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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.Sc, The University 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 i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PLANT SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1981 (c") Andrea Joanne Smulders, 1981 In presenting t h i s thesis 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 that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis f o r scholarly purposes may be granted by the Head of my Department or by h i s or her representative. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed with-out my written permission. Department of Plant Science The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, B.C., Canada V6T 1W5 September, 1981 ABSTRACT Indigenous Rhizobium i s o l a t e s from naturally-formed bean root nodules were antagonistic to some of the root r o t t i n g pathogens of snap bean. Rhizobium 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 species i n dual culture agar plate tests but were not i n h i b i t o r y to Rhizoctonia s o l a n i or Pythium i s o l a t e s . With one exception, a l l indigenous Rhizobium i s o l a t e s showed some degree of antagonism towards F. so l a n i f. sp. phaseoli i n v i t r o . 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 technique u t i l i z e d . 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 -v i t y was recorded i n 38% of the Rhizobium i s o l a t e s tested where wide zones of i n h i b i t i o n formed between the test i s o l a t e s and persisted f or 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 nodulating Rhizobium i s o l a t e s to Fusarium root rot of snap bean was observed i n growth pouch experi-ments. Protection of bean plants from severe Fusarium root rot occurred i n combinations where the inoculum concentration of Rhizobium ( 1 0 \ 10 6 cells/pouch) was equal to or greater than the inoculum concentration of F_. s o l a n i (10 2 , 1 0 v spores/pouch). Ten Rhizobium i s o l a t e s , which were highly antagonistic 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 root rot i n vi v o . Two Rhizobium i s o l a t e s , RCC324 and RCC607, i n h i b i t o r y to Fusarium root rot did not reduce Rhizoctonia root rot of bean. S o i l experiments supported the r e s u l t s of growth pouch experiments whereby ino c u l a t i o n of bean seed with a high concentration of Rhizobium (RCC106 at 10 8 cells/seed) e f f e c t i v e l y reduced bean root 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, F. so l a n i (inoculum: s o i l , 1:10"* or 1:120). These r e s u l t s indicated the p o t e n t i a l exists f o r f i e l d control of Fusarium root rot of snap bean by a highly antagonistic nodulating i s o l a t e of Rhizobium. - i i i -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 v i i i DEDICATION x ACKNOWLEDGEMENTS x i INTRODUCTION AND LITERATURE REVIEW . . 1 Introduction 1 The Disease 1 Control of Bean Root Rots 3 Objectives 10 MATERIALS AND METHODS 11 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 . . . . 11 1. F i e l d Survey 11 2. Growth Pouch Technique (GPT) 12 3. Pathogenicity Tests 15 I I . Isolation 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 I I I . In Vi t r o Studies 19 1. Standardized Agar Plate Technique (SAPT) 19 2. Screening of Survey Isolates In Vi t r o 20 3. Basis of the In V i t r o I n h i b i t i o n 21 IV. In Vivo Studies 23 1. Screening of Rhizobium i n Growth Pouches 23 2. S o i l Tests 25 - i v -Page RESULTS 28 I. Isolation and I d e n t i f i c a t i o n of Pathogenic Fungi . . . . 28 I I . I s o l a tion and I d e n t i f i c a t i o n of Rhizobium 34 I I I . In Vi t r o Studies 36 1. Screening of Survey Isolates In Vi t r o 36 2. Basis of the In Vi t r o I n h i b i t i o n . 40 IV. In Vivo Studies . . 40 1. Screening of Rhizobium i n Growth Pouches 40 2. S o i l Tests 48 DISCUSSION 51 SUMMARY AND CONCLUSIONS 58 LITERATURE CITED 59 APPENDIX I • . . .' 64. LIST OF TABLES Table 1 Root rot fungi obtained from other source Table 2 Nitrogen-deficient nutrient s o l u t i o n used to water 'Topcrop' snap bean i n growth . pouches. Table 3 Authenticated Rhizobium species obtained from other sources. Table 4 Rhizobium and fungal i s o l a t e s tested i n dual culture agar pla t e s . Table 5 Rhizobium i s o l a t e s evaluated f o r i n h i b i t i o n of Fusarium s o l a n i (FS911) and Rhizoctonia  so l a n i (RSI) root rots i n growth pouches of 'Topcrop' snap bean. Table 6 Rhizobium i s o l a t e s evaluated f o r i n h i b i t i o n of Fusarium s o l a n i (FS911) root rot of 'Topcrop' snap bean i n growth pouches. Table 7 Fungi i s o l a t e d from snap bean roots from commercial f i e l d s i n the Fraser Valley, B r i t i s h Columbia. Table 8 Root disease index and shoot and root dry weights of 'Topcrop* snap bean grown i n growth pouches and inoculated at four d i f f e r e n t stages with Fusarium sol a n i 2 at 10 7 spores/pouch. Table 9 Root disease index and shoot and root dry weights of 'Topcrop' snap bean grown i n growth pouches and inoculated at f i v e d i f f e r e n t stages with Fusarium sol a n i 911 at 10 6 spaces/pouch. Table 10 Root disease index of three snap bean c u l t i v a r s ('Harvester', 'Stringless Greenpod', 'Topcrop') and 'Lincoln' pea grown i n growth pouches. Table 11 Pathogenicity of two Rhizoctonia s o l a n i i s o l a t e s to 'Topcrop' snap bean i n growth pouches. Page 12 15 19 21 24 25 28 29 30 32 34 Table 12 Source of Rhizobium i s o l a t e s from snap bean root nodules. 35 - v i -Page Table 13 Table 14 Nodulating 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. The e f f e c t of Rhizobium i s o l a t e s on fungal growth as determined by dual culture agar plate t e s t s . 35 37 Table 15 Level 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 dual culture agar plate tests with Fusarium s o l a n i 2. Table 16 I n h i b i t i o n zones rated+++ in- dual culture agar plate test of Rhizobium and Fusarium  so l a n i 2. Table 17 _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 so l a n i (FS2, FS911) and Rhizoctonia s o l a n i (RSI, RS2, RS3) i n dual culture agar plate t e s t s . 37 39 39 Table 18 Hydrogen ion concentration of dual culture agar plates inoculated with Rhizobium and Fusarium so l a n i 2. 41 Table 19 E f f e c t of Rhizobium 106 and Fusarium so l a n i 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. 43 Table 20 Level of antagonism of nodulating Rhizobium i s o l a t e s to Fusarium s o l a n i (FS2, FS911) i n dual culture agar plate tests and growth pouches of 'Topcrop' snap bean. Table 21 Interaction of Rhizobium i s o l a t e s and Fusarium so l a n i 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. Table 22 Nodulating 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 dual culture agar plate tests and i n growth pouches of 'Topcrop' snap bean. 45 46 47 Table 23 The l e v e l of antagonism of nodulating Rhizobium i s o l a t e s to Fusarium s o l a n i (FS2, FS91) i n dual agar plate tests and i n growth pouches of 'Topcrop' snap bean. 49 - v i i -LIST OF FIGURES Figure 1 'Topcrop' snap bean grown by a growth pouch technique. Figure 2 Fusarium solani 911 root rot of 'Topcrop' snap bean grown by a grouch pouch technique Figure 3 a. Rhizoctonia solani root rot and b. hypocotyl rot of 'Topcrop' snap bean i n growth pouches. Figure 4 Level of i n h i b i t i o n i n dual culture agar plate tests: a. Fusarium solani (FS2) inhibited (+. + +) by Rhizobium, i s o l a t e RCC326; b. Rhizoctonia solani 1 not inhibited (-) by RCC326. Figure 5 a. Fusarium solani 911 (P 3 •= 10^ spores/ pouch) root rot of 'Topcrop' snap bean i n growth pouches. b. Suppressed by inoculation with Rhizobium, iso l a t e RCC816 (R A = 10 6 cells/pouch). Figure 6 a. Fusarium solani 911 (P 2 = inoculum: s o i l 1:10) root rot of 'Topcrop' snap bean grown i n pasteurized s o i l . b. Suppressed by inoculation with Rhizobium, iso l a t e RCC106 (R^ = 10 8 cells/seed). - v i i i -LIST OF APPENDIX TABLES Page Table 1 Effect of Rhizobium 816 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. Table 2 Effect of Rhizobium 324 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. Table 3 Effect of Rhizobium 321 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. Table 4 Effect of Rhizobium 607 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. Table 5 Effect of Rhizobium 812 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. Table 6 Effect of Rhizobium 106, Fusarium solani (FS911) and Rhizoctonia solani (RSI) on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. Table 7 Interaction of Rhizobium isolates and Rhizoctonia solani (RSI, RS2, RS3) on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches. Table 8 Interaction of Rhizobium isolates and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean in growth pouches. Table 9 Effect of Rhizobium 106 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean grown i n pasteurized greenhouse s o i l . 63 64 65 66 67 68 69 70 71 - i x -Table 10 Effect of Rhizobium 106 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean grown i n pasteurized greenhouse s o i l . Page 72 Table 11 Effect of Rhizobium 816 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean grown i n pasteurized greenhouse s o i l . 73 - x -To my parents and Peter who gave me the moral support necessary to complete th i s t h e s i s . - x i -ACKNOWLEDGMENTS I would l i k e to si n c e r e l y thank both of my major supervisors, Dr. H.S. Pepin, A g r i c u l t u r e Canada, Research Station, Vancouver, and Dr. R.J. Copeman, Associate Professor, Department of Plant Science, University of B r i t i s h Columbia, f o r t h e i r invaluable guidance and en-couragement throughout t h i s project. Appreciation i s also extended to the other members of my thesis committee: Dr. V.C. Runeckles, Dr. F.B. H o l l and Dr. R.J. Bandoni f o r th e i r useful suggestions and constructive c r i t i c i s m s pertaining to th i s t h e s i s . The Director and s t a f f members of the Agr i c u l t u r e Canada, Research Station, Vancouver are g r a t e f u l l y acknowledged f o r t h e i r assistance and for providing the f a c i l i t i e s necessary to carry out th i s research project. In p a r t i c u l a r , I wish to thank Mrs. A. MacPherson f or her patience and per s i s t e n t a id i n the s t a t i s t i c a l aspects of th i s study. F i n a n c i a l assistance was provided by a grant from the Natural, Applied, and Health Sciences Grants Committee of the Univ e r s i t y of B r i t i s h Columbia and a Summer Student Scholarship awarded by the University of B r i t i s h Columbia. INTRODUCTION AND LITERATURE REVIEW Introduction Increasing attention i s being focused on vegetable proteins, especially those derived from legumes, i n an attempt to a l l e v i a t e world protein shortages (Neergaard, 1977; Freytag, 1975). Phaseolus vulgaris L. i s an exemplary legume with a protein content of 15-31% (Neergaard, 1975) and a valuable source of calcium, r i b o f l a v i n and iron (Zaumeyer, 1957). Bean i s v i r t u a l l y universal 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 tropics and subtropics, bean has been a major dietary staple since pre-colonial 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 pr o f i t a b l e and the land must be abandoned (Zaumeyer and Thomas, 1957). Less severe infestations of root r o t t i n g organisms may be responsible for unaccountable losses that l i m i t the y i e l d potential of the crop. If yields are to be increased to meet world protein demands, steps must be taken to e f f e c t i v e l y control root disease, preferably by means requiring a minimal expenditure of energy. The Disease Root rots of snap bean, i n c i t e d by Fusarium solani (Mart.) App. & Wr. F. sp. phaseoli (Burkh.) Snyd. & Hans, and Rhizoctonia solani Kuehn are ubiquitous i n cultivated 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; Christou, 1962; Reyes & M i t c h e l l , 1962). Depending upon when infection occurs, seed rot, pre-- 2 -and post-emergence damping off , foot rot (hypocotyl r o t ) , or root rot may result (MacSwan & Koepsell, 1981). Affected plants show non-specific symptoms such as poor emergence and growth, chlorotic leaves, premature de f o l i a t i o n and stunting (Walker, 1952). Severe root rot reduces the number of surviving roots available for 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 certain areas the crop has been completely decimated (Neergaard, 1975; MacSwan & Koepsell, 1981). I n i t i a l l y , Fusarium root rot of bean i s distinguished by a s l i g h t reddish discoloration of the taproot which gradually increases i n inten-s i t y to cover the root (Zaumeyer & Thomas, 1957). Elongate streaks of i n d e f i n i t e size and margin extend down the hypocotyl and taproot. Dis-eased tissue 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 rot' (Burkholder, 1919; Westcott, 1971). Lateral roots are destroyed but adventitious roots developing above the lesions can support the crop to harvest (MacSwan & Koepsell, 1981). Fusarium root rot i s generally favored by warm, dr i e r weather i n mid-season (Chupp & Sherf, 1960) Rhizoctonia root rot of bean i s distinguished from Fusarium root rot by the type of lesions that develop on the hypocotyl and taproot. Lesions of Rhizoctonia are varying shades of brown, sunken, oval to elongate regions with a d e f i n i t e margin (Chupp & Sherf, 1960). Lesions can g i r d l e the root and diseased