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Plant/bacteria coadaptation in a grass/legume pasture Chanway, Christopher Peter 1987

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V PLANT/BACTERIA COADAPTATION IN A GRASS/LEGUME PASTURE by CHRISTOPHER PETER CHANWAY B . S c , The U n i v e r s i t y of Winnipeg, 1978 B . S . A g r i c , The U n i v e r s i t y of Manitoba, 1980 M.Sc, The U n i v e r s i t y of B r i t i s h Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of P l a n t Science) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA ©Ch r i s t o p h e r Peter Chanway, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PLANT SCIENCE The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D a t e 22 June, 1987 DE-6(3/81) ABSTRACT The r e l a t i o n s h i p between p l a n t s and rh i z o s p h e r e b a c t e r i a c o l l e c t e d from a 45 year o l d permanent pasture was i n v e s t i g a t e d . S e v e r a l methods of s t r a i n i d e n t i f i c a t i o n w i t h i n Rhizobium trifolii were ev a l u a t e d . S e p a r a t i o n of b a c t e r i a l i s o l a t e s based on d i f f e r e n c e s i n i n t r i n s i c a n t i b i o t i c r e s i s t a n c e was not a p p r o p r i a t e because s t r a i n s developed h y b r i d r e s i s t a n c e p a t t e r n s when grown i n a common bro t h . S e r o l o g i c a l a n a lyses of b a c t e r i a l a n t i g e n s using p o l y c l o n a l antiserum y i e l d e d two r e l i a b l e methods f o r i d e n t i f y i n g R. trifolii i s o l a t e s . A g g l u t i n a t i o n and immunofluorescence procedures were not u s e f u l i n d i s t i n g u i s h i n g these s t r a i n s but immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) were h i g h l y s u i t a b l e . Adaptation of the ELISA allowed i s o l a t e s to be i d e n t i f i e d d i r e c t l y from i n d i v i d u a l root nodules without f i r s t s u b c u l t u r i n g the b a c t e r i a . A s t r a i n of Bacillus polymyxa i s o l a t e d from the same pasture was shown to s t i m u l a t e growth of c r e s t e d wheatgrass (Agropyron cristatum L.) and white c l o v e r (Trifolium repens L . ) . The primary m a n i f e s t a t i o n of the e f f e c t was i n c r e a s e d root weight (P < 0.05), but shoot responses were a l s o observed. P e r e n n i a l ryegrass (Lolium perenne L.) g e n e r a l l y r e a c t e d n e g a t i v e l y to i n o c u l a t i o n with t h i s bacterium. F u r t h e r s t i m u l a t i o n of growth was noted when ramets of the white c l o v e r genotype homologous to ( s h a r i n g a common o r i g i n ) B. pol ymyxa were i n o c u l a t e d i n pure stands (P < 0.05). Clones of the homologous p e r e n n i a l ryegrass genotype a l s o showed a y i e l d i n c r e a s e from s l i g h t l y below c o n t r o l l e v e l s to s l i g h t l y above them when t e s t e d i n a s i m i l a r manner. D e t a i l e d a n a l y s i s of the c r e s t e d wheatgrass response to i n o c u l a t i o n r e v e a l e d that b a c t e r i a l p r o d u c t i o n of i n d o l e a c e t i c a c i d was the most l i k e l y cause of the growth s t i m u l a t i o n . Other b a c t e r i a l c h a r a c t e r i s t i c s such as the a b i l i t y to f i x atmospheric n i t r o g e n or to s o l u b i l i z e organic phosphorus were concluded to be u n r e l a t e d to the growth response. Co-adaptive c o m p a t i b i l i t y between genotypes of L. perenne and T. repens was not apparent when the e f f e c t of R. trifolii was ignored. However, when c l o n e s of pasture p l a n t s that had been neighbours i n the f i e l d were i n o c u l a t e d with R. trifolii i s o l a t e d from root nodules of the " p a r e n t a l " c l o v e r genotype, b i o t i c s p e c i a l i z a t i o n between the pasture p l a n t s became e v i d e n t . The magnitude