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The interaction between Rhizobium and Fusarium Solani F. Sp. Phaseoli and Rhizoctonia Solani Smulders, Andrea Joanne 1981

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THE INTERACTION BETWEEN RHIZOBIUM AND FUSARIUM SOLANI F. SP. PHASEOLI AND RHIZOCTONIA SOLANI  by ANDREA JOANNE SMULDERS B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1978  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PLANT SCIENCE  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1981 (c") Andrea Joanne Smulders, 1981  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 of the  require-  ments f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e study.  I f u r t h e r agree t h a t p e r m i s s i o n  f o r extensive  c o p y i n g of  t h e s i s f o r s c h o l a r l y purposes may  be granted by  or by h i s or her  I t i s understood t h a t c o p y i n g  representative.  the Head of my  p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be out my  written  permission.  Department of P l a n t Science The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, B.C., Canada V6T 1W5 September,  1981  and  this  Department or  allowed  with-  ABSTRACT  Indigenous Rhizobium  i s o l a t e s from n a t u r a l l y - f o r m e d  bean r o o t  nodules were a n t a g o n i s t i c to some of the r o o t r o t t i n g pathogens bean.  Rhizobium  i n dual  i s o l a t e s i n h i b i t e d the r a d i a l growth of Fusarium  c u l t u r e agar p l a t e t e s t s but were not i n h i b i t o r y to  s o l a n i or Pythium  isolates.  With one e x c e p t i o n ,  i s o l a t e s showed some degree of antagonism phaseoli i n v i t r o .  recorded  Rhizoctonia  a l l indigenous  towards  species  Rhizobium  F. s o l a n i f . sp.  The l e v e l of i n v i t r o i n h i b i t i o n depended upon the  agar p l a t e t e c h n i q u e u t i l i z e d . v i t y was  o f snap  A high  l e v e l of i n v i t r o i n h i b i t o r y a c t i -  i n 38% of the Rhizobium  i s o l a t e s t e s t e d where wide zones  of i n h i b i t i o n formed between the t e s t i s o l a t e s and p e r s i s t e d f o r more than 1 week.  A s i m i l a r i n h i b i t o r y e f f e c t of 8/17  to Fusarium r o o t r o t of snap bean was ments.  nodulating  observed i n growth pouch e x p e r i -  P r o t e c t i o n of bean p l a n t s from severe Fusarium r o o t r o t  i n combinations where the inoculum c o n c e n t r a t i o n cells/pouch)  was  equal to or greater  F_. s o l a n i (10 , 1 0 2  v  highly antagonistic root r o t i n v i v o .  spores/pouch).  experiments  of Rhizobium  Ten Rhizobium  (10\  10  6  of  i s o l a t e s , which were  i n v i t r o , had no apparent i n h i b i t o r y e f f e c t on Fusarium Two  Rhizobium  i s o l a t e s , RCC324 and RCC607, i n h i b i t o r y r o o t r o t of bean.  Soil  supported the r e s u l t s of growth pouch experiments whereby  i n o c u l a t i o n of bean seed w i t h a h i g h c o n c e n t r a t i o n 8  occurred  than the inoculum c o n c e n t r a t i o n  to Fusarium r o o t r o t d i d not reduce R h i z o c t o n i a  10  Rhizobium i s o l a t e s  of Rhizobium  (RCC106 a t  c e l l s / s e e d ) e f f e c t i v e l y reduced bean r o o t r o t i n c i t e d by a low  inoculum p o t e n t i a l of the pathogen, 1:120).  F. s o l a n i (inoculum:  soil,  These r e s u l t s i n d i c a t e d the p o t e n t i a l e x i s t s f o r f i e l d  Fusarium r o o t r o t of snap bean by a h i g h l y a n t a g o n i s t i c i s o l a t e of  Rhizobium.  1:10"* or c o n t r o l of  nodulating  - iii -  TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  i i i  LIST OF TABLES  v  LIST OF FIGURES  . ..  v i i  LIST OF APPENDIX I TABLES  viii  DEDICATION  x  ACKNOWLEDGEMENTS  xi  INTRODUCTION AND LITERATURE REVIEW . .  1  Introduction The Disease Control of Bean Root Rots Objectives  1 1 3 10  MATERIALS AND METHODS I.  I s o l a t i o n and I d e n t i f i c a t i o n of Pathogenic Fungi 1. 2. 3.  II.  III.  IV.  F i e l d Survey Growth Pouch Technique (GPT) Pathogenicity Tests  11 ....  11 11 12 15  I s o l a t i o n and I d e n t i f i c a t i o n of Rhizobium  17  1.  F i e l d Survey  17  2.  Nodulation Tests  18  In V i t r o Studies  19  1. 2.  Standardized Agar P l a t e Technique (SAPT) Screening of Survey Isolates In V i t r o  19 20  3.  Basis of the I n V i t r o I n h i b i t i o n  21  I n Vivo Studies  23  1. 2.  23 25  Screening of Rhizobium i n Growth Pouches S o i l Tests  - iv-  Page RESULTS I. II. III.  IV.  28 I s o l a t i o n and I d e n t i f i c a t i o n of Pathogenic Fungi . . . .  28  I s o l a t i o n and I d e n t i f i c a t i o n of Rhizobium  34  In V i t r o Studies  36  1. 2.  36 40  Screening of Survey I s o l a t e s I n V i t r o Basis of the In V i t r o I n h i b i t i o n .  In Vivo Studies  . .  1.  Screening of Rhizobium i n Growth Pouches  2.  S o i l Tests  40 40 48  DISCUSSION  51  SUMMARY AND CONCLUSIONS  58  LITERATURE CITED  59  APPENDIX I  • . . .'  64.  LIST OF TABLES Page Table  1  Root r o t f u n g i o b t a i n e d from o t h e r source  12  Table  2  N i t r o g e n - d e f i c i e n t n u t r i e n t s o l u t i o n used to water 'Topcrop' snap bean i n growth . pouches.  15  A u t h e n t i c a t e d Rhizobium s p e c i e s o b t a i n e d from o t h e r s o u r c e s .  19  Rhizobium and f u n g a l i s o l a t e s t e s t e d i n d u a l c u l t u r e agar p l a t e s .  21  Rhizobium i s o l a t e s e v a l u a t e d f o r i n h i b i t i o n of Fusarium s o l a n i (FS911) and R h i z o c t o n i a s o l a n i (RSI) r o o t r o t s i n growth pouches of 'Topcrop' snap bean.  24  Rhizobium i s o l a t e s e v a l u a t e d f o r i n h i b i t i o n of Fusarium s o l a n i (FS911) r o o t r o t of 'Topcrop' snap bean i n growth pouches.  25  F u n g i i s o l a t e d from snap bean r o o t s from commercial f i e l d s i n the F r a s e r V a l l e y , B r i t i s h Columbia.  28  Root d i s e a s e index and shoot and r o o t d r y weights of 'Topcrop* snap bean grown i n growth pouches and i n o c u l a t e d a t f o u r d i f f e r e n t stages w i t h Fusarium s o l a n i 2 at 1 0 spores/pouch.  29  Root d i s e a s e index and shoot and r o o t d r y weights of 'Topcrop' snap bean grown i n growth pouches and i n o c u l a t e d a t f i v e d i f f e r e n t stages w i t h Fusarium s o l a n i 911 at 10 spaces/pouch.  30  Root d i s e a s e index of t h r e e snap bean c u l t i v a r s ('Harvester', ' S t r i n g l e s s Greenpod', 'Topcrop') and ' L i n c o l n ' pea grown i n growth pouches.  32  P a t h o g e n i c i t y of two R h i z o c t o n i a s o l a n i i s o l a t e s to 'Topcrop' snap bean i n growth pouches.  34  Source of Rhizobium i s o l a t e s from snap bean root nodules.  35  Table  Table  Table  Table  Table  Table  3  4  5  6  7  8  7  Table  9  6  T a b l e 10  T a b l e 11  Table  12  - vi Page T a b l e 13  Table  14  T a b l e 15  T a b l e 16  T a b l e 17  T a b l e 18  T a b l e 19  T a b l e 20  T a b l e 21  T a b l e 22  T a b l e 23  N o d u l a t i n g a b i l i t y of Rhizobium i s o l a t e s to 'Topcrop' snap bean i n growth pouches.  35  The e f f e c t of Rhizobium i s o l a t e s on f u n g a l growth as determined by d u a l c u l t u r e agar plate tests.  37  L e v e l of jin v i t r o i n h i b i t o r y a c t i v i t y of Rhizobium i n d u a l c u l t u r e agar p l a t e t e s t s w i t h Fusarium s o l a n i 2.  37  I n h i b i t i o n zones r a t e d + + + in- dual c u l t u r e agar p l a t e t e s t of Rhizobium and Fusarium s o l a n i 2.  39  _In v i t r o i n h i b i t o r y a c t i v i t y of seven Rhizobium i s o l a t e s to Fusarium s o l a n i (FS2, FS911) and R h i z o c t o n i a s o l a n i (RSI, RS2, RS3) i n d u a l c u l t u r e agar plate tests.  39  Hydrogen i o n c o n c e n t r a t i o n of d u a l c u l t u r e agar p l a t e s i n o c u l a t e d w i t h Rhizobium and Fusarium s o l a n i 2.  41  E f f e c t of Rhizobium 106 and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and dry weight of 'Topcrop' snap bean i n growth pouches.  43  L e v e l of antagonism of n o d u l a t i n g Rhizobium i s o l a t e s t o Fusarium s o l a n i (FS2, FS911) i n d u a l c u l t u r e agar p l a t e t e s t s and growth pouches of 'Topcrop' snap bean.  45  I n t e r a c t i o n of Rhizobium i s o l a t e s and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and dry weight of 'Topcrop' snap bean i n growth pouches.  46  N o d u l a t i n g a b i l i t y of Rhizobium i s o l a t e s and l e v e l of antagonism to Fusarium s o l a n i (FS2, FS911) i n d u a l c u l t u r e agar p l a t e t e s t s and i n growth pouches of 'Topcrop' snap bean.  47  The l e v e l of antagonism o f n o d u l a t i n g Rhizobium i s o l a t e s to Fusarium s o l a n i (FS2, FS91) i n d u a l agar p l a t e t e s t s and i n growth pouches of 'Topcrop' snap bean.  49  - vii  -  LIST OF FIGURES  Figure 1  'Topcrop' snap bean grown by a growth pouch technique.  Figure 2  Fusarium s o l a n i 911 root r o t of 'Topcrop' snap bean grown by a grouch pouch technique  Figure 3  a. Rhizoctonia s o l a n i root r o t and b.  Figure 4  Figure 5  hypocotyl r o t of 'Topcrop' snap bean i n growth pouches.  Level of i n h i b i t i o n i n dual c u l t u r e agar plate t e s t s : a.  Fusarium s o l a n i (FS2) i n h i b i t e d (+. + +) by Rhizobium, i s o l a t e RCC326;  b.  Rhizoctonia s o l a n i 1 not i n h i b i t e d (-) by RCC326.  a.  Fusarium s o l a n i 911 ( P •= 10^ spores/ pouch) root r o t of 'Topcrop' snap bean i n growth pouches.  b.  Suppressed by i n o c u l a t i o n with Rhizobium, i s o l a t e RCC816 ( R = 10 cells/pouch).  3  6  A  Figure 6  a.  Fusarium s o l a n i 911 ( P = inoculum: s o i l 1:10) root r o t of 'Topcrop' snap bean grown i n pasteurized s o i l .  b.  Suppressed by i n o c u l a t i o n with Rhizobium, i s o l a t e RCC106 (R^ = 10 c e l l s / s e e d ) .  2  8  - viii -  LIST OF APPENDIX TABLES Page Table  Table  Table  Table  Table  Table  Table  Table  Table  1  2  3  4  5  6  7  8  9  E f f e c t of Rhizobium 816 and Fusarium s o l a n i 911 on root disease and nodulation i n d i c e s and dry weight of 'Topcrop' snap bean i n growth pouches.  63  E f f e c t of Rhizobium 324 and Fusarium s o l a n i 911 on root disease and nodulation i n d i c e s and dry weight of 'Topcrop' snap bean i n growth pouches.  64  E f f e c t of Rhizobium 321 and Fusarium s o l a n i 911 on root disease and nodulation i n d i c e s and dry weight of 'Topcrop' snap bean i n growth pouches.  65  E f f e c t of Rhizobium 607 and Fusarium s o l a n i 911 on root disease and nodulation i n d i c e s and dry weight of 'Topcrop' snap bean i n growth pouches.  66  E f f e c t of Rhizobium 812 and Fusarium s o l a n i 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches.  67  E f f e c t of Rhizobium 106, Fusarium s o l a n i (FS911) and Rhizoctonia s o l a n i (RSI) on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches.  68  I n t e r a c t i o n of Rhizobium i s o l a t e s and Rhizoctonia s o l a n i (RSI, RS2, RS3) on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches.  69  I n t e r a c t i o n of Rhizobium i s o l a t e s and Fusarium s o l a n i 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches.  70  E f f e c t of Rhizobium 106 and Fusarium s o l a n i 911 on root disease and nodulation i n d i c e s and dry weight of 'Topcrop' snap bean grown i n pasteurized greenhouse s o i l .  71  -  ix  -  Page Table  Table  10  11  E f f e c t of Rhizobium 106 and Fusarium s o l a n i 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean grown i n pasteurized greenhouse s o i l . E f f e c t of Rhizobium 816 and Fusarium s o l a n i 911 on root disease and nodulation i n d i c e s and dry weight of 'Topcrop' snap bean grown i n pasteurized greenhouse s o i l .  72  73  - x -  To my p a r e n t s and P e t e r who  gave  me  the moral support n e c e s s a r y to complete this  thesis.  - xi -  ACKNOWLEDGMENTS  I would l i k e Dr. H.S. P e p i n , Dr.  to s i n c e r e l y thank b o t h o f my major  supervisors,  A g r i c u l t u r e Canada, Research S t a t i o n , Vancouver, and  R.J. Copeman, A s s o c i a t e P r o f e s s o r , Department of P l a n t  Science,  U n i v e r s i t y of B r i t i s h Columbia, f o r t h e i r i n v a l u a b l e guidance and encouragement throughout t h i s p r o j e c t . Appreciation committee:  i s a l s o extended t o t h e o t h e r members o f my t h e s i s  Dr. V.C. Runeckles, Dr. F.B. H o l l and D r . R.J. Bandoni f o r  t h e i r u s e f u l suggestions  and c o n s t r u c t i v e c r i t i c i s m s p e r t a i n i n g t o t h i s  thesis. The  D i r e c t o r and s t a f f members o f t h e A g r i c u l t u r e Canada, Research  S t a t i o n , Vancouver a r e g r a t e f u l l y acknowledged f o r t h e i r a s s i s t a n c e and f o r p r o v i d i n g the f a c i l i t i e s n e c e s s a r y to c a r r y out t h i s r e s e a r c h In p a r t i c u l a r ,  I wish t o thank Mrs. A. MacPherson f o r h e r p a t i e n c e and  p e r s i s t e n t a i d i n the s t a t i s t i c a l a s p e c t s F i n a n c i a l a s s i s t a n c e was p r o v i d e d Applied,  project.  and H e a l t h  Sciences  of t h i s  study.  by a grant  from the N a t u r a l ,  Grants Committee o f the U n i v e r s i t y o f B r i t i s h  Columbia and a Summer Student S c h o l a r s h i p awarded by the U n i v e r s i t y of British  Columbia.  INTRODUCTION AND LITERATURE REVIEW Introduction Increasing a t t e n t i o n i s being focused on vegetable p r o t e i n s , e s p e c i a l l y those derived from legumes, i n an attempt to a l l e v i a t e world p r o t e i n shortages (Neergaard, 1977; Freytag, 1975).  Phaseolus v u l g a r i s  L. i s an exemplary legume with a p r o t e i n content of 15-31% (Neergaard, 1975) and a valuable source of calcium, r i b o f l a v i n and i r o n (Zaumeyer, 1957).  Bean i s v i r t u a l l y u n i v e r s a l i n i t s d i s t r i b u t i o n and i s an  important food crop throughout the world (Zaumeyer and Thomas, 1957).  In  the American t r o p i c s and subtropics, bean has been a major d i e t a r y staple since p r e - c o l o n i a l days (Freytag, 1975).  Bean root rots are known to  increase i n areas under intense c u l t i v a t i o n u n t i l production i s no longer p r o f i t a b l e and the land must be abandoned (Zaumeyer and Thomas, 1957). Less severe i n f e s t a t i o n s of root r o t t i n g organisms may be responsible f o r unaccountable losses that l i m i t the y i e l d p o t e n t i a l of the crop.  If  y i e l d s are to be increased to meet world p r o t e i n demands, steps must be taken to e f f e c t i v e l y c o n t r o l root disease, preferably by means r e q u i r i n g a minimal expenditure of energy. The Disease Root rots of snap bean, i n c i t e d by Fusarium s o l a n i (Mart.) App. & Wr. F. sp. phaseoli (Burkh.) Snyd. & Hans, and Rhizoctonia s o l a n i Kuehn are ubiquitous i n c u l t i v a t e d s o i l (Chupp & Sherf, 1960; Neergaard, 1975). They survive as dormant propagules and can increase i n the rhizosphere of host and non-host crops(Schroth & Hendrix, 1962; C h r i s t o u , 1962; Reyes & M i t c h e l l , 1962).  Depending upon when i n f e c t i o n occurs, seed r o t , pre-  - 2 and post-emergence damping o f f , foot r o t (hypocotyl r o t ) , or root r o t may r e s u l t (MacSwan & Koepsell, 1981).  Affected plants show non-specific  symptoms such as poor emergence and growth, c h l o r o t i c leaves, premature d e f o l i a t i o n and stunting (Walker, 1952).  Severe root r o t reduces the  number of s u r v i v i n g roots a v a i l a b l e f o r symbiotic nodulation (Tu, 1978). The diseases are s u f f i c i e n t l y d e b i l i t a t i n g that i n c e r t a i n areas the crop has been completely decimated (Neergaard, 1975; MacSwan & K o e p s e l l , 1981). I n i t i a l l y , Fusarium root r o t of bean i s d i s t i n g u i s h e d by a s l i g h t reddish d i s c o l o r a t i o n of the taproot which gradually increases i n i n t e n s i t y to cover the root (Zaumeyer & Thomas, 1957).  Elongate streaks of  i n d e f i n i t e s i z e and margin extend down the hypocotyl and taproot.  Dis-  eased t i s s u e eventually becomes dark brown and i s c h a r a c t e r i s t i c a l l y a 'dry r o t ' (Burkholder, 1919; Westcott, 1971).  L a t e r a l roots are destroyed  but adventitious roots developing above the l e s i o n s can support the crop to harvest (MacSwan & K o e p s e l l , 1981).  Fusarium root r o t i s generally  favored by warm, d r i e r weather i n mid-season (Chupp & Sherf,  1960)  Rhizoctonia root r o t of bean i s d i s t i n g u i s h e d from Fusarium root rot by the type of l e s i o n s that develop on the hypocotyl and taproot. Lesions of Rhizoctonia are varying shades of brown, sunken, oval to elongate regions w i t h a d e f i n i t e margin (Chupp & Sherf, 1960). can g i r d l e the root and diseased t i s s u e takes on a predominantly  Lesions reddish-  brown d i s c o l o r a t i o n before becoming completely n e c r o t i c . A sign of Rhizoctonia root r o t i s brown to black s c l e r o t i a of v a r i a b l e s i z e and form which develop i n the n e c r o t i c t i s s u e and s i g n i f y the end of the p a r a s i t i c phase of the pathogen (Christou, 1962).  