tissue takes on a predominantly reddish-brown discoloration before becoming completely necrotic. A sign of Rhizoctonia root rot i s brown to black s c l e r o t i a of variable size and form which develop i n the necrotic tissue and sig 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 rot of bean i s generally considered an early season problem i n cooler condi-tions (Yang & Hagedorn, 1966). - 3 -Fusarium and Rhizoctonia attack bean roots and hypocotyls by multiple infections (Schrothand Snyder, 1961; Christou, 1962). These pathogens can enter root tissues by direct penetration or i n d i r e c t l y v i a wounds caused by mechanical injury due to c u l t i v a t i o n practices, nematodes, insects or at the points where l a t e r a l roots emerge (Christou, 1962. Garrett, 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 tissues 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 (conidia, chlamydospores, mycelial fragments and sclerotia) can be carried as contaminants on the seed coat, i n crop debris or i n s o i l humus pa r t i c l e s (Nash et a l . , 1961; MacSwan and Koepsell, 1981). Transmission of the root rot organisms to other areas i s f a c i l i t a t e d by r a i n splashing, flooding, wind, c u l t i v a t i o n practices and s o i l insects (Chupp and Sherf, 1960). Control of Bean Root Rots Control methods of Fusarium and Rhizoctonia root rots are not adequate even though these diseases are prevalent and s i g n i f i c a n t l y destructive ( M i l l e r and Burke, 1974;. MacSwan and Koepsell, 1981). Bean cu 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 rot. The age of the plant, vigour and root secretions also influence root disease severity. Generally, root disease i s worse on younger plants stressed by unfavorable growing conditions (Zaumeyer and Thomas, 1957). Immunity to Fusarium and Rhizoctonia root rot does not exist i n any bean c u l t i v a r and breeding for resistance i s impeded by the great v a r i a b i l i t y among pathogenic strains (Chupp and Sherf, 1960). Most commercial c u l t i v a r s (e.g. 1Slenderwhite', 'Tenderwhite', 'Topcrop'), while moderately susceptible to Fusarium root - 4 -rot, can s t i l l be pr o f i t a b l y grown i n infested s o i l under environmental conditions favourable to crop growth (Hagedorn and Rand, 1974). Phaseolus  coccineus L. (Scarlet runner bean), some cu l t i v a r s of lima bean, the bean cu l t i v a r 'Tendergreen' and Plant Introduction Accession Line N203 show tolerance to Fusarium root rot (Booth and Waterson, 1964;, Hagedorn and Rand, 1974). Venezuela 54 and dark-seeded Plant Introduction Accession Lines 109859, 163583, 165426 show tolerance to Rhizoctonia root rot (Prasad and Weigle, 1969, 1970). Chemical control of bean root rots i s of dubious value (Maloy and Burkholder, 1959). Fungicide, steam or hot water seed treatments and furrow sprays with fungicides have reduced root rot but are not en t i r e l y effective (Papavizas and Lewis, 1975; MacSwan and Koepsell, 1981). S o i l fumigation i s not a viable economical control practice for bean root rots (Zaumeyer and Thomas, 1957; Westcott, 1971). Root diseases are influenced by abiot i c and b i o t i c factors i n the environment. Cultural practices can be implemented to al t e r these conditions to increase host plant vigour and supppress root rot (Zaumeyer and Thomas, 1957). Any practice 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 infested area or by producing new roots above the points of in f e c t i o n (Burke, 1968). Root tissues of vigorous plants are less susceptible to disease because they consist of more mature, thicker walled, l i g n i f i e d c e l l s which are not as readily penetrated and destroyed by the pathogen as weak plants with thin 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); Abiotic factors of paramount importance i n determining the incidence and severity of root rot are s o i l physical 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; Garrett, 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 cold, wet early season weather (Chupp and Sherf, 1960; Burke, 1964; Westcott, 1971). 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 analysis so that an adequate pH and nutrient levels are provided for'vigorous crop growth. Y i e l d increases of bean grown i n s o i l infested with J _ . solani and R. solani were achieved by subsoiling to a depth of 18 inches below the d r i l l row prior to planting (Burke, 1968. Cook, 1977). This enhanced root development and penetration especially 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 also provided a b e n e f i c i a l effect by allowing adventitious roots to develop above the infected root tissue (MacSwan and Koepsell, 1981). S t r i c t sanitation should be practiced by removing or deep plowing the inf ected crop debris after harvest (Chupp and Sherf, 1960). Long rotations of 5 to 6 years with a grain such as wheat or barley i s necessary to reduce inoculum potential of the fungus i n the s o i l (Maloy and Burkholder, 1959; Westcott, 1971). Accidental introduction of v i r u l e n t strains 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, 1960; Neergaard, 1977). Organic amendments (mature barley or wheat straw, C h i t i n , cellulose) can reduce root rot 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 limited due to the lack of exogenous nutrients required for either chlamydospore germination or fungal growth. Control of Fusarium root rot of bean by the application of cellulose amendments was reported by Adams - 6 -et a l , 1968. Amendments must be used care 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 rot (Toussoun and Patrick, 1963). B i o t i c factors i n the environment that play a key role i n deter-mining the incidence and severity of root rot are attributes of the surrounding populations of s o i l organisms. In the environment, organisms exhibit a multitude of interactions as they progress toward a b i o l o g i c a l l y balanced state or equilibrium (Baker and Cook, 1974). Rhizosphere organ-isms, closely associated with root pathogens and the host plant, may have no effect on root rot or they may act as synergists or antagonists. Potential synergists to root diseases are nematodes, viruses, other plant pathogens and root rot fungi which may greatly intensify plant losses i n the f i e l d . Peas infected with bean yellow mosaic virus or common pea mosaic virus released more root exudate than uninfected plants (Baker and Cook, 1974). As a result the inoculum concentration and number of root infections due to J_. solani (Mart.) Appel & Wr. F. sp. p i s i (F.R. Jones) Snyd. & Hans, and Aphanomyces euteiches Drechs. increased. I n t e n s i f i c a t i o n of R. solani root rot of sweet pea also occurred following early infection by pea enation mosaic vi r u s . Antagonistic microorganisms can reduce root rot and function as nonchemical means for plant disease control (Henis, 1970). Beirne (1967) broadly defined a b i o l o g i c a l control agent as "any l i v i n g organism that can be manipulated by man for pest control purposes." (Baker and Cook, 1974). B i o l o g i c a l control i s an integral part of a pest control program and should be used i n conjunction with sound c u l t u r a l practices 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 result of antagonism ( a n t i b i o s i s , mycoparasitism, l y s i s ) or competition (Anderson and Ruber, 1965; Huber et a l . , 1966). 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 incited by Sclerotium r o l f s i i Sacc. and Rhizoctonia solani i n f i e l d experiments with bean, cotton or tomato. A s i g n i f i c a n t increase i n bean y i e l d was also attained. T_. harzianum i s mycoparasitic and was capable of lysing mycelia of the pathogens i n dual culture. Rhizosphere bacteria are p o t e n t i a l l y important antagonists to fungal root disease because of the i r abundance and association with plant roots. Competitive interactions between s o i l microorganisms for nutrients, space, oxygen and other requirements could be the major factors governing bio-l o g i c a l control of soilborne fungi (Marshall and Alexander, 1960). Nutrient competition between t y p i c a l s o i l bacteria 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 (Beijerinck & van Delden) Conn d r a s t i c a l l y limited fungal growth. Huber et a l . (1966) used a plate 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 active i n the b i o l o g i c a l control of F_. solani f. sp. phaseoli and R. solani root rot of bean. Their results indicated that the most important mechanism of b i o l o g i c a l control of bean root rot was fungal necrosis (hyphal death) due to s o i l bacteria. Rhizobium species are s o i l - i n h a b i t i n g , gram negative bacteria of economic importance i n the legume rhizosphere because of the i r role i n root nodulation and symbiotic nitrogen f i x a t i o n (Beringer et a l . , 1980; Vance and Johnson,, 1981). As a result of this symbiosis, legume crops can be grown without expensive chemical f e r t i l i z e r s . The antifungal a c t i v i t y of Rhizobium was demonstrated by Drapeau et a l . (1973) using an agar plate - 8 -technique. The r a d i a l growth of phytopathogenic Fusarium spp. and Phytophthora cactorum (Leb. & Cohn) Schroet. on yeast mannitol agar plated, was inhibited by three different strains of Rhizobium. Rhizoctonia solani and Pythium ultimum Trow were not affected by Rhizobium  in v i t r o . Similar 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 strains of R. leguminosarum. The Rhizobium strains showed varying degrees of a c t i v i t y towards F. solani f. sp. p i s i , a major incita n t of pea root rot. 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 tests. Rhizobial parasitism of root rot fungi was reported by Tu (1978b, 1979). Inoculation of culture plates of the test fungi with a r h i z o b i a l suspension caused a reduction i n fungal sporulation and extensive internal and external colonization of the hyphae by Rhizobium japonicum (Kirchner) Buchanan. The aseptate fungi, 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 fungi, Fusarium oxysporum and Ascochyta imperfecta Pk. Interactions between Rhizobium and root rot pathogens i j i s i t u may not be reproducible or detectable i n v i t r o , therefore i n vivo studies are necessary. Chi and Hanson (1961) f i r s t reported a b e n e f i c i a l effect of Rhizobium t r i f o l i i Dangeard on red clover (Trifolium pratense L.) exposed to F_. oxysporum, F_. roseum (Lk.) emend. Snyd. & Hans, and F_. solani w i l t and root rot fungi. Red clover plants, grown i n a sand-nutrient solution medium i n the greenhouse, were consistently more vigorous and developed less root rot when inoculated with Rhizobium. Mew and Howard (1969) proposed the use of an acid-tolerant, effective s t r a i n of R. japonicum to control F_. oxysporum root rot of soybean. Soybeans were grown i n a sand-nutrient solution medium, at pH 7 or 7.6. Root rot was - 9 -reduced to a trace or absent when R. japonicum was present but the same effect was not observed at pH 5. In similar experiments Orellana e_t a l . (1976) suggested that nodulation of soybean with e f f i c i e n t strains of R. japonicum could a l l e v i a t e the detrimental effect of R. solani root r ot. The b e n e f i c i a l effect of R. japonicum and Endogone mosseae Mosse i n sup-pressing Phytophthora megasperma root rot 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 rot was reduced as the concentration of rhizobia 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 rot, a predisposing factor to winter injury. The effect of Rhizobium on root rot was not eradicative but provided a s i g n i f i c a n t degree of protection from severe root rot (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 N-f i x a t i o n , but also for the added protection 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 correlation between the incidence and severity of F. solani root rot and root nodulation by R. leguminosarum. However, Gray and Hine (1976) reported that a l f a l f a root nodules in c i t e d by R. m e l i l o t i were the primary sites of infection by P_. megasperma early 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 effect of R. japonicum and Endogone mosseae on root rot of soybean caused by Pythiuiii ultimum. - 10 -S i m i l a r l y , Gray and Sackston (1980) did not observe any reduction i n Fusarium s o l a n i root r o t of pea by r h i z o b i a l i n o c u l a t i o n although anta-gonism had been observed jin v i t r o . Objectives Rhi zobium i n addition to i t s t r a d i t i o n a l r o l e i n root nodulation and symbiotic N-fi x a t i o n , has recently been implicated as an antagonist suppressing root rot or as a synergist, enhancing root rot of legumes. This discrepancy i n the l i t e r a t u r e warrants further i n v e s t i g a t i o n to ascertain the nature of the i n t e r a c t i o n between Rhizobium and root r o t t i n g fungi. The objectives of t h i s thesis were to investigate the e f f e c t of Rhizobium on bean root r o t t i n g fungi and to determine the r o l of Rhizobium i n the development of root rots i n c i t e d by J _ . s o l a n i f. sp phaseoli and R. s o l a n i . MATERIALS AND METHODS I. ISOLATION AND IDENTIFICATION OF PATHOGENIC FUNGI 1. F i e l d Survey In order to investigate the nature of the i n t e r a c t i o n between Rhizobium and bean root rot fungi a f i e l d survey was f i r s t undertaken to c