of the e f f e c t , which was c h a r a c t e r i z e d by s u p e r i o r white c l o v e r y i e l d s (P < 0.05), c o u l d be l a r g e l y accounted for by the presence of the adapted L. perenne/R. trifolii combinations, r e g a r d l e s s of the white c l o v e r genotype. Since T. repens was the dominant component in the s p e c i e s mixture, these trends were a l s o apparent when t o t a l forage biomass was analyzed (P < 0.05). However, e c o l o g i c a l combining a b i l i t y was found to be lowest in these a s s o c i a t i o n s (P < 0.05). S i m i l a r experimentation with i s o l a t e s of B. pol ymyxa (or B. p o l y m y x a - l i k e organisms) was performed. Again the g r a s s / b a c t e r i a combination was shown to be i n f l u e n t i a l i n the growth response as the presence of homologous L. perenne/B. pol ymyxa combinations r e s u l t e d i n s u p e r i o r white c l o v e r and p e r e n n i a l ryegrass performance (P < 0.05). When T. repens was i n o c u l a t e d with a mixture of R. t r i f o l i i s t r a i n s , u n r e l a t e d i s o l a t e s formed more root nodules than d i d homologous ones (P < 0.05). The presence of p e r e n n i a l ryegrass d i d not m i t i g a t e t h i s e f f e c t . However, when homologous R. t r i f o l i i was a d m i n i s t e r e d as a s i n g l e s t r a i n inoculum, y i e l d advantages i n white c l o v e r were observed (P < 0.05). If B. pol ymyxa was present, homologous s t r a i n s of R. t r i f o l i i tended to form most of the root nodules r e g a r d l e s s of the T. repens or L. perenne genotypes. The s i g n i f i c a n c e of the y i e l d advantages observed i n v a r i o u s two and three-way plant/microbe genotype combinations i s d i s c u s s e d with respect to above ground p l a n t performance. V TABLE OF CONTENTS A b s t r a c t i i Table of Contents v L i s t of Tables v i i i L i s t of F i g u r e s x Acknowledgements x i i i G eneral I n t r o d u c t i o n 1 CHAPTER 1 STRAIN IDENTIFICATION IN Rhizobium t r i f o l i i . . . . 8 I n t r o d u c t i o n 9 M a t e r i a l s and Methods 20 Microorganisms 20 Pl a n t I n f e c t i o n Assay 20 P r e p a r a t i o n of C e l l E x t r a c t s f o r E l e c t r o p h o r e s i s 21 E l e c t r o p h o r e s i s 22 Pro d u c t i o n of Antiserum 23 I s o l a t i o n of IgG from Antiserum 24 Cross A d s o r p t i o n of IgG 24 Root Nodules with Standard S t r a i n s 25 Antigen P r e p a r a t i o n s f o r Immunoassays 25 Drop A g g l u t i n a t i o n Procedure 26 Gel Immune D i f f u s i o n 26 Immunofluorescent Antibody S t a i n i n g (IFAS) 27 Enzyme-Linked Immunosorbent Assay (ELISA) 28 A n t i b i o t i c Disk Assay 30 v i A n t i b i o t i c Gradient Agar P l a t e Assay 31 R e s u l t s 33 D i s c u s s i o n 51 CHAPTER 2 PLANT GROWTH PROMOTING BACTERIA 62 I n t r o d u c t i o n 63 Phosphorus S o l u b i l i z a t i o n ... 65 Disease Suppression 71 Plan t Growth Substances 76 A s s o c i a t i v e N i t r o g e n F i x a t i o n 80 M a t e r i a l s and Methods 91 B a c t e r i a l C u l t u r e and E v a l u a t i o n 91 Pl a n t Growth and I n o c u l a t i o n 92 Nitr o g e n F i x a t i o n 95 Supplemental Phosphorus and Growth Regulators 95 I s o l a t i o n of Plant Growth Promoting Substances 96 Bioassays 97 Gas Chromatography 98 Experimental Design and S t a t i s t i c a l A n a l y s i s 99 R e s u l t s 100 B i o l o g i c a l N i trogen F i x a t i o n 113 Phosphate S o l u b i l i z a t i o n 113 Growth Promoting A c t i v i t y 116 D i s c u s s i o n 118 P l a n t Growth Promotion i n Mixed Forage Stands 118 v i i Y i e l d Response using Homologous Organisms 119 Mechanisms of P l a n t Growth Promotion 121 CHAPTER 3 GENOTYPE SPECIFICITY BETWEEN PLANTS AND MICROORGANISMS 128 I n t r o d u c t i o n 129 M a t e r i a l s and Methods 157 P l a n t s and Microorganisms 157 Plan t Growth and I n o c u l a t i o n 158 I d e n t i f i c a t i o n of the Microsymbiont i n Root Nodules.. 