Rhizoctonia root r o t  of bean i s generally considered an e a r l y season problem i n cooler conditions (Yang & Hagedorn, 1966).  - 3 -  Fusarium and Rhizoctonia attack bean roots and hypocotyls by m u l t i p l e i n f e c t i o n s (Schrothand  Snyder, 1961;  Christou, 1962).  These  pathogens can enter root t i s s u e s by d i r e c t penetration or i n d i r e c t l y v i a wounds caused by mechanical i n j u r y due to c u l t i v a t i o n p r a c t i c e s , nematodes, i n s e c t s or at the points where l a t e r a l roots emerge (Christou, 1962. G a r r e t t , 1970).  Only epidermal and c o r t i c a l c e l l s of the roots are  invaded by these fungi which ramify i n these host t i s s u e s both i n t e r c e l l u l a r l y and i n t r a c e l l u l a r l y (Christou, 1962;  Chi j i t a i . , 1964).  Fungal propagules ( c o n i d i a , chlamydospores, m y c e l i a l fragments and s c l e r o t i a ) can be c a r r i e d as contaminants on the seed coat, i n crop debris or i n s o i l humus p a r t i c l e s (Nash et a l . , 1961; MacSwan and Koepsell, 1981).  Transmission of the root r o t organisms to other areas  i s f a c i l i t a t e d by r a i n splashing, f l o o d i n g , wind, c u l t i v a t i o n p r a c t i c e s and s o i l i n s e c t s (Chupp and Sherf, 1960). Control of Bean Root Rots Control methods of Fusarium and Rhizoctonia root r o t s are not adequate even though these diseases are prevalent and s i g n i f i c a n t l y d e s t r u c t i v e ( M i l l e r and Burke, 1974;. MacSwan and K o e p s e l l , 1981). c u l t i v a r s vary i n s u s c e p t i b i l i t y to root r o t .  Bean  The age of the p l a n t ,  vigour and root secretions also i n f l u e n c e root disease s e v e r i t y . Generally, root disease i s worse on younger plants stressed by unfavorable growing conditions (Zaumeyer and Thomas, 1957).  Immunity to Fusarium and  Rhizoctonia root r o t does not e x i s t i n any bean c u l t i v a r and breeding f o r r e s i s t a n c e i s impeded by the great v a r i a b i l i t y among pathogenic (Chupp and Sherf, 1960).  strains  Most commercial c u l t i v a r s (e.g. Slenderwhite', 1  'Tenderwhite', 'Topcrop'), while moderately susceptible to Fusarium root  - 4 rot,  can s t i l l be p r o f i t a b l y grown i n i n f e s t e d s o i l under environmental  conditions favourable to crop growth (Hagedorn and Rand, 1974).  Phaseolus  coccineus L. (Scarlet runner bean), some c u l t i v a r s of lima bean, the bean c u l t i v a r 'Tendergreen' and P l a n t Introduction Accession Line N203 show tolerance to Fusarium root r o t (Booth and Waterson, 1964;, Rand, 1974).  Hagedorn and  Venezuela 54 and dark-seeded Plant Introduction Accession  Lines 109859, 163583, 165426 show tolerance to Rhizoctonia root r o t (Prasad and Weigle, 1969, 1970).  Chemical c o n t r o l of bean root r o t s i s  of dubious value (Maloy and Burkholder, 1959).  Fungicide, steam or hot  water seed treatments and furrow sprays w i t h fungicides have reduced root rot but are not e n t i r e l y e f f e c t i v e (Papavizas and Lewis, 1975; and K o e p s e l l , 1981).  MacSwan  S o i l fumigation i s not a v i a b l e economical c o n t r o l  p r a c t i c e f o r bean root r o t s (Zaumeyer and Thomas, 1957; Westcott, 1971). Root diseases are influenced by a b i o t i c and b i o t i c factors i n the environment.  C u l t u r a l p r a c t i c e s can be implemented to a l t e r these  conditions to increase host plant vigour and supppress root r o t (Zaumeyer and Thomas, 1957).  Any p r a c t i c e that improves the growth and b i o l o g i c a l  e f f i c i e n c y of the plant w i l l improve i t s general resistance to disease (Garrett, 1970).  Vigorous plants can escape i n f e c t i o n from soilborne  phytopathogens by growing out of an i n f e s t e d area or by producing new roots above the points of i n f e c t i o n (Burke, 1968).  Root t i s s u e s of  vigorous plants are l e s s susceptible to disease because they consist of more mature, t h i c k e r walled, l i g n i f i e d  c e l l s which are not as r e a d i l y  penetrated and destroyed by the pathogen as weak plants with t h i n walled, immature,- u n l i g n i f l e d c e l l s (Chi and Hansen, 1961; Tang and Hagedorn, 196.6); A b i o t i c f a c t o r s of paramount importance i n determining the incidence and s e v e r i t y of root r o t are s o i l p h y s i c a l conditions such as the pH,  - 5 -  oxygen tension, bulk density, moisture, drainage, temperature, f e r t i l i t y and s o i l type (Baker and Maurer, 1967; G a r r e t t , 1970).  Bean should  be  planted i n warm, well-drained, f e r t i l e s o i l and seeding delayed to avoid c o l d , wet e a r l y season weather (Chupp and Sherf, 1960; Burke, Westcott, 1971).  1964;  F e r t i l i z e r and lime should be applied to the s o i l  according to a s o i l a n a l y s i s so that an adequate pH and n u t r i e n t l e v e l s are provided for'vigorous  crop growth.  Y i e l d increases of bean grown i n  s o i l i n f e s t e d with J _ . s o l a n i and R. s o l a n i were achieved by s u b s o i l i n g to a depth of 18 inches below the d r i l l row p r i o r to p l a n t i n g (Burke, Cook, 1977).  1968.  This enhanced root development and penetration e s p e c i a l l y  i n heavy, compacted s o i l .  Shallow c u l t i v a t i o n and h i l l i n g a l s o provided  a b e n e f i c i a l e f f e c t by allowing adventitious roots to develop above the infected root t i s s u e (MacSwan and K o e p s e l l , 1981).  Strict sanitation  should be p r a c t i c e d by removing or deep plowing the inf ected crop debris a f t e r harvest (Chupp and Sherf, 1960).  Long r o t a t i o n s of 5 to 6 years  with a grain such as wheat or barley i s necessary to reduce inoculum p o t e n t i a l of the fungus i n the s o i l (Maloy and Burkholder, 1971).  1959; Westcott,  Accidental i n t r o d u c t i o n of v i r u l e n t s t r a i n s of the root r o t t i n g  fungi can be avoided by using uncontaminated seed and cleaning farm machinery before entering a disease-free area (Chupp and Sherf, Neergaard, 1977).  1960;  Organic amendments (mature barley or wheat straw,  C h i t i n , c e l l u l o s e ) can reduce root r o t by increasing the C:.N r a t i o of the s o i l and immobilizing nitrogen (Snyder et a l , , 1959; Papavizas et a l . , 1968).  The invasive capacity of Fusarium and Rhizoctonia reportedly  becomes l i m i t e d due to the lack of exogenous n u t r i e n t s required f o r e i t h e r chlamydospore germination or fungal growth.  Control of Fusarium root r o t  of bean by the a p p l i c a t i o n of c e l l u l o s e amendments was reported by Adams  - 6 et a l , 1968.  Amendments must be used c a r e f u l l y as under cold wet, }  anaerobic conditions phytotoxins are released which increase the host plant's s u s c e p t i b i l i t y to root r o t (Toussoun and P a t r i c k , 1963). B i o t i c f a c t o r s i n the environment that play a key r o l e i n determining the incidence and s e v e r i t y of root r o t are a t t r i b u t e s of the surrounding populations of s o i l organisms.  In the environment, organisms  e x h i b i t a multitude of i n t e r a c t i o n s as they progress toward a b i o l o g i c a l l y balanced state or e q u i l i b r i u m (Baker and Cook, 1974).  Rhizosphere organ-  isms, c l o s e l y associated with root pathogens and the host p l a n t , may have no e f f e c t on root r o t or they may act as synergists or antagonists. P o t e n t i a l synergists to root diseases are nematodes, v i r u s e s , other plant pathogens and root r o t fungi which may g r e a t l y i n t e n s i f y plant losses i n the f i e l d .  Peas i n f e c t e d with bean yellow mosaic v i r u s or common pea  mosaic v i r u s released more root exudate than uninfected plants (Baker Cook, 1974).  and  As a r e s u l t the inoculum concentration and number of root  i n f e c t i o n s due to J_. s o l a n i (Mart.) Appel & Wr. F. sp. p i s i (F.R. Jones) Snyd. & Hans, and Aphanomyces euteiches Drechs. increased.  Intensification  of R. s o l a n i root r o t of sweet pea a l s o occurred f o l l o w i n g e a r l y i n f e c t i o n by pea enation mosaic v i r u s . Antagonistic microorganisms can reduce root r o t and f u n c t i o n as nonchemical means f o r plant disease c o n t r o l (Henis, 1970). Beirne (1967) broadly defined a b i o l o g i c a l c o n t r o l agent as "any l i v i n g organism that can be manipulated 1974).  by man f o r pest c o n t r o l purposes."  (Baker and Cook,  B i o l o g i c a l c o n t r o l i s an i n t e g r a l part of a pest c o n t r o l program  and should be used i n conjunction with sound c u l t u r a l p r a c t i c e s and not treated as a separate d i s c i p l i n e .  Suppression of root diseases by micro-  organisms i s generally considered to be the r e s u l t of antagonism  ( a n t i b i o s i s , mycoparasitism, 1965; Huber et a l . , 1966).  l y s i s ) or competition (Anderson and Ruber, Elad et a l . (1980) reported that an antagon-  i s t i c s t r a i n of Trichoderma harzianum R i f a i s i g n i f i c a n t l y decreased the diseases i n c i t e d by Sclerotium r o l f s i i Sacc. and Rhizoctonia s o l a n i i n f i e l d experiments with bean, cotton or tomato. bean y i e l d was also a t t a i n e d .  A s i g n i f i c a n t increase i n  T_. harzianum i s mycoparasitic and was  capable  of l y s i n g mycelia of the pathogens i n dual c u l t u r e . Rhizosphere b a c t e r i a are p o t e n t i a l l y important antagonists to fungal root disease because of t h e i r abundance and a s s o c i a t i o n with plant roots. Competitive i n t e r a c t i o n s between s o i l microorganisms f o r n u t r i e n t s , space, oxygen and other requirements could be the major f a c t o r s governing b i o l o g i c a l c o n t r o l of soilborne fungi (Marshall and Alexander,  1960).  Nutrient competition between t y p i c a l s o i l b a c t e r i a and F. oxysporum f. sp. cubense (E.F.S.) Snyd. & Hans, both i n l i q u i d and s t e r i l e s o i l media was demonstrated by Marshall and Alexander (1980).  Agrobacterium radiobacter  ( B e i j e r i n c k & van Delden) Conn d r a s t i c a l l y l i m i t e d fungal growth.  Huber  et a l . (1966) used a p l a t e p r o f i l e technique to d i r e c t l y i s o l a t e and study s o i l microorganisms a c t i v e i n the b i o l o g i c a l c o n t r o l of F_. s o l a n i f. sp. phaseoli and R. s o l a n i root r o t of bean.  Their r e s u l t s i n d i c a t e d  that the most important mechanism of b i o l o g i c a l c o n t r o l of bean root r o t was fungal necrosis (hyphal death) due to s o i l b a c t e r i a . Rhizobium species are s o i l - i n h a b i t i n g , gram negative b a c t e r i a of economic importance i n the legume rhizosphere because of t h e i r r o l e i n root nodulation and symbiotic nitrogen f i x a t i o n (Beringer et a l . , Vance and Johnson,, 1981).  1980;  As a r e s u l t of t h i s symbiosis, legume crops  be grown without expensive chemical f e r t i l i z e r s .  can  The a n t i f u n g a l a c t i v i t y  of Rhizobium was demonstrated by Drapeau et a l . (1973) using an agar p l a t e  - 8 technique.  The r a d i a l growth of phytopathogenic Fusarium spp. and  Phytophthora cactorum (Leb. & Cohn) Schroet. on yeast mannitol agar p l a t e d , was i n h i b i t e d by three d i f f e r e n t s t r a i n s of Rhizobium. Rhizoctonia s o l a n i and Pythium ultimum Trow were not affected by Rhizobium in v i t r o .  S i m i l a r j\n v i t r o i n h i b i t i o n was demonstrated by Gray and  Sackston (1980) using 10 s t r a i n s of R. leguminosarum.  The Rhizobium  s t r a i n s showed varying degrees of a c t i v i t y towards F. s o l a n i f. sp. p i s i , a major i n c i t a n t of pea root r o t . Anfcoun et a l . (1978a) also observed i n h i b i t i o n of F_. oxysporum by Rhizobium m e l i l o t i i n agar plate t e s t s . R h i z o b i a l parasitism of root r o t fungi was reported by Tu (1978b, 1979).  Inoculation of c u l t u r e plates of the t e s t fungi with a r h i z o b i a l  suspension caused a reduction i n fungal sporulation and  extensive  i n t e r n a l and external c o l o n i z a t i o n of the hyphae by Rhizobium japonicum (Kirchner) Buchanan.  The aseptate f u n g i , Phytophthora megasperma Drechs.  and Pythium ultimum were more susceptible to r h i z o b i a l parasitism i n v i t r o than the septate f u n g i , Fusarium oxysporum and Ascochyta imperfecta  Pk.  Interactions between Rhizobium and root r o t pathogens i j i s i t u may not be reproducible or detectable i n v i t r o , therefore i n v i v o studies are necessary.  Chi and Hanson (1961) f i r s t reported a b e n e f i c i a l e f f e c t  of Rhizobium t r i f o l i i Dangeard on red clover ( T r i f o l i u m pratense L.) exposed to F_. oxysporum, F_. roseum (Lk.) emend. Snyd. & Hans, and F_. s o l a n i w i l t and root r o t f u n g i .  Red clover p l a n t s , grown i n a sand-  n u t r i e n t s o l u t i o n medium i n the greenhouse, were c o n s i s t e n t l y more vigorous and developed l e s s root r o t when inoculated with Rhizobium.  Mew  and  Howard (1969) proposed the use of an a c i d - t o l e r a n t , e f f e c t i v e s t r a i n of R. japonicum to c o n t r o l F_. oxysporum root r o t of soybean.  Soybeans were  grown i n a sand-nutrient  Root r o t was  s o l u t i o n medium, at pH 7 or 7.6.  - 9 reduced to a trace or absent when R. japonicum was present but the same e f f e c t was not observed at pH 5.  In s i m i l a r experiments Orellana e_t a l .  (1976) suggested that nodulation of soybean with e f f i c i e n t s t r a i n s of R. japonicum could a l l e v i a t e the detrimental e f f e c t of R. s o l a n i root r o t . The b e n e f i c i a l e f f e c t of R. japonicum and Endogone mosseae Mosse i n suppressing Phytophthora megasperma root r o t of soybean has been claimed (Chow and Schmitthenner,. 1974).  In greenhouse and f i e l d experiments, Tu  (1978, 1980) observed that at a given concentration of F. oxysporum or P_. megasperma, a l f a l f a or soybean root r o t was reduced as the concentration of r h i z o b i a i n the s o i l increased.  Rhizobia protected a l f a l f a from severe  w i n t e r - k i l l i n g by reducing root r o t , a predisposing f a c t o r to winter injury.  The e f f e c t of Rhizobium on root r o t was not e r a d i c a t i v e but  provided a s i g n i f i c a n t degree of p r o t e c t i o n from severe root r o t (Tu, 1978a,b, 1980). While most of the l i t e r a t u r e indicates that legumes should be inoculated with Rhizobium, not only to increase root nodulation and Nf i x a t i o n , but a l s o f o r the added p r o t e c t i o n from root diseases, there have been a few reports to the contrary.  Gray and Sackston (1979) surveyed  44 pea (Pisum sativum L.) f i e l d s i n Quebec and found no c o r r e l a t i o n between the incidence and s e v e r i t y of F. s o l a n i root r o t and root nodulation by R. leguminosarum.  However, Gray and Hine (1976) reported that a l f a l f a  root nodules i n c i t e d by R. m e l i l o t i were the primary s i t e s of i n f e c t i o n by P_. megasperma e a r l y i n the growing season.  In greenhouse experiments  with pasteurized f i e l d s o i l , they also reported that seedling death was 24% greater when R. m e l i l o t i was present i n combination with P_. megasperma. Chow and Schmitthenner (1974) reported no apparent e f f e c t of R. japonicum and Endogone mosseae on root r o t of soybean caused by Pythiuiii ultimum.  -  S i m i l a r l y , Gray and  10  Sackston (1980) d i d not  Fusarium s o l a n i r o o t r o t of pea gonism had  -  by  observe any  reduction  in  r h i z o b i a l i n o c u l a t i o n although anta-  been observed jin v i t r o .  Objectives Rhi zobium i n a d d i t i o n t o i t s t r a d i t i o n a l r o l e i n r o o t and  s y m b i o t i c N - f i x a t i o n , has  r e c e n t l y been i m p l i c a t e d  s u p p r e s s i n g r o o t r o t or as a s y n e r g i s t , T h i s d i s c r e p a n c y i n the ascertain  The  antagonist  enhancing r o o t r o t of legumes.  l i t e r a t u r e warrants f u r t h e r i n v e s t i g a t i o n to  the n a t u r e of the  rotting fungi.  as an  nodulation  i n t e r a c t i o n between Rhizobium and  objectives  root  of t h i s t h e s i s were to i n v e s t i g a t e  e f f e c t of Rhizobium on bean r o o t r o t t i n g f u n g i and  the  to determine the r o l  of Rhizobium i n the development of r o o t r o t s i n c i t e d by J _ . s o l a n i f . phaseoli  and  R.  solani.  sp  MATERIALS AND METHODS  I.  1.  