o l l e c t i s o l a t e s of these micro-organisms. Ten bean (Phaseolus v u l g a r i s L.) f i e l d s , located throughout the Fraser Valley, B r i t i s h Columbia, were surveyed i n the summer of 1979. Samples of diseased and healthy, nodulated plants were c o l l e c t e d . P o t e n t i a l l y pathogenic fungi were i s o l a t e d from diseased bean roots showing reddish-brown to dark brown d i s c o l o r a t i o n or le s i o n s . Diseased root t i s s u e was sectioned into 0.25 to 0.5 cm segments and surface s t e r i l i z e d by immersion i n 95% ethyl alcohol f o r 1 minute followed by immersion i n a 1:10 commercial bleach s o l u t i o n f o r 2 to 5 minutes. The root segments were transferred through 5 rinses of s t e r i l e d i s t i l l e d water, plated onto potato dextrose agar (PDA, D i f c o ) , tapwater agar, or Nash and Snyder's PCNB media (Tuite, 1969) and incubated at 21 to 24°C. Subcultures of representative fungal colonies were established on PDA plate s . Tentative i d e n t i f i c a t i o n s were made on the basis of colony morphology a f t e r approximately 1 week. Microscopic examination of mycelia and spores were compared to standard mycological keys f o r the Fungi Imperfecti (Barnett, 1960) to i d e n t i f y the i s o l a t e s to genus. Single 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 species according to the scheme of Toussoun and Nelson (1968). The c o l l e c t i o n of fungal i s o l a t e s was maintained on PDA slants both at room temperature and at 4°C. - 12 -Authenticated i s o l a t e s of F. s o l a n i and R. so l a n i were obtained from other sources (Table 1). Table 1. Root rot fungi obtained from other sources Species Fusarium so l a n i f. sp. phaseoli (FS2) F. so l a n i f. sp. phaseoli (FS911) F. so l a n i (FSIV) Rhizoctonia s o l a n i f. sp. phaseoli IV (RSI) R. s o l a n i (RS2) R. so l a n i (RS3) Source Dr. J.C. Tu, Harrow, Ontario Dr. R. H a l l , Guelph, Ontario Prosser, Washington Dr. Wells, Davis, C a l i f o r n i a Dr. R.J. Copeman, Vancouver, B.C. Dr. J.C. Tu, Harrow, Ontario 2. Growth Pouch Technique (GPT) Bean seedlings were grown i n growth pouches (DiSPo Seed Packs, Northrup, King &Co., Minneapolis, MN 55413) as previously described by Weaver and Frederick (1972). These s t e r i l e , c l e a r , p l a s t i c packets (18 x 16.5 cm) contained a wide paper wick folded into a trough at the top to accommodate the seed. A flame s t e r i l i z e d scapel was used to cut a 0.5 to 0.75 cm hole i n the bottom of the trough to permit the downward passage of the large bean r a d i c l e along the paper wick. The pouches were then placed i n s p e c i a l wooden support boxes (outer dimensions 45 x 19.5 x 15.5 cm) that held 60 to 70 pouches. Press board d i v i d e r s (17.5 x 14 cm) were spaced at 5 cm i n t e r v a l s along the length of the box to support the pouches i n a v e r t i c a l p o s i t i o n ( F i g . 1). Bean seeds (Phaseolus v u l g a r i s L. 'Topcrop') were used i n a l l - 13 -Figure 1. 'Topcrop' snap bean grown by a growth pouch technique. - 14 -experiments unless otherwise s p e c i f i e d . Seeds were surface d i s i n f e s t e d by placing them for 1 minute i n 95% ethyl alcohol followed by a 10 minute immersion i n a 1:10 commercial bleach s o l u t i o n . The seeds were rinsed with 5 changes of s t e r i l e d i s t i l l e d water. One surface s t e r i l i z e d bean seed was placed into the trough of each growth pouch so that the micro-pylar side was d i r e c t l y over the hole made previously i n the trough. A p l a s t i c drinking straw was placed into the side of each pouch and i n i t i a l l y 20 ml of s t e r i l e d i s t i l l e d water was added through the straw with a syringe. Subsequently, water was supplied as necessary and 10 ml of a n i t r o g e n - d e f i c i e n t nutrient s o l u t i o n with microelements at pH 6.5, was added weekly to each pouch (Table 2) (Hoagland and Arnon, 1950). Inoculated pouches were placed i n a completely randomized design i n the support boxes on benches i n the greenhouse for 4 weeks unless otherwise stated. Supplemental l i g h t was provided for 12 hr/day by c o o l -white fluorescent tubes producing approximately 2,800 l x . Temperature i n the greenhouse ranged from 18 to 24°C. Where s p e c i f i e d boxes of seedling growth pouches were placed i n an environmental control chamber at 21 ± 2°C and exposed to approximately 1,850 l x from a mixture of incandescent bulbs and cool-white fluorescent tubes for 16 hr/day. At the end of the growth period the plants were rated for root rot severity according to an equal increment disease index described by Tu (1978a). Healthy-appearing roots, creamy-white i n colour, were given a disease index of 0 while completely rotted, dead roots were given a disease index of 9. Shoot and root dry weights of i n d i v i d u a l plants were determined where stated. S t a t i s t i c a l analysis of the disease index and root and shoot dry weights involved a m u l t i f a c t o r i a l analysis of variance, Duncan's multiple range test (p = 0.05) and sets of contrasts. Individual - 15 -Table 2. Nitrogen-deficient nutrient s o l u t i o n used to water 'Topcrop' snap bean i n growth pouches Solution a: ml/L nutrient s o l u t i o n 0.5 M K2SO4 1.0 M MgS04 0.05 M C a ( H 2 P 0 4 ) 2 0.01 M CaSOij 5 2 10 200 Solution b: (Add 1 ml/L to nutrient s o l u t i o n (a)) H3BO3 MnCl 2 ' 4H 20 ZnSOu • 7H20 C u S o i t • 5H20 H 2Mo0 4 • H 20 g/L 2.86 1.81 0.22 0.08 0.02 Solution c: (Add 1 ml/L to nutrient s o l u t i o n (a)) 0.5% Fe (tartrate) experiments were not repeated unless otherwise s p e c i f i e d . 3. Pathogenicity Tests Bean seedlings at the crook stage of development were inoculated with one of the test fungi. Two types of inocula were prepared for patho-g e n i c i t y tests according to whether or not the test fungus r e a d i l y produced spores. Inoculum c o n s i s t i n g of a spore d i l u t i o n was prepared for Fusarium i s o l a t e s . The test fungus was grown on PDA plates 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 into the plates and the surface scraped l i g h t l y with a s t e r i l e s c a l p e l . The supernatant was c o l l e c t e d and the spore concentration determined with a hemocytometer. The spore - 16 -d i l u t i o n was prepared with s t e r i l e d i s t i l l e d water. A s t e r i l e pipette was used to add 1 ml of inoculum to each pouch. Inoculum con s i s t i n g of mycelia was used i n pathogenicity tests with Rhizoctonia. One plate of R. s o l a n i , grown on PDA for 1 week was mixed with 200 ml of s t e r i l e d i s t i l l e d water i n a Waring Blender at low speed f o r one minute. Mycelial inoculum was added at 1 to 10 ml/pouch with a s t e r i l e p i p ette. Preliminary pathogenicity tests were performed using the above procedure to i d e n t i f y v i r u l e n t i s o l a t e s which could then be used i n subsequent experiments. Concentration of the spore inoculum was not standardized but was at le a s t 10 5 spores/ml. Each treatment was re p l i c a t e d 3 times. The presence or absence of root rot was recorded a f t e r 4 weeks. To determine the e f f e c t of seedling age on pathogenicity, bean plants, grown according to the GPT, were inoculated at 1. the seed stage; 2. the r a d i c l e stage, when only the primary root was present; 3. the crook stage, when the hypocotyl was s t i l l bent; and 4. the f i r s t , t r ue-leaf stage, when the leaves were not yet unfolded. F_. so l a n i 2 (FS2) was used at a concentration of 10 7 spores/pouch. Control pouches of bean were treated with s t e r i l e d i s t i l l e d water only. Each treatment was r e p l i c a t e d 10 times. A s i m i l a r experiment was performed with F. so l a n i 911 (FS911) with two deviations. An inoculum concentration of 10 6 spores/pouch was used and an add i t i o n a l inoculation stage, the f i r s t , t r i f o l i a t e - l e a f stage was included. Each treatment was r e p l i c a t e d 7 times and the plants were rated a f t e r a growth period of 31 days. The pathogenicity of FS911 and FSIV to three bean c u l t i v a r s ('Harvester', 'Stringless Greenpod', 'Topcrop') and one pea c u l t i v a r (Pisum sativum L. 'Lincoln') was tested according to the GPT. A l l c u l t i -vars were inoculated at the seed stage with 10 6 spores/pouch of FS911 or FSIV except 'Topcrop', which was inoculated at the crook stage. Each treatment was r e p l i c a t e d 4 times and plants were rated a f t e r a growth period of 3 weeks. The virulence of three R. s o l a n i i s o l a t e s (RSI, RS2, RS3) was tested by inoculating 'Topcrop' seedlings at the crook stage with ei t h e r 10 or 20 ml/pouch of mycelial inoculum. Each treatment was r e p l i c a t e d 3 times and plants were rated a f t e r a growth period of 3 weeks. Two of these i s o l a t e s , RSI and RS2, were subsequently tested by the GPT for virulence at three inoculum concentrations (1, 5, 10 ml/pouch). Seedling were inoculated at the f i r s t , true-leaf stage with the mycelial inoculum with each treatment r e p l i c a t e d 4 times. Af t e r a growth period of 32 days plants were rated. I I . ISOLATION AND IDENTIFICATION OF RHIZOBIUM 1. F i e l d Survey Rhizobium i s o l a t e s were recovered from bean root nodules c o l l e c t e d during the f i e l d survey. Healthy-appearing nodules were excised from bean roots and surface s t e r i l i z e d i n 95% ethyl alcohol f o r one minute followed by immersion i n a 1:10 commercial bleach s o l u t i o n or a 1% mercuric chloride s o l u t i o n f o r 2 to 5 minutes. The nodules were then transferred a s e p t i c a l l y , through 5 rinses of s t e r i l e d i s t i l l e d water. Flame s t e r i l i z e d forceps -were used to crush i n d i v i d u a l nodules. A drop of r h i z o b i a l c e l l s from the crushed nodule was streaked across a yeast - 18 -mannitol agar (YMA) plate (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 bacteria 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 after 3 to 5 days on YMA plates were selected. Subcultures were established on YMA plates. Isolates were tested for dye absorption on YMA plus Congo red. Rhizobium isolates remain colourless or only weakly absorb the dye (Vincent, 1970). A gram stain test and microscopic examination were performed to v e r i f y that isolates were gram negative, small to medium sized rods (0.5 - 0.9 x 1.2 - 3 jam) occurring singly or i n pairs. Younger cultures were checked for m o t i l i t y while older cultures were checked for the presence of prominent, highly r e f r a c t i l e granules of polymerized g-hydroxybutyrate (PHB). Cultures were also examined to v e r i f y that endospores were not produced. Bacterial isolates conforming to these characteristics were maintained i n the Rhizobium Culture Collection (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 ba c t e r i a l isolates (RCC) as Rhizobium ultimately r e l i e d on their a b i l i t y to nodulate 'Topcrop' snap bean. Only nodulating isolates were used i n subsequent studies. Nodulation tests were performed using the GPT. Bean seedlings were inoculated at the crook stage with 1 ml/pouch of a turbid b a c t e r i a l suspension. The bac t e r i a l inoculum was prepared from 1 week old cultures on YMA plates. - 19 -Inoculum concentration was not standardized but was at l e a s t 10 6 c e l l s / m l . Each treatment was r e p l i c a t e d 4 times and the presence or absence of root nodules was recorded a f t e r 4 weeks. Nine authenticated Rhizobium species, obtained from other sources were also used i n nodulation tests (Table 3). Table 3. Authenticated Rhizobium species obtained from other sources. Rhizobium species Source R. japonicum (RJ1A, RJ1B) Dr. J.C. Tu, Harrow, Ontario R. phaseoli (RP1) Dr. F.B. H o l l , Vancouver, B.C. R. phaseoli (RP2) Dr. J.C. Tu, Harrow, Ontario R. leguminosarum (RLl, RL2) Dr. F.B. H o l l , Vancouver, B.C. R. leguminosarum (RCR1045) Rothamsted C o l l e c t i o n of R. l u p i n i (RCR3211) .Rhizobium, Harpenden, R. t r i f o l i i (RCR5) Hertfordshire, England I I I . IN VITRO STUDIES 1. Standardized Agar Plate Technique (SAPT) The agar plate technique of Drapeau et^ a l . (1973) was used i n these experiments except that YMA plates were inoculated with Rhizobium 2 days p r i o r to addition of the test fungus. A streak of Rhizobium was made i n a l i n e down one side of the agar plate and incubated at room temperature (21 to 24°C) for 2 days. Inoculum of the t e s t fungi, from the f i e l d survey, was prepared by growing the fungi a week on PDA plates at room temperature (21 to 24°C). Discs, 5 mm i n diameter, were cut from the outer margins of the fungal colony with a flame s t e r i l i z e d cork borer and transferred a s e p t i c a l l y to the preinoculated Rhizobium p l a t e s . - 20 -The fungal inoculum di s c was placed 5 cm away from the Rhizobium streak. Each agar te s t plate was r e p l i c a t e d 3 times unless otherwise s p e c i f i e d . The inoculated plates were incubated at room temperature (21 to 24°C) for 7 to 10 days u n t i l the mycelial fronts began to approach the Rhizobium streak. Signs of fungal growth i n h i b i t i o n could be observed when the fungus was within 1 cm of the Rhizobium streak. The i n t e r a c t i o n between Rhizobium and the test fungus was rated according to the system devised by Drapeau ejt a l . (1973) . 