160 Experimental Design and S t a t i s t i c a l A n a l y s i s 160 R e s u l t s 162 D i s c u s s i o n 214 Summary 236 L i t e r a t u r e C i t e d 240 v i i i LIST OF TABLES 1-1. S e n s i t i v i t y of antigen/antibody combinations using the drop a g g l u t i n a t i o n procedure with four s t r a i n s of Rhizobium trifolii 35 1-2. S p e c i f i c i t y of antigen/antibody combinations using the g e l immunodiffusion procedure with four s t r a i n s of Rhizobium t r i f o l i i . . . 37 1-3. ELISA using unadsorbed IgG r a i s e d a g a i n s t Rhizobium trifolii s t r a i n s L1, L5, and L6, compared with ELISA using L5 and L6 IgG c r o s s adsorbed with L6 and L5 c e l l s , r e s p e c t i v e l y 41 1-4. Percentage c r o s s r e a c t i o n of root nodule e x t r a c t s i n f e c t e d with standard s t r a i n s of Rhizobium trifolii by heterologous antiserum 42 1-5. Percentage (v/v) of m i n o r i t y a n t i g e n component in a n t i g e n mixtures r e q u i r e d f o r d e t e c t i o n by ELISA 44 1-6. Standard s t r a i n s of Rhizobium trifolii most c l o s e l y resembling the response of unknown i s o l a t e s to a n t i b i o t i c s and i d e n t i f i c a t i o n of unknowns using IAR p a t t e r n s and double d i f f u s i o n 45 1-7. Diameter of growth i n h i b i t i o n zone of Rhizobium trifolii s t r a i n s L3, L5, and L6 i n response to a n t i b i o t i c d i s k s when grown as r e f e r e n c e c u l t u r e s i n the same experiment as unknown i s o l a t e s , and i d e n t i f i c a t i o n of unknowns using IAR p a t t e r n s and double d i f f u s i o n . . 46 1-8. Diameter of growth i n h i b i t i o n zone i n response to a n t i b i o t i c d i s k s and r e a c t i o n s of known and unknown i s o l a t e s with s t r a i n s p e c i f i c a n t i s e r a 47 1- 9. Growth response of four s t r a i n s of Rhizobium trifolii and unknown "progeny" i s o l a t e s to a n t i b i o t i c c o n c e n t r a t i o n g r a d i e n t s 49 2- 1. P h y s i o l o g i c a l and bio c h e m i c a l c h a r a c t e r i s t i c s of Bacillus pol ymyxa s t r a i n L6 101 2-2. E f f e c t of Bacillus pol ymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o i n pure stands of genotypic mixtures of p e r e n n i a l ryegrass or white c l o v e r 102 ix 2-3. E f f e c t of Bacillus pol ymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o in pure stands of p e r e n n i a l r y e g r a s s or white c l o v e r genotypes homologous to B. pol ymyxa 103 2-4. E f f e c t of Bacillus polymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o i n a 50:50 (by numbers) genotypic and s p e c i e s mixture of p e r e n n i a l ryegrass and white c l o v e r 104 2-5. E f f e c t of Bacillus polymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o in a 50:50 (by numbers) mixture of p e r e n n i a l ryegrass and white c l o v e r 105 2-6. E f f e c t of Bacillus polymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o in a 50:50 (by numbers) mixture of p e r e n n i a l ryegrass and white c l o v e r c l o n e s homologous to each other and B. pol ymyxa 106 2-7. E f f e c t of Bacillus polymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o i n a 50:50 (by numbers) mixture of p e r e n n i a l ryegrass and white c l o v e r 107 2-8. E f f e c t of Bacillus polymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o i n a 50:50 (by numbers) mixture of p e r e n n i a l ryegrass and white c l o v e r 108 2-9. E f f e c t of p l a n t growth r e g u l a t o r s , s o l u b l e phosphorus and i n o c u l a t i o n with Bacillus polymyxa on emergence of c r e s t e d wheatgrass 5 to 17 days a f t e r sowing 112 2-10. E f f e c t of p l a n t growth r e g u l a t o r s , s o l u b l e