ISOLATION AND IDENTIFICATION OF PATHOGENIC FUNGI  Field  Survey  In o r d e r to i n v e s t i g a t e the n a t u r e of the i n t e r a c t i o n between Rhizobium collect  and bean r o o t r o t f u n g i a f i e l d  survey was f i r s t undertaken t o  i s o l a t e s of these micro-organisms.  L.) f i e l d s ,  l o c a t e d throughout  Ten bean (Phaseolus v u l g a r i s  the F r a s e r V a l l e y , B r i t i s h Columbia,  surveyed i n the summer o f 1979.  were  Samples o f d i s e a s e d and h e a l t h y ,  n o d u l a t e d p l a n t s were c o l l e c t e d . P o t e n t i a l l y pathogenic f u n g i were i s o l a t e d from d i s e a s e d bean r o o t s showing reddish-brown  t o dark brown d i s c o l o r a t i o n or l e s i o n s .  Diseased  r o o t t i s s u e was s e c t i o n e d i n t o 0.25 t o 0.5 cm segments and s u r f a c e s t e r i l i z e d by immersion immersion  i n 95% e t h y l a l c o h o l f o r 1 minute f o l l o w e d by  i n a 1:10 commercial  b l e a c h s o l u t i o n f o r 2 t o 5 minutes.  r o o t segments were t r a n s f e r r e d through 5 r i n s e s of s t e r i l e water,  p l a t e d onto p o t a t o d e x t r o s e agar  Nash and Snyder's  The  distilled  (PDA, D i f c o ) , tapwater agar, o r  PCNB media ( T u i t e , 1969) and i n c u b a t e d a t 21 t o 24°C.  S u b c u l t u r e s o f r e p r e s e n t a t i v e f u n g a l c o l o n i e s were e s t a b l i s h e d on PDA plates.  T e n t a t i v e i d e n t i f i c a t i o n s were made on the b a s i s o f c o l o n y  morphology a f t e r a p p r o x i m a t e l y 1 week.  M i c r o s c o p i c examination o f m y c e l i a  and spores were compared to standard m y c o l o g i c a l keys f o r the F u n g i Imperfecti  ( B a r n e t t , 1960) t o i d e n t i f y the i s o l a t e s t o genus.  spore i s o l a t e s of Fusarium were made and i d e n t i f i e d to the scheme of Toussoun and Nelson  (1968).  Single  to s p e c i e s a c c o r d i n g  The c o l l e c t i o n of f u n g a l  i s o l a t e s was m a i n t a i n e d on PDA s l a n t s both a t room temperature  and a t 4°C.  - 12 -  A u t h e n t i c a t e d i s o l a t e s of F. s o l a n i and R. sources  s o l a n i were o b t a i n e d from other  (Table 1 ) .  T a b l e 1.  Root r o t f u n g i o b t a i n e d from other  Species Fusarium (FS2)  sources  Source  s o l a n i f . sp. p h a s e o l i  Dr. J.C.  Tu, Harrow, O n t a r i o  F. s o l a n i f . sp. p h a s e o l i (FS911)  Dr. R. H a l l , Guelph, O n t a r i o  F. s o l a n i  P r o s s e r , Washington  (FSIV)  R h i z o c t o n i a s o l a n i f . sp. p h a s e o l i IV (RSI)  Dr. W e l l s , D a v i s ,  R.  solani  (RS2)  Dr. R.J.  Copeman, Vancouver,  R.  solani  (RS3)  Dr. J.C.  Tu, Harrow, O n t a r i o  2.  Growth Pouch Technique  King  &Co.,  M i n n e a p o l i s , MN  Weaver and F r e d e r i c k (1972). (18 x 16.5  cm)  to 0.75  Seed Packs,  55413) as p r e v i o u s l y d e s c r i b e d by  These s t e r i l e , c l e a r , p l a s t i c  packets  c o n t a i n e d a wide paper wick f o l d e d i n t o a trough a t the  top to accommodate the seed. a 0.5  B.C.  (GPT)  Bean s e e d l i n g s were grown i n growth pouches (DiSPo Northrup,  California  A flame  sterilized  cm h o l e i n the bottom of the trough  s c a p e l was to permit  passage of the l a r g e bean r a d i c l e along the paper wick.  used to cut the downward  The pouches were  then p l a c e d i n s p e c i a l wooden support boxes (outer dimensions 45 x 19.5 15.5  cm)  t h a t h e l d 60 to 70 pouches.  were spaced  a t 5 cm  Press board  dividers  i n t e r v a l s along the l e n g t h of the box  pouches i n a v e r t i c a l p o s i t i o n  (17.5 x 14 to support  ( F i g . 1).  Bean seeds (Phaseolus v u l g a r i s L.  'Topcrop') were used i n a l l  x cm)  the  -  F i g u r e 1.  13  -  'Topcrop' snap bean grown by a growth pouch t e c h n i q u e .  - 14 -  experiments u n l e s s  otherwise s p e c i f i e d .  by p l a c i n g them f o r 1 minute i n 95% immersion i n a 1:10  Seeds were s u r f a c e d i s i n f e s t e d  ethyl alcohol followed  commercial b l e a c h  solution.  w i t h 5 changes of s t e r i l e d i s t i l l e d water. seed was  placed  p y l a r s i d e was  20 ml  One  seeds were r i n s e d  surface  sterilized  bean  i n t o the trough of each growth pouch so t h a t the m i c r o d i r e c t l y over the h o l e made p r e v i o u s l y i n the t r o u g h .  p l a s t i c d r i n k i n g straw was initially  The  by a 10 minute  placed  i n t o the s i d e of each pouch  of s t e r i l e d i s t i l l e d water was  with a syringe.  Subsequently, water was  and  added through the  straw  s u p p l i e d as n e c e s s a r y and  10  of a n i t r o g e n - d e f i c i e n t n u t r i e n t s o l u t i o n w i t h microelements a t pH was  added weekly to each pouch (Table Inoculated  pouches were p l a c e d  2)  (Hoagland and  i n a c o m p l e t e l y randomized  Supplemental l i g h t was  provided  to 24°C.  s e e d l i n g growth pouches were p l a c e d a t 21 ± 2°C  and  At the end  cool-white  Temperature  l x from a mixture of  f l u o r e s c e n t tubes f o r 16 hr/day. the p l a n t s were r a t e d f o r r o o t r o t  to an equal increment d i s e a s e  index d e s c r i b e d  by  (1978a).  H e a l t h y - a p p e a r i n g r o o t s , creamy-white i n c o l o u r , were  a disease  index of 0 w h i l e c o m p l e t e l y r o t t e d , dead r o o t s were g i v e n  disease  index of 9.  Shoot and  determined where s t a t e d . r o o t and  Tu  given a  r o o t dry weights of i n d i v i d u a l p l a n t s were  S t a t i s t i c a l a n a l y s i s of the d i s e a s e  index  shoot dry weights i n v o l v e d a m u l t i f a c t o r i a l a n a l y s i s of  Duncan's m u l t i p l e range t e s t  cool-  i n an environmental c o n t r o l chamber  of the growth p e r i o d  s e v e r i t y according  unless  Where s p e c i f i e d boxes of  exposed to a p p r o x i m a t e l y 1,850  i n c a n d e s c e n t b u l b s and  6.5,  design  f o r 12 hr/day by  white f l u o r e s c e n t tubes p r o d u c i n g a p p r o x i m a t e l y 2,800 l x . i n the greenhouse ranged from 18  ml  Arnon, 1950).  i n the support boxes on benches i n the greenhouse f o r 4 weeks otherwise s t a t e d .  A  (p = 0.05)  and  s e t s of c o n t r a s t s .  and  variance, Individual  - 15 T a b l e 2.  N i t r o g e n - d e f i c i e n t n u t r i e n t s o l u t i o n used to water 'Topcrop' snap bean i n growth pouches  S o l u t i o n a:  ml/L  nutrient  solution  0.5 M K2SO4  5  1.0 M MgS0  2  4  0.05 M  Ca(H P0 )  0.01  CaSOij  M  2  4  10  2  200  S o l u t i o n b:  g/L  (Add 1 ml/L t o n u t r i e n t  s o l u t i o n (a)) 2.86  H3BO3 MnCl  1.81 ' 4H 0  2  2  ZnSOu  • 7H 0  CuSoit  • 5H 0  0.08  2  H Mo0 2  0.22  2  4  0.02  • H 0 2  S o l u t i o n c: (Add 1 ml/L t o n u t r i e n t 0.5%  experiments  3.  s o l u t i o n (a))  Fe ( t a r t r a t e )  were n o t r e p e a t e d u n l e s s o t h e r w i s e s p e c i f i e d .  Pathogenicity Tests Bean s e e d l i n g s a t the crook stage o f development were  w i t h one of the t e s t f u n g i .  Two types o f i n o c u l a were p r e p a r e d f o r patho-  g e n i c i t y t e s t s a c c o r d i n g t o whether o r n o t the t e s t fungus produced  spores.  f o r Fusarium  inoculated  readily  Inoculum c o n s i s t i n g o f a spore d i l u t i o n was p r e p a r e d  isolates.  The t e s t fungus was grown on PDA p l a t e s f o r 1 week.  S t e r i l e d i s t i l l e d water was then poured  i n t o the p l a t e s and the s u r f a c e  scraped l i g h t l y w i t h a s t e r i l e s c a l p e l .  The supernatant  and  was c o l l e c t e d  the spore c o n c e n t r a t i o n determined w i t h a hemocytometer.  The spore  - 16 -  d i l u t i o n was prepared w i t h s t e r i l e d i s t i l l e d water. was used  t o add 1 ml of inoculum t o each pouch.  A sterile  Inoculum  c o n s i s t i n g of  m y c e l i a was used i n p a t h o g e n i c i t y t e s t s w i t h R h i z o c t o n i a . R. s o l a n i , grown on PDA f o r 1 week  pipette  One p l a t e of  was mixed w i t h 200 ml o f s t e r i l e  d i s t i l l e d water i n a Waring B l e n d e r a t low speed f o r one minute. inoculum was added a t 1 t o 10 ml/pouch w i t h a s t e r i l e  pipette.  P r e l i m i n a r y p a t h o g e n i c i t y t e s t s were performed procedure t o i d e n t i f y v i r u l e n t subsequent  experiments.  u s i n g the above  i s o l a t e s which c o u l d then be used i n  C o n c e n t r a t i o n o f the spore inoculum was n o t  s t a n d a r d i z e d but was a t l e a s t 1 0 r e p l i c a t e d 3 times.  Mycelial  5  spores/ml.  The presence o r absence  Each treatment was o f r o o t r o t was r e c o r d e d  a f t e r 4 weeks. To determine  the e f f e c t of s e e d l i n g age on p a t h o g e n i c i t y , bean  p l a n t s , grown a c c o r d i n g t o the GPT, were i n o c u l a t e d a t 1.  the seed stage;  2.  the r a d i c l e s t a g e , when o n l y the primary r o o t was present;  3.  the crook s t a g e , when the h y p o c o t y l was bent; and  4.  the f i r s t , t r u e - l e a f not y e t unfolded.  still  stage, when the l e a v e s were  F_. s o l a n i 2 (FS2) was used a t a c o n c e n t r a t i o n of 1 0  7  spores/pouch.  C o n t r o l pouches of bean were t r e a t e d w i t h s t e r i l e d i s t i l l e d water o n l y . Each treatment was r e p l i c a t e d 10 times.  A s i m i l a r experiment  performed w i t h F. s o l a n i 911 (FS911) w i t h two d e v i a t i o n s . c o n c e n t r a t i o n of 1 0 stage, the f i r s t ,  6  An inoculum  spores/pouch was used and an a d d i t i o n a l  trifoliate-leaf  stage was i n c l u d e d .  Each  was  inoculation treatment  was r e p l i c a t e d 7 times and the p l a n t s were r a t e d a f t e r a growth p e r i o d o f 31  days.  The p a t h o g e n i c i t y of FS911 and FSIV ('Harvester',  ' S t r i n g l e s s Greenpod',  to t h r e e bean  'Topcrop')  cultivars  and one pea c u l t i v a r  (Pisum sativum L. ' L i n c o l n ' ) was t e s t e d a c c o r d i n g to the GPT. v a r s were i n o c u l a t e d a t the seed stage w i t h 1 0 FSIV except  6  spores/pouch  A l l cultiof FS911 o r  'Topcrop', which was i n o c u l a t e d a t the crook s t a g e .  Each  treatment was r e p l i c a t e d 4 times and p l a n t s were r a t e d a f t e r a growth p e r i o d of 3 weeks. The v i r u l e n c e of t h r e e R. s o l a n i i s o l a t e s t e s t e d by i n o c u l a t i n g  'Topcrop'  (RSI, RS2, RS3) was  s e e d l i n g s a t the crook stage w i t h  10 o r 20 ml/pouch of m y c e l i a l inoculum.  Each treatment was  either  replicated  3 times and p l a n t s were r a t e d a f t e r a growth p e r i o d of 3 weeks.  Two of  these i s o l a t e s , RSI and RS2, were subsequently t e s t e d by the GPT f o r v i r u l e n c e a t t h r e e inoculum c o n c e n t r a t i o n s (1, 5, 10 ml/pouch).  Seedling  were i n o c u l a t e d a t the f i r s t ,  inoculum  true-leaf  w i t h each treatment r e p l i c a t e d 4 t i m e s .  stage w i t h the m y c e l i a l  A f t e r a growth p e r i o d of 32 days  p l a n t s were r a t e d .  II.  1.  Field  ISOLATION AND  IDENTIFICATION OF RHIZOBIUM  Survey  Rhizobium d u r i n g the f i e l d  i s o l a t e s were r e c o v e r e d from bean r o o t nodules survey.  H e a l t h y - a p p e a r i n g nodules were e x c i s e d from  bean r o o t s and s u r f a c e s t e r i l i z e d f o l l o w e d by immersion  i n 95% e t h y l a l c o h o l f o r one minute  i n a 1:10 commercial  b l e a c h s o l u t i o n o r a 1%  m e r c u r i c c h l o r i d e s o l u t i o n f o r 2 to 5 minutes.  The nodules were then  t r a n s f e r r e d a s e p t i c a l l y , through 5 r i n s e s of s t e r i l e d i s t i l l e d Flame s t e r i l i z e d  collected  water.  f o r c e p s -were used t o c r u s h i n d i v i d u a l n o d u l e s .  A drop  of r h i z o b i a l c e l l s from the crushed nodule was s t r e a k e d a c r o s s a y e a s t  -  18 -  mannitol agar (YMA) p l a t e (Vincent, 1970).  Inoculated plates were  incubated at root temperature (21 to 24°C) u n t i l d i s t i n c t b a c t e r i a l colonies developed. Single colonies of b a c t e r i a were selected as possible Rhizobium on the basis of colony morphology and growth rate on YMA.  Colonies 2 to  4 mm i n diameter, convex, c i r c u l a r to oval-shaped, semi-transluscent, cream-coloured or white with moderate to abundant polysaccharide, developing a f t e r 3 to 5 days on YMA plates were selected.  Subcultures  were established on YMA p l a t e s . I s o l a t e s were tested f o r dye absorption on YMA plus Congo red. Rhizobium i s o l a t e s remain c o l o u r l e s s or only weakly absorb the dye (Vincent, 1970).  A gram s t a i n t e s t and microscopic examination were  performed to v e r i f y that i s o l a t e s were gram negative, small to medium sized rods (0.5 - 0.9 x 1.2 - 3 jam) occurring s i n g l y or i n p a i r s .  Younger  cultures were checked f o r m o t i l i t y while older cultures were checked f o r the presence of prominent, h i g h l y r e f r a c t i l e granules of polymerized g-hydroxybutyrate (PHB).  Cultures were a l s o examined to v e r i f y that  endospores were not produced.  B a c t e r i a l i s o l a t e s conforming to these  c h a r a c t e r i s t i c s were maintained i n the Rhizobium Culture C o l l e c t i o n (RCC) on YMA slants at room temperature or 4°C. 2.  Nodulation Tests I d e n t i f i c a t i o n of the b a c t e r i a l i s o l a t e s (RCC) as Rhizobium  u l t i m a t e l y r e l i e d on t h e i r a b i l i t y to nodulate 'Topcrop' snap bean. nodulating i s o l a t e s were used i n subsequent studies. were performed using the GPT.  Only  Nodulation t e s t s  Bean seedlings were inoculated at the  crook stage with 1 ml/pouch of a t u r b i d b a c t e r i a l suspension.  The  b a c t e r i a l inoculum was prepared from 1 week o l d cultures on YMA p l a t e s .  -  Inoculum  c o n c e n t r a t i o n was  Each treatment was nodules was  19  -  not s t a n d a r d i z e d but was  a t l e a s t 10  Nine a u t h e n t i c a t e d Rhizobium  o b t a i n e d from o t h e r sources were a l s o used i n n o d u l a t i o n t e s t s  T a b l e 3.  R. japonicum  cells/ml.  r e p l i c a t e d 4 times and the presence or absence of r o o t  r e c o r d e d a f t e r 4 weeks.  Rhizobium  6  species,  (Table 3 ) .  A u t h e n t i c a t e d Rhizobium s p e c i e s o b t a i n e d from o t h e r s o u r c e s .  species  (RJ1A,  Source  RJ1B)  Dr. J.C. Tu, Harrow, O n t a r i o  R. p h a s e o l i  (RP1)  Dr. F.B. H o l l , Vancouver,  R. p h a s e o l i  (RP2)  Dr. J.C. Tu, Harrow, O n t a r i o  R. leguminosarum  ( R L l , RL2)  Dr. F.B. H o l l , Vancouver,  R. leguminosarum (RCR1045) R. l u p i n i (RCR3211) R. t r i f o l i i (RCR5)  III.  1.  S t a n d a r d i z e d Agar P l a t e  B.C.  B.C.  Rothamsted C o l l e c t i o n of .Rhizobium, Harpenden, H e r t f o r d s h i r e , England  IN VITRO STUDIES  Technique (SAPT)  The agar p l a t e technique of Drapeau these experiments except t h a t YMA  et^ a l .  (1973) was  used i n  p l a t e s were i n o c u l a t e d w i t h Rhizobium  2 days p r i o r to a d d i t i o n of the t e s t fungus.  A s t r e a k of Rhizobium  was  made i n a l i n e down one s i d e of the agar p l a t e and i n c u b a t e d a t room temperature the f i e l d  (21 to 24°C) f o r 2 days.  survey, was  at room temperature the o u t e r margins  Inoculum  prepared by growing  (21 t o 2 4 ° C ) .  of the t e s t f u n g i , from  the f u n g i a week on PDA  D i s c s , 5 mm  plates  i n diameter, were cut from  of the f u n g a l c o l o n y w i t h a flame s t e r i l i z e d  and t r a n s f e r r e d a s e p t i c a l l y to the p r e i n o c u l a t e d Rhizobium  cork b o r e r  plates.  - 20 -  The  f u n g a l inoculum d i s c was  Each agar t e s t p l a t e was  p l a c e d 5 cm away from the Rhizobium  streak.  r e p l i c a t e d 3 times u n l e s s o t h e r w i s e s p e c i f i e d .  The i n o c u l a t e d p l a t e s were i n c u b a t e d a t room temperature (21 to 24°C) f o r 7 t o 10 days u n t i l streak.  the m y c e l i a l f r o n t s began to approach the Rhizobium  Signs of f u n g a l growth  i n h i b i t i o n c o u l d be observed when the  fungus was w i t h i n 1 cm of the Rhizobium  streak.  The i n t e r a c t i o n between Rhizobium and the t e s t fungus was a c c o r d i n g t o the system d e v i s e d by Drapeau of was  i n v i t r o i n h i b i t i o n were r e c o g n i z e d :  ejt a l . (1973) .  Four  rated levels  -, no i n h i b i t i o n of the fungus  observed; +, a v e r y s l i g h t degree of f u n g a l i n h i b i t i o n was  observed  as a zone of g r a d u a l l y evanescent hyphae near the Rhizobium s t r e a k , but the  fungus q u i c k l y c o l o n i z e d the agar plate;++-, a c l e a r zone of  i n h i b i t i o n was  observed and became c o l o n i z e d by s m a l l masses of hyphae  w i t h i n 3 to 5 days; + + -l-, a c l e a r zone of i n h i b i t i o n was  observed and  p e r s i s t e d a t l e a s t 1 week a f t e r i t s i n i t i a l  A measurement of  the  zone w i d t h was  2.  