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 recognized: -, no i n h i b i t i o n of the fungus was observed; +, a very s l i g h t degree of fungal i n h i b i t i o n was observed as a zone of gradually evanescent hyphae near the Rhizobium streak, but the fungus quickly colonized 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 colonized by small masses of hyphae within 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 persisted at l e a s t 1 week a f t e r i t s i n i t i a l formation. A measurement of the zone width was taken at t h i s time. 2. Screening of Survey Isolates In V i t r o In preliminary dual culture agar plate tests Rhizobium i s o l a t e s were tested f o r t h e i r antagonistic a c t i v i t y towards representative fungal i s o l a t e s from the culture c o l l e c t i o n (Table 4). Each test plate was r e p l i c a t e d twice. Subsequently, a l l 51 nodulating and non-nodulating b a c t e r i a l i s o l a t e s were screened by the SAPT for antifungal a c t i v i t y to the s e n s i t i v e s t r a i n F_. s o l a n i (FS2) . This experiment was repeated twice. I n h i b i t i o n zones, rated + + +', were measured 1 week a f t e r t h e i r i n i t i a l formation. - 21 -Table 4. Rhizobium and fungal i s o l a t e s tested i n dual culture agar pla t e s . Rhizobium i s o l a t e s Fungal i s o l a t e s 100 FMl, PSl, F01, PS2, F02, PS3, FRl, PUl, FS1, PU2, FS2, RSI, FS3, RS2 101 F01, FS4, F02, FS5, F03, RSI F04, F05, FS2, FS3, 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, 115 FS6, RSI FM, Fusarium moniliforme FO, F. oxysporum FR, F. roseum FS, F. so l a n i PS, Pythium sylvaticum PU, Pythium ultimum RS, Rhizoctonia s o l a n i 3. Basis of the In V i t r o I n h i b i t i o n To determine whether or not i n h i b i t i o n of Fusarium by Rhizobium was due to a pH change, measurement of the pH of agar test plates was taken at the end of the screening experiments. A 1.5 to 2 cm s t r i p of pH i n d i c a t o r paper was inserted into the i n h i b i t i o n zone, the fungal - 22 -colony (FS2) and the opposite side of the Rhizobium streak. As a pre-liminary test only one r e p l i c a t e of each test plate was examined i n two of the screening experiments. The pH of YMA plates was i n i t i a l l y 6.5. In an attempt to demonstrate that the i n v i t r o i n h i b i t i o n of Fusarium was due to some d i f f u s i b l e , metabolite produced by Rhizobium, segments of the i n h i b i t i o n zone were used to replace the Rhizobium streak i n the SAPT. I n h i b i t i o n zone segments ( 2 x 4 mm) were removed from SAP tests of FS2 and Rhizobium i s o l a t e s RCC100, 107, 109,111, 121, 319, 321, 326, 812 and 815 when zones were 7 days old. Zones were placed .5 to 2.5 cm away from an inoculum d i s c of FS2. Each te s t plate was r e p l i c a t e d twice and the experiment repeated twice. The plates were checked d a i l y for the development of i n h i b i t i o n zones. Cut out segments of i n h i b i t i o n zones were also placed d i r e c t l y onto YMA plates seeded with the same Rhizobium i s o l a t e or spores of FS2. Zones of i n h i b i t i o n were removed from SAP screening tests of FS2 and RCC107, 121 or 326. Each test plate was r e p l i c a t e d twice and the experiment repeated twice. C e l l - f r e e extracts of Rhizobium were tested for t h e i r a b i l i t y to i n h i b i t the growth of FS2 and FS911 on YMA plates. Rhizobium i s o l a t e s (RCC100, 106, 107, 116, 121, 607, 811) were grown i n yeast mannitol shake culture for 3 to 31 days at room temperature (21 to 24°C). The cultures were centrifuged i n a S o r v a l l Superspeed centrifuge, SS-34, at 12,100 g for 30 minutes. The supernatants were s t e r i l i z e d by m i l l i p o r e f i l t r a t i o n . A drop or streak of the c e l l - f r e e extract was placed opposite a 5 mm inoculum di s c of FS2 and FS911. The c e l l - f r e e extract replaced the Rhizobium streak i n the SAPT. Each agar test plate was r e p l i c a t e d twice. C e l l - f r e e extracts of RCC106 or RCC607, stored at 4°C, were also placed i n 5 mm 'wells' cut out of the agar 1 cm away from inoculum discs of FS2. The 'wells' were replenished d a i l y with the c e l l - f r e e extract. Each test p l a t e was r e p l i c a t e d twice and checked d a i l y for the development of i n h i -b i t i o n zones. A c e l l - f r e e extract of RCC607, concentrated (1:10) by d i a l y s i s (tubing pore s i z e 4.8 mm) against polyethylene g l y c o l (6000 MW), was tested as previously i n 5 mm agar 'wells'. Two r e p l i c a t e s were made and the experiment repeated twice. IV. IN VIVO STUDIES 1. Screening 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 (Table 5) i n v o l v i n g varying concentrations of Rhizobium and _F. s o l a n i (FS911) to determine whether or not i n h i b i t i o n of root r o t occurred i n vivo. Bean seedlings were simultaneously inoculated at either the crook stage or the f i r s t , t rue-leaf stage with four l e v e l s of Rhizobium (0, 10 2, 101*, 10 6 c e l l s / pouch) i n combination with FS911 (0, 102', 1 0 \ 10 6 spores/pouch). Inoculum was prepared as previously described. 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 repeated except that involving RCC106. Plants were grown for 4 weeks and then assigned a root disease index, nodulation index (0, no nodules ?to 3, abundant nodules) and root and shoot dry weights were determined. The e f f e c t of Rhizobium on the root r o t complex of _F. s o l a n i (FS911) and R. s o l a n i (RSI) was investigated i n a 3 x 4 f a c t o r i a l experiment s i m i l a r to the previous experiments. Bean seedlings at the f i r s t , true-l e a f stage were simultaneously inoculated with RCC106 (0, 10 s, 10 6, 10 7 cells/pouch) and inoculum mixture of FS911 at ei t h e r 0 or 10 6 spores/pouch with RSI at ei t h e r 2 or 4 ml/pouch (Table 5). Each treatment was r e p l i c a -ted 5 times. Table 5. Rhizobium i s o l a t e s evaluated for i n h i b i t i o n of Fusarium s o l a n i (FS911) and Rhizoctonia  s o l a n i (RSI) root rots i n growth pouches of 'Topcrop' snap bean. Rhizobium In v i t r o i n h i b i t i o n Pathogen tested Seedling i s o l a t e of _F. s o l a n i 2* ±n vivo inoculation stage 106 + + + FS911 crook 607 + + + FS911 crook 812 + + + FS911 f i r s t , t rue-leaf 321 + + + FS911 f i r s t , t rue-leaf 324 + FS911 crook 816 - FS911 crook 106 + + + FS911 + RSI f i r s t , t r u e-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 recognized: -, 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 ; + +, clear zone of i n h i b i t i o n , colonized a f t e r 3 to 5 days; + + +, clear zone of i n h i b i t i o n p ersisted at l e ast 1 week. A s i m i l a r 4 x 4 f a c t o r i a l experiment was performed to study the e f f e c t of three Rhizobium i s o l a t e s (RCC107, 324, or 613) on R. s o l a n i root r o t of bean. Bean seedlings, grown by the GPT, were inoculated at the crook stage with Rhizobium at 10 8 cells/pouch. Two days l a t e r 3 ml/ pouch of R. s o l a n i inoculum (RSI, RS2, or RS3 was added. Each treatment was r e p l i c a t e d 4 times and the pouches were grown for a period of 3 weeks. Large scale screening experiments were c a r r i e d out using the GPT (Table 6). Bean seedlings, grown i n an environmental control chamber, were inoculated at the crook stage with FS911 at 0, 10 2, or 101* spores/ - 25 -pouch. Two Rhizobium i s o l a t e s at 0, I0h, or 10 6 cells/pouch were tested i n each experiment. Rhizobium was added the pouches at the same time as FS911. Table 6. Rhizobium i s o l a t e s evaluated f o r i n h i b i t i o n of Fusarium s o l a n i (FS911) root rot i n growth pouches of 'Topcrop' snap bean. Rhizobium Nodulating 'In v i t r o i n h i b i t i o n i s o l a t e a b i l i t y of F. sola n i 2* 107, 109 + , + + + + + + 111, 115 + + + + , + + + 116, 118 + , + + + + , + + + 319, 321 + + + + , + + + 323, 812 + + + + + + 816, RL1 +, - + 815, RCR1045 + + + + + + *Four l e v e l s of in 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 was observed; +, s l i g h t i n h i b i t i o n ; ++, cl e a r zone of i n h i b i t i o n , colonized a f t e r 3 to 5 days; + + +, clear zone of i n h i b i t i o n p ersisted at le a s t 1 week. 2. S o i l Tests Similar f a c t o r i a l experiments involving four concentrations of both Rhizobium and of F_. so l a n i (FS911) were performed i n greenhouse s o i l pH 6.5, The s o i l was pasteurized twice, once on each of two consecutive days, infested with FS911 inoculum and placed into 15 cm pots. The FS911 inocu-lum was grown i n s t e r i l i z e d v ermiculite and yeast mannitol l i q u i d f o r - 26 -2 or 3 weeks at room temperature (21 to 24°C). Cultures were shaken d a i l y to loosen clumps. The four l e v e l s of FS911 inoculum were made up to a standardized volume with s t e r i l i z e d v e r m i c u l i t e then thoroughly mixed with the pasteurized s o i l i n volume to volume r a t i o s indicated below. Each pot of infested s o i l was seeded with 5 surface s t e r i l i z e d bean seeds. Five holes, 2 to 3 cm deep, were made i n the surface of the s o i l of each pot and one seed was placed into each hole. Rhizobium inoculum at 0, IO 4, 10 6 or 10 8 c e l l s / m l was prepared as described previously from YMA stock cultures. Each seed was treated with 1 ml of Rhizobium inoculum while controls were treated with 1 ml of s t e r i l e , d i s t i l l e d water. The s o i l was pushed back into the holes to cover the seeds. Each of the 16 t r e a t -ments was r e p l i c a t e d 4 times and the 64 pots were placed i n a completely randomized design on a bench i n the greenhouse for 4 weeks. Pots were i n d i v i d u a l l y watered with ordinary tapwater as required. Temperatures i n the greenhouse ranged from 18 to 24°C, Unless otherwise s p e c i f i e d supplemental l i g h t was provided by cool-white fluorescent tubes providing 2,800 l x for 12 hr/day. At the end of the experiment the plants were c a r e f u l l y l i f t e d from the s o i l and the roots passed through 5 ri n s e s with tapwater. The number of plants that emerged per pot was recorded and each plant was assigned a root disease index, nodulation index and root and shoot dry weights were determined. S t a t i s t i c a l analysis was based on an average per pot value of the disease index, root nodulation index and dry weights. A m u l t i f a c t o r i a l analysis of variance was performed on the plant emergence data. In several treatments plants did not emerge which resulted i n missing data. The computer program BMD10V was used to generate dummy va r i a b l e s for the analysis of variance, Duncan's multiple range t e s t and contrasts (Dixon and Brown, 1977). - 27 -Rhizobium i s o l a t e RCC106 was tested f o r antagonism to FS911 root rot of bean i n pasteurized s o i l as described above. Inoculum of FS911 was used a f t e r a 3 week incubation period. The volume:volume r a t i o s of FS911 to pasteurized s o i l were 0, 1:10**, 1:103 and 1:102. Control pots contained a standardized volume of s t e r i l i z e d vermieulite. A s i m i l a r experiment was performed with RCC106 except that FS911 inoculum was used a f t e r a 2 week incubation period. The r a t i o s of FS911 inoculum to pasteurized s o i l were 0, 1:120, 1:60, and 1:30. Supplemental l i g h t was not provided i n t h i s experiment. The f i n a l s o i l test involved RCC816 using the same condition described f o r the second test except that supplemental l i g h t from fluorescent tubes was provided. - 28 -RESULTS I. ISOLATION AND IDENTIFICATION OF PATHOGENIC FUNGI A t o t a l of 151 fungal i s o l a t e s was recovered and i d e n t i f i e d from bean roots c o l l e c t e d during the f i e l d survey. P o t e n t i a l l y pathogenic species of Fusarium, Pythium and Th i e l a v i o p s i s were c o l l e c t e d but Rhizoctonia s o l a n i was not recovered (Table 7). Most of the fungal i s o l a t e s (144/151) were i d e n t i f i e d as species of Fusarium although only F_. oxysporum (FO) and F. sola n i (FS) were considered p o t e n t i a l l y important root pathogens of snap bean. Preliminary pathogenicity tests demonstra-ted that most of the 17. oxysporum and F. so l a n i i s o l a t e s tested were a v i r u -lent (F02, F03, F04, F05, F06, F07, F08, FS1, FS4, FS5, FS6, FS9, FS10) and only FS2, FS3 and FS8 i n c i t e d a mild degree of bean root r o t . Authenticated i s o l a t e s of F. sola n i (FS911, FSIV) and R. sola n i (RSI, RS2, RS3) were pathogenic to bean except RS3. Table 7. Fungi i s o l a t e d from snap bean roots from commercial f i e l d s i n the Fraser Valley, B r i t i s h Columbia. Species Number of Isolations Fusarium moniliforme (FM) 35 F. oxysporum (FO) 100 F. roseum (FR) 3 F. s o l a n i (FS) 6 Pythium spp. (PY) 2 Thi e l a v i o p s i s (TH) 5 - 29 -Younger seedings were more susceptible than older seedlings to FS2 root rot (Table 8). The r a d i c l e stage was the most susceptible stage while plants inoculated at the seed or crook stage developed s i g n i f i c a n t l y less root r o t . Plants inoculated at the f i r s t , true-leaf stage were not s i g n i f i c a n t l y d i f f e r e n t from controls. Because FS2 was Table 8. Root disease index and shoot and root dry weights of 'Topcrop' snap bean grown i n growth pouches and inoculated at four d i f f e r e n t stages with Fusarium  s o l a n i 2 at 10 7 spores/pouch Growth stage Root Disease Shoot dry Root dry inoculated index* weight 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 F i r s t , t rue-leaf 1.2 ab 0. .339 a 