phosphorus and i n o c u l a t i o n with Bacillus polymyxa on p l a n t dry weight and root/shoot r a t i o of c r e s t e d wheatgrass grown i n s o i l under greenhouse c o n d i t i o n s 114 2-11. Survey of c h a r a c t e r i s t i c s of Bacillus polymyxa s t r a i n L6 used i n these experiments which may account f o r the observed p l a n t growth response 115 X LIST OF FIGURES 1-1. S o l u b l e p r o t e i n s of s i x i s o l a t e s of Rhizobium trifolii determined by sodium dodecyl s u l f a t e p o l y a c r y l a m i d e g r a d i e n t g e l e l e c t r o p h o r e s i s (SDS-PAGGE) 34 1-2. D e t e c t i o n of homologous s t r a i n s of Rhizobium trifolii whole c e l l s u s i n g the ELISA 38 1-3. D e t e c t i o n of homologous s t r a i n s of Rhizobium trifolii i n root nodule e x t r a c t s using the ELISA 39 3-1. E f f e c t of L. perenne/T. repens genotype combinations on the y i e l d of T. repens 163 3-2. Advantage observed i n growth of T. repens when the y i e l d of homologous genotype combinations i s expressed as a percentage of the y i e l d observed i n heterologous combinations 164 3-3. E f f e c t of R. trifolii/L. per enne/T. repens genotype combinations on the y i e l d of T. repens.... 165 3-4. E f f e c t of R. trifolii/L. perenne/T. repens genotype combinations on the y i e l d of T. repens.... 166 3-5. E f f e c t of L. perenne/T. repens genotype combinations on the y i e l d of T. repens 168 3-6. E f f e c t of L. perenne/T. repens genotype combinations on the y i e l d of L. perenne 169 3-7. Advantage observed i n growth of L. perenne when the y i e l d of homologous genotype combinations i s expressed as a percentage of the y i e l d observed in heterologous combinations 170 3-8. E f f e c t of R. trifolii/L. perenne/T. repens genotype combinations on the y i e l d of L. perenne 171 3-9. E f f e c t of R. trifolii/L. perenne/T. repens genotype combinations on the y i e l d of L. perenne 172 3-10. E f f e c t of L. perenne/T. repens genotype combinations on combined forage y i e l d 174 3-11. Advantage observed i n combined forage growth when the y i e l d of homologous genotype combinations i s expressed as a percentage of the y i e l d observed i n heterologous combinations 175 x i 3-12. E f f e c t of R. trifolii/L. perenne/T. repens genotype combinations on combined forage y i e l d . . . . . 176 3-13. E f f e c t of R. trifolii/L. perenne/T. repens genotype combinations on combined forage y i e l d 177 3-14. E f f e c t of R. trifolii/L. perenne genotype combinations on the y i e l d of T. repens 178 3-15..Effect of R. trifolii/L. perenne genotype combinations on the y i e l d of L. perenne 179 3-16. E f f e c t of R. trifolii/L. perenne genotype combinations on combined forage y i e l d 180 3-17. E f f e c t of R. trifolii/T. repens genotype combinations on the y i e l d of T. repens 182 3 - 1 8 . E f f e c t of R. trifolii/T. repens genotype combinations on the y i e l d of L. perenne 183 3-19. E f f e c t of R. trifolii/T. repens genotype combinations on combined forage y i e l d 184 3-20. E f f e c t of genotype combinations on e c o l o g i c a l combining a b i l i t y between L. perenne and T. r epens 185 3 - 2 1 . E f f e c t of R. trifolii/L. perenne/T. repens genotype combinations on e c o l o g i c a l combining a b i l i t y between L. perenne and T. repens 186 3-22. E f f e c t of p l a n t genotypes on R. trifolii s t r a i n s e l e c t i o n by T. repens 188 3-23. E f f e c t of L. perenne/T. repens genotype combinations on R. trifolii s t r a i n s e l e c t i o n by T. repens 189 3-24. R e l a t i o n s h i p between R. trifolii i s o l a t e s and T. repens genotypes when grown without L. per enne 190 3-25. E f f e c t of B. polymyxa/T. repens genotype combinations on the y i e l d of T. repens 193 3-26. E f f e c t of B. pol ymyxa/L. perenne/T. repens genotype combinations on the y i e l d of T. r epens 194 3-27. E f f e c t of B. polymyxa/L. perenne/T. repens genotype combinations on the y i e l d of T. repens 195 x i i 3-28. E f f e c t of L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of T. repens 196 3 -29 . E f f e c t of B. pol ymyxa/L. perenne genotype combinat ions on the y i e l d of T. repens 197 3-30 . E f f e c t of genotype combinat ions on root nodule weight of T. repens 198 3~31. E f f e c t of B. pol ymyxa/L. perenne/T. repens genotype c o m b i n a t i o n s on r o o t nodule weight of T. r epe ns . 