S c r e e n i n g of Survey I s o l a t e s In V i t r o In  formation.  taken a t t h i s time.  p r e l i m i n a r y d u a l c u l t u r e agar p l a t e t e s t s Rhizobium  were t e s t e d f o r t h e i r a n t a g o n i s t i c a c t i v i t y  towards  f u n g a l i s o l a t e s from the c u l t u r e c o l l e c t i o n  (Table 4 ) .  was  replicated  bacterial the  twice.  isolates  representative Each t e s t  plate  Subsequently, a l l 51 n o d u l a t i n g and n o n - n o d u l a t i n g  i s o l a t e s were screened by the SAPT f o r a n t i f u n g a l a c t i v i t y to  s e n s i t i v e s t r a i n F_. s o l a n i  repeated t w i c e .  (FS2) .  T h i s experiment  was  I n h i b i t i o n zones, r a t e d + + +', were measured 1 week a f t e r  t h e i r i n i t i a l formation.  - 21 -  T a b l e 4.  Rhizobium and f u n g a l i s o l a t e s t e s t e d i n d u a l c u l t u r e agar p l a t e s .  Rhizobium  isolates  100  FMl, F01, F02, F R l , FS1, FS2, FS3, P S l , PS2, PS3, P U l , PU2, RSI, RS2  101  F01, F02, F03, F04, F05, FS2, FS3, FS4, FS5, RSI  102,  103  FS3, FS4, FS5, RSI, RS2  105  FS2, FS3, FS4, FS5, RSI, RS2  106  FS4, FS5, RSI  107  FS2, FS4, FS5, RSI  108  FS4, FS5  109  FS2, FS3, FS4, FS6  114,  FM,  Fungal i s o l a t e s  115  FS6, RSI  Fusarium m o n i l i f o r m e  FO, F. oxysporum FR, F. roseum FS, F.  solani  PS, Pythium  sylvaticum  PU, Pythium  ultimum  RS, R h i z o c t o n i a  3.  solani  B a s i s of the In V i t r o  Inhibition  To determine whether or not i n h i b i t i o n of Fusarium by was  Rhizobium  due to a pH change, measurement of the pH of agar t e s t p l a t e s  taken a t the end of the s c r e e n i n g experiments. pH i n d i c a t o r paper was  inserted  A 1.5  was  to 2 cm s t r i p of  i n t o the i n h i b i t i o n zone, the f u n g a l  - 22 -  colony  (FS2) and the o p p o s i t e s i d e of the Rhizobium s t r e a k .  As a p r e -  l i m i n a r y t e s t o n l y one r e p l i c a t e of each t e s t p l a t e was  examined  of the s c r e e n i n g experiments.  initially  The pH of YMA  p l a t e s was  i n two 6.5.  In an attempt to demonstrate t h a t the i n v i t r o i n h i b i t i o n o f Fusarium was  due to some d i f f u s i b l e , m e t a b o l i t e produced by  Rhizobium,  segments of the i n h i b i t i o n zone were used to r e p l a c e the Rhizobium i n the SAPT.  I n h i b i t i o n zone segments ( 2 x 4  mm)  streak  were removed from  SAP  t e s t s of FS2 and Rhizobium i s o l a t e s RCC100, 107, 109,111, 121, 319, 326, 812 and 815 when zones were 7 days o l d . 2.5  cm away from an inoculum d i s c of FS2.  Zones were p l a c e d  Each t e s t p l a t e was  321,  .5 to replicated  twice and the experiment r e p e a t e d t w i c e .  The p l a t e s were checked  f o r the development  Cut out segments of i n h i b i t i o n  of i n h i b i t i o n zones.  zones were a l s o p l a c e d d i r e c t l y onto YMA Rhizobium i s o l a t e or spores of FS2. SAP  daily  p l a t e s seeded w i t h the same  Zones of i n h i b i t i o n were removed from  s c r e e n i n g t e s t s of FS2 and RCC107, 121 or 326.  Each t e s t p l a t e  was  r e p l i c a t e d t w i c e and the experiment r e p e a t e d t w i c e . C e l l - f r e e e x t r a c t s of Rhizobium were t e s t e d f o r t h e i r a b i l i t y to i n h i b i t t h e growth of FS2 and FS911 (RCC100, 106, 107, 116, 121,  on YMA  plates.  Rhizobium  isolates  607, 811) were grown i n y e a s t m a n n i t o l shake  c u l t u r e f o r 3 t o 31 days a t room  temperature (21 t o 24°C).  The  cultures  were c e n t r i f u g e d i n a S o r v a l l Superspeed c e n t r i f u g e , SS-34, a t 12,100 g f o r 30 minutes.  The s u p e r n a t a n t s were s t e r i l i z e d by m i l l i p o r e  A drop or s t r e a k of the c e l l - f r e e e x t r a c t was inoculum d i s c of FS2 and FS911. Rhizobium s t r e a k i n the SAPT.  filtration.  placed opposite a 5  mm  The c e l l - f r e e e x t r a c t r e p l a c e d the Each agar t e s t p l a t e was r e p l i c a t e d  twice.  C e l l - f r e e e x t r a c t s of RCC106 or RCC607, s t o r e d a t 4°C, were a l s o p l a c e d i n 5 mm  ' w e l l s ' c u t out of the agar 1 cm away from inoculum d i s c s of FS2.  The  ' w e l l s ' were r e p l e n i s h e d d a i l y w i t h the c e l l - f r e e e x t r a c t .  p l a t e was  r e p l i c a t e d t w i c e and checked d a i l y f o r the development  b i t i o n zones. dialysis was  Each  test  of i n h i -  A c e l l - f r e e e x t r a c t of RCC607, c o n c e n t r a t e d (1:10) by  ( t u b i n g pore s i z e 4.8 mm)  t e s t e d as p r e v i o u s l y i n 5 mm  against polyethylene g l y c o l  agar  'wells'.  Two  (6000  MW),  r e p l i c a t e s were made  and the experiment repeated t w i c e .  IV.  1.  IN VIVO STUDIES  S c r e e n i n g of JBhi z o b i i r m i n Growth Pouches The GPT was  used i n f a c t o r i a l experiments  ( T a b l e 5)  v a r y i n g c o n c e n t r a t i o n s of Rhizobium and _F. s o l a n i  involving  (FS911) t o determine  whether or not i n h i b i t i o n of r o o t r o t o c c u r r e d i n v i v o .  Bean s e e d l i n g s  were s i m u l t a n e o u s l y i n o c u l a t e d a t e i t h e r the crook stage or the  first,  t r u e - l e a f s t a g e w i t h f o u r l e v e l s of Rhizobium  cells/  pouch) i n combination w i t h FS911 was  (0, 1 0 ,  (0, 10 ', 1 0 \  prepared as p r e v i o u s l y d e s c r i b e d .  2  2  10  6  10 *, 1 0 1  6  spores/pouch).  Inoculum  Each of the 16 treatments  was  r e p l i c a t e d 4 times but i n d i v i d u a l experiments were not r e p e a t e d except t h a t i n v o l v i n g RCC106.  P l a n t s were grown f o r 4 weeks and then a s s i g n e d a  r o o t d i s e a s e i n d e x , n o d u l a t i o n index (0, no n o d u l e s t o 3, abundant nodules) ?  and r o o t and  shoot d r y weights were determined.  The e f f e c t of Rhizobium and R.  solani  (RSI) was  on the r o o t r o t complex of _F. s o l a n i  investigated i n a 3 x 4 f a c t o r i a l  s i m i l a r t o the p r e v i o u s experiments.  (FS911)  experiment  Bean s e e d l i n g s a t the f i r s t ,  true-  l e a f s t a g e were s i m u l t a n e o u s l y i n o c u l a t e d w i t h RCC106 (0, 1 0 ,  10 ,  c e l l s / p o u c h ) and  spores/pouch  s  inoculum m i x t u r e of FS911  a t e i t h e r 0 or 1 0  w i t h RSI a t e i t h e r 2 or 4 ml/pouch ( T a b l e 5 ) . ted  5 times.  6  6  Each treatment was  10  7  replica-  T a b l e 5.  Rhizobium isolate  Rhizobium i s o l a t e s e v a l u a t e d f o r i n h i b i t i o n of Fusarium s o l a n i (FS911) and R h i z o c t o n i a s o l a n i (RSI) r o o t r o t s i n growth pouches of 'Topcrop' snap bean.  In v i t r o i n h i b i t i o n of _F. s o l a n i 2*  Pathogen t e s t e d ±n v i v o  Seedling i n o c u l a t i o n stage  106  + + +  FS911  crook  607  + + +  FS911  crook  812  + + +  FS911  first,  true-leaf  321  + + +  FS911  first,  true-leaf  324  +  FS911  crook  816  -  FS911  crook  106  + + +  FS911  +  RSI  first,  true-leaf  *Four l e v e l s of jin v i t r o i n h i b i t i o n were r e c o g n i z e d : -, no i n h i b i t i o n was observed; +, s l i g h t i n h i b i t i o n ; + +, c l e a r zone of i n h i b i t i o n , c o l o n i z e d a f t e r 3 t o 5 days; + + +, c l e a r zone of i n h i b i t i o n p e r s i s t e d at l e a s t 1 week.  A similar 4 x 4  f a c t o r i a l experiment  e f f e c t of t h r e e Rhizobium r o o t r o t of bean.  isolates  was  performed  (RCC107, 324,  or 613)  Bean s e e d l i n g s , grown by the GPT,  the crook stage w i t h Rhizobium pouch of R.  was  s o l a n i inoculum  a t 10  8  on R.  solani  were i n o c u l a t e d a t  cells/pouch.  Two  or RS3 was  added.  (RSI, RS2,  t o study the  days l a t e r Each  3 ml/  treatment  r e p l i c a t e d 4 times and the pouches were grown f o r a p e r i o d of 3  weeks. Large s c a l e s c r e e n i n g experiments were c a r r i e d out u s i n g the (Table 6 ) .  Bean s e e d l i n g s , grown i n an  environmental c o n t r o l  were i n o c u l a t e d a t the crook stage w i t h FS911  a t 0, 1 0 , 2  GPT  chamber,  or 10 * 1  spores/  - 25 -  pouch.  Two Rhizobium i s o l a t e s  i n each experiment.  a t 0, I0 , h  Rhizobium was added  or 10  6  c e l l s / p o u c h were t e s t e d  the pouches  a t the same time as  FS911.  T a b l e 6.  Rhizobium i s o l a t e s e v a l u a t e d f o r i n h i b i t i o n of Fusarium s o l a n i (FS911) r o o t r o t i n growth pouches of 'Topcrop' snap bean.  Rhizobium isolate  Nodulating ability  'In v i t r o i n h i b i t i o n o f F. s o l a n i 2*  107, 109  +, +  111, 115  +  + + +,+ + +  118  +, +  + + +, + + +  319, 321  +  + + +, + + +  323, 812  +  + +  816, RL1  +, -  116,  815, RCR1045  + +  + + +  + + + +  +  + +  + + +  *Four l e v e l s o f in v i t r o i n h i b i t i o n were r e c o g n i z e d : -, no i n h i b i t i o n was observed; +, s l i g h t i n h i b i t i o n ; + + , c l e a r zone o f i n h i b i t i o n , c o l o n i z e d a f t e r 3 to 5 days; + + +, c l e a r zone of i n h i b i t i o n p e r s i s t e d a t l e a s t 1 week.  2.  S o i l Tests  S i m i l a r f a c t o r i a l experiments i n v o l v i n g f o u r c o n c e n t r a t i o n s of both Rhizobium and of F_. s o l a n i  (FS911) were performed i n greenhouse  s o i l pH 6.5,  The s o i l was p a s t e u r i z e d twice, once on each of two c o n s e c u t i v e days, infested  w i t h FS911 inoculum and p l a c e d i n t o  15 cm p o t s .  The FS911 i n o c u -  lum was grown i n s t e r i l i z e d v e r m i c u l i t e and y e a s t m a n n i t o l l i q u i d f o r  - 26 -  2 or 3 weeks a t room temperature to  l o o s e n clumps.  (21 t o 24°C).  C u l t u r e s were shaken  daily  The f o u r l e v e l s o f FS911 inoculum were made up t o a  s t a n d a r d i z e d volume w i t h s t e r i l i z e d  v e r m i c u l i t e then t h o r o u g h l y mixed  w i t h t h e p a s t e u r i z e d s o i l i n volume t o volume r a t i o s i n d i c a t e d below. Each pot o f i n f e s t e d s o i l was seeded w i t h 5 s u r f a c e s t e r i l i z e d  bean seeds.  F i v e h o l e s , 2 t o 3 cm deep, were made i n t h e s u r f a c e o f t h e s o i l of each pot  and one seed was p l a c e d i n t o each h o l e .  10  or 1 0  6  cultures.  8  pushed  inoculum a t 0, I O , 4  c e l l s / m l was prepared as d e s c r i b e d p r e v i o u s l y from YMA s t o c k Each seed was t r e a t e d w i t h 1 ml of Rhizobium  c o n t r o l s were t r e a t e d w i t h 1 ml of s t e r i l e , was  Rhizobium  distilled  back i n t o t h e h o l e s t o cover t h e seeds.  inoculum w h i l e  water.  The s o i l  Each of t h e 16 t r e a t -  ments was r e p l i c a t e d 4 times and t h e 64 p o t s were p l a c e d i n a c o m p l e t e l y randomized  d e s i g n on a bench i n t h e greenhouse  f o r 4 weeks.  i n d i v i d u a l l y watered w i t h o r d i n a r y tapwater as r e q u i r e d . the  greenhouse  ranged from 18 t o 24°C,  supplemental l i g h t  Temperatures i n  Unless otherwise s p e c i f i e d  was p r o v i d e d by c o o l - w h i t e f l u o r e s c e n t tubes p r o v i d i n g  2,800 l x f o r 12 hr/day.  A t t h e end of t h e experiment  c a r e f u l l y l i f t e d from t h e s o i l and t h e r o o t s passed tapwater.  Pots were  t h e p l a n t s were through 5 r i n s e s w i t h  The number o f p l a n t s t h a t emerged per pot was r e c o r d e d and each  p l a n t was a s s i g n e d a r o o t d i s e a s e index, n o d u l a t i o n index and r o o t and shoot d r y weights were determined.  Statistical  a n a l y s i s was based on an  average per pot v a l u e o f t h e d i s e a s e index, r o o t n o d u l a t i o n index and d r y weights.  A m u l t i f a c t o r i a l a n a l y s i s o f v a r i a n c e was performed  emergence d a t a .  on t h e p l a n t  I n s e v e r a l treatments p l a n t s d i d not emerge which  r e s u l t e d i n missing data.  The computer program BMD10V was used t o generate  dummy v a r i a b l e s f o r t h e a n a l y s i s of v a r i a n c e , Duncan's m u l t i p l e range and c o n t r a s t s  (Dixon and Brown,  1977).  test  - 27 -  Rhizobium i s o l a t e RCC106 was t e s t e d f o r antagonism t o FS911 r o o t r o t o f bean i n p a s t e u r i z e d  s o i l as d e s c r i b e d  used a f t e r a 3 week i n c u b a t i o n p e r i o d . to pasteurized contained  s o i l were 0, 1:10**, 1:10  a standardized  above.  Inoculum  of FS911 was  The volume:volume r a t i o s of FS911 3  and 1:10 .  volume o f s t e r i l i z e d  2  Control pots  vermieulite.  A similar  experiment was performed w i t h RCC106 except t h a t FS911 inoculum was used a f t e r a 2 week i n c u b a t i o n p e r i o d . pasteurized not p r o v i d e d  s o i l were 0, 1:120, 1:60, and 1:30. i n t h i s experiment.  the same c o n d i t i o n d e s c r i b e d light  The r a t i o s of FS911 inoculum t o  The f i n a l  Supplemental l i g h t  s o i l t e s t i n v o l v e d RCC816 u s i n g  f o r the second t e s t except t h a t  from f l u o r e s c e n t tubes was p r o v i d e d .  was  supplemental  - 28 -  RESULTS  I.  ISOLATION AND IDENTIFICATION OF PATHOGENIC FUNGI  A t o t a l of 151 f u n g a l i s o l a t e s was bean r o o t s c o l l e c t e d d u r i n g the f i e l d  recovered  survey.  s p e c i e s of Fusarium, Pythium and T h i e l a v i o p s i s Rhizoctonia isolates  s o l a n i was n o t r e c o v e r e d  r o o t pathogens of snap bean.  lent and  pathogenic  were c o l l e c t e d but  (Table 7 ) . Most o f the f u n g a l  (FS) were c o n s i d e r e d  potentially  only important  P r e l i m i n a r y p a t h o g e n i c i t y t e s t s demonstra-  t h a t most of the 17. oxysporum and F. s o l a n i i s o l a t e s t e s t e d were a v i r u (F02, F03, F04, F05, F06, F07, F08, FS1, FS4, FS5, FS6, FS9, FS10) o n l y FS2, FS3 and FS8 i n c i t e d a m i l d degree of bean r o o t r o t .  Authenticated RS2,  Potentially  (144/151) were i d e n t i f i e d as s p e c i e s of Fusarium although  F_. oxysporum (FO) and F. s o l a n i  ted  and i d e n t i f i e d from  i s o l a t e s of F. s o l a n i  RS3) were pathogenic  Table  7.  (FS911, FSIV) and R. s o l a n i (RSI,  t o bean except RS3.  Fungi i s o l a t e d from snap bean r o o t s from commercial f i e l d s i n the F r a s e r V a l l e y , B r i t i s h Columbia.  Species Fusarium m o n i l i f o r m e F. oxysporum (FO)  Number o f I s o l a t i o n s (FM)  35 100  F. roseum (FR)  3  F. s o l a n i (FS)  6  Pythium spp. (PY)  2  Thielaviopsis  5  (TH)  - 29  -  Younger seedings were more s u s c e p t i b l e than o l d e r s e e d l i n g s FS2  r o o t r o t (Table 8 ) .  The  r a d i c l e stage was  the most  to  susceptible  stage w h i l e p l a n t s i n o c u l a t e d a t the seed or crook stage developed significantly  l e s s root r o t .  stage were not  Table 8.  Plants  i n o c u l a t e d at the f i r s t ,  s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l s .  true-leaf  Because FS2  was  Root d i s e a s e index and shoot and r o o t dry weights of 'Topcrop' snap bean grown i n growth pouches and i n o c u l a t e d a t f o u r d i f f e r e n t stages w i t h Fusarium s o l a n i 2 at 10 spores/pouch 7  Growth stage inoculated  Root D i s e a s e index*  Shoot dry weight  Root dry weight  (g)  (g)  Seed  2.2  b**  0,.295  a  0,.091  a  Radicle  6.7  c  0..214  a  0..075  a  Crook  1.9  b  0..290  a  0..101  a  1.2  ab  0..339  a  0..106  a  0.2  a  0..342  a  0.,104  a  First,  true-leaf  Uninoculated  *Root r o t s e v e r i t y was based on an equal increment d i s e a s e 0, h e a l t h y r o o t s , to 9, c o m p l e t e l y r o t t e d , dead r o o t s .  index from  **Duncan's m u l t i p l e range t e s t : v a l u e s f o l l o w e d by the same l e t t e r are not s i g n i f i c a n t , P = 0.05. Each v a l u e was an average of 10 r e p l i c a t e s .  only  s l i g h t l y v i r u l e n t , at h i g h  o n l y moderate to low d i s e a s e weights were not bean p l a n t s  i n d i c e s were r e c o r d e d .  s i g n i f i c a n t l y d i f f e r e n t i n any  i n o c u l a t e d at the f i r s t  v i r u l e n t FS911 first,  inoculum c o n c e n t r a t i o n  and  stage and  7  spores/pouch)  Shoot and  treatment.  root  dry  Similarly,  f o u r growth stages w i t h h i g h l y  s i g n i f i c a n t l y greater  trifoliate-leaf  (10  controls  root disease (Table 9 ) .  i n d i c e s than  the  Fusarium r o o t r o t  - 30 -  was c h a r a c t e r i z e d by b r i g h t r e d s t r e a k s of i n d e f i n i t e s i z e and margin e x t e n d i n g down t h e h y p o c o t y l and t a p r o o t . brown and n e c r o t i c ,  These l e s i o n s became dark  l a t e r a l r o o t s d i e d back and w i t h i n 3 weeks the e n t i r e  r o o t system was dead ( F i g .  2).  