0. .106 a Uninoculated 0.2 a 0. .342 a 0. ,104 a *Root rot severity was based on an equal increment disease index from 0, healthy roots, to 9, completely rotted, dead roots. **Duncan's multiple range t e s t : values followed 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 value 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 high inoculum concentration (10 7 spores/pouch) only moderate to low disease indices were recorded. Shoot and root dry weights were not s i g n i f i c a n t l y d i f f e r e n t i n any treatment. S i m i l a r l y , bean plants inoculated at the f i r s t four growth stages with highly v i r u l e n t FS911 and s i g n i f i c a n t l y greater root disease indices than the f i r s t , t r i f o l i a t e - l e a f stage and controls (Table 9). Fusarium root rot - 30 -was characterized by bright red streaks of i n d e f i n i t e s i z e and margin extending down the hypocotyl and taproot. These lesions became dark brown and necr o t i c , l a t e r a l roots died back and within 3 weeks the ent i r e root system was dead (Fig. 2). Shoot and root dry weights of seedlings inoculated at 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 less than other treatments. Table 9. Root disease index and shoot and root dry weights of 'Topcrop' snap bean grown i n growth pouches and inoculated at f i v e d i f f e r e n t stages with Fusarium  sola n i 911 at 10 6 spores/pouch Growth stage Root disease Shoot dry Root dry inoculated index* weight 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 F i r s t , true-leaf 7.7 c 0.282 b 0.088 b F i r s t , t r i f o l i a t e l eaf 4.9 b 0.261 b 0.088 b Uninoculated 0.0 a 0.330 b 0.091 b *Root rot severity was based on an equal increment disease index from 0, healthy roots, to 9, completely rotted, dead roots. **Duncan's multiple range t e s t : values followed 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 value was an average of 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 (FS911, FSIV) were highly v i r u l e n t to the three bean c u l t i v a r s 'Harvester', 'Stringless Greenpod* and 'Topcrop' but were avi r u l e n t to 'Lincoln' pea (Table 10). A l l three bean c u l t i v a r s were equally susceptible to FS911. 'Stringless Greenpod' was s l i g h t l y less susceptible to FSIV than the other two c u l t i v a r s . - 31 -Figure 2 - Fusarium s o l a n i 911 root rot of 'Topcrop' snap bean grown by a growth pouch technique. - 32 -Table 10. Root.disease index of three snap bean c u l t i v a r s , 'Harvester', 'Stringless Greenpod', 'Topcrop , : and 'Lincoln' pea grown i n growth pouches * Pathogen Root Disease Index** Snap bean Pea 'Harvester' 'S. Greenpod' 'Topcrop' 'Lincoln' Means Fusarium s o l a n i IV 8;8c*** 7.8b 9.0c 0.0a 6.4b F. so l a n i 911 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 *Plants at the seed stage were inoculated with F. so l a n i i s o l a t e s at 10 6 spores/pouch except 'Topcrop' which was inoculated at the seed stage. **Root rot se v e r i t y was based on an equal increment disease index from 0, healthy roots to 9, completely rotted, dead roots. ***Duncan's multiple range t e s t : values followed 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 value was an average of 4 r e p l i c a t e s . Regardless of the inoculum concentration, severe root rot developed when snap bean was inoculated with R. s o l a n i i s o l a t e s (RSI, RS2). RS3 caused l i t t l e to no root rot and was considered a v i r u l e n t to snap bean. Rhizoctonia root rot was characterized by d i s t i n c t , oval to elongate, sunken lesions of d e f i n i t e s i z e (.5 to .75 cm) and margin, appearing on the hypocotyl and taproot. The root became completely g i r d l e d with brown to black s c l e r o t i a of R. so l a n i forming on the surface of decaying root tis s u e ( F i g . 3). The disease index increased as the inoculum concentra-t i o n of RSI or RS2 increased. Bean seedlings inoculated at the f i r s t , t rue-leaf stage with 10 ml of R. so l a n i inoculum developed a moderate degree of root rot while at 1 ml of inoculum only a very s l i g h t degree root rot developed (Table 11). - 33 -Figure 3 - Rhizoctonia s o l a n i (a) root rot and (b) hypocotyl rot of 'Topcrop' snap bean i n growth pouches. - 34 -Table 11. Pathogenicity of two Rhizoctonia s o l a n i i s o l a t e s to 'Topcrop' snap bean i n growth pouches Inoculum concentration (ml/pouch) Root Disease R. so l a n i 1 Index* R. s o l a n i 2 0 0 0 1 3 2 5 3 3 10 6 5 *Root rot severity was based on an equal increment disease.index from 0, healthy roots to 9, completely rotted, dead roots. Each value was an average of 4 r e p l i c a t e s . I I . 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 selected from bean root nodule i s o l a t i o n s resembling Rhizobium according to morphological c r i t e r i a and growth rate on YMA (Table 12). P o t e n t i a l Rhizobium colonies on YMA-congo red plates did not absorb the dye. Isolates were gram negative, motile, small to medium s i z e rod-shaped c e l l s . Isolates did not produce endospores. Unequivocal evidence that the b a c t e r i a l i s o l a t e s were Rhizobium spp. depended on t h e i r a b i l i t y to nodulate snap bean (P. vu l g a r i s 'Topcrop'). However only 40/51 b a c t e r i a l i s o l a t e s i n the c o l l e c -t i o n were capable of nodulating 'Topcrop' snap bean (Table 13). Healthy -appearing, spherical root nodules (2 to 4 mm i n diameter) developed on primary and secondary roots. Authenticated Rhizobium species i n c i t i n g root nodulation of snap bean were R. leguminosarum (RCR1045), R. t r i f o l i i (RCR5) and R. phaseoli (RP1, RP2) but not R. japonicum (RJIA, RJIB), R. leguminosarum (RL1, RL2) or R. l u p i n i (RCR3211). According to Vincent - 35 -(1970), R. phaseoli always nodulates bean while R. leguminosarum, R. l u p i n i and R. t r i f o l i i only very r a r e l y nodulate bean. Table 12. Source of Rhizobium i s o l a t e s from snap bean root nodules* Isolate Number Source 100, 108, 118, 101, 109, 119, 102, 111, 121, 103, 113, 123 105, 114, 219 319, 321, 323, 324, 325, 610, 611, 603, 615, 604, 637, 606, 638, 607, 644 811, 812, 814, 815, 816 18 1 6 12 5 f i e l d 1 f i e l d 2 f i e l d 3 f i e l d 6 f i e l d 8 *Bean plants were c o l l e c t e d from commercial f i e l d s i n the Fraser Valley, B r i t i s h Columbia. Table 13. Nodulating 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 Nodulating Rhizobium i s o l a t e s Non-nodulating Rhizobium i s o l a t e s 100, 101, 102, 105, 106, 107 108, 109, 111, 113, 114, 115 116, 118, 119, 123 319, 321, 324, 326 601, 603, 604, 606, 607, 608, 609 611, 615, 637, 638, 644 811, 812, 814, 816 R. leguminosarum (RCR1045) R. phaseoli (RP1, RP2) R. t r i f o l i i (RCR5) 103, 121 219 323, 325 815 R. leguminosarum (RL1, RL2) R. l u p i n i (RCR3211) R. japonicum (RJIA, RJIB) - 36 -I I I . IN VITRO STUDIES 1. Screening of Survey Isolates i n In.-vitro Preliminary experiments demonstrated that the i n h i b i t i o n of fungal growth hi v i t r o depended on the Rhizobium and fungal i s o l a t e tested. Fusarium species were the most widely i n h i b i t e d by Rhizobium while Pythium and R. sol a n i i s o l a t e s were not i n h i b i t e d by any of the Rhizobium i s o l a t e s tested (Table 14. F i g . 4). Pythium and R. sol a n i i s o l a t e s r a p i d l y colonized the YMA plate i n 2 to 3 days as compared to Fusarium which required at least one week to reach the Rhizobium streak. Because F. so l a n i 2 was one of the more s e n s i t i v e i s o l a t e s to Rhizobium i t was selected f o r agar plate screening t e s t s . With the exception of RCC816, a l l RCC i s o l a t e s showed some degree of antagonism towards FS2 i n v i t r o (Table 15) . I n h i b i t i o n zones of 17 Rhizobium i s o l a t e s , rated + + +, were measured 1 week a f t e r the zones had formed. Zone width varied from 0.14 to 0.59 cm (Table 16). The l e v e l of i n h i b i t o r y a c t i v i t y towards FS2 did not depend on the nodulating a b i l i t y of the Rhizobium i s o l a t e . R. leguminosarum (RCR1045), which very r a r e l y nodulates bean, rated + + + i n agar plate t e s t . Non-nodulating i s o l a t e s (RCC103, 121, 219, 323, 325, RL1, RL2) also showed varying degrees of antifungal a c t i v i t y towards FS2 i n v i t r o . The agar plate screening test was repeated on 3 separate occasions with e s s e n t i a l l y the same r e s u l t s . Seven Rhizobium i s o l a t e s (RL1, RCC603, 219, 644, 814, 121 and 321) i n h i b i t o r y to F. s o l a n i (FS2, FS911) had no e f f e c t on the growth of R. s o l a n i (RSI, RS2, RS3) i n agar plate tests (Table 17). FS2 was more widely i n h i b i t e d by Rhizobium than FS911 i n t h i s experiment. When the experiment was repeated the r e s u l t s were the same. - 37 -Table 14. The e f f e c t of Rhizobium i s o l a t e s * on fungal growth as determined by dual culture agar plate tests Rhizobium Fungal i s o l a t e s * * i s o l a t e s Inhibited Not i n h i b i t e d 100 FMl, FS3 FOl, F02, FR1, FS1, FS2, PSl, RSI, PS2, RS2 PS3, PU1, PU2, 101 FS2, FS3, FS4, FS5 FOl, RSI F02, F03, F04, F05, 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, 115 FS6 RSI *Results were based on 2 r e p l i c a t e s . **FM, Fusarium moniliforme; FO, F. oxysporum; FR, F. roseum; FS, F. s o l a n i ; PS, Pythium sylvaticum; PU, P. ultimum; RS, Rhizoctonia s o l a n i . Table 15. Level of 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 dual culture agar plate tests with Fusarium so l a n i Level of i n h i b i t i o n * Rhizobium i s o l a t e * * - 816, RCR3211, RJIA, RJIB, RP1, RP2 + 102, 103, 113, 114, 601, 603, 606, 608, RCR5, RLl, RL2 119, 123, 324, 609, 611, 615, + + 101, 105, 108, 219, 638, 644, 811, 814 325, 604, 637, + + + 100, 106, 107, 109, 118, 121, 319, 321, 812, 815, RCR1045 111, 115, 116, 323, 326, 607, *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 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 ; ++, cl e a r zone of i n h i b i t i o n , colonized 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 persisted at l e a s t one week. Levels 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. phaseoli. - 38 -F i gu re 4 - L e v e l of i n h i b i t i o n i n dua l c u l t u r e agar p l a t e 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) R h i z o c t o n i a s o l a n i 1 not i n h i b i t e d (-) by RCC326. - 39 -Table 16. I n h i b i t i o n zones rated + + + i n dual culture agar pl a t e t e s t s of Rhizobium and Fusarium s o l a n i 2* Rhizobium Average width Rhizobium Average width i s o l a t e * of zone (cm) i s o l a t e of zone (cm) 319 0.14 107 0.23 RCR1045 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 *Zones were measured 1 week a f t e r i n i t i a l formation. **Average of 2 r e p l i c a t e s , a l l others included 3 r e p l i c a t e s . Table : 17. 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 Rhizoctonia s o l a n i (RSI, RS2, RS3) i n dual culture . agar plate t e s t s . ' ' Fungal Rhizobium i s o l a t e i s o l a t e 121 321 644 814 603 219 RL2* FS2 + + +** + + + ++ ++ + + + + FS911 + + + + + + + + + + + + -RSI _ _ _ _ _ -RS2 - -RS3 - - - - - -*R. 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 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 ; + +, clear zone of i n h i b i t i o n , colonized i n 3 to 5 days; + + +, clear zone of i n h i b i t i o n persisted at le a s t one week. Levels 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 . - 40 -2. Basis of the In V i t r o I n h i b i t i o n In v i t r o experiments did not indicate the mechanism(s) responsible f o r fungal i n h i b i t i o n i n dual culture agar plate t e s t s . Measurements of the pH of the i n h i b i t i o n zone, the fungal colony and the opposite side of the Rhizobium streak did not show any large differences (Table 18). In v i t r o antagonism between Rhizobium and Fusarium was not considered to be due to a pH change of the media. No i n h i b i t i o n zones developed between cut out segments of the i n h i b i t i o n zone placed opposite inoculum discs of FS2 or placed onto YMA plates seeded with Rhizobium or FS2. C e l l - f r e e extracts (RCC100, 106, 107, 116, 121, 607, or 811) and concentrated c e l l -free extracts (RCC607) or Rhizobium did not i n h i b i t the growth of FS2 or FS911. The existence of an i n h i b i t o r y substance produced by Rhizobium and i n h i b i t o r y to Fusarium i n v i t r o was not substantiated. IV. IN VIVO STUDIES 1. •Screening of Rhizobium i n Growth Pouches A s i g n i f i c a n t reduction i n the root disease index occurred when 'Topcrop' bean seedlings were inoculated with RCC 106 at 10 6 cells/pouch and the concentration of the pathogen, FS911, was 10"* spores/pouch (Table 19). RCC 106 caused s i g n i f i c a n t root nodulation which was then reduced at high concentration of FS911 (10 6 spores/pouch). Root and shoot dry weights were reduced by FS911 at IO 4 and 10 6 spores/pouch as compared to uninoculated controls of plants treated with RCC 106. This experiment was repeated once and the same r e s u l t s were observed. Similar growth pouch experiments with nodulating Rhizobium i s o l a t e s 106, 321, 607, 324 and 816 (Fig. 5) rated + + + , + + + , + + +, + and - i n dual culture agar plate t e s t s , also showed a s i g n i f i c a n t reduction i n FS911 - 41 -Table 18. Hydrogen imconcentration of d i f f e r e n t areas of dual culture agar plate tests inoculated v i t h