199 3-32. E f f e c t of B. pol ymyxa/L. perenne genotype combinat ions on the y i e l d of L. perenne 200 3-33 . E f f e c t of B. pol ymyxa/T. repens genotype combinat ions on the y i e l d of L. perenne 201 3-34. E f f e c t of L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of L. perenne 202 3~35. E f f e c t of B. polymyxa/L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of L. perenne.... 204 3~36. E f f e c t of B. pol ymyxa/L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of L. per e nne 205 3-37 . E f f e c t of B. pol ymyxa/T. repens genotype c o m b i n a t i o n s on combined forage y i e l d 206 3-38. E f f e c t of L. perenne/T. repens genotype combinat ions on combined forage y i e l d 207 3-39 . E f f e c t of B. pol ymyxa/L. perenne genotype c o m b i n a t i o n s on combined forage y i e l d 208 3-40 . E f f e c t of B. pol ymyxa/L. perenne/T. repens genotype c o m b i n a t i o n s on combined forage y i e l d 209 3 -41 . E f f e c t of B. pol ymyxa/L. perenne/T. repens genotype c o m b i n a t i o n s on combined forage y i e l d 210 3-42. E f f e c t of p l a n t and m i c r o o r g a n i s m genotypes on R. trifolii s t r a i n s e l e c t i o n by T. repens. 211 3 -43 . E f f e c t of B. pol ymyxa/L. perenne genotype c o m b i n a t i o n s on R. trifolii s t r a i n s e l e c t i o n by T. repens 212 x i i i ACKNOWLEDGEMENTS I would l i k e t o e x p r e s s s i n c e r e t h a n k s t o D r . F.B. H o l l f o r h i s g u i d a n c e and s u p p o r t t h r o u g h o u t t h i s p r o g r a m . I am a l s o t o i n d e b t e d t o Dr. R. T u r k i n g t o n f o r h i s a d v i c e and e n t h u s i a s m d u r i n g t h e p r o j e c t . I would l i k e t o thank D r . R . J . Copeman f o r h i s d i r e c t i o n and e x p e r t i s e i n m i c r o b i o l o g i c a l t e c h n i q u e s and f o r g e n e r o u s l y a l l o w i n g me use of h i s equipment d u r i n g my work. F i n a l l y , I am g r a t e f u l t o R. R a d l e y , who p e r f o r m e d t h e a n a l y s i s f o r p l a n t g r o w t h s u b s t a n c e s , L. S c r u b b f o r h e l p w i t h e l e c t r o p h o r e t i c t e c h n i q u e s , and M.D. W r i g h t f o r t e c h n i c a l a s s i s t a n c e t h r o u g h o u t t h i s r e s e a r c h . 1 GENERAL INTRODUCTION P l a n t s with a h i s t o r y of c o e x i s t e n c e have been shown to perform b e t t e r i n a s s o c i a t i o n than when grown with p r e v i o u s l y "unknown" neighbours ( A l l a r d and Adams, 1969; Joy and L a i t i n e n , 1980). T h i s f i n e - s c a l e b i o t i c s p e c i a l i z a t i o n a l s o occurs between genotypes of white c l o v e r and i n d i v i d u a l s of s e v e r a l s p e c i e s of pasture grasses (Turkington and Harper, 1979) as w e l l as between i n d i v i d u a l genotypes of white c l o v e r and p e r e n n i a l r y e g r a s s when grown i n mixture (Aarssen- and T u r k i n g t o n , 1985; Evans et a l . , 1985) The b e n e f i c i a l e f f e c t s of such c o - e x i s t e n c e are manifested by s u p e r i o r white c l o v e r y i e l d s (Aarssen and T u r k i n g t o n , 1985) and may i n c l u d e enhanced ryegrass performance i n the second year of growth (Evans et a l . , 1985). P l a n t s i n v o l v e d i n these a s s o c i a t i o n