Shoot and r o o t d r y weights o f s e e d l i n g s  i n o c u l a t e d a t the seed and r a d i c l e stage weighed s i g n i f i c a n t l y l e s s than other treatments.  Table 9.  Root d i s e a s e index and shoot and r o o t d r y weights of 'Topcrop' snap bean grown i n growth pouches and i n o c u l a t e d a t f i v e d i f f e r e n t stages w i t h Fusarium s o l a n i 911 a t 1 0 spores/pouch 6  Growth stage inoculated  Root d i s e a s e index*  Shoot d r y weight  Root d r y weight  (g)  (g)  Seed  7.6 c**  0.081 a  0.029 a  Radicle  8.9 c  0.100 a  0.027 a  Crook  7.9 c  0.244 b  0.082 b  First,  true-leaf  7.7 c  0.282 b  0.088 b  First,  t r i f o l i a t e l e a f 4.9 b  0.261 b  0.088 b  0.330 b  0.091 b  Uninoculated  0.0 a  *Root r o t s e v e r i t y was based on an equal increment d i s e a s e index from 0, h e a l t h y r o o t s , t o 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e range t e s t : v a l u e s f o l l o w e d by the same l e t t e r a r e not s i g n i f i c a n t , P = 0.05. Each v a l u e was an average o f 7 r e p l i c a t e s .  F. s o l a n i i s o l a t e s bean c u l t i v a r s  (FS911, FSIV) were h i g h l y v i r u l e n t  'Harvester',  t o the t h r e e  ' S t r i n g l e s s Greenpod* and 'Topcrop' but were  a v i r u l e n t t o ' L i n c o l n ' pea ( T a b l e 1 0 ) . A l l t h r e e bean c u l t i v a r s were e q u a l l y s u s c e p t i b l e t o FS911.  ' S t r i n g l e s s Greenpod' was s l i g h t l y  s u s c e p t i b l e t o FSIV than the o t h e r two c u l t i v a r s .  less  - 31 -  Figure  2 - Fusarium s o l a n i 911 r o o t r o t of 'Topcrop' snap bean grown by a growth pouch t e c h n i q u e .  - 32  Table 10.  -  R o o t . d i s e a s e index of t h r e e snap bean c u l t i v a r s , 'Harvester', ' S t r i n g l e s s Greenpod', 'Topcrop and ' L i n c o l n ' pea grown i n growth pouches * ,:  Pathogen  Root D i s e a s e  Snap bean 'S. Greenpod' 'Topcrop'  'Harvester' Fusarium  solani  IV  Index** Pea 'Lincoln'  Means  8;8c***  7.8b  9.0c  0.0a  6.4b  9.0c  8.8c  8.8c  0.0a  6.0b  Uninoculated  0.0a  0.0a  0.0a  0.0a  0.0a  Means  5.9c  5.5c  5.9c  0.0a  F. s o l a n i  911  * P l a n t s a t the seed stage were i n o c u l a t e d w i t h F. s o l a n i i s o l a t e s a t 10 spores/pouch except 'Topcrop' which was i n o c u l a t e d a t the seed stage. 6  **Root r o t s e v e r i t y was based on an equal increment d i s e a s e index 0, h e a l t h y r o o t s to 9, c o m p l e t e l y r o t t e d , dead r o o t s .  from  ***Duncan's m u l t i p l e range t e s t : v a l u e s f o l l o w e d by the same l e t t e r are not s i g n i f i c a n t , P = 0.05. Each v a l u e was an average of 4 r e p l i c a t e s .  Regardless when snap bean was caused  little  of the inoculum  c o n c e n t r a t i o n , severe r o o t r o t  i n o c u l a t e d w i t h R.  to no r o o t r o t and was  R h i z o c t o n i a r o o t r o t was  solani isolates  the h y p o c o t y l and  r o o t became c o m p l e t e l y  ( F i g . 3).  t i o n of RSI true-leaf  or RS2  taproot.  The  to snap bean.  to .75  cm)  and margin, appearing  The d i s e a s e index i n c r e a s e d as the inoculum increased.  degree of r o o t r o t w h i l e a t 1 ml of inoculum (Table  11).  developed  root  concentra-  Bean s e e d l i n g s i n o c u l a t e d at the s o l a n i inoculum  on  g i r d l e d w i t h brown  s o l a n i forming on the s u r f a c e of decaying  stage w i t h 10 ml of R.  r o o t r o t developed  RS3  c h a r a c t e r i z e d by d i s t i n c t , o v a l to e l o n g a t e , (.5  tissue  (RSI, RS2).  considered a v i r u l e n t  sunken l e s i o n s of d e f i n i t e s i z e  to b l a c k s c l e r o t i a of R.  developed  first,  a moderate  o n l y a v e r y s l i g h t degree  - 33 -  F i g u r e 3 - R h i z o c t o n i a s o l a n i (a) r o o t r o t and (b) h y p o c o t y l r o t of 'Topcrop' snap bean i n growth pouches.  - 34 -  T a b l e 11.  P a t h o g e n i c i t y of two R h i z o c t o n i a s o l a n i i s o l a t e s to 'Topcrop' snap bean i n growth pouches  Inoculum c o n c e n t r a t i o n (ml/pouch)  Root D i s e a s e Index* R.  solani 1  R. s o l a n i 2  0  0  0  1  3  2  5  3  3  10  6  5  *Root r o t s e v e r i t y was based on an equal increment d i s e a s e . i n d e x from 0, h e a l t h y r o o t s t o 9, c o m p l e t e l y r o t t e d , dead r o o t s . Each v a l u e was an average of 4 r e p l i c a t e s .  II.  ISOLATION AND  IDENTIFICATION OF RHIZOBIUM  A t o t a l of 42 b a c t e r i a l  i s o l a t e s was  s e l e c t e d from bean r o o t  nodule i s o l a t i o n s r e s e m b l i n g Rhizobium a c c o r d i n g to m o r p h o l o g i c a l c r i t e r i a and growth r a t e on YMA  (Table 12).  P o t e n t i a l Rhizobium c o l o n i e s on  congo r e d p l a t e s d i d not absorb the dye.  I s o l a t e s were gram n e g a t i v e ,  m o t i l e , s m a l l to medium s i z e rod-shaped c e l l s . endospores. Rhizobium vulgaris  I s o l a t e s d i d not  produce  U n e q u i v o c a l evidence t h a t the b a c t e r i a l i s o l a t e s were  spp. depended 'Topcrop').  on t h e i r a b i l i t y  However o n l y 40/51  t i o n were c a p a b l e of n o d u l a t i n g  primary and secondary r o o t s .  to n o d u l a t e snap bean (P. bacterial isolates  (2 to 4 mm  A u t h e n t i c a t e d Rhizobium  leguminosarum  (RP1, RP2)  (RL1, RL2)  species  (RCR3211).  inciting  (RCR1045), R.  but not R. japonicum  or R. l u p i n i  Healthy -  i n diameter) developed on  r o o t n o d u l a t i o n of snap bean were R. leguminosarum (RCR5) and R. p h a s e o l i  i n the c o l l e c -  'Topcrop' snap bean (Table 13).  a p p e a r i n g , s p h e r i c a l r o o t nodules  R.  YMA-  trifolii  (RJIA, R J I B ) ,  A c c o r d i n g to V i n c e n t  - 35 -  (1970), R. p h a s e o l i always n o d u l a t e s bean w h i l e R. leguminosarum, l u p i n i and R. t r i f o l i i  T a b l e 12.  R.  o n l y v e r y r a r e l y n o d u l a t e bean.  Source of Rhizobium i s o l a t e s root nodules*  Isolate  from snap bean  Number  100, 101, 102, 103, 105, 108, 109, 111, 113, 114, 118, 119, 121, 123  Source  18  field 1  219  1  field  2  319, 321, 323, 324, 325,  6  field  3  610, 603, 604, 606, 607, 611, 615, 637, 638, 644  12  811, 812, 814, 815, 816  5  *Bean p l a n t s were c o l l e c t e d B r i t i s h Columbia.  T a b l e 13.  8  N o d u l a t i n g a b i l i t y of Rhizobium i s o l a t e s t o 'Topcrop' snap bean i n growth pouches  Non-nodulating Rhizobium isolates  100, 101, 102, 105, 106,  107  103,  108, 109, 111, 113, 114,  115  219  116, 118, 119,  123  323,  319, 321, 324,  326  815  601, 603, 604, 606, 607, 608, 611, 615, 637, 638,  644  816  R. leguminosarum  field  from commercial f i e l d s i n the F r a s e r V a l l e y ,  N o d u l a t i n g Rhizobium isolates  811, 812, 814,  field 6  (RP1,  R. t r i f o l i i  (RCR5)  325  R. leguminosarum  (RL1, RL2)  R. l u p i n i (RCR3211) R. japonicum (RJIA, RJIB)  (RCR1045)  R. p h a s e o l i  609  121  RP2)  - 36 -  III.  1.  IN VITRO STUDIES  S c r e e n i n g o f Survey I s o l a t e s  i n In.-vitro  P r e l i m i n a r y experiments demonstrated t h a t the i n h i b i t i o n of f u n g a l growth  hi v i t r o depended  on the Rhizobium and f u n g a l i s o l a t e  tested.  Fusarium s p e c i e s were the most w i d e l y i n h i b i t e d by Rhizobium w h i l e Pythium and R.  solani  i s o l a t e s were not i n h i b i t e d by any of the Rhizobium  tested  (Table 14. F i g . 4 ) .  c o l o n i z e d the YMA  plate  Pythium and R. s o l a n i  isolates  2 was  selected a l l RCC  one of the more s e n s i t i v e  f o r agar p l a t e isolates  (Table 15) .  not  cm  depend  isolates  screening t e s t s .  Because  F.  to Rhizobium i t was  With the e x c e p t i o n of RCC816,  showed some degree of antagonism towards FS2 i n v i t r o  Inhibition  measured 1 week a f t e r t o 0.59  rapidly  i n 2 to 3 days as compared to Fusarium which  r e q u i r e d a t l e a s t one week to r e a c h the Rhizobium s t r e a k . solani  isolates  zones of 17 Rhizobium i s o l a t e s , r a t e d + + +, were  the zones had formed.  ( T a b l e 16).  Zone w i d t h v a r i e d  The l e v e l of i n h i b i t o r y  on the n o d u l a t i n g a b i l i t y  from  0.14  a c t i v i t y towards FS2 d i d  of the Rhizobium i s o l a t e .  R.  leguminosarum  (RCR1045), which v e r y r a r e l y n o d u l a t e s bean, r a t e d + + +  i n agar p l a t e  test.  RL1, RL2) in v i t r o .  also  Non-nodulating i s o l a t e s  showed v a r y i n g degrees of a n t i f u n g a l a c t i v i t y  The agar p l a t e  occasions with  s c r e e n i n g t e s t was  essentially  solani  to F. s o l a n i  (RSI, RS2,  RS3)  FS2  r e p e a t e d on 3 s e p a r a t e  (RL1, RCC603, 219, 644, 814,  121 and  321)  (FS2, FS911) had no e f f e c t on the growth of R. i n agar p l a t e  tests  w i d e l y i n h i b i t e d by Rhizobium than FS911 experiment was  towards  325,  the same r e s u l t s .  Seven Rhizobium i s o l a t e s inhibitory  (RCC103, 121, 219, 323,  (Table 17).  FS2 was more  i n t h i s experiment.  r e p e a t e d the r e s u l t s were the same.  When the  - 37 -  T a b l e 14.  Rhizobium isolates  The e f f e c t of Rhizobium i s o l a t e s * on f u n g a l growth as determined by d u a l c u l t u r e agar p l a t e t e s t s  Fungal  Not  100  FMl, FS3  FOl,  F02,  FR1,  101  FS2,  FS3,  FS4,  FS5  FS3,  FS4,  FS5  105  FS2,  FS3,  FS4,  106  FS4,  FS5  107  FS2,  FS4,  108  FS4,  FS5  109  FS2,  FS3,  102,  114,  isolates**  Inhibited  103  115  FS1, FS2,  FS5  inhibited  PSl, RSI,  PS2, RS2  PS3,  PU1, PU2,  FOl, RSI  F02,  F03,  F04, F05,  RSI,  RS2  RSI,  RS2  RSI FS5  FS4,  RSI  FS6  FS6  * R e s u l t s were based on 2  RSI replicates.  **FM, Fusarium m o n i l i f o r m e ; FO, F. oxysporum; FR, F. roseum; FS, F. s o l a n i ; PS, Pythium s y l v a t i c u m ; PU, P. ultimum; RS, R h i z o c t o n i a s o l a n i .  T a b l e 15.  L e v e l o f in v i t r o i n h i b i t o r y a c t i v i t y of Rhizobium i n d u a l c u l t u r e agar p l a t e t e s t s w i t h Fusarium solani  L e v e l of i n h i b i t i o n *  Rhizobium  isolate**  -  816,  +  102, 103, 113, 114, 119, 601, 603, 606, 608, 609, RCR5, R L l , RL2  123, 324, 611, 615,  + +  101, 638,  105, 108, 219, 325, 644, 811, 814  604, 637,  + ++  100, 118, 812,  106, 107, 109, 111, 121, 319, 321, 323, 815, RCR1045  115, 116, 326, 607,  RCR3211, RJIA, RJIB, RP1, RP2  *Four l e v e l s o f i n v i t r o i n h i b i t i o n were recognized:. -, no i n h i b i t i o n ; +, s l i g h t i n h i b i t i o n ; ++, c l e a r zone of i n h i b i t i o n , c o l o n i z e d a f t e r 3 to 5 days; + + +, c l e a r zone o f i n h i b i t i o n p e r s i s t e d a t l e a s t one week. L e v e l s of i n h i b i t i o n were averages of 3 r e p l i c a t e s . *RCR1045 (R. l.eguminoarum), RCR3211 (R. l u p i n i ) , RCR5 (R. t r i f o l i i ) . RJ, R. japonicum. RL, R. leguminosarum. RP, R. p h a s e o l i .  -  38  -  F i g u r e 4 - L e v e l of i n h i b i t i o n i n d u a l c u l t u r e agar p l a t e tests: (a) F u s a r i u m s o l a n i (FS2) i n h i b i t e d (+ + +) by Rhizobium i s o l a t e RCC326; (b) R h i z o c t o n i a s o l a n i 1 n o t i n h i b i t e d (-) by RCC326.  - 39 -  T a b l e 16.  I n h i b i t i o n zones r a t e d + + + i n d u a l c u l t u r e agar p l a t e t e s t s o f Rhizobium and Fusarium s o l a n i 2*  Rhizobium isolate*  Average w i d t h of zone (cm)  319  Rhizobium isolate  Average w i d t h of zone (cm)  0.14  107  0.23  0.14  116  0.24  100  0.15  106  0.25  321  0.17  326  0.26  323  0.17  115  0.31  812  0.18**  109  0.53**  607  0.18  815  0.54  111  0.19  121  0.59  118  0.22  RCR1045  *Zones were measured 1 week a f t e r **Average of 2 r e p l i c a t e s ,  T a b l e :17.  Fungal isolate  a l l others included 3  replicates.  I n v i t r o i n h i b i t o r y a c t i v i t y o f seven Rhizobium i s o l a t e s t o Fusarium s o l a n i (FS2, FS911) and R h i z o c t o n i a s o l a n i (RSI, RS2, RS3) i n d u a l c u l t u r e . agar p l a t e t e s t s . ' '  Rhizobium i s o l a t e 644 814 603  219  + + +  ++  ++  +  + +  + + +  + + +  +  ++  -  121  321  FS2  + + +**  FS911  + + +  RSI  _  RS2  -  RS3  -  *R.  i n i t i a l formation.  _  _  _  _  RL2*  +  -  -  -  -  -  -  leguminosarum.  **Four l e v e l s of i n v i t r o i n h i b i t i o n were r e c o g n i z e d : -, no i n h i b i t i o n ; +, s l i g h t i n h i b i t i o n ; + +, c l e a r zone o f i n h i b i t i o n , c o l o n i z e d i n 3 t o 5 days; + + +, c l e a r zone of i n h i b i t i o n p e r s i s t e d a t l e a s t one week. L e v e l s o f i n h i b i t i o n were averages of 3 r e p l i c a t e s .  - 40  2.  B a s i s of the In V i t r o  -  Inhibition  In v i t r o experiments d i d not  i n d i c a t e the mechanism(s) r e s p o n s i b l e  f o r f u n g a l i n h i b i t i o n i n d u a l c u l t u r e agar p l a t e t e s t s . the pH of the i n h i b i t i o n zone, the f u n g a l colony the Rhizobium s t r e a k d i d not  show any  and  to a pH change of the media.  cut out  No  or p l a c e d onto YMA  extracts  (RCC100, 106,  116,  121,  607,  or 811)  f r e e e x t r a c t s (RCC607) or Rhizobium d i d not FS911.  The  1.  i n h i b i t o r y to Fusarium i n v i t r o was  •Screening  to be  inoculum d i s c s  and  inhibit  not  Cell-free  concentrated  Rhizobium  IN VIVO STUDIES  of Rhizobium i n Growth Pouches index  o c c u r r e d when  'Topcrop' bean s e e d l i n g s were i n o c u l a t e d w i t h RCC  106  at 10  and  10"*  spores/pouch  the c o n c e n t r a t i o n of the pathogen, FS911, was RCC  106  shoot dry weights were reduced by FS911 compared to u n i n o c u l a t e d experiment was  6  cells/pouch  caused s i g n i f i c a n t r o o t n o d u l a t i o n which was  reduced at h i g h c o n c e n t r a t i o n of FS911  repeated  (10  6  spores/pouch).  at IO  4  and  10  6  Root  c o n t r o l s of p l a n t s t r e a t e d w i t h RCC once and  324  and  816  then and  spores/pouch as 106.  the same r e s u l t s were observed.  growth pouch experiments w i t h n o d u l a t i n g Rhizobium i s o l a t e s 106, 607,  or  substantiated.  A s i g n i f i c a n t r e d u c t i o n i n the r o o t d i s e a s e  (Table 19).  cell-  the growth of FS2  e x i s t e n c e of an i n h i b i t o r y substance produced by  IV.  In  i n h i b i t i o n zones developed between  p l a t e s seeded w i t h Rhizobium or FS2.  107,  s i d e of  not c o n s i d e r e d  segments of the i n h i b i t i o n zone p l a c e d o p p o s i t e  of FS2  and  the o p p o s i t e  l a r g e d i f f e r e n c e s (Table 18).  v i t r o antagonism between Rhizobium and Fusarium was due  Measurements of  ( F i g . 5) r a t e d + + + , + + + , + + +, + and  This Similar 321,  - i n dual  c u l t u r e agar p l a t e t e s t s , a l s o showed a s i g n i f i c a n t r e d u c t i o n i n  FS911  - 41 -  Table 18.  Rhizobium isolate  Hydrogen i m c o n c e n t r a t i o n o f d i f f e r e n t areas o f d u a l c u l t u r e agar p l a t e t e s t s i n o c u l a t e d v i t h Fusarium s o l a n i 2 and Rhizobium*  Level of i n v i t r o inhibition**  Inhibition zone  pH of Opposite s i d e o f Rhizobial streak  FS2  100  + + +  6.1  5.2  106  + + +  6.2  5.6  6.2  107  + + +  6.1  6.1  6.4  109  + + +  6.8  7.0  6.5  111  + + +  5.4  4.8  6.8  116  + + +  6.2  5.5  6.5  118  + + +  6.2  5.6  6.8  121  + + +  5.0  5.6  6.8  6.5  319  + + +  5.9  5.3  6.8  321  + + +  5.9  5.3  6.8  323  + + +  6.2  5.3  6.5  812  + + +  5.7  5.6  6.5  815  + + +  5.9  5.3  6.5  RCR1045***  + + +  6.2  6.2  6.8  101  + +  5.9  5.3  5.9  105  + +  5.0  5.0  6.2  108  + +  6.2  6.2  6.6  219  + +  5.9  5.6  6.5  604  + +  6.2  6.2  6.8  637  + +  6.5  6.5  6.8  638  + +  6.5  6.2  6.8  644  .+ +  6.8  6.5  6.2  102  +  5.9  5.9  5.9  103  +  6.5  6.2  6.5 6.5  113  +  5.6  5.3  114  +  5.6  5.3  6.5  119  +  6.2  6.2  6.8  123  +  5.6  5.3  6.8  324  +  5.6  5.0  6.8  601  +  5.9  5.6  6.8  603  +  6.5  6.5  606  +  6.2  5.3  6.8  608  +  6.6  5.9  6.8  609  +  6.6  5.9  6.8  611  +  6.5  6.2  6.8  615  +  6.2  5.9  6.8  RCR5***  +  5.6  5.0  5.9  RL1***  +  7.1  7.1  7.1  816  -  6.8  6.5  6.8  7.1  7.1  7.1  RCR3211***  -  6.8  *Values based on 1 r e p l i c a t e **Four l e v e l s of i n v i t r o i n h i b i t i o n were r e c o g n i z e d : no f u n g a l i n h i b i t i o n ; +, s l i g h t i n h i b i t i o n ; + + a c l e a r zone o f i n h i b i t i o n , c o l o n i z e d a f t e r 3-5 days; + + + a c l e a r zone o f i n h i b i t i o n p e r s i s t e d at l e a s t one week. RCR1045, RL1 (R. leguminosarum); RCR5 (R. t r i f p l i i ) ; RCR3211 (R. l u p i n i ) • :  - 42 -  r o o t r o t of bean.  Results  a r e summarized i n Table  v i d u a l experiments are l o c a t e d i n Appendix 1. FS911  20 and d a t a o f i n d i -  The a b i l i t y  to i n h i b i t  r o o t r o t i n v i v o d i d n o t depend on the i n h i b i t o r y a c t i v i t y  f o r the Rhizobium i s o l a t e i n v i t r o .  