Fusarium s o l a n i 2 and Rhizobium* Rhizobium i s o l a t e Level of i n v i t r o pH of i n h i b i t i o n * * I n h i b i t i o n zone Opposite side of Rhizobial streak FS2 100 + + + 6.1 5.2 6.5 106 + + + 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 6.8 319 + + + 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 . + + 6.5 6.2 6.8 102 + 5.9 5.9 5.9 103 + 6.5 6.2 6.5 113 + 5.6 5.3 6.5 114 + 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 - 6.8 606 + 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 RCR3211*** - 7.1 7.1 7.1 *Values based on 1 re 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 recognized: no fungal 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 clear zone of i n h i b i t i o n , colonized a f t e r 3-5 days; + + + a clear zone of i n h i b i t i o n persisted at least 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 -root rot of bean. Results are summarized i n Table 20 and data of i n d i -v i d u a l experiments are located i n Appendix 1. The a b i l i t y to i n h i b i t FS911 root r ot i n vivo did not depend on the i n h i b i t o r y a c t i v i t y recorded for the Rhizobium i s o l a t e i n v i t r o . Only one Rhizobium i s o l a t e , RCC 812 did not protect beans from FS911 root rot i n th i s set of experiments although i t rated + + + i n duo culture agar plate t e s t . Another seri e s of screening experiments demonstrated a s i g n i f i c a n t reduction i n the severity of FS911 root r ot when bean plants were inoculated with nodulating Rhizobium i s o l a t e s , 107 and 109. At the lowest concentration of FS911 (10 2 spores/pouch), inoculation with RCC 107 at 10 6 cells/pouch or RCC 109 at 10k and 10 6 cells/pouch s i g n i f i c a n t l y reduced root r o t (Table 21). At the highest concentration of FS911 (10 6 spores/pouch) only an equally high l e v e l of RCC 107 (10 e c e l l s / pouch) reduced root r o t . The nodulating a b i l i t y of the Rhizobium i s o l a t e could be a pre r e q u i s i t e to i n h i b i t o r y a c t i v i t y and suppression of root rot..Rhizobium -inoculation tended to increase the root and shoot dry weights while inoculation with the pathogen, FS911, had the opposite e f f e c t . A reduction i n FS911 root rot was not found when bean plants were inoculated with 11 other Rhizobium test i s o l a t e s . These r e s u l t s are summarized i n Table 22 and the s i g n i f i c a n t data of i n d i v i d u a l experiments are located i n Appendix 1. Pathogenicity tests demonstrated that R. s o l a n i 1 and 2 were highly v i r u l e n t pathogens of bean while R. s o l a n i 3 was av i r u l e n t to bean. Nodulating Rhizobium i s o l a t e s 107, 324, and 607 did not reduce RS2 root r o t . A s l i g h t reduction i n RSI root r ot was recorded when bean plants were inoculated with a high concentration (10 8 cells/pouch) of RCC 107. RSI was less v i r u l e n t to bean than RS2. Data of th i s experiment are also located i n Appendix 1. - 43 -Table 19. 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 F_. s o l a n i 911 Rhizobium 106 (cells/pouch)  (spores/ml) 0 10 2 10 ^  10 6 Means Root disease index* 0 10 2 10* 10 6 Means 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 Nodulation index*** 0 10 2 10" 10 6 Means 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 3.0d 2.0bcd 2.5cd l.Oabc 2.1c 1.1a 1.0a 0.8a 0.8a Root dry weight (g) 0 10 2 10 k 10 6 Means .101b .073ab .030a .025a ,057a .064ab .047ab .059ab ,075ab' ,061a .058ab ,069ab ,056ab ,057ab ,060a .087ab .085ab .071ab .070ab .078a • 078a .068a .054a .057a Shoot dry weight (g) 0 10 2 10 * 10 6 Means ,367b ,285ab ,102a ,101a ,214a •221ab .146ab .253ab .255ab .219a .193ab .280ab .195ab .259ab • 232a .376b .340ab .280ab .203ab .300a .289a .263a .207a .204a *Each value was an average of four r e p l i c a t e s . Root rot sev e r i t y was based on an equal increment disease index from 0, no root rot to 9, completely rotted, dead roots. **Duncan's multiple range t e s t : values followed 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. **Nodulation index was based on a scale of 0, no root nodules to 3, abundant nodules. - 44 -Figure 5 - (a) Fusarium so l a n i 911 (IO1* spores/pouch) root rot 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 (10 6 cells/pouch). - 45 -Table 20. Level of antagonism of nodulating Rhizobium i s o l a t e s to Fusarium s o l a n i (FS2, FS911) i n dual culture agar plate tests and growth pouches of 'Topcrop' snap bean -Rhizobium i s o l a t e In v i t r o i n h i b i t i o n of FS2* In vivo i n h i b i t i o n .': Combination at which :'. i n h i b i t i o n of FS911 root r ot was observed 816 - + R2,3 ,V.P3** 324 + + R2,4:P2 106 + + + + Rif :P3 321 + + + + R2:P2, R i f . P i t 607 + + + + R<t:P2 812 + + + -106*** + + + + R 3 . P 3 , 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 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 ; + +, clear zone of i n h i b i t i o n , colonized i n 3 to 5 days; + + +, clear zone of i n h i b i t i o n persisted at le a s t 1 week. **Four inoculum concentrations of Rhizobium were tested: Ri = 0; R 2 = 10 2; R3 = 101*; Ri+ = 10 6 cells/pouch. Four inoculum concentra-tions of the pathogen, FS911 were tested: Pi = 0; P 2 = 10 2; P 3 = 10 4; Pit = 10 6 spores/pouch. I n h i b i t i o n of FS911 root r ot was observed at the inoculum l e v e l s indicated. ***Four inoculum concentrations of Rhizobium were tested: Ri = 0; R2 = 10 5; R3 = 10 6; R.+ = 10 7 cells/pouch. Three pathogen treatments were tested; FS911 at.10 6 cells/pouch and three l e v e l s of Rhizoctonia  s o l a n i 1. - Pi = 0';" P 2 = FS911 + 2 ml RSI; P 3 = FS911 + 4 ml RSI, I n h i b i t i o n of root rot was observed at the inoculum l e v e l s indicated. - 46 -Table 21. Interactions of Rhizobium i s o l a t e s and Fusarium  s o l a n i 911, on root and nodulation indices and dry weight of snap bean i n growth pouches Rhizobium Isolates (cells/pouch) F. s o l a n i 911 107 109 (spores/pouch) 0 10* 10 b 10* 10° Means Root disease index* 0 0.3a** 0.0a ,0.3a 2.0ab 0.0a 0.5a 10 2 3.5b 1.3ab 0.5a 0.3a 0.0a 1.1a 10 * 8.3c 2.5ab 7.3c 8.5c 6.3c 6.6b Means 4.0c 1.3a 2.7abc 3.6bc 2. lab Nodulation index*** 0 0.0a 2. 3de 2.5de 0.3ab 0.3ab 1.1a 10 2 0.0a 1.3abcd 1.8cde 0.3ab 1.5bcd 1.0a 10" 0.0a 3.0e 0.3ab 0.0a 0.8abc 0.8a Means 0.0a 2.2d 1.5cd 0.2ab 0.8bc Root dry weight (s) 0 .083bcd .074abcd .085bcd .043abc .058abcd .069a 10 2 • 024a .045abc .069abcd .094cd .112d .069a 10" .057abcd .096cd .031ab .062abcd .087bcd .067a Means .054a .072a .062a .066a .085a Shoot dry weight (s) 0 .326bcd •297abcd .353cd .189abc .202abc .273a 10 2 • 081a .169abc .277abcd ,370cd .45 Id .279a 10 * •218abcd .372cd .115ab .242abcd .361cd .261a Means .208a . 2 7;9ab .248ab .267ab .338b *Each value was an average of 4 r e p l i c a t e s . Root rot sev e r i t y was based on an equal increment disease index from 0, no root rot to 9, completely rotted, dead roots. **Duncan's multiple range t e s t : values followed 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. ***Nodulation index was based on a scale of 0, no root nodules, to 3, abundant nodules. - 47 -Table 22. Nodulating 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 dual culture agar p l a t e tests and i n growth pouches 'Topcrop' snap bean Rhizobium i s o l a t e Nodulating a b i l i t y In v i t r o i n h i b i t i o n FS2* In vivo i n h i b i t i o n 107 + + + + + 109 + + + + + 812 + + + + + 323 - + + + -111 + + + + -115 + + + + -116 + + + + -118 + + + + -319 + + + + -321 + + + + -815 - + + + -816 + - -R L l * * * - + -RCR1045*** + + + + -Combination at which i n h i b i t i o n of FS911 root rot was observed** R 3 :P 2, R 2 $ R-2 > 3 '^2 Ro :Po *Four l e v e l s of i n v i t r o 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 ; + +, clear i n h i b i t i o n zones, colonized a f t e r 3 to 5 days; + + +, clear zones of i n h i b i t i o n p e r s i s t e d at least 1 week. **Three inoculum concentrations of Rhizobium were tested: Ri = 0, R 2 = 10k, R 3 = 10 6 cells/pouch. Three inoculum concentrations of FS911 were tested: P x = 0, P 2 = 10 2, P 3 f= I O 4 spores/pouch. I n h i b i t i o n of FS911 root rot was observed at the inoculum l e v e l s indicated. ***Rhizobium leguminosarum. - 48 -2. S o i l Tests The root disease index of 'Topcrop' snap bean, grown i n pots of FS911 infested s o i l , was s i g n i f i c a n t l y reduced by RCC106 at 10 8 c e l l s / seed (Table 23). The best protection against FS911 root rot occurred at the lowest inoculum p o t e n t i a l of FS911 (inoculum: s o i l , 1:10^ of 1:120) at the highest RCC106 inoculum leve 1 (10 8 c e l l s / s e e d ) . ( F i g . 6). These r e s u l t s supported previous r e s u l t s of growth pouch experiments. By comparison, RCC816 had no apparent e f f e c t on FS911 root rot of snap grown i n s o i l . Data of i n d i v i d u a l s o i l tests are located i n Appendix 1. Inoculation of 'Topcrop' bean seed with RCC106 gave s i g n i f i c a n t l y greater nodulation than uninoculated controls. Control plants developed root nodules i n the absence of Rhizobium i n o c u l a t i o n . The presence of FS911 did not a f f e c t the degree of root nodulation, seedling emergence or root dry weight but did tend to lower the shoot dry weight. Inoculation with RCC106 or RCC816 tended to increase the shoot dry weight but did not have any s i g n i f i c a n t e f f e c t on seedling emergence or root dry weight. Table 23. The l e v e l of antagonism of nodulating Rhizobium i s o l a t e s to Fusarium solan i (FS2, FS91) i n dual agar plate tests and i n growth pouches of 'Topcrop' snap bean Rhizobium i s o l a t e In v i t r o i n h i b i t i o n of FS2* Inh i b i t i o n of FS911 root rot i n growth pouches Combination at which i n h i b i t i o n of FS911 root rot was observed i n growth pouches** I n h i b i t i o n FS911 root i n s o i l of rot Combination at which i n h i b i t i o n of FS911 root rot was observed i n s o i l * * * 106 ++4 + R„:P 3 R„:P 2 (1:10'*)**** 106 +++ + R<.:P3 + R.,:P2 (1:120)***** 816 - R2, R3, R-,:P3 -*Four le v e l s of i n v i t r o fungal i n h i b i t i o n due to Rhizobium 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 ; + +, clear zone of i n h i b i t i o n , colonized 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 persisted at least 1 week. Levels of i n h i b i t i o n were averages of three re p l i c a t e s . **Four inoculum concentrations of Rhizobium were tested: Ri = 0; R 2 = 10 2; R 3 = IO1*; Ri, = 10 6 cells/pouch. Four inoculum concentrations of the pathogen, FS911, were tested: Pi = 0; P 2 = 10 2; P3 = IO"*; P"t = 10 6 spores/pouch. ***Four le v e l s of Rhizobium were tested: Ri = 0; R 2 = IO1*; R3 = 10 6; Ri, = 10 8 cells/seed. ****Four le v e l s of the pathogen, FS911, inoculum to s o i l (volume:volume) were tested: P i = 0; P 2 = 1:10 ; P 3 = 1:103; P* = 10 2. *****Four le v e l s of the pathogen, FS911, inoculum to s o i l (volume:volume) were tested: Pi = 0; P 2 = 1:120; P 3 = 1:60; P„ = 1:30. - 50 -Figure 6 - (a) Fusarium s o l a n i 911 ( P 2 = inoculum:soil, 1:10H) root rot of 'Topcrop' snap bean, grown i n pasteurized s o i l , (b) suppressed by in o c u l a t i o n with Rhizobium, i s o l a t e RCC106 (R 4 = 10 8 cells/seed) DISCUSSION The antagonistic e f f e c t of Rhizobium to some, but not a l l of the root pathogens of snap bean was demonstrated f o r the f i r s t time i n t h i s work. I n h i b i t i o n was observed between Rhizobium and Fusarium i n dual culture agar plate t e s t s . 