s a l s o show s u p e r i o r e c o l o g i c a l combining a b i l i t y as i n d i c a t e d by the r a t i o of the minimum y i e l d i n g mixture component to the maximum one. Q u o t i e n t s approaching u n i t y imply a high p r o b a b i l i t y of c o e x i s t e n c e due to niche divergence and subsequent avoidance of c o m p e t i t i v e pressures (Harper, 1977). C o m p a t i b i l i t y between neighbouring p l a n t s has not been e x p l a i n e d by a n a l y s i s of the a b i o t i c components which may a f f e c t v e g e t a t i v e performance (Aarssen, 1983). S i m i l a r l y , examination of shoot c h a r a c t e r i s t i c s of the p l a n t s i n v o l v e d i n these p a r t n e r s h i p s y i e l d e d no apparent e x p l a n a t i o n f o r such behavior (Turkington and Harper, 1979; Aarssen, 1983; 2 Aarssen and Turking t o n , 1985). R h i z o s p h e r i c i n t e r a c t i o n s between p l a n t s and microorganisms are important and can have s u b s t a n t i a l e f f e c t s on p l a n t growth (Rovira and Davey, 1974; Vose and Ruschel, 1981; Gaskins et a l . , 1985; C u r l and True l o v e , 1986). Consequently, a study of some r o o t -a s s o c i a t e d phenomena may y i e l d i n s i g h t s i n t o b i o l o g i c a l accommodation between i n d i v i d u a l p l a n t s . The p o t e n t i a l e f f e c t that r h i z o s p h e r e microorganisms may exert on p l a n t performance i s re v e a l e d by the vast s i z e of t h e i r p o p u l a t i o n s . While nonrhizosphere f i e l d s o i l may co n t a i n up to 100 m i l l i o n b a c t e r i a l c e l l s g 1 dry weight of s o i l ( G r i f f i n , 1972), root a s s o c i a t e d samples have been shown to c o n t a i n over 1 b i l l i o n b a c t e r i a when expressed on a comparable b a s i s (Rouatt and Katznelson, 1961). Since a l l s o i l - d e r i v e d n u t r i e n t s i n p l a n t s must pass through the rhi z o s p h e r e , the p o t e n t i a l f o r m i c r o b i a l a l t e r a t i o n of these compounds and subsequent i n d i r e c t e f f e c t s on p l a n t growth are gre a t . I f m i c r o b i o l o g i c a l phenomena are r e l a t e d to genotype c o m p a t i b i l i t y between pasture p l a n t s , then symbiotic n i t r o g e n f i x a t i o n may be very i n f l u e n t i a l , p a r t i c u l a r l y where legume s p e c i e s form a component of the p o p u l a t i o n . For example, the cl o v e r / R h i zobi um symbiosis has been estimated to f i x up to 300 kg ha 1 of n i t r o g e n per year (Postgate, 1982). Since v a r i a b i l i t y between s p e c i f i c plant/microbe a s s o c i a t i o n s f o r ni t r o g e n f i x a t i o n i s w e l l documented (Mytton, 1975; Mytton 3 and De F e l i c e , 1977; Hagedorn and C a l d w e l l , 1981; Smith et a l . , 1982), d i f f e r e n c e s i n the e f f i c i e n c y of such combinations c o u l d have s u b s t a n t i a l e f f e c t s on p l a n t growth. The general o b j e c t i v e of t h i s work was to evaluate the importance of v a r i o u s r h i z o s p h e r i c i n t e r a c t i o n s i n the genotype c o m p a t i b i l i t y observed by Aarssen and Turkington (1985). S p e c i f i c a l l y : (1) T. repens performance was s t u d i e d when i n o c u l a t e d with d i f f e r e n t microsymbiont s t r a i n s . T h i s allowed f o r the growth of T. repens, when i n o c u l a t e d with R. trifolii that i t n a t u r a l l y harbors i n root nodules (homologous s t r a i n s ) , to be compared with i t s performance when growing with u n r e l a t e d s t r a i n s . (2) Since s t r a i n s of R. trifolii d i f f e r i n c o m p e t i t i v e a b i l i t y to form root nodules (Vincent and Waters, 1953; Robinson, 1969; Masterton and Sherwood, 1974; Marques P i n t o et a l . , 1974; Mytton and De F e l i c e , 1977), an assessment