recorded  Only one Rhizobium i s o l a t e , RCC 812  d i d n o t p r o t e c t beans from FS911 r o o t r o t i n t h i s s e t of experiments a l t h o u g h i t r a t e d + + + i n duo c u l t u r e agar p l a t e Another s e r i e s o f s c r e e n i n g  test.  experiments demonstrated a s i g n i f i c a n t  r e d u c t i o n i n the s e v e r i t y o f FS911 r o o t r o t when bean p l a n t s were inoculated with nodulating  Rhizobium i s o l a t e s ,  concentration  2  10  k  and 1 0  6  cells/pouch  reduced r o o t r o t (Table 21). A t t h e h i g h e s t (10  6  spores/pouch) o n l y an e q u a l l y h i g h  pouch) reduced r o o t r o t .  significantly  concentration  of r o o t r o t . . R h i z o b i u m dry weights w h i l e  o f FS911  l e v e l o f RCC 107 ( 1 0  The n o d u l a t i n g  e  cells/  a b i l i t y of the Rhizobium  i s o l a t e c o u l d be a p r e r e q u i s i t e t o i n h i b i t o r y a c t i v i t y and  effect.  A t the lowest  o f FS911 ( 1 0 spores/pouch), i n o c u l a t i o n w i t h RCC 107 a t  c e l l s / p o u c h or RCC 109 a t 10  6  107 and 109.  suppression  - i n o c u l a t i o n tended to i n c r e a s e the r o o t and shoot  i n o c u l a t i o n with  the pathogen, FS911, had the o p p o s i t e  A r e d u c t i o n i n FS911 r o o t r o t was n o t found when bean p l a n t s were  inoculated with  11 o t h e r Rhizobium t e s t  summarized i n T a b l e  isolates.  These r e s u l t s a r e  22 and the s i g n i f i c a n t d a t a of i n d i v i d u a l experiments  are l o c a t e d i n Appendix 1. Pathogenicity  t e s t s demonstrated t h a t R. s o l a n i 1 and 2 were h i g h l y  v i r u l e n t pathogens of bean w h i l e R. s o l a n i 3 was a v i r u l e n t t o bean. Nodulating rot.  Rhizobium i s o l a t e s 107, 324, and 607 d i d n o t reduce RS2 r o o t  A s l i g h t r e d u c t i o n i n RSI r o o t r o t was recorded  were i n o c u l a t e d w i t h a h i g h c o n c e n t r a t i o n RSI  was l e s s v i r u l e n t  l o c a t e d i n Appendix 1.  to bean than RS2.  when bean p l a n t s  (10 cells/pouch) 8  o f RCC 107.  Data of t h i s experiment a r e a l s o  - 43 -  T a b l e 19.  F_. s o l a n i 911 (spores/ml)  E f f e c t o f Rhizobium 106 and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight o f 'Topcrop' snap bean  0  Rhizobium 106 ( c e l l s / p o u c h ) 10 10 ^ 2  10  6  Means  Root d i s e a s e i n d e x * 0 10 10* 10 Means 2  6  0.0a** 4.8bc . 7.3cd 8.5d 5.1b  0.0a 5.0bc . 4.8bc 5.8bcd 3.9ab  0.0a 5.3bcd 6. 3cd 7.3cd 4.7b  0.0a 2.5ab 1.3a 7.5cd 2.8a  0.0a 4.4b 4.9b 7.3c  3.0d 2.0bcd 2.5cd l.Oabc 2.1c  1.1a 1.0a 0.8a 0.8a  Nodulation index*** 0 10 10" 10 Means 2  6  0.0a 0.0a 0.0a 0.0a 0.0a  0.8ab 1.3abc 0.3a l.Oabc 0.8b  0.5ab 0.8ab 0.5ab l.Oabc 0.7ab  Root d r y weight (g) 0 10 10 10 Means 2  k  6  .101b .073ab .030a .025a ,057a  .064ab .047ab .059ab ,075ab' ,061a  .058ab ,069ab ,056ab ,057ab ,060a  .087ab .085ab .071ab .070ab .078a  • 078a .068a .054a .057a  .376b .340ab .280ab .203ab .300a  .289a .263a .207a .204a  Shoot d r y weight (g) 0 10 10 * 10 Means 2  6  ,367b ,285ab ,102a ,101a ,214a  •221ab .146ab .253ab .255ab .219a  .193ab .280ab .195ab .259ab • 232a  *Each v a l u e was an average of f o u r r e p l i c a t e s . Root r o t s e v e r i t y was based on an e q u a l increment d i s e a s e index from 0, no r o o t r o t t o 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e range t e s t : not s i g n i f i c a n t , P = 0.05.  v a l u e s f o l l o w e d by the same l e t t e r a r e  * * N o d u l a t i o n i n d e x was based on a s c a l e o f 0, no r o o t nodules to 3, abundant n o d u l e s .  - 44 -  F i g u r e 5 - (a) Fusarium s o l a n i 911 (IO * spores/pouch) r o o t r o t of 'Topcrop' snap bean i n growth pouches, (b) suppressed by i n o c u l a t i o n w i t h Rhizobium, i s o l a t e RCC816 ( 1 0 c e l l s / p o u c h ) . 1  6  - 45 -  T a b l e 20.  Rhizobium isolate  L e v e l of antagonism of n o d u l a t i n g Rhizobium i s o l a t e s to Fusarium s o l a n i (FS2, FS911) i n d u a l c u l t u r e agar p l a t e t e s t s and growth pouches of 'Topcrop' snap bean -  In v i t r o i n h i b i t i o n of FS2*  In v i v o Combination a t which i n h i b i t i o n .': :'. i n h i b i t i o n of FS911 r o o t r o t was observed  816  -  +  R2,3 ,V.P3**  324  +  +  R2,4:P2  106  + ++  +  Rif :P3  321  + ++  +  R2:P2,  607  + ++  +  R<t:P2  812  + ++  -  106***  + ++  +  R3.P3,  Rif.Pit  R4:P2  *Four l e v e l s of i n v i t r o i n h i b i t i o n were r e c o g n i z e d : -, no i n h i b i t i o n ; + , s l i g h t i n h i b i t i o n ; + +, c l e a r zone of i n h i b i t i o n , c o l o n i z e d i n 3 to 5 days; + + +, c l e a r zone of i n h i b i t i o n p e r s i s t e d a t l e a s t 1 week. **Four inoculum c o n c e n t r a t i o n s of Rhizobium were t e s t e d : R i = 0; R = 1 0 ; R 3 = 10 *; Ri+ = 1 0 c e l l s / p o u c h . Four inoculum c o n c e n t r a t i o n s of t h e pathogen, FS911 were t e s t e d : P i = 0; P = 1 0 ; P = 1 0 ; Pit = 1 0 spores/pouch. I n h i b i t i o n of FS911 r o o t r o t was observed a t t h e inoculum l e v e l s i n d i c a t e d . 2  1  6  2  2  2  4  6  3  ***Four inoculum c o n c e n t r a t i o n s of Rhizobium were t e s t e d : R i = 0; R2 = 1 0 ; R3 = 1 0 ; R.+ = 1 0 c e l l s / p o u c h . Three pathogen t r e a t m e n t s were t e s t e d ; FS911 a t . 1 0 c e l l s / p o u c h and t h r e e l e v e l s of R h i z o c t o n i a s o l a n i 1. - P i = 0';" P = FS911 + 2 ml RSI; P = FS911 + 4 ml RSI, I n h i b i t i o n of r o o t r o t was observed a t the inoculum l e v e l s i n d i c a t e d . 5  6  7  6  2  3  - 46 T a b l e 21.  I n t e r a c t i o n s o f Rhizobium i s o l a t e s and Fusarium s o l a n i 911, on r o o t and n o d u l a t i o n i n d i c e s and dry weight of snap bean i n growth pouches Rhizobium I s o l a t e s  F. s o l a n i 911 (spores/pouch)  0  107 10*  (cells/pouch) 109  10  b  10*  10°  Means  Root d i s e a s e i n d e x * 0 10 10 * Means 2  0.3a** 3.5b 8.3c 4.0c  0.0a 1.3ab 2.5ab 1.3a  ,0.3a 0.5a 7.3c 2.7abc  2.0ab 0.3a 8.5c 3.6bc  0.0a 0.0a 6.3c 2. l a b  0.5a 1.1a 6.6b  0.3ab 1.5bcd 0.8abc 0.8bc  1.1a 1.0a 0.8a  Nodulation index*** 0 10 10" Means 2  0.0a 0.0a 0.0a 0.0a  2. 3de 1.3abcd 3.0e 2.2d  2.5de 1.8cde 0.3ab 1.5cd  Root dry weight 0 10 10" Means 2  .083bcd • 024a .057abcd .054a  .074abcd .045abc .096cd .072a  .085bcd .069abcd .031ab .062a  Shoot dry weight 0 10 10 * Means 2  .326bcd • 081a •218abcd .208a  •297abcd .169abc .372cd . 2 7 9ab ;  .353cd .277abcd .115ab .248ab  0.3ab 0.3ab 0.0a 0.2ab  (s) .043abc .094cd .062abcd .066a  .058abcd .112d .087bcd .085a  .069a .069a .067a  .202abc .45 Id .361cd .338b  .273a .279a .261a  (s) .189abc ,370cd .242abcd .267ab  *Each v a l u e was an average o f 4 r e p l i c a t e s . Root r o t s e v e r i t y was based on an e q u a l increment d i s e a s e index from 0, no r o o t r o t t o 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e range t e s t : not s i g n i f i c a n t , P = 0.05.  v a l u e s f o l l o w e d by the same l e t t e r a r e  * * * N o d u l a t i o n index was based on a s c a l e of 0, no r o o t n o d u l e s , t o 3, abundant n o d u l e s .  - 47 T a b l e 22.  N o d u l a t i n g a b i l i t y of Rhizobium i s o l a t e s and l e v e l of antagonism to Fusarium s o l a n i (FS2, FS911) i n d u a l c u l t u r e agar p l a t e t e s t s and i n growth pouches 'Topcrop' snap bean  Combination a t which i n h i b i t i o n of FS911 r o o t r o t was observed**  Nodulating ability  In v i t r o inhibition FS2*  In v i v o inhibition  107  +  + + +  +  R  109  +  + + +  +  R-2 > 3 '^2  812  +  + + +  +  Ro :Po  323  -  + + +  -  111  +  + + +  -  115  +  + + +  -  116  +  + + +  -  118  +  + + +  -  319  +  + + +  -  321  +  + + +  -  815  -  + + +  -  816  +  -  -  RLl***  -  +  -  RCR1045***  +  + + +  -  Rhizobium isolate  3  :P , R 2  2  $  *Four l e v e l s of i n v i t r o were r e c o g n i z e d : -, no i n h i b i t i o n ; +, s l i g h t i n h i b i t i o n ; + +, c l e a r i n h i b i t i o n zones, c o l o n i z e d a f t e r 3 t o 5 days; + + +, c l e a r zones of i n h i b i t i o n p e r s i s t e d a t l e a s t 1 week. **Three inoculum c o n c e n t r a t i o n s o f Rhizobium were t e s t e d : R i = 0, R = 10 , R = 1 0 c e l l s / p o u c h . Three inoculum c o n c e n t r a t i o n s of FS911 were t e s t e d : P = 0, P = 1 0 , P f= I O spores/pouch. I n h i b i t i o n of FS911 r o o t r o t was observed a t the inoculum l e v e l s indicated. k  6  2  3  x  2  2  ***Rhizobium  4  3  leguminosarum.  - 48 -  2.  S o i l Tests The r o o t d i s e a s e index of 'Topcrop' snap bean, grown i n pots of  FS911 seed the at  infested  s o i l , was s i g n i f i c a n t l y reduced by RCC106 a t 1 0  (Table 23).  8  The b e s t p r o t e c t i o n a g a i n s t FS911 r o o t r o t o c c u r r e d a t  lowest inoculum p o t e n t i a l o f FS911 (inoculum: s o i l , the h i g h e s t RCC106 inoculum l e v e 1  (10 cells/seed). 8  1:10^ of 1:120) ( F i g . 6).  r e s u l t s supported p r e v i o u s r e s u l t s of growth pouch experiments. comparison, in  soil.  cells/  These  By  RCC816 had no apparent e f f e c t on FS911 r o o t r o t of snap grown  Data of i n d i v i d u a l s o i l  t e s t s a r e l o c a t e d i n Appendix  1.  I n o c u l a t i o n o f 'Topcrop' bean seed w i t h RCC106 gave s i g n i f i c a n t l y g r e a t e r n o d u l a t i o n than u n i n o c u l a t e d c o n t r o l s . nodules i n the absence  of Rhizobium  C o n t r o l p l a n t s developed  inoculation.  root  The presence of FS911  did  n o t a f f e c t the degree o f r o o t n o d u l a t i o n , s e e d l i n g emergence o r r o o t  dry  weight but d i d tend t o lower the shoot d r y weight.  Inoculation with  RCC106 o r RCC816 tended t o i n c r e a s e the shoot d r y weight b u t d i d not have any s i g n i f i c a n t e f f e c t on s e e d l i n g emergence o r r o o t d r y weight.  Table 23.  Rhizobium isolate  The l e v e l of antagonism of n o d u l a t i n g Rhizobium i s o l a t e s to Fusarium s o l a n i (FS2, FS91) i n d u a l agar p l a t e t e s t s and i n growth pouches of 'Topcrop' snap bean  In v i t r o inhibition of FS2*  I n h i b i t i o n of FS911 r o o t r o t i n growth pouches  Combination at which i n h i b i t i o n of FS911 r o o t r o t was observed i n growth pouches**  106  ++4  +  R„:P  106  +++  +  R<.:P  816  -  I n h i b i t i o n of FS911 r o o t r o t in soil  R„:P  3  +  3  R, 2  Combination a t which i n h i b i t i o n of FS911 r o o t r o t was observed i n soil***  R,  R.,:P  (1:120)*****  2  -  R-,:P  3  (1:10'*)****  2  3  *Four l e v e l s of i n v i t r o f u n g a l i n h i b i t i o n due to Rhizobium were r e c o g n i z e d : -, no i n h i b i t i o n ; +, s l i g h t i n h i b i t i o n ; + +, c l e a r zone of i n h i b i t i o n , c o l o n i z e d a f t e r 3 t o 5 days; + + +, c l e a r zone of i n h i b i t i o n p e r s i s t e d at l e a s t 1 week. L e v e l s of i n h i b i t i o n were averages of three replicates. **Four inoculum c o n c e n t r a t i o n s of Rhizobium were t e s t e d : cells/pouch.  P3 = IO"*; P"t = 10 ***Four l e v e l s  6  of Rhizobium were t e s t e d :  ; P 3 = 1:10 ; P* =  *****Four l e v e l s P  2  = 1 0 ; R 3 = IO *; Ri, = 2  2  1  3  R i = 0; R  = IO *; R 1  2  3  = 1:60;  P„ =  = 1 0 ; Ri, = 1 0 6  3  8  cells/seed.  (volume:volume) were t e s t e d :  P i = 0;  (volume:volume) were t e s t e d :  P i = 0;  10 . 2  of the pathogen, FS911, inoculum t o s o i l  = 1:120; P  10  P i = 0; P  spores/pouch.  ****Four l e v e l s of the pathogen, FS911, inoculum to s o i l P 2 = 1:10  R i = 0; R  Four inoculum c o n c e n t r a t i o n s of the pathogen, FS911, were t e s t e d :  1:30.  2  6  =  10 ; 2  - 50 -  Figure  6 - (a) Fusarium s o l a n i 911 ( P 2 = i n o c u l u m : s o i l , 1:10 ) r o o t r o t of 'Topcrop' snap bean, grown i n p a s t e u r i z e d soil, (b) suppressed by i n o c u l a t i o n w i t h Rhizobium, i s o l a t e RCC106 ( R = 1 0 c e l l s / s e e d ) H  8  4  DISCUSSION  The a n t a g o n i s t i c e f f e c t of Rhizobium to some, but not a l l of the r o o t pathogens of snap bean was work.  I n h i b i t i o n was  but R.  The l e v e l of i n v i t r o i n h i b i t i o n depended  combination of Rhizobium and f u n g a l i s o l a t e s t e s t e d .  isolates  time i n t h i s  observed between Rhizobium and Fusarium i n d u a l  c u l t u r e agar p l a t e t e s t s . the  demonstrated f o r the f i r s t  on  Rhizobium .  i n h i b i t e d F. m o n i l i f o r m e , F_. oxysporum, F. roseum and F_. s o l a n i  d i d not i n h i b i t R h i z o c t o n i a s o l a n i or Pythium  isolates.  s o l a n i and Pythium i s o l a t e s r a p i d l y c o l o n i z e d the agar p l a t e i n 2 t o 3  days w h i l e Fusarium i s o l a t e s r e q u i r e d a t l e a s t 7 days to r e a c h the Rhizobium s t r e a k .  Growth r a t e of the t e s t fungus c o u l d be a f a c t o r i n  d e t e r m i n i n g the l e v e l of i n v i t r o  inhibition.  Drapeau  e t al_.  (1973)  observed the same phenomenon and suggested t h a t the r a p i d growth r a t e of R. s o l a n i and Pythium may  not have a l l o w e d s u f f i c i e n t  time f o r Rhizobium  to a c t . The m a j o r i t y o f i n d i g e n o u s Rhizobium i s o l a t e s s e l e c t e d from n a t u r a l l y - f o r m e d bean r o o t nodules e x h i b i t e d some degree of i n h i b i t o r y activity  to the s e n s i t i v e i s o l a t e , F. S o l a n i 2.  g o n i s t i c a c t i v i t y was moderate  r e c o r d e d i n 38% of the Rhizobium i s o l a t e s ; a  l e v e l of antagonism was  gonism was  r e c o r d e d i n 26%; a low l e v e l of a n t a -  r e c o r d e d i n 33% and o n l y one i s o l a t e , RCC  apparent a n t a g o n i s t i c a c t i v i t y of  A h i g h l e v e l of a n t a -  towards FS2  816,  (Table 15).  showed no  Further  screening  i n d i g e n o u s s o i l m i c r o f l o r a c o u l d l e a d to the d i s c o v e r y of a s u p e r i o r ,  a n t a g o n i s t i c Rhizobium i s o l a t e which c o u l d be used to c o n t r o l r o o t r o t . A u t h e n t i c a t e d Rhizobium s p e c i e s R. l u p i n i , R. p h a s e o l i and R.  (R. japonicum, R.  leguminosarum,  t r i f o l i i ) were e i t h e r v e r y poor a n t a g o n i s t s  - 52 -  or  had no i n h i b i t o r y e f f e c t on FS2.  RCR3211, RJIA, RJIB, R L l , RL2, RP1,  These Rhizobium i s o l a t e s  (RCR5,  RP2) had been i n c u l t u r e f o r a l o n g e r  p e r i o d of time than the i s o l a t e s from bean r o o t nodules which might t h e i r l a c k of i n h i b i t o r y a c t i v i t y leguminosarum  (RCR1045) o n l y RCC  in vitro. isolates,  explain  With the e x c e p t i o n of R. tentatively  i d e n t i f i e d as R.  p h a s e o l i , from bean r o o t nodules p o s s e s s e d a h i g h l e v e l of i n h i b i t o r y a c t i v i t y (+++)  in v i t r o .  Gray and Sackston (1980) s i m i l a r l y  t h a t i n h i b i t i o n of F. s o l a n i f . sp. p i s i s t r a i n of R. leguminosarum  i n agar p l a t e s depended  on the  tested.  The l e v e l of i n v i t r o p l a t e technique u t i l i z e d .  reported  i n h i b i t i o n was  a l s o a f u n c t i o n of the agar  The d u a l c u l t u r e agar p l a t e t e c h n i q u e was  adopted as a f a s t and d i r e c t method of s c r e e n i n g Rhizobium i s o l a t e s f o r antagonism to bean r o o t pathogens. between Rhizobium and Fusarium. than 7 days.  Wide zones of i n h i b i t i o n  formed  These zones o f t e n p e r s i s t e d f o r more  P r e l i m i n a r y agar p l a t e t e s t s showed that a change i n the  agar medium, the d i s t a n c e s e p a r a t i n g the t e s t organisms and the p r e i n o c u l a t i o n p e r i o d w i t h Rhizobium a l t e r e d the degree of f u n g a l  inhibition  observed. The i n h i b i t o r y e f f e c t of Rhizobium i s o l a t e s t o F_. s o l a n i observed i n v i t r o was  s i m i l a r l y observed i n growth pouch and s o i l experiments.  l e v e l o f r o o t r o t i n h i b i t i o n depended Rhizobium and the pathogen, FS911. h i g h c o n c e n t r a t i o n s of Rhizobium t r a t i o n s of pathogen, FS911 10  6  c e l l s / p o u c h , was  FS911  (10  6  upon the c o n c e n t r a t i o n of b o t h  G e n e r a l l y , r o o t r o t was  ( I O , 10 4  The  6  c e l l s / p o u c h ) to low concen-  ( 1 0 , 10** spores/pouch). 2  suppressed by  I s o l a t e RCC324, a t  the only i s o l a t e e f f e c t i v e a g a i n s t a h i g h l e v e l of  spores/pouch).  Inoculum  l e v e l s of Rhizobium and the pathogen  were h i g h e r than those expected under f i e l d  conditions.  However, i t i s  - 53  -  f e a s i b l e to i n o c u l a t e the seed w i t h order  to e s t a b l i s h the a n t a g o n i s t  bean p l a n t s i s an a r t i f i c i a l  a high concentration  i n the s o i l .  system but had  and  2)  r o o t s c o u l d be  pouch method of growing  s e v e r a l advantages:  l a r g e number of Rhizobium i s o l a t e s c o u l d be activity;  The  of Rhizobium i n  1)  a  screened f o r a n t i f u n g a l ..  