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 on the combination of Rhizobium and fungal i s o l a t e s tested. Rhizobium . i s o l a t e s i n h i b i t e d F. moniliforme, F_. oxysporum, F. roseum and F_. so l a n i but did not i n h i b i t Rhizoctonia s o l a n i or Pythium i s o l a t e s . R. so l a n i and Pythium i s o l a t e s r a p i d l y colonized the agar plate i n 2 to 3 days while Fusarium i s o l a t e s required at l e a s t 7 days to reach the Rhizobium streak. Growth rate of the test fungus could be a factor i n determining the l e v e l of i n v i t r o i n h i b i t i o n . Drapeau et al_. (1973) observed the same phenomenon and suggested that the rapid growth rate of R. so l a n i and Pythium may not have allowed s u f f i c i e n t time for Rhizobium to act. The majority of indigenous Rhizobium i s o l a t e s selected from naturally-formed bean root nodules exhibited some degree of i n h i b i t o r y a c t i v i t y to the s e n s i t i v e i s o l a t e , F. Solani 2. A high l e v e l of anta-g o n i s t i c a c t i v i t y was recorded i n 38% of the Rhizobium i s o l a t e s ; a moderate l e v e l of antagonism was recorded i n 26%; a low l e v e l of anta-gonism was recorded i n 33% and only one i s o l a t e , RCC 816, showed no apparent antagonistic a c t i v i t y towards FS2 (Table 15). Further screening of indigenous s o i l m i c roflora could lead to the discovery of a superior, antagonistic Rhizobium i s o l a t e which could be used to control root r o t . Authenticated Rhizobium species (R. japonicum, R. leguminosarum, R. l u p i n i , R. phaseoli and R. t r i f o l i i ) were either very poor antagonists - 52 -or had no i n h i b i t o r y e f f e c t on FS2. These Rhizobium i s o l a t e s (RCR5, RCR3211, RJIA, RJIB, RLl, RL2, RP1, RP2) had been i n culture f o r a longer period of time than the i s o l a t e s from bean root nodules which might explain t h e i r lack of i n h i b i t o r y a c t i v i t y i n v i t r o . With the exception of R. leguminosarum (RCR1045) only RCC i s o l a t e s , t e n t a t i v e l y i d e n t i f i e d as R. phaseoli, from bean root nodules possessed a high 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 reported that i n h i b i t i o n of F. s o l a n i f . sp. p i s i i n agar plates depended on the s t r a i n of R. leguminosarum tested. The l e v e l of i n v i t r o i n h i b i t i o n was also a function of the agar plate technique u t i l i z e d . The dual culture agar plate technique was adopted as a f a s t and d i r e c t method of screening Rhizobium i s o l a t e s f o r antagonism to bean root pathogens. Wide zones of i n h i b i t i o n formed between Rhizobium and Fusarium. These zones often p e r s i s t e d f o r more than 7 days. Preliminary agar plate tests showed that a change i n the agar medium, the distance separating the test organisms and the pre-ino c u l a t i o n period with Rhizobium a l t e r e d the degree of fungal i n h i b i t i o n 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 to F_. so 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. The l e v e l of root r o t i n h i b i t i o n depended upon the concentration of both Rhizobium and the pathogen, FS911. Generally, root rot was suppressed by high concentrations of Rhizobium (IO 4, 10 6 cells/pouch) to low concen-tr a t i o n s of pathogen, FS911 (10 2, 10** spores/pouch). Isolate RCC324, at 10 6 cells/pouch, was the only i s o l a t e e f f e c t i v e against a high l e v e l of FS911 (10 6 spores/pouch). Inoculum l e v e l s of Rhizobium and the pathogen were higher 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 inoculate the seed with a high concentration of Rhizobium i n order to e s t a b l i s h the antagonist i n the s o i l . The pouch method of growing bean plants i s an a r t i f i c i a l system but had several advantages: 1) a large number of Rhizobium i s o l a t e s could be screened for antifungal .. a c t i v i t y ; 2) roots could be inoculated without mechanical in j u r y ; 3) and the development of root r o t could be d i r e c t l y observed without the complexity and interference of s o i l f a c t o r s . A s i g n i f i c a n t reduction i n Fusarium root rot of bean was obtained with 8/17 nodulating Rhizobium i s o l a t e s (RCC 106, 107, 109, 321, 324, 607, 812, 816) tested i n growth pouches. Nodulation of bean roots could be a p r e r e q u i s i t e f o r antagonism to root rot 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 root rot i n growth pouches although they rated+ + +,+++, and + i n dual culture agar plate t e s t s . RCC324 (rated +) and RCC816 (rated -) i n v i t r o were s t i l l i n h i b i t o r y to Fusarium root rot i n vivo. On the contrary, nodulating and non-nodulating i s o l a t e s rated +++ i n v i t r o (RCC111, 115, 116, 118, 319, 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 tested ^n vivo. In v i t r o i n h i b i t o r y a c t i v i t y was not a r e l i a b l e i n d i c a t o r of i n vivo a c t i v i t y . The v a l i d i t y of conclusions based s o l e l y on the r e s u l t s of agar plate tests has been questioned (Huber and Watson, 1966). Gray and Sackston (1980) s i m i l a r l y tested s t r a i n s of R. leguminosarum against _F. s o l a n i f. sp. p i s i and did not 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 i n v ivo r e s u l t s . Variable r e s u l t s were obtained with one Rhizobium i s o l a t e , RCC816, which reduced Fusarium root rot when bean plants were grown i n growth pouches i n the greenhouse but not when plants were grown i n an environ-- 54 -mental control chamber. Subtle differences i n temperature, water p o t e n t i a l , pH, l i g h t i n t e n s i t y or some other fa c t o r ( s ) might account f o r t h i s d i s -crepancy i n r e s u l t s . These factors and t h e i r impact on the Rhizobium -root pathogen i n t e r a c t i o n are by no means completely understood. Further experiments would be necessary to define the environmental parameters optimal to Fusarium root rot i n h i b i t i o n by Rhizobium. S o i l experiments, performed i n the greenhouse, supported the findings of growth pouch experiments. A s i g n i f i c a n t reduction i n the root disease index occurred with the highest inoculum p o t e n t i a l of RCC106 (10 8 cells/seed) only at the lowest inoculum p o t e n t i a l of FS911 (inoculum: s o i l , l ^ O 4 or 1:120). RCC106 caused s i g n i f i c a n t l y greater root.nodu-l a t i o n than untreated controls. Nodulation of untreated control plants could have been due to indigenous r h i z o b i a that survived on the seed coat or i n the s o i l . By comparison, RCG816 had no apparent i n h i b i t o r y e f f e c t on Fusarium root rot i n s o i l . This supported ^n v i t r o evidence where RCC816 rated - i n dual culture agar plate t e s t s . Growth pouch experiments showed that RCC816 was not a r e l i a b l e antagonist to Fusarium root r o t . Other reports i n the l i t e r a t u r e have also given evidence of a reduction i n legume root rot by r h i z o b i a l i noculation (Chi and Hansen, 1961. Mew and Howard, 1969;, Chou and Schmitthenner, 1974; Tu, 1978a,b, 1980), Tu s i m i l a r l y found that at a given concentration of the pathogen, higher concentrations of Rhizobium were more e f f e c t i v e i n reducing root rot 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 root rot pathogens of bean varied i n t h e i r s e n s i t i v i t y to Rhizobium. The reduction of Rhizoctonia root rot was not as great as the reduction of Fusarium root r o t . Only a s l i g h t reduction i n RSI root rot of bean plants i n growth pouches occurred with RCC107 although - 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 root rot (RCC324, 607) were not e f f e c t i v e i n reducing Rhizoctonia root r o t . The present work has confirmed e a r l i e r work with root rots of other legumes where only some of the diseases were reduced by Rhizobium (Mew and Howard, 1969; Chow and Schmitthenner, 1974 Tu, 1980). Timing of Rhizobium i n o c u l a t i o n was also a f a c t o r i n determining the l e v e l of root rot reduction by Rhizobium. The severity of root rot i n c i t e d by a combination of Fusarium and Rhizoctonia was reduced when bean plants were inoculated with RCC106 2 days before the pathogens were added to the pouches. Fusarium root rot was reduced when bean plants, i n growth pouches, were inoculated at the crook stage with RCC812. However, when bean plants were inoculated at the f i r s t , true-leaf stage with RCC812 no reduction i n root rot occurred. The establishment of Rhizobium i n the rhizosphere of young seedling or p r i o r to i n o c u l a t i o n with the pathogen provided better protection against severe root r o t . Tu (1980) found that s i g n i f i c a n t l y less root rot developed when a l f a l f a plants were inoculated simultaneously with Rhizobium and F. oxysporum or _P. megasperma rather than sequentially with Rhizobium being added several weeks a f t e r the pathogen. Unfortunately, preinoculation of legume roots with Rhizobium to e s t a b l i s h the antagonist i n the rhizosphere i s not possible i n the f i e l d s i t u a t i o n where inoculum of the pathogen may reside adjacent to the seed. The basis for fungal i n h i b i t i o n observed i n v i t r o was not deter-mined. Experiments demonstrated that i n h i b i t i o n was not due to a change i n the pH of the agar media. Involvement of an i n h i b i t o r y substance as suggested by Drapeau tit al_. (1973) or some type of n u t r i t i o n a l pheno-menon as suggested by Antoun et a l . (1978b) that could account for fungal - 56 -i n h i b i t i o n was not substantiated i n t h i s t h e s i s . Mechanisms proposed for i n v i t r o i n h i b i t i o n might also explain the i n h i b i t i o n of root rot observed i n vivo but they could involve d i f f e r e n t mechanisms because there was not a perfect c o r r e l a t i o n between i n v i t r o and i n vivo r e s u l t s . Suppression of Fusarium root rot by Rhizobium could be the r e s u l t of antagonism ( a n t i b i o s i s , mycoparasitism, l y s i s ) or competition. Further studies are required to determine which of these mechanisms, i f any, i s responsible and involved i n root r o t suppression. Rhizobium could be mycoparasitic causing hyphal necrosis of the pathogen. Fusarium spp. produce germ tubes which invade host plant roots by multiple penetration s i t e s . Rhizobium are s u f f i c i e n t l y numerous i n the s o i l to be important antagonists by intercepting pathogen germlings and reducing root i n f e c t i o n . 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 could prevent contact between the host plant roots and the pathogen. Tu (1978) described r h i z o b i a l parasitism of root rot fungi i n v i t r o which could be the mechanism involved i n root rot suppression i n vivo. Rhizobium colonized the hyphae of JP. megasperma, P_. ultimum, A. imperfecta and F. oxysporum, both i n t e r n a l l y and externally and reduced fungal sporulation. Aseptate fungi (PM, PU) were found to be more susceptible to r h i z o b i a l parasitism than the septate fungi (AI, FO). In vivo greenhouse experiments also performed by Tu (1978) supported these i n v i t r o observations. Results i n t h i s work have shown that Rhizobium caused a s i g n i f i c a n t reduction i n root rot i n c i t e d by the septate fungus F. s o l a n i f. sp, phaseoli. The existence of mycoparasitism i n vivo has not yet been observed. Rhizobium could be s u c c e s s f u l l y competing with root pathogens f o r nutrients and space i n the s o i l and l a t e r within the rhizosphere of the host plant. A high competitive a c t i v i t y of Rhizobium could account for - 57 -the suppression of the pathogen and root rot development. Successful Rhizobium inoculation has been reported to improve the degree of root nodulation, host plant vigour and general resistance to disease (Chi and Hansen, 1961; Yang and Hagedorn, 1966). Environmental factors i n f l u e n -cing the i n t e r a c t i o n between Rhizobium and root pathogens adds yet another unknown dimension of complexity. The influence of temperature, oxygen tension, water p o t e n t i a l , nutrients (root secretions), s o i l type and pH can s e l e c t i v e l y favour the growth of Rhizobium or the root pathogen. 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 with Rhizobium and the p r o v i s i o n of optimal growing conditions can encourage the Rhizobium-1egume symbiosis and p o t e n t i a l l y reduce root r o t . B i o l o g i c a l control of Fusarium root rot by i n o c u l a t i o n of bean with a s u f f i c i e n t concentration of an antagonistic, nodulating Rhizobium i s o l a t e i s f e a s i b l e . Implementation of the control i s f a c i l i t a t e d by the established p r a c t i c e of legume seed in o c u l a t i o n with Rhizobium. Instead 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 the basis of nodulating a b i l i t y , i s o l a t e s could also be screened for t h e i r antagonistic a c t i v i t y to root r o t pathogens. A highly antagonistic, nodulating Rhizobium i s o l a t e could then be grown i n a peat medium and commercial seed inoculated p r i o r to planting i n the f i e l d . The antagonistic Rhizobium i s o l a t e must be able to survive, r a p i d l y multiply and p e r s i s t i n the s o i l i n order to achieve suppression of root r o t . F i e l d studies should now be c a r r i e d out to see i f a high enough concentration of Rhizobium can be applied, by seed i n -oculation, to give i n h i b i t i o n of root rot under the e x i s t i n g inoculum po t e n t i a l of F. s o l a n i i n s o i l . - 58 -SUMMARY AND CONCLUSIONS Nodulating Rhizobium i s o l a t e s were antagonistic to Fusarium  so l a n i i n v i t r o and i n vivo. The degree of antagonism depended on the Rhizobium i s o l a t e and Fusarium i s o l a t e tested and the screening tech-nique u t i l i z e d . Isolates of Rhizoctonia s o l a n i were not s e n s i t i v e to Rhizobium i n v i t r o but a trace of i n h i b i t i o n was observed i n vivo . Rhizobium acted as a b i o l o g i c a l control agent for Fusarium root rot of snap bean. 1. The antagonism between Rhizobium and Fusarium so l a n i observed i n v i t r o was not always observed i n vivo . Results of the dual culture agar plate tests were not always i n d i c a t i v e of the performance of 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 correlated with the re s u l t s of greenhouse s o i l experiments. Growth pouches provided a useful method of screening many Rhizobium i s o l a t e s f o r antagonism to Fusarium root r o t . 3. A l l e v i a t i o n of Fusarium root rot was achieved by inoculating bean roots with a high concentration (IO1*, 10 6, 10 8) of a nodulating Rhizobium i s o l a t e . Increasing the inoculum l e v e l of Rhizobium and decreasing the inoculum l e v e l of the pathogen gave a corresponding decrease i n Fusarium root rot of snap bean. LITERATURE CITED Adams, P.B., J.A. Lewis, and G.C. Papavizas. 1968. Survival of root-infecting fungi i n s o i l . IV. 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Tuite, John. 1969. Plant pathological methods. Fungi and bacteria. Burgess Publishing Company, Minneapolis, Minn. 239 pp. Vance, CP., and L.E.B. Johns on. 1981. Nodulation. A plant disease perspective. Plant Dis., 65: 118-124. Vincent, J.M. 1970. A manual for the p r a c t i c a l study of root-nodule b a c t e r i a . 