a t o x i c l e v e l as a r e s u l t o f the r e s p i r a -t i o n o f the other organisms in the s o i l . However, C_. puteana was a b l e t o grow on the s u r f a c e of the s o i l in a l l t r ea tments , reduc ing the p o s s i b i l i t y tha t a e r a t i o n i s a major f a c t o r . The f a c t o r which seems t o be most import -ant in i n t e r p r e t i n g the r e s u l t s presented here is that many f u n g i s t a t i c or a n t i b i o t i c substances produced by s o i l microorganisms are w a t e r - s o l u b l e and d i f f u s e through the water f i l m in the s o i l . When the re i s s u f f i c i e n t mo is tu re the substances d i f f u s e r e a d i l y and become cont inuous in the s o i l . When the water content i s lower the water f i l m in s o i l i s d i scon t inuous so t h e r e may be areas of c o n c e n t r a t i o n of a n t i b i o t i c s around the s i t e of p r o d u c t i o n . Park (25), in a d i s c u s s i o n of antagonisms in a s o i l , says "One must v i s u a l i z e not a g e n e r a l l y un i form h a b i t a t in s o i l , but ' i s l a n d s ' o f , f o r example, d i f f e r e n t l e v e l s of a c t i v i t y and d i f f e r e n t l e v e l s of i n h i b i t i o n o c c u r r i n g in l o c a l i z e d a reas . It i s in some of these i s l a n d s that n u t r i e n t s w i l l occur at a h igh l e v e l , and thus permit the o p e r a t i o n of a n t a g o n i s t i c mechanisms other than c o m p e t i t i o n . " Thus m i c r o - h a b i t a t s ( i n terms of the c o n d i t i o n s encountered by invading hyphal t i p s ) may be more v a r i a b l e in a d r i e r s o i l than in a wet one because the re i s l ess movement of w a t e r - s o l u b l e products in the d r i e r s o i l . The i s l a n d s where a n t a g o n i s t i c mechanisms operate may remain as i s l a n d s , w i t h a l o c a l i z e d d i s t r i b u t i o n of the a n t a g o n i s t i c p r i n c i p l e s . There may a l s o be areas where a fungus which can t o l e r a t e a lower s o i l mo is tu re can spread f r e e l y . Maciejowska and W i l l i a m s (22) have shown that Trichoderma develops b e t t e r at h igh s o i l mo is tu re l e v e l s . Thus t h e r e may a l s o be a g rea te r p roduct ion as w e l l as a g rea te r d i s p e r s a l of the a n t a g o n i s t i c e f f e c t in wet ter s o i l s . 89 Recent ly Webster and Lomas (31) have shown that the I so la tes used by B r i a n and Hemming (4) and B r i a n and McGowan (5) t o o b t a i n g l i o t o x i n and v i r i d i n a re not Trichoderma v i r i d e as o r i g i n a l l y d e s c r i b e d , but G l i o c l a d i u m v i r e n s M i l l e r , Giddens and F o s t e r . Tests by Webster and Lomas demonstrated a n t i b i o t i c a c t i v i t y in i s o l a t e s of G, v i r e n s but not in i s o l a t e s of T. v i r i d e . They d i s c u s s the nature of the antagonism of T. v i r i d e to s o i l fung i and p o s s i b l e exp lanat ions f o r i t . The fungus i s o l a t e used in the present study was i d e n t i f i e d by the author as T. v i r i d e . U n f o r t u n a t e l y the i s o l a t e is no longer a v a i l a b l e f o r a check of the i d e n t i f i c a t i o n , but i t was undoubtedly a n t a g o n i s t i c t o C. puteana both in agar c u l t u r e and in s o i l - w h e t h e r by means of a n t i b i o t i c p roduct ion or by some other means. The f a c t that c o n d i t i o n s e x i s t when a r o o t - r o t organism such as C. puteana can grow and spread through the s o i l may be s i g n i f i c a n t in an understanding of root d i s e a s e development in f o r e s t s t a n d s . McMinn (23) has shown that t h e r e are pe r iods du r ing the growing season in most s i t e s in the D o u g l a s - f i r r eg ion of eastern Vancouver i s l a n d when at l e a s t the s u r f a c e s o i l l aye rs ( to a depth of 20 cm) had very low mo is tu re c o n t e n t s , in s e v e r a l cases below the w i l t i n g percentage . T h i s was in a study area w i t h a mean annual p r e c i p i t a t i o n from 54 t o 107 i nches . Thus the re may i n -deed be c o n d i t i o n s s u i t a b l e f o r the spread of C. puteana through s o i l in many f o r e s t areas at some t ime du r ing most y e a r s . The c o n d i t i o n s may not be c o n t i n u o u s l y s u i t a b l e , but i f a w o o d - r o t t i n g fungus can c o l o n i z e new s u b s t r a t e s such as broken branches or other s l a s h i t may spread v e g e t a t i v e l y over a c o n s i d e r a b l e d i s t a n c e in s e v e r a l y e a r s . The spread of a r o o t - r o t t i n g fungus is not n e c e s s a r i l y r e l a t e d t o root i n f e c t i o n i f i t can c o l o n i z e a new woody s u b s t r a t e s a p r o p h y t i c a l l y . The c o n d i t i o n s f avou rab le f o r spread through the s o i l a re not n e c e s s a r i l y 9 0 the same as those f o r i n f e c t i o n of r o o t s , where host r e s i s t a n c e or s u s c -e p t i b i l i t y i s a major f a c t o r a l s o i n f l uenced by s o i l c o n d i t i o n s . Most s t u d i e s in the past have been concerned w i t h d i s e a s e development, which i s the r e s u l t of a combinat ion of f a c t o r s favour ing fungus development and those f avou r ing host r e s i s t a n c e . The present study has been concerned on ly w i t h f a c t o r s f a v o u r i n g fungus development. In the case of pathogens caus ing root r o t s of t r e e s , the fungus may spread through s o i l when c o n d i -t i o n s are s u i t a b l e f o r such sp read . It may then be in contact w i t h t r e e roots when c o n d i t i o n s are f avou rab le f o r i n f e c t i o n . The inoculum p o t e n t i a l would be renewed by sap rophy t i c c o l o n i z a t i o n of new food bases along the way. S tud ies w i t h other w o o d - r o t t i n g root pathogens should be c a r r i e d o u t , w i t h a s e p a r a t i o n of the c o n d i t i o n s f a v o u r i n g fungus spread through s o i l from those f a v o u r i n g root i n f e c t i o n . The e f f e c t of s o i l mo is tu re should be cons ide red c a r e f u l l y because of i t s v a r i a b i l i t y under n a t u r a l c o n d i t i o n s and i t s c o n t r o l l i n g e f f e c t on o ther s o i l f a c t o r s . 91 BIBLIOGRAPHY 1. Basham, J . T . and Z . J . R . Morawski . 1964. C u l l s t u d i e s , the d e f e c t s and a s s o c i a t e d basidiomycete fungi in the heartwood of l i v i n g t r e e s in the f o r e s t s of O n t a r i o . Canada Dept. of F o r e s t r y P u b l . No. 1072, 69 p. 2 . B i e r , J . E . and M.H. Rowat. 1962. 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Trans . 47: 535-540.
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Botany
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Some interrelations of root-rotting Basidiomycetes and soil-inhabiting Fungi imperfecti
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