i n o c u l a t e d w i t h o u t mechanical i n j u r y ;  the development of r o o t r o t c o u l d be d i r e c t l y observed without  complexity  and  i n t e r f e r e n c e of s o i l  Fusarium r o o t r o t of bean was isolates  (RCC  106,  107,  109,  factors.  obtained 321,  324,  3) the  A s i g n i f i c a n t reduction i n  with  8/17  nodulating  607,  812,  816)  Rhizobium  t e s t e d i n growth  pouches. Nodulation  of bean r o o t s c o u l d be a p r e r e q u i s i t e f o r antagonism  to r o o t r o t i n v i v o .  Non-nodulating i s o l a t e s , RCC323, 815,  RL1,  had  no  apparent i n h i b i t o r y e f f e c t of Fusarium r o o t r o t i n growth pouches although they r a t e d + + +,+++, and + i n d u a l c u l t u r e agar p l a t e t e s t s . ( r a t e d +)  and  RCC816 ( r a t e d -)  i n v i t r o were s t i l l  root rot i n vivo.  On  r a t e d +++  (RCC111, 115,  in vitro  the c o n t r a r y , n o d u l a t i n g 116,  118,  319,  and  RCC324  i n h i b i t o r y to Fusarium non-nodulating  321,  323,  812,  815,  RCR1045) showed no apparent i n h i b i t o r y e f f e c t when t e s t e d ^ n v i v o . v i t r o i n h i b i t o r y a c t i v i t y was The v a l i d i t y t e s t s has  of c o n c l u s i o n s  been questioned  not  a r e l i a b l e i n d i c a t o r of i n v i v o  sp. p i s i  in vivo  and  d i d not  In activity.  based s o l e l y on the r e s u l t s of agar p l a t e  (Huber and Watson, 1966).  (1980) s i m i l a r l y t e s t e d s t r a i n s of R. f.  isolates  Gray and  Sackston  leguminosarum a g a i n s t _F. s o l a n i  f i n d a d i r e c t c o r r e l a t i o n between i n v i t r o  and  results. V a r i a b l e r e s u l t s were o b t a i n e d  w i t h one Rhizobium i s o l a t e , RCC816,  which reduced Fusarium r o o t r o t when bean p l a n t s were grown i n growth pouches i n the greenhouse but not when p l a n t s were grown i n an  environ-  - 54 -  mental c o n t r o l chamber. pH,  S u b t l e d i f f e r e n c e s i n temperature, water  potential,  l i g h t i n t e n s i t y or some o t h e r f a c t o r ( s ) might account f o r t h i s  crepancy i n r e s u l t s . r o o t pathogen  dis-  These f a c t o r s and t h e i r impact on the Rhizobium  i n t e r a c t i o n are by no means c o m p l e t e l y understood.  -  Further  experiments would be n e c e s s a r y to d e f i n e the environmental parameters o p t i m a l to Fusarium r o o t r o t i n h i b i t i o n by S o i l experiments, performed f i n d i n g s of growth pouch  Rhizobium.  i n the greenhouse,  experiments.  supported the  A s i g n i f i c a n t r e d u c t i o n i n the  r o o t d i s e a s e index o c c u r r e d w i t h the h i g h e s t inoculum p o t e n t i a l of RCC106 (10  c e l l s / s e e d ) o n l y a t the lowest inoculum p o t e n t i a l of FS911  8  soil,  l^O  4  or 1:120).  RCC106 caused s i g n i f i c a n t l y g r e a t e r root.nodu-  l a t i o n than u n t r e a t e d c o n t r o l s . c o u l d have been due or  i n the s o i l .  (inoculum:  N o d u l a t i o n of u n t r e a t e d c o n t r o l  plants  to indigenous r h i z o b i a t h a t s u r v i v e d on the seed coat  By comparison, RCG816 had no apparent i n h i b i t o r y  on Fusarium r o o t r o t i n s o i l .  T h i s supported ^ n v i t r o evidence where  RCC816 r a t e d - i n d u a l c u l t u r e agar p l a t e t e s t s . showed t h a t RCC816 was  effect  Growth pouch  experiments  not a r e l i a b l e a n t a g o n i s t t o Fusarium r o o t r o t .  Other r e p o r t s i n the l i t e r a t u r e have a l s o g i v e n e v i d e n c e of a r e d u c t i o n i n legume r o o t r o t by r h i z o b i a l and Howard, 1969;,  inoculation  ( C h i and Hansen, 1961.  Chou and Schmitthenner, 1974;  Mew  Tu, 1978a,b, 1980),  Tu  s i m i l a r l y found t h a t a t a g i v e n c o n c e n t r a t i o n of the pathogen, h i g h e r c o n c e n t r a t i o n s of Rhizobium were more e f f e c t i v e i n r e d u c i n g r o o t r o t of a l f a l f a and soybean  i n c i t e d by F. oxysporum and P. megasperma.  D i f f e r e n t r o o t r o t pathogens to  Rhizobium.  of bean v a r i e d i n t h e i r  The r e d u c t i o n of R h i z o c t o n i a r o o t r o t was  as the r e d u c t i o n of Fusarium r o o t r o t .  sensitivity  not as g r e a t  Only a s l i g h t r e d u c t i o n i n RSI  r o o t r o t of bean p l a n t s i n growth pouches  o c c u r r e d w i t h RCC107 a l t h o u g h  - 55 -  RSI  was  not  i n h i b i t e d by Rhizobium i n v i t r o .  Rhizobium i s o l a t e s e f f e c t i v e  i n reducing  Fusarium r o o t r o t (RCC324, 607)  were not  Rhizoctonia  root r o t .  confirmed e a r l i e r work w i t h  r o o t r o t s of o t h e r by Rhizobium (Mew  The  present  work has  legumes where o n l y and  Howard, 1969;  some of the d i s e a s e s  Chow and  Timing of Rhizobium i n o c u l a t i o n was  a combination of Fusarium and  were reduced Tu,  1980).  a l s o a f a c t o r i n determining The  s e v e r i t y of r o o t r o t  Rhizoctonia  was  bean p l a n t s were i n o c u l a t e d w i t h RCC106 2 days b e f o r e added to the pouches.  reducing  Schmitthenner, 1974  the l e v e l of r o o t r o t r e d u c t i o n by Rhizobium. i n c i t e d by  effective in  Fusarium r o o t r o t was  reduced when  the pathogens were  reduced when bean p l a n t s ,  i n growth pouches, were i n o c u l a t e d a t the crook stage w i t h RCC812. However, when bean p l a n t s were i n o c u l a t e d a t the f i r s t , w i t h RCC812 no  reduction  i n root rot occurred.  Rhizobium i n the r h i z o s p h e r e w i t h the pathogen p r o v i d e d  The  t r u e - l e a f stage  establishment  of young s e e d l i n g or p r i o r to i n o c u l a t i o n  better protection against  (1980) found t h a t s i g n i f i c a n t l y  severe root r o t .  w i t h Rhizobium and F.  oxysporum  or _P. megasperma r a t h e r than s e q u e n t i a l l y w i t h Rhizobium being s e v e r a l weeks a f t e r the pathogen.  Unfortunately,  may  r e s i d e adjacent The  mined.  to the  i n the  rhizosphere  s i t u a t i o n where inoculum of the pathogen  seed.  basis f o r fungal  i n h i b i t i o n observed i n v i t r o was  Experiments demonstrated t h a t i n h i b i t i o n was  i n the pH  added  p r e i n o c u l a t i o n of  legume r o o t s w i t h Rhizobium to e s t a b l i s h the a n t a g o n i s t p o s s i b l e i n the f i e l d  Tu  l e s s r o o t r o t developed when a l f a l f a  p l a n t s were i n o c u l a t e d s i m u l t a n e o u s l y  i s not  of  of the agar media.  suggested by Drapeau tit al_.  not due  not  deter-  to a change  Involvement of an i n h i b i t o r y substance as (1973) o r some type of n u t r i t i o n a l pheno-  menon as suggested by Antoun et a l .  (1978b) t h a t c o u l d account f o r  fungal  - 56 -  i n h i b i t i o n was  not  substantiated  in this thesis.  Mechanisms proposed f o r  i n v i t r o i n h i b i t i o n might a l s o e x p l a i n the i n h i b i t i o n of r o o t r o t observed i n v i v o but  they c o u l d i n v o l v e d i f f e r e n t mechanisms because t h e r e was  a p e r f e c t c o r r e l a t i o n between i n v i t r o and  i n vivo results.  of Fusarium r o o t r o t by Rhizobium c o u l d be  the r e s u l t of antagonism  ( a n t i b i o s i s , m y c o p a r a s i t i s m , l y s i s ) or c o m p e t i t i o n .  Further  r e q u i r e d to determine which of these mechanisms, i f any, and  involved i n root rot  Fusarium spp.  by m u l t i p l e p e n e t r a t i o n the s o i l and  to be  reducing  studies  are  i s responsible  c a u s i n g hyphal n e c r o s i s of  the  produce germ tubes which invade host p l a n t sites.  important a n t a g o n i s t s  root i n f e c t i o n .  roots  Rhizobium are s u f f i c i e n t l y numerous i n by  i n t e r c e p t i n g pathogen germlings  By c o l o n i z i n g the hyphal t i p s r h i z o b i a c o u l d  p r e v e n t c o n t a c t between the host p l a n t r o o t s and described  Suppression  suppression.  Rhizobium c o u l d be m y c o p a r a s i t i c pathogen.  not  the pathogen.  Tu  (1978)  r h i z o b i a l p a r a s i t i s m of r o o t r o t f u n g i i n v i t r o which c o u l d  the mechanism i n v o l v e d i n r o o t r o t s u p p r e s s i o n  in vivo.  c o l o n i z e d the hyphae of JP. megasperma, P_. ultimum, A. oxysporum, both i n t e r n a l l y and Aseptate fungi  (PM,  PU)  Rhizobium  imperfecta  reduced f u n g a l  and  F.  sporulation.  were found to be more s u s c e p t i b l e to r h i z o b i a l  p a r a s i t i s m than the s e p t a t e a l s o performed by Tu  e x t e r n a l l y and  be  fungi  (AI, FO).  In v i v o greenhouse experiments  (1978) supported these i n v i t r o observations.  Results  i n t h i s work have shown t h a t Rhizobium caused a s i g n i f i c a n t r e d u c t i o n i n r o o t r o t i n c i t e d by  the s e p t a t e  fungus F.  e x i s t e n c e of m y c o p a r a s i t i s m i n v i v o has Rhizobium c o u l d be n u t r i e n t s and host p l a n t .  s o l a n i f . sp, p h a s e o l i .  not y e t been observed.  s u c c e s s f u l l y competing w i t h  space i n the s o i l and A high competitive  The  r o o t pathogens f o r  l a t e r w i t h i n the r h i z o s p h e r e  of  the  a c t i v i t y of Rhizobium c o u l d account f o r  - 57 -  the s u p p r e s s i o n  of the pathogen and  Rhizobium i n o c u l a t i o n has nodulation,  host  Hansen, 1961;  been r e p o r t e d  plant vigour  Yang  and  r o o t r o t development.  and  to improve the degree of  general  Hagedorn, 1966).  c i n g the i n t e r a c t i o n between Rhizobium and unknown dimension of c o m p l e x i t y .  The  t e n s i o n , water p o t e n t i a l , n u t r i e n t s can  s e l e c t i v e l y favour  Successful  r e s i s t a n c e to d i s e a s e  symbiosis and  (Chi  r o o t pathogens adds y e t  (root s e c r e t i o n s ) , s o i l  type and  the  growing c o n d i t i o n s can encourage the Rhizobium-1egume  p o t e n t i a l l y reduce r o o t r o t .  isolate is feasible.  i n o c u l a t i o n of bean w i t h  of an a n t a g o n i s t i c , n o d u l a t i n g  Rhizobium  Implementation of the c o n t r o l i s f a c i l i t a t e d by  e s t a b l i s h e d p r a c t i c e of legume seed i n o c u l a t i o n w i t h Rhizobium. of s e l e c t i n g Rhizobium i s o l a t e s s o l e l y on i s o l a t e s c o u l d a l s o be r o t pathogens.  A highly antagonistic, nodulating  p l a n t i n g i n the f i e l d .  The  of r o o t r o t .  Field  enough c o n c e n t r a t i o n  o c u l a t i o n , to g i v e p o t e n t i a l of F.  ability,  to r o o t  Rhizobium i s o l a t e  could  commercial seed i n o c u l a t e d p r i o r t o  a n t a g o n i s t i c Rhizobium i s o l a t e must be  to s u r v i v e , r a p i d l y m u l t i p l y and  p e r s i s t i n the s o i l s t u d i e s should  now  of Rhizobium can be  be  i n order  to a c h i e v e  c a r r i e d out  a p p l i e d , by  able  to  soil.  see  seed i n -  i n h i b i t i o n of r o o t r o t under the e x i s t i n g inoculum  solani in  the  Instead  the b a s i s of n o d u l a t i n g  screened f o r t h e i r a n t a g o n i s t i c a c t i v i t y  then be grown i n a peat medium and  a high  pH  the growth of Rhizobium or the r o o t pathogen.  a s u f f i c i e n t concentration  if  another  i n f l u e n c e of temperature, oxygen  B i o l o g i c a l c o n t r o l of Fusarium r o o t r o t by  suppression  and  Environmental f a c t o r s i n f l u e n -  C u l t u r a l p r a c t i c e s such as seed i n o c u l a t i o n w i t h Rhizobium and p r o v i s i o n of o p t i m a l  root  - 58 -  SUMMARY AND CONCLUSIONS  N o d u l a t i n g Rhizobium i s o l a t e s were a n t a g o n i s t i c t o Fusarium s o l a n i i n v i t r o and i n v i v o .  The degree o f antagonism depended  on the  Rhizobium i s o l a t e and Fusarium i s o l a t e t e s t e d and the s c r e e n i n g nique u t i l i z e d .  tech-  I s o l a t e s o f R h i z o c t o n i a s o l a n i were n o t s e n s i t i v e t o  Rhizobium i n v i t r o but a t r a c e of i n h i b i t i o n was observed i n v i v o . Rhizobium a c t e d as a b i o l o g i c a l c o n t r o l agent f o r Fusarium r o o t r o t of snap bean. 1.  The antagonism between Rhizobium and Fusarium s o l a n i observed i n v i t r o was n o t always observed i n v i v o .  R e s u l t s of the d u a l  culture  agar p l a t e t e s t s were n o t always i n d i c a t i v e of the performance o f Rhizobium i s o l a t e s i n v i v o . 2.  The r e s u l t s of growth pouch experiments c o r r e l a t e d w i t h the r e s u l t s of greenhouse  soil  experiments.  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Extension Services of Oregon State University, Washington State University and the University of Idaho. 259 pp. Mair, CR. 1968. Influence of nitrogen n u t r i t i o n on Fusarium root rot of Pinto bean and i t s suppression by barley straw. Phytopathology, 58: 620-625. Maloy, O.C, and W.H. Burkholder. 1959. on the Fusarium root rot of bean.  Some effects of crop rotation Phytopathology, 49:: 583-587,  Marshall, K.C., and M. Alexander. 1960, Competition between s o i l bacteria and Fusarium. PI. S o i l , 12: 143-153. Mew,  T., and F.L. Howard. 1969. Root r o t of Soybean (Glycine max) i n r e l a t i o n to antagonism of Rhizobium japonicum and Fusarium oxysporum. Phytopathology, 59: 401. (Abstr.) "  M i l l e r , D.E., and D.W. Burke. 1974. Influence of s o i l bulk density and water potential on Fusarium root rot of bean. Phytopathology, 64: 526-529. Nash, S.M., T. Christou, and W. Snyder. 1961. Existence of Fusarium solani f. phaseoli as chlamydospores i n s o i l . Phytopathology, 51: 308-312. Neergaard P.  1977.  Seed pathology.  Halsted Press, New York.  1187 pp.  Orellana, R.G., C. Sloger, and V.L. M i l l e r . 1976. Rhizoctonia-Rhizobium interactions i n r e l a t i o n to y i e l d parameters of soybean. Phytopathology, 66: 464-467. Papavizas, G.C, J.A. Lewis, and P.B. Adams. 1968. Survival of root-. \:-~ infecting fungi i n s o i l . I I . Influence of amendment and s o i l carbon-to-nitrogen balance on Fusarium root rot of beans. Phytopathology, 58: 365-372.  -  62  -  Papavizas, G.C. and J.A. Lewis. 1975. E f f e c t of seed treatment with fungicides on bean root rots. PI. Dis. Reptr., 59: 24^28. Prasad, K., and J.L. Weigle. 1969. Resistance to Rhizoctonia solani i n Phaseolus vulgaris (snap bean). PI. Dis. Reptr., 53: 350-352. Prasad, K., and J.L. Weigle. 1970. Screening for resistance to Rhizoctonia solani i n Phaseolus v u l g a r i s . PI. Dis. Reptr., 54: 40-44. Reyes, A., and J.E. M i t c h e l l . 1962. Growth response of several isolates of Fusarium i n rhizospheres of host and non-host plants. Phytopathology, 52: 1196-1200. Schroth, M.N., and F.F. Hendrix, JR. 1962. Influence of nonsusceptible plants on the survival of Fusarium solani f . phaseoli i n s o i l . Phytopathology, 52: 906-909. Schroth, M.N.,' and W.C. Snyder. 1961. E f f e c t of host exudates on chamydospore germination of the bean root rot fungus, Fusarium solani f . phaseoli. Phytopathology, 51: 389-393. Snyder, W.C, M.N. Schroth, and J . Christou. 1959. E f f e c t of plant residues on root rot of bean. Phytopathology, 49: 755-756, Toussoun, T.A., and P.E. Nelson. 1968. A .pictorial guide to the i d e n t i f i c a t i o n of Fusarium species. Pennsylvania State University Press, University Park. 51 pp. Toussoun, T.A., and L.A. Patrick. 1963. E f f e c t of phytotoxic substances from decomposing plant residues on root rot of bean. Phytopathology, 53: 265-270. Tu, J.C. 1978a. Protection of soybean .from severe Phytophthora root r o t by Rhizobium. Physiol. Plant Pathol. 12: 233-240. Tu, J.C. 1978b. Prevention of soybean root nodulation by tetracycline and i t s effect on soybean root rot caused by an a l f a l f a s t r a i n of Fusarium oxysporunv. • Phytopathology, 68: 1303-1306. Tu, J.C. 1979. Evidence of d i f f e r e n t i a l tolerance among some root rot fungi to r h i z o b i a l parasitism i n v i t r o . Physiol. Plant Pathol., 14: 171-177. Tu, J.C. 1980. Incidence of root rot and overwintering of a l f a l f a as influenced by rhizobia. Phytopath. 2. 97: 97-108. Tuite, John. 1969. Plant pathological methods. Fungi and bacteria. Burgess Publishing Company, Minneapolis, Minn. 239 pp.  Vance, C P . , and L.E.B. Johns on. 1981. Nodulation. perspective. P l a n t D i s . , 65: 118-124.  A plant disease  V i n c e n t , J.M. 1970. A manual f o r the p r a c t i c a l study of r o o t - n o d u l e bacteria. IBP Handbook No. 15. Blackwell S c i e n t i f i c Publications, Oxford and Edinburgh. 163 pp. Walker, J.C. 1952. 529 pp.  D i s e a s e s of v e g e t a b l e c r o p s .  McGraw-Hill,  New  York.  Weaver, R.W., and L.R. F r e d e r i c k . 1972. A new technique f o r mostprobable-number counts of r h i z o b i a . P I . S o i l , 36: 219-222. Westcott, C. 1971. 3rd ed. 843  P l a n t d i s e a s e handbook. pp.  