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. Diseases of vegetable crops. McGraw-Hill, New York. 529 pp. Weaver, R.W., and L.R. Frederick. 1972. A new technique for most-probable-number counts of r h i z o b i a . PI. S o i l , 36: 219-222. Westcott, C. 1971. Plant disease handbook. Van Nostrand, Reinhold Co., 3rd ed. 843 pp. Yang, S., and D.J. Hagedorn. 1966. Root rot of processing bean i n Wisconsin. P i . Dis. Reptr., 50: 578-580. Zaumeyer, W.J., and H.R. Thomas. 1957. A monographic study of bean diseases and methods for t h e i r c o n t r o l . U.S. Dept. Agric. Tech. B u l l . 868, 255 pp. - :64"-APPENDIX I Table 1. E f f e c t of Rhizobium 816 and Fusarium so l a n i 911 on root desease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches J_. s o l a n i 911 Rhizobium?816 (cells/pouch) (spores/pouch) 0 IO 2 10* To5" Means Root disease index* 0 10 2 10* 10 6 Means 0.0a 3.Obcd 7.0e 8.8e 4.7b 0.3a 1.5ab 4.3d 7.3e 3. 3a 0.0a 4.5d 4.0cd 7.5e 4.0ab 0.0a 3.3bcd 1.8abc 8.5e 3.4a 0.1a 3.1b 4.3c 8.0d Nodulation index*** 10 2 10* 10 6 Means 0.0a 0.0a 0.0a 0.0a 0.0a 1.5ab 2.3b 1.5ab 2.0ab 1.8b 2.3b 1.5ab 1.3ab 0.0a 1.3b 2.0ab 2.3b 1.3ab 0.0a 1.4b 1.4b 1.5b l.Oab 0.5a Root dry weight (g) 10" 10* 10 6 Means .083ab .037a .071ab • 034a .056a .082ab .084ab .053a .096ab .078a .097ab .076ab .061a .033a .067a .056a ,091ab .104b .032a • 096a .079a .072a .097a .049a Shoot dry weight (g) 0 .433b .329ab .399ab .267ab .357b 10 2 .226ab .353ab .305ab .399ab .321ab 10* .237ab .254ab .299ab .258ab .262ab 10 6 .109a .423b .170ab .163ab .216a Means .251a .340a .293a .272a *A11 values were averages of four r e p l i c a t e s . Severity of root rot 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 t e s t : values followed 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. ***Nodulation index was based on a scale from 0, no root nodules, tp 3 abundant nodules. - 66 -Table 2. Effect of Rhizobium 324 and Fusarium solani 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean in growth pouches F. solani 911 _ (Spores/pouch) 0 Rhizobium 324 (cells/pouch) 10 10c Means Root disease index* 102 IO* 106 Means 10z 10* 106 Means 0.0a 0.0a 0.0a 0.0a 0.0a 8.3d 2. 8ab 7.5cd 4.3bc 5.7b 5.0bcd 7.5cd 2.3ab 6.3bcd 5.4b 8.3d 8.3d 6.5cd 8,0d 7,8c. 5.4a 4.6a 4.2a 4.6a Nodulation index 0.0a 2.3bc 2.0bc 1. 3abc 1.4a 0.0a 0.8ab 0.8ab 2.8c 1.1a 0.0a 0.0a 2.3bc 0.8ab 0.8a 0.0a 0.5ab 1.3abc 1.5abc 0.8a 0.0a 0.96 1.6b 1.6b Root dry weight (g) 10* 10" 106 Means .lllab .076ab ,085ab . 052ab ,081a .130b ,059ab .044a .072ab .076a .075ab ,049a .091ab ,092ab ,077a .06lab .098ab .071ab ,063ab ,073a .094a .071a .073a .070a 102 10" 106 Means Shoot dry weight (g) ,365ab ,212ab ,299ab ,166a ,260a .415b .164a .119a .230ab . 232a 194ab 140a 315ab 316ab 241a ,187ab ,317ab ,200ab ,204ab ,227a .290a .208a .233a .229a *A11 values were averages of four replicates. Root rot severity was based on an equal increment root disease index from 0, no root rot, to 9, completely rotted, dead roots. **Duncan's multiple range test: values followed by the same letter are not significant, P = 0.05. ***Nodulation index was based on a scale from 0, no root nodules, to 3, abundant nodules. - 67 -Table 3. E f f e c t of Rhizobium 321 and Fusarium so l a n i 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches F. s o l a n i 911 Rhizobium 321 (cells/pouch)  (Spores/pouch) 0 IO 7 10** To* Means Root disease index* 10 z 10* 10 6 Means 0. 0a 3.7bcd 1. 7ab 7,0e. 3.1b 0.0a 0.3a 2.0ab 5,7cde 2,0ab 0.3a 0.7ab 5,3cde 6,3de 3.2b 0.0a 1.7ab l«7ab. 3 ..Oabc 1,6a 0.1a 1.6b 2..7b 5.5c. Nodulation index*** 10 z 10* 10 6 Means 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 0.7ab 0.3ab 0.3ab 0.3ab 0.4ab 0.5a 0.6a 0.2a 0.2a Root dry weight (g) 0 10 2 10* 10 6 Means ,069a ,057a ,059a . 054a ,060a .050a .051a .050a • 061a .053a .066a .063a .073a .063a .066a ,070a ,056a ,046a ,054a ,057a .064a .057a .057a .058a Shoot dry weight (g) 10^ 10* 10 6 Means ,376a ,293a ,345a ,335a ,337a ,267a ,296a ,359a ,274a ,299a .417a .399a • 461a • 305a .395a ,447a ,391a ,256a ,311a ,351a ,377a ,345a ,355a ,306a *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:, values followed 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. ***Nodulation index was based on a scale from 0, no root nodules, to 3, abundant nodules. - 68 -Table 4. E f f e c t of Rhizobium 607 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 F. s o l a n i 911 _ (Spores/pouch) 0 Rhizobium 607 (cells/pouch) pr:-iO^" 10* 10s Means Root disease index* 10 z 10* 10 6 Means 0.0a 5.8c 7.0cde 8.8e 5.4a 0.0a 6.0cd 8.0cde 7.0cde 5.3a 0.0a 6.8cde 8.3de 8.3de 5.8a 0.0a 3.3b 8.0cde 8.8e 5.0a 0.0a 5.4b 7.8c 8.2c Nodulation index 0 10 2 10* 10 6 Means 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 dry weight (g) 10" 10* 10 6 Means ,099a ,108a ,097a ,070a ,093a -067a .076a .063a • 097a .076a .097a • 059a .070a • 068a .073a ,089a .121a .087a .060a .089a .088a .091a .079a .074a Shoot dry weight (g) 10 2 10* 10 6 Means ,341a ,326a ,304a ,107a ,269a .203a • 256a .166a .310a . 234a .269a .162a .137a .222a .197a ,294a .344a ,152a .123a ,228a ,276a ,272a ,190a ,190a *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 t e s t : values not followed 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. ***Nodulation index was based on a scale from o, no root nodules, to 3 abundant nodules. - 6 9 -Table 5. E f f e c t of Rhizobium 812 and Fusarium sol a n i 911 on root disease and nodulation indices and dry weight 'Topcrop' snap bean i n growth pouches F. s o l a n i 911 _ (Spores/pouch) 0 Rhizobium 812 (cells/pouch) 1CV 1(T 10 € Means .Root disease index* i o z 10". i o 6 Means 0.0a** 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 0.0a 2.0ab 7.3def 8.5f 4.4a 0.0a 2,8b 5.6c 7.9d Nodulation index*** 10 z 10* 10 6 Means 1.3abc 0.3ab 0.0a 0.8ab 0.6ab 0.8ab 0.3ab 0.0a 0.0a 0.3a 0.5ab 0.3ab 0.5ab 0.5ab 0.4ab 2.3c 1.8bc 0.5ab 0. 0a 1. lb> 1.2b 0.6ab 0.3a 0.3a Root dry weight (g) 10 z 10* 10 6 Means ,121c ,103abc ,063abc ,039a ,081a .063abc .088abc .040a .069abc .065a .118bc .087abc .088abc .050a .086a ,088abc ,124c ,058ab ,045a .079a .098b .100b ,062a .051a Shoot dry weight (g) 10 z 10* 10 6 Means .452cd .331bcd .199ab • 088a .268a .184ab ,324bcd ,138ab ,236abc .220a .328bcd .300abcd .275abcd .150ab .263a ,330bcd ,471d ,176ab ,114ab ,273a .323b .356b ,197a .147a *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. - 70 -Table 6. 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 FS911 (Spores/pouch) RSI (ml/pouch) Rhizobium 106 (cells/pouch) 10^ 10fc 10' Means Root disease index 0 10 6 10 6 Means 0 2 4, 0.0a 8.0cd 8.6d 5,5b 0.0a 7.4cd 7,6cd 5.0ab 0.0a 7.2cd 7.0bc. 4,7a 0.0a 5.8b 7,6cd 4.5a 0.0a 7.1b 7,7b Nodulation index 0 10 6 10 6 Means 0 2 4 0.0a 0.0a 0.0a 0.0a 1.6cd 0.6abc 0.2ab 0.8b l.Oabcd 0.4abc 1.Oabcd 0.8b 2.2d 1.4bcd 1.4bcd 1.7c 1.2a 0.6a 0.7a 0 10 6 10 6 Means 0 2 4 Root dry weight (g) ,109ab .074ab .087ab .090a .118b .087ab .084ab • 096a .067a .079ab .074ab .073a .102ab .095ab .091ab .096a. .099a .084a ,084a 0 10 6 10 6 Means Shoot dry weight (g) 0 2 4 ,416c ,160a ,176a ,251ab ,450c ,227ab , 206ab ,294ab ,243ab ,213ab ,197a ,218a ,366bc ,369bc ,194a ,310b ,369b ,242a ,193a *A11 values were averages of f i v e 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 an scale from 0, no root nodules, to 3, abundant nodules. - 7 1 -Table 7. Interaction 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 R. s o l a n i Rhizobium i s o l a t e • i s o l a t e (I0 a cells/ml/pouch)  (3 mis/pouch) 0 107 324 603 Means Root disease index** 0 0.0a 0.0a 0.8a 0.0a 0.2a RS3 0.5a 0.0a 0.5a 0.3a 0.3a RSI 7.3cd 5.0b 6.5bc 7,5cd 6.6b RS2 9.0d 8.0cd 8,3cd 8.3cd 8.4c Mean 4.2a 3.3a 4.0a 4.0a Nodulation index 0 0.3ab 2.0cd 2.8d 2.5d 1,9b RS3 0.0a 1.5abcd 1.3abcd 1.8bcd 1.1a RSI 0.0a 1.8cd 2.0cd 1.3abcd 1.3ab RS2 0,0a 1.8bcd 1.5bcd 0.8abc 1.0a Mean 0.1a 1.8b 1.6b 1.9b Root dry weight (g) 0 , 103b ,078ab •084ab ,075ab , 085b RS3 ,061ab .054a ,078ab ,067ab .065al RSI .057ab •066ab .086ab • 049a . 064al RS2 ..051a .052a .066ab •055ab .056 Mean • 068a • 062a .079a .061a Shoot dry weight (g) 0 ,372c . 326abc .347bc .328abc .343b RS3 .247abc .228abc . 331abc ,268abc .268a RSI .203abc . 233abc. .323abc .192ab ,238a RS2 .163a .227abc .250abc ,252abc Mean .246a .253a .260a .313a *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 t e s t : values followed 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. ***Nodulation index was based on a scale from 0, no root nodules, to 3, abundant nodules. - 72 -Table 8. Interaction of Rhizobium i s o l a t e s and Fusarium sol a n i 911 on root disease and nodulation indices and dry weight of 'Topcrop' snap bean i n growth pouches F_. .Solani 911 Rhizobium (cells/pouch) (Spores/pouch) 107 109 10* 7~10B' IO1* 10 b Means Root disease index* 10 2 10* Means 0.0a 4.0abcd 9.0c 4.3a 0.0a 1.8ab 5.Obcde 2.3a 0.3a 3.3abc 7.3cde 3. 6a 0.0a 4. 5abcd Q. 8.3de 4.3a 0.0a 4.0abcd 3 .5abc 2.5a 0.1a 3.5b 6.6c Nodulation index*** 0 10 2 10* Means 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 3.0c 2.3bc 2.3bc 2.5b 0.6a 0.8a 0.5a 0 10 2 10* Means Root dry weight (g) .075ab .063ab .036a .056a .064ab .096ab •073ab .077ab •077ab .068ab .049a .065ab .037a .075ab .032a ,048a .130b .086ab .080ab .099b .076a .077a .054a 0 10 2 10* Means Shoot dry weight (g) ,261ab ,243ab ,098a ,200a ,189ab ,325ab ,254ab ,256ab .241ab .237ab .143ab .207a ,095a ,248ab ,078a .140a ,406b ,326ab .296ab ,343b ,238a ,276a ,174a *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. **Duncans multiple range te s t : values followed 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. **Nodulation index based on a scale from 0, no root nodules, to 3, abundant nodules. - 73 -Table 9. E f f e c t of Rhizob ium 106 and Fusarium so l a n i 911, on root disease and nodulation indices and dry weight of 'Topcrop' snap bean grown i n pasteurized greenhouse soil,. F. s o l a n i 911:soil Rhizobium 106 (cells/pouch) (volume:volume) 0 10" 10 b 10 b Means Root disease index* 0 1.2abc** 0.3ab 0.2ab 0.8a 0.4a 1:10* 5.8d 3.0bcd 3.5bcd 1.7abc 3.5b 1:10 3 4.led 5.5d 4.3cd 1.8abc 3.9bc 1:10 2 5.7d 5.7d 4.3cd 4.3cd 5.0c Means 4.2b 3.6b 3.lab 2.0a Nodulation index*** 0 1.5a 2.7a 2.4a 2.0a 2.1a 1:10* 1.8a 1.9a 1.5a 1.7a 2.4a 1:10 3 1.6a 2.5a 1.8a 3.0a 2.2a 1:10 2 2.3a 2.5a 2.1a 2.7a 1.7a Means 1.8a 2.4a 2.0a 2.3a Root dry weight (g) 0 •188a .243a .193a .157a • 195a 1:10* .221a .214a .136a .181a .188a 1:103 •334a .249a .207a • 215a .251a 1:10 2 .416a .265a .143a .199a .256a Means .290a .243a .170a .188a Shoot dry weight (g) 0 .938a 1.08a .855a .754a .906a 1:10* .770a .944a .680a .840a .924a 1:103 .941a .940a .794a 1.09a .942a 1:10 2 1.01a 1.11a .672a .909a .809a Means ,915a 1.02a .750a • 899a *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 t e s t : values followed 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. ***Nodulation index was based on a scale from 0, no root nodules, to 3, abundant nodules. - 74 -Table 10. 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 weights of 'topcrop' snap bean growth i n pasteurized greenhouse s o i l F. s o l a n i 911: s o i l Rhizobium 106 feel 1 s / s p . p r f ) Means (volume:volume) o 10 * 10 6 10 8 Root disease index* 0 0.5ab** 0.5ab 0.3a 0.0a 0.3a 1:120 5.4 ef 2.7abcde 3. 2abedef 2.3abcd 3.4b 1:60 5.8ef 3.5bcdef 3.6cdef 2. 7abcde 3.9b 1:30 6.2f 3.labedef 3.3abcdef 4.6def 4.3b Means 4.5b 2.4a 2.6a 2.4a Nodulation index*** 0 0.1a l.Oabcd 1.5bcde 1.7bcde 1.1a 1:120 0.5ab 1.3bcde 1.3bcde 2.4de 1.4a 1:60 0.5ab 1.5bcde l. l a b c d 2.6e 1.4a 1:30 0.7abc 1.3abcd 2.3de 1.9cde 1.5a Means .4a 1.3b 1.6b 2.2c Root dry weight 0 0 0 • 095a .111a .147a .146a .125a 1:120 • 129a . 154a .112a .161a .139a 1:60 .105a .172a • 112a • 144a .133a 1:30 .078a • 148a .137a .116a .120a Means .102a .146a .127a .142a Shoot dry weight (8) 0 .374a .600ab .927ab 1.05b .738a 1:120 .518ab .536ab •628ab .772ab .627a 1:60 .461 ab •645ab .777ab .555ab .609a 1:30 .446ab .511ab .651ab .576ab • 546a Means .449a .573ab .759b .738b *Values were averages of four r e p l i c a t e s . Root r o t sev e r i t y was based on an equal increments root disease index from 0, no root rot, to 9, completely rotted, dead roots. **Duncan s multiple range t e s t : values followed by the same tests are not s i g n i f i c a n t , P = 0.05. ***Nodulation index was based on a scale from 0, no root nodules, to 3, abundant nodules. - 75 -Table 11. E f f e c t of Rhizobium 816 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 F. s o l a n i 911:soil (volume:volume) 0 Rhizobium 816 (cells/seed). 10 10' 10 c Means Root disease index* 0 1:120 1:60 1:30 Means 1.2ab** 1.9abc 4.8def 5.8f 3.4a Oabc 6cde 5cde 5def 4a 0. 3. 3. 5. 3. l a lbcd 8cdef 2def l a 0.1a 3.0bcd 5.6ef 5.3ef 3.5a 0.8a 2.9b 4.4c 5.2c 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 1.9a 1.5a 1.6a 1.2a 1.6a 1.4a 1.9a 1.4 a 1.6a 0 1:120 1:60 1:30 Means Root dry weight (g) .121a .129a .133a ,224a ,158a .136a ,180a .265a ,123a ,142a ,131a .101a .167a .171a .123a • 150a .252a ,150a ,128a ,170a .133a .192a .179a ,126a 0 1:120 1:60 1:30 Means Shoot dry weight (g) •647a .588a .745a . 869ab 1.03ab .906ab •871ab .732a .953ab 609a .635a .614a 756a .743a ,804a .945ab 1.74c 1.33bc .501a 1.13b .731a 1.14b .968b .590a *Values were averages of four r e p l i c a t e s . Root r o t 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 t e s t : values followed 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. ***Nodulation index was based on a scale from 0, no root nodules, to 3, abundant nodules. 

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