of microsymbiont s t r a i n s e l e c t i o n by white c l o v e r genotypes was made. R e s u l t s from t h i s type of experiment y i e l d e d i n f o r m a t i o n r e g a r d i n g the a f f i n i t y of homologous R. trifolii s t r a i n s f o r t h e i r corresponding white c l o v e r genotypes. If host and microsymbiont have undergone coadaptive p r o c e s s e s , i t i s reasonable to hypothesize that a white c l o v e r genotype c o u l d " s e l e c t " i t s homologous R. trifolii s t r a i n from a mixture and perform b e t t e r with i t . The development of a r e l a t i v e l y simple yet r e l i a b l e method of i d e n t i f y i n g R. trifolii s t r a i n s was r e q u i r e d . S e v e r a l methods have been employed for i d e n t i f i c a t i o n (Schwinghamer and Dudman, 1980; V i n c e n t , 1982), but s e p a r a t i o n of c l o s e l y r e l a t e d s t r a i n s can be a problem. Since a l l s t r a i n s used in t h i s work were i s o l a t e d from the same permanent pasture, i t was a n t i c i p a t e d t h a t c e r t a i n c h a r a c t e r i s t i c s , which would be u s e f u l i n s t r a i n s e p a r a t i o n , were shared by d i f f e r e n t i s o l a t e s . Furthermore, to be of use i n an e c o l o g i c a l study, d i r e c t i d e n t i f i c a t i o n of the microsymbiont from i n d i v i d u a l root nodules i s r e q u i r e d to f a c i l i t a t e the examination of l a r g e samples. S u b c u l t u r i n g of b a c t e r i a l s t r a i n s from nodule t i s s u e before i d e n t i f i c a t i o n would r e s u l t i n p r o h i b i t i v e time and labor c o n s t r a i n t s and y i e l d l e s s r e l i a b l e r e s u l t s . Consequently, s e v e r a l methods of s t r a i n i d e n t i f i c a t i o n w i t h i n R. trifolii were . i n v e s t i g a t e d which r e s u l t e d i n the s u c c e s s f u l use of the enzyme-linked immunosorbent assay f o r t h i s purpose. 5 (4) An assessment of the e f f e c t s that r o o t - a s s o c i a t e d asymbiotic b a c t e r i a exert on p l a n t performance was undertaken because there are s e v e r a l r e p o r t s of st i m u l a t e d p l a n t growth due to i n o c u l a t i o n with a broad taxonomic range of b a c t e r i a (Brown, 1974; Suslow, 1982; Gaskins et a l . , 1985) r e s u l t i n g i n y i e l d advantages of 97% to 300% i n vegeta b l e and c e r e a l crops (Rennie and Larson, 1979; Suslow and Schroth, 1982). (5) A study of genotype i n t e r a c t i o n s between s o i l b a c t e r i a and pasture p l a n t s was a l s o c a r r i e d out. Examination of t h i s phenomenon i s p a r t i c u l a r l y r e l e v a n t to the problem at hand because i t p r o v i d e s a mechanism by which a grass p l a n t may s t i m u l a t e the growth of a neighbouring legume. I f the a s s o c i a t e d monocot harbored a plant-growth-promoting s t r a i n of b a c t e r i a that had s t i m u l a t o r y e f f e c t s on white c l o v e r , one c o u l d envisage a mutually b e n e f i c i a l r e l a t i o n s h i p between T. repens/R. trifolii a s s o c i a t i o n s and g r a s s / b a c t e r i a combinations. The plant-growth-promoting bacterium c o u l d s t i m u l a t e white c l o v e r growth d i r e c t l y or a f f e c t the processes of n o d u l a t i o n or n i t r o g e n f i x a t i o n and t h e r e f o r e subsequent growth. S e v e r a l 6 authors have demonstrated that f r e e - l i v i n g s o i l microbes may confer b e n e f i c i a l e f f e c t s to n o d u l a t i o n and growth of legumes when c o i n o c u l a t e d with Rhizobium (Singh and Subba Rao, 1979; Burns et a l , 1981; El-Bahrawy, 1983). S i m i l a r l y , the grass component may p r o v i d e a f a v o r a b l e environment f o r the s u r v i v a l or growth of R. trifolii when not enagaged i n symbiosis. The d e t a i l s of s e v e r a l methods of s t r a i n i d e n t i f i c a t i o n w i t h i n Rhizobium trifolii are presented in Chapter 1. In t h i s s e c t i o n , an assessment of s e r o l o g i c a l and other techniques has been made. Plan t growth promoting s t r a i n s of Bacillus polymyxa or B. polymyxa-like microorganisms were i s o l a t e d from s o i l samples in which white c l o v e r and p e r e n n i a l r y e g r a s s r o o t s were i n t e r m i n g l e d . The p l a n t growth s t i m u l a t i n g e f f e c t s of one of these s t r a i n s was c h a r a c t e r i z e d and i s presented in Chapter 2. P o s s i b l e mechanisms by which the growth response occu r r e d were a l s o i n v e s t i g a t e d i n t h i s s e c t i o n . F a c t o r i a l experiments were c a r r i e d out to assess the importance of i n t e r a c t i o n s between c o e x i s t i n g components of a permanent pasture to b i o l o g i c a l accommodation between i n d i v i d u a l p l a n t s . In these experiments p l a n t y i e l d was measured when c l o n e s of organisms were i n v o l v e d i n v a r i o u s two- and three-way homologous genotype combinations. The f a c t o r s s t u d i e d i n c l u d e d genotypes of the pasture p l a n t s 7 Trifolium repens and Lol i um perenne and t h e i r r o o t - a s s o c i a t e d s o i l microbes Rhizobium trifolii and Bacillus pol ymyxa. T h i s work a l l o w e d for an assessment of the e f f e c t of v a r i o u s p l a n t / m i c r o b e combinat ions on above ground genotype s p e c i f i c i t y between i n d i v i d u a l white c l o v e r and p e r e n n i a l r y e g r a s s p l a n t s . The i n f l u e n c e of these f a c t o r s was a l s o s t u d i e d i n r e l a t i o n to R. trifolii s t r a i n s e l e c t i o n by genotypes of T. repens u s i n g the m i c r o b i a l i d e n t i f i c a t i o n t e c h n i q u e s d i s c u s s e d i n Chapter 1. R e s u l t s from these exper iments and an i n t e r p r e t a t i o n of the data are p r e s e n t e d i n Chapter 3. 8 CHAPTER 1 STRAIN IDENTIFICATION IN Rhizobium trifolii 9 INTRODUCTION Heterogeneity between i s o l a t e s of Rhizobium trifolii has been known f o r decades (Hughes and V i n c e n t , 1942; Purchase and V i n c e n t , 1949). These e a r l y s t u d i e s d e t e c t e d d i f f e r e n c e s between c e l l s u r f a c e antigens using s t r a i n s p e c i f i c a n t i -R h i z o b i urn antiserum i n a whole c e l l tube a g g l u t i n a t i o n assay (Vincent, 1941). T h i s procedure, which i s r e l a t i v e l y quick and simple compared with other s e r o l o g i c a l techniques, p r o v i d e s some in f o r m a t i o n about somatic and f l a g e l l a r a n t i g e n s . However, a g g l u t i n a t i o n independent of a n t i b o d y / a n t i g e n r e a c t i o n s , which can r e s u l t i n f a l s e p o s i t i v e r eadings, may occur between c e l l s t h a t are unstable i n s a l i n e . T h i s phenomenon, known as a u t o a g g l u t i n a t i o n , c o n t r i b u t e s to a m b i g u i t i e s in d e t e c t i n g t r u e a g g l u t i n a t i o n of c e l l s ; the assay i s not a good method f o r d i s t i n g u i s h i n g c l o s e l y r e l a t e d , yet d i f f e r e n t a n t i g e n s (Schwinghamer and Dudman, 1980). Furthermore, some s u b c u l t u r e s of Rhizobium s t r a i n s can become no n - a g g l u t i n a b l e (Vincent, 1982) f u r t h e r d e t r a c t i n g from the e f f i c a c y of the t e s t as a primary s t r a i n i d e n t i f i c a t i o n technique. A s u b s t a n t i a l improvement over a g g l u t i n a t i o n procedures was acheived by g e l immunodiffusion or double d i f f u s i o n a n a l y s i s (Ouchterlony, 1968). Bands of p r e c i p i t a t i o n ( p r e c i p i t i n bands) can be seen where homologous antiserum/antigen r e a