Van Nostrand, R e i n h o l d  Co.,  Yang, S., and D.J. Hagedorn. 1966. Root r o t of p r o c e s s i n g bean i n Wisconsin. P i . D i s . Reptr., 50: 578-580. Zaumeyer, W.J., and H.R. Thomas. 1957. A monographic study of bean d i s e a s e s and methods f o r t h e i r c o n t r o l . U.S. Dept. A g r i c . Tech. Bull. 868, 255 pp.  - 64":  APPENDIX I  T a b l e 1.  J_. s o l a n i 911 (spores/pouch)  E f f e c t of Rhizobium 816 and Fusarium s o l a n i 911 on r o o t desease and n o d u l a t i o n i n d i c e s and d r y weight of 'Topcrop' snap bean i n growth pouches  Rhizobium?816 ( c e l l s / p o u c h ) IO 10*  0  2  Root d i s e a s e 0 10 10* 10 Means 2  6  0.0a 3.Obcd 7.0e 8.8e 4.7b  0.3a 1.5ab 4.3d 7.3e 3. 3a  Nodulation 10 10* 10 Means 2  6  0.0a 0.0a 0.0a 0.0a 0.0a  To "  Means  0.0a 3.3bcd 1.8abc 8.5e 3.4a  0.1a 3.1b 4.3c 8.0d  2.0ab 2.3b 1.3ab 0.0a 1.4b  1.4b 1.5b l.Oab 0.5a  5  index* 0.0a 4.5d 4.0cd 7.5e 4.0ab  index***  1.5ab 2.3b 1.5ab 2.0ab 1.8b  2.3b 1.5ab 1.3ab 0.0a 1.3b  Root d r y weight (g) 10" 10* 10 Means 6  .083ab .037a .071ab • 034a .056a  .082ab .084ab .053a .096ab .078a  .097ab .076ab .061a .033a .067a  .056a ,091ab .104b .032a • 096a  .079a .072a .097a .049a  .267ab .399ab .258ab .163ab .272a  .357b .321ab .262ab .216a  Shoot d r y weight (g) 0 10 10* 10 Means 2  6  .433b .226ab .237ab .109a .251a  .329ab .353ab .254ab .423b .340a  .399ab .305ab .299ab .170ab .293a  *A11 v a l u e s were averages of f o u r r e p l i c a t e s . S e v e r i t y of r o o t r o t was based on an equal increment r o o t d i s e a s e index from 0, no r o o t r o t ; to 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e range t e s t : not s i g n i f i c a n t , P .= 0.05.  v a l u e s f o l l o w e d by the same l e t t e r a r e  * * * N o d u l a t i o n index was based on a s c a l e from 0, no r o o t n o d u l e s , tp 3 abundant n o d u l e s .  - 66 Table 2.  F. solani 911 (Spores/pouch)  E f f e c t of Rhizobium 324 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches _ 0  Rhizobium 324 (cells/pouch)  10  10  c  Means  Root disease index* 10 IO* 10 Means 2  6  0.0a 8.3d 5.0bcd 8.3d 5.4a  0.0a 2. 8ab 7.5cd 8.3d 4.6a  0.0a 7.5cd 2.3ab 6.5cd 4.2a  0.0a 4.3bc 6.3bcd 8,0d 4.6a  0.0a 5.7b 5.4b 7,8c.  2.0bc 0.8ab 2.3bc 1.3abc 1.6b  1. 3abc 2.8c 0.8ab 1.5abc 1.6b  1.4a 1.1a 0.8a 0.8a  Nodulation index 10 10* 10 Means z  6  0.0a 0.0a 0.0a 0.0a 0.0a  2.3bc 0.8ab 0.0a 0.5ab 0.96  Root dry weight (g) 10* 10" 10 Means 6  .lllab .076ab ,085ab . 052ab ,081a  .130b ,059ab .044a .072ab .076a  .075ab ,049a .091ab ,092ab ,077a  .06lab .098ab .071ab ,063ab ,073a  .094a .071a .073a .070a  ,187ab ,317ab ,200ab ,204ab ,227a  .290a .208a .233a .229a  Shoot dry weight (g) 10 10" 10 Means 2  6  ,365ab ,212ab ,299ab ,166a ,260a  .415b .164a .119a .230ab . 232a  194ab 140a 315ab 316ab 241a  *A11 values were averages of four r e p l i c a t e s . Root rot severity was based on an equal increment root disease index from 0, no root r o t , to 9, completely rotted, dead roots. **Duncan's multiple range test: not s i g n i f i c a n t , P = 0.05.  values followed by the same l e t t e r are  ***Nodulation index was based on a scale from 0, no root nodules, to 3, abundant nodules.  - 67 -  T a b l e 3.  F. s o l a n i 911 (Spores/pouch)  E f f e c t of Rhizobium 321 and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight of 'Topcrop' snap bean i n growth pouches  0  Rhizobium 321 ( c e l l s / p o u c h ) IO 10** 7  Root d i s e a s e 10 10* 10 Means z  6  0. 0a 3.7bcd 1. 7ab 7,0e. 3.1b  0.0a 0.3a 2.0ab 5,7cde 2,0ab  To*  Means  0.0a 1.7ab l«7ab. 3 ..Oabc 1,6a  0.1a 1.6b 2..7b 5.5c.  0.7ab 0.3ab 0.3ab 0.3ab 0.4ab  0.5a 0.6a 0.2a 0.2a  index* 0.3a 0.7ab 5,3cde 6,3de 3.2b  N o d u l a t i o n index*** 10 10* 10 Means z  6  0.7ab 0.7ab 0.0a 0.3ab 0.4ab  0.7ab 1.3b 0.3ab 0.0a 0.6b  0.0a 0.0a 0.0a 0.0a 0.0a  Root d r y weight (g) 0 10 10* 10 Means 2  6  ,069a ,057a ,059a . 054a ,060a  .050a .051a .050a • 061a .053a  .066a .063a .073a .063a .066a  ,070a ,056a ,046a ,054a ,057a  .064a .057a .057a .058a  ,447a ,391a ,256a ,311a ,351a  ,377a ,345a ,355a ,306a  Shoot d r y weight (g) 10^ 10* 10 Means 6  ,376a ,293a ,345a ,335a ,337a  ,267a ,296a ,359a ,274a ,299a  .417a .399a • 461a • 305a .395a  *A11 v a l u e s were averages o f f o u r r e p l i c a t e s . Root r o t s e v e r i t y was based on an equal increment r o o t d i s e a s e index from 0, no r o o t r o t , to 9, c o m p l e t e l y r o t t e d dead r o o t s . ^Duncan's m u l t i p l e range test:, not s i g n i f i c a n t , P = 0,05.  v a l u e s f o l l o w e d by the same l e t t e r a r e  * * * N o d u l a t i o n index was based on a s c a l e from 0, no r o o t n o d u l e s , t o 3, abundant n o d u l e s .  - 68 T a b l e 4.  F. s o l a n i 911 (Spores/pouch)  10 10* 10 z  6  Means  E f f e c t o f Rhizobium 607 and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight of 'Topcrop' snap bean i n growth pouches  _ 0  Rhizobium 607 ( c e l l s / p o u c h ) pr:iO^" 10*  Root disease index* 0.0a 0.0a 0.0a 5.8c 6.0cd 6.8cde 7.0cde 8.0cde 8.3de 8.8e 7.0cde 8.3de 5.4a 5.3a 5.8a  10  s  0.0a 3.3b  8.0cde 8.8e 5.0a  Means  0.0a 5.4b 7.8c 8.2c  Nodulation index 0 10 10* 10 Means 2  6  0.0a 0.0a 0.0a 0.0a 0.0a  0.0a 0.0a 0.0a 0.0a 0.0a  0.8abc 0.5abc 0.3ab 0.8abc 0.6a  1.8c 1.5bc 1.3abc 0.3ab 1.2b  0.6a 0.5a 0.4a 0.3a  Root d r y weight (g) 10" 10* 10 Means 6  ,099a ,108a ,097a ,070a ,093a  -067a .076a .063a • 097a .076a  .097a • 059a .070a • 068a .073a  ,089a .121a .087a .060a .089a  .088a .091a .079a .074a  ,294a .344a ,152a .123a ,228a  ,276a ,272a ,190a ,190a  Shoot d r y weight (g) 10 10* 10 Means 2  6  ,341a ,326a ,304a ,107a ,269a  .203a • 256a .166a .310a . 234a  .269a .162a .137a .222a .197a  *A11 v a l u e s were averages of f o u r r e p l i c a t e s . Root r o t s e v e r i t y was based on an equal increment r o o t d i s e a s e index from 0, no r o o t r o t , to 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e range t e s t : v a l u e s n o t f o l l o w e d by the same l e t t e r a r e not s i g n i f i c a n t , P = 0.05. * * * N o d u l a t i o n index was based on a s c a l e from o, no r o o t n o d u l e s , t o 3 abundant n o d u l e s .  -69 T a b l e 5.  F. s o l a n i 911 (Spores/pouch)  -  E f f e c t o f Rhizobium 812 and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight 'Topcrop' snap bean i n growth pouches  _ 0  Rhizobium 812 ( c e l l s / p o u c h )  1CV  1(T  10  €  Means  .Root d i s e a s e index* 0.0a**  io 10". io z  6  Means  2.8ab 6.0cdef 7.8ef  4,1a  0.0a 3.0abc 4.3bcd 7.3def 3.6a  0.0a 3,3bc 5.0bcde 8.0ef  4.1a  Nodulation 10 10* 10 Means z  6  1.3abc 0.3ab 0.0a 0.8ab 0.6ab  0.8ab 0.3ab 0.0a 0.0a 0.3a  0.0a 2.0ab 7.3def 8.5f 4.4a  0.0a 2,8b 5.6c 7.9d  2.3c 1.8bc 0.5ab 0. 0a 1. lb>  1.2b 0.6ab 0.3a 0.3a  index***  0.5ab 0.3ab 0.5ab 0.5ab 0.4ab  Root d r y weight (g) 10 10* 10 Means z  6  ,121c ,103abc ,063abc ,039a ,081a  .063abc .088abc .040a .069abc .065a  .118bc .087abc .088abc .050a .086a  ,088abc ,124c ,058ab ,045a .079a  .098b .100b ,062a .051a  ,330bcd ,471d ,176ab ,114ab ,273a  .323b .356b ,197a .147a  Shoot d r y weight (g) 10 10* 10 Means z  6  .452cd .331bcd .199ab • 088a .268a  .184ab ,324bcd ,138ab ,236abc .220a  .328bcd .300abcd .275abcd .150ab .263a  *A11 v a l u e s were averages of f o u r r e p l i c a t e s . Root r o t s e v e r i t y was based on an equal increment r o o t d i s e a s e index from 0, no r o o t r o t , to 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e range t e s t : not s i g n i f i c a n t , P = 0.05.  v a l u e s f o l l o w e d by the same l e t t e r a r e  * * * N o d u l a t i o n index was based on a s c a l e from 0, no r o o t n o d u l e s , t o 3 abundant n o d u l e s .  - 70 -  T a b l e 6.  FS911 (Spores/pouch)  E f f e c t of Rhizobium 106, Fusarium s o l a n i (FS911) and R h i z o c t o n i a s o l a n i (RSI) on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight of 'Topcrop' snap bean i n growth pouches Rhizobium 106 10^  RSI (ml/pouch)  Root d i s e a s e 0 10 10 Means 6  6  0 2 4,  0.0a 8.0cd 8.6d  5,5b  (cells/pouch) 10 10' fc  Means  index 0.0a 7.4cd 7,6cd 5.0ab  0.0a 7.2cd 7.0bc. 4,7a  0.0a 5.8b  0.0a 7.1b  7,6cd 4.5a  7,7b  l.Oabcd 0.4abc 1.Oabcd 0.8b  2.2d 1.4bcd 1.4bcd 1.7c  1.2a 0.6a 0.7a  N o d u l a t i o n index 0 10 10 Means 6  6  0 2 4  0.0a 0.0a 0.0a 0.0a  1.6cd 0.6abc 0.2ab 0.8b  Root d r y weight (g) 0 10 10 Means 6  6  0 2 4  ,109ab .074ab .087ab .090a  .118b .087ab .084ab • 096a  .067a .079ab .074ab .073a  .102ab .095ab .091ab .096a.  .099a .084a ,084a  ,243ab ,213ab ,197a ,218a  ,366bc ,369bc ,194a ,310b  ,369b ,242a ,193a  Shoot dry weight (g) 0 10 10 Means 6  6  0 2 4  ,416c ,160a ,176a ,251ab  ,450c ,227ab , 206ab ,294ab  *A11 v a l u e s were averages of f i v e r e p l i c a t e s r o o t r o t s e v e r i t y was based on an equal increment r o o t d i s e a s e index from 0, no r o o t r o t , to 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e range t e s t : not s i g n i f i c a n t , P = 0.05.  v a l u e s f o l l o w e d by the same l e t t e r a r e  * * * N o d u l a t i o n index was based on an s c a l e from 0, no r o o t n o d u l e s , to 3, abundant n o d u l e s .  -71 T a b l e 7.  R. s o l a n i • isolate (3 mis/pouch)  I n t e r a c t i o n of Rhizobium i s o l a t e s and Rhizoctonia s o l a n i (RSI, RS2, RS3) on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight o f 'Topcrop' snap bean i n growth pouches  Rhizobium i s o l a t e ( I 0 cells/ml/pouch) 107 324 a  0  Root d i s e a s e 0 RS3 RSI RS2 Mean  0.0a 0.5a 7.3cd 9.0d 4.2a  0 RS3 RSI RS2 Mean  0.3ab 0.0a 0.0a 0,0a 0.1a  Means  0.8a 0.5a  0.0a 0.3a  0.2a 0.3a  6.5bc 8,3cd 4.0a  7,5cd 8.3cd 4.0a  6.6b 8.4c  2.8d 1.3abcd 2.0cd 1.5bcd 1.6b  2.5d 1.8bcd 1.3abcd 0.8abc 1.9b  1,9b 1.1a 1.3ab 1.0a  index**  0.0a 0.0a 5.0b 8.0cd 3.3a  Nodulation  603  index  2.0cd 1.5abcd 1.8cd 1.8bcd 1.8b  Root dry weight (g) 0 RS3 RSI RS2 Mean  , 103b ,061ab .057ab ..051a • 068a  ,078ab .054a •066ab .052a • 062a  •084ab ,078ab .086ab .066ab .079a  ,075ab ,067ab • 049a •055ab .061a  , 085b .065al . 064al .056  .328abc ,268abc .192ab ,252abc .313a  .343b .268a ,238a  Shoot dry weight (g) 0 RS3 RSI RS2 Mean  ,372c .247abc .203abc .163a .246a  . 326abc .228abc . 233abc. .227abc .253a  .347bc . 331abc .323abc .250abc .260a  *A11 V a l u e s were averages o f f o u r r e p l i c a t e s . Root r o t s e v e r i t y was based on an equal increment r o o t d i s e a s e index from 0, no r o o t r o t , to 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e range t e s t : not s i g n i f i c a n t , P = 0.05.  v a l u e s f o l l o w e d by the same l e t t e r a r e  * * * N o d u l a t i o n index was based on a s c a l e from 0, no r o o t n o d u l e s , t o 3, abundant n o d u l e s .  - 72 T a b l e 8.  I n t e r a c t i o n of Rhizobium i s o l a t e s and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight of 'Topcrop' snap bean i n growth pouches  F_. .Solani 911 (Spores/pouch)  Rhizobium ( c e l l s / p o u c h ) 107 10*  109 7~10 ' B  Root d i s e a s e 10 10* Means 2  0.0a 4.0abcd 9.0c 4.3a  0.0a 1.8ab 5.Obcde 2.3a  IO *  10  0.0a 4. 5abcd  0.0a 4.0abcd 3 .5abc 2.5a  0.1a 3.5b 6.6c  3.0c 2.3bc 2.3bc 2.5b  0.6a 0.8a 0.5a  1  b  Means  index* 0.3a 3.3abc 7.3cde 3. 6a  Q.  8.3de 4.3a  N o d u l a t i o n index*** 0 10 10* Means 2  0.0a 0.0a 0.0a 0.0a  0.0a 0.0a 0.0a 0.0a  0.0a 0.3a 0.0a 0.1a  0.0a 1.5b 0.0a 0.5a  Root d r y weight (g) 0 10 10* Means 2  .075ab .063ab .036a .056a  .064ab .096ab •073ab .077ab  •077ab .068ab .049a .065ab  .037a .075ab .032a ,048a  .130b .086ab .080ab .099b  .076a .077a .054a  ,095a ,248ab ,078a .140a  ,406b ,326ab .296ab ,343b  ,238a ,276a ,174a  Shoot d r y weight (g) 0 10 10* Means 2  ,261ab ,243ab ,098a ,200a  ,189ab ,325ab ,254ab ,256ab  .241ab .237ab .143ab .207a  *A11 v a l u e s were averages o f f o u r r e p l i c a t e s Root r o t s e v e r i t y was based on an equal increment r o o t d i s e a s e index from 0, no r o o t r o t , to 9, c o m p l e t e l y r o t t e d , dead r o o t s . :  **Duncans m u l t i p l e range t e s t : not s i g n i f i c a n t , P = 0.05.  v a l u e s f o l l o w e d by the same l e t t e r a r e  * * N o d u l a t i o n index based on a s c a l e from 0, no r o o t n o d u l e s , t o 3, abundant n o d u l e s .  - 73 -  Table 9.  E f f e c t o f Rhizob ium 106 and Fusarium s o l a n i 911, on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight o f 'Topcrop' snap bean grown i n p a s t e u r i z e d greenhouse s o i l , .  F. s o l a n i 9 1 1 : s o i l (volume:volume) 0  Rhizobium 106 ( c e l l s / p o u c h ) 10" 10 b  Root d i s e a s e 0 1:10* 1:10 1:10 Means 3  2  1.2abc** 5.8d 4.led 5.7d 4.2b  0.3ab 3.0bcd 5.5d 5.7d 3.6b  Nodulation 0 1:10* 1:10 1:10 Means 3  2  1.5a 1.8a 1.6a 2.3a 1.8a  2.7a 1.9a 2.5a 2.5a 2.4a  10  b  Means  index* 0.2ab 3.5bcd 4.3cd 4.3cd 3.lab  0.8a 1.7abc 1.8abc 4.3cd 2.0a  0.4a 3.5b 3.9bc 5.0c  2.0a 1.7a 3.0a 2.7a 2.3a  2.1a 2.4a 2.2a 1.7a  index*** 2.4a 1.5a 1.8a 2.1a 2.0a  Root d r y weight (g) 0 1:10* 1:10 1:10 Means 3  2  •188a .221a •334a .416a .290a  .243a .214a .249a .265a .243a  .193a .136a .207a .143a .170a  .157a .181a • 215a .199a .188a  • 195a .188a .251a .256a  Shoot d r y weight (g) 0 1:10* 1:10 1:10 Means 3  2  .938a .770a .941a 1.01a ,915a  1.08a .944a .940a 1.11a 1.02a  .855a .680a .794a .672a .750a  .754a .840a 1.09a .909a • 899a  .906a .924a .942a .809a  *Values were averages of f o u r r e p l i c a t e s . Root r o t s e v e r i t y was based on an equal increment r o o t ; d i s e a s e . i n d e x from 0,'<no r o o t r o t , t o 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan's m u l t i p l e . r a n g e t e s t : not s i g n i f i c a n t , P = 0.05. * * * N o d u l a t i o n index was based abundant nodules.  v a l u e s f o l l o w e d by the same l e t t e r a r e  on a s c a l e from 0, no r o o t nodules,  t o 3,  - 74 -  Table 10.  E f f e c t of Rhizobium 106 and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weights o f 'topcrop' snap bean growth i n p a s t e u r i z e d greenhouse s o i l  F. s o l a n i 911: s o i l (volume:volume)  Rhizobium 106 feel 1 s 10 * 10  o  /sp.prf) 10  Means  0.0a 2.3abcd 2. 7abcde 4.6def 2.4a  0.3a 3.4b 3.9b 4.3b  1.5bcde 1.3bcde l.labcd 2.3de 1.6b  1.7bcde 2.4de 2.6e 1.9cde 2.2c  1.1a 1.4a 1.4a 1.5a  .147a .112a • 112a .137a .127a  .146a .161a • 144a .116a .142a  .125a .139a .133a .120a  .927ab •628ab .777ab .651ab .759b  1.05b .772ab .555ab .576ab .738b  .738a .627a .609a • 546a  6  8  Root d i s e a s e i n d e x * 0 1:120 1:60 1:30 Means  0.5ab** 5.4 ef 5.8ef 6.2f 4.5b  0.5ab 2.7abcde 3.5bcdef 3.labedef 2.4a  Nodulation 0 1:120 1:60 1:30 Means  0.1a 0.5ab 0.5ab 0.7abc .4a  index***  l.Oabcd 1.3bcde 1.5bcde 1.3abcd 1.3b  Root dry weight 0 1:120 1:60 1:30 Means  0.3a 3. 2abedef 3.6cdef 3.3abcdef 2.6a  • 095a • 129a .105a .078a .102a  0 0  .111a . 154a .172a • 148a .146a  Shoot d r y weight (8) 0 1:120 1:60 1:30 Means  .374a .518ab .461 ab .446ab .449a  .600ab .536ab •645ab .511ab .573ab  *Values were averages o f f o u r r e p l i c a t e s . Root r o t s e v e r i t y was based on an e q u a l increments r o o t d i s e a s e from 0, no r o o t r o t , t o 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan s m u l t i p l e range t e s t : not s i g n i f i c a n t , P = 0.05.  index  v a l u e s f o l l o w e d by the same t e s t s a r e  * * * N o d u l a t i o n index was based on a s c a l e from 0, no r o o t n o d u l e s , t o 3, abundant n o d u l e s .  - 75 T a b l e 11.  E f f e c t of Rhizobium 816 and Fusarium s o l a n i 911 on r o o t d i s e a s e and n o d u l a t i o n i n d i c e s and d r y weight of 'Topcrop' snap bean grown i n p a s t e u r i z e d greenhouse s o i l  F. s o l a n i 9 1 1 : s o i l (volume:volume) 0  Rhizobium 816 10  (cells/seed).  10'  10  0. l a 3. l b c d 3. 8cdef 5. 2def 3. l a  0.1a 3.0bcd 5.6ef 5.3ef 3.5a  0.8a 2.9b 4.4c 5.2c  1.9a 1.5a 1.6a 1.2a 1.6a  1.4a 1.9a 1.4 a 1.6a  c  Means  Root d i s e a s e i n d e x * 0 1:120 1:60 1:30 Means  1.2ab** 1.9abc 4.8def 5.8f 3.4a  Oabc 6cde 5cde 5def 4a  Nodulation index*** 0 1:120 1:60 1:30 Means  1.1a 2.1a 1.0a 1.7a 1.5a  0.8a 1.9a 1.5a 1.8a 1.5a  2.0a 2.0a 1.5a 1.8a 1.8a  Root d r y weight (g) 0 1:120 1:60 1:30 Means  .121a ,224a ,180a ,142a .167a  .129a ,158a .265a ,131a .171a  .133a .136a ,123a .101a .123a  • 150a .252a ,150a ,128a ,170a  .133a .192a .179a ,126a  .945ab 1.74c 1.33bc .501a 1.13b  .731a 1.14b .968b .590a  Shoot d r y weight (g) 0 1:120 1:60 1:30 Means  •647a . 869ab •871ab 609a 756a  .588a 1.03ab .732a .635a .743a  .745a .906ab .953ab .614a ,804a  *Values were averages of f o u r r e p l i c a t e s . Root r o t s e v e r i t y was based on an e q u a l i n c r e m e n t . r o o t d i s e a s e i n d e x from 0, no r o o t r o t ; to 9, c o m p l e t e l y r o t t e d , dead r o o t s . **Duncan s m u l t i p l e range t e s t : v a l u e s f o l l o w e d by the same l e t t e r a r e n o t s i g n i f i c a n t , P = 0.05. * * * N o d u l a t i o n index was based on a s c a l e from 0, no r o o t n o d u l e s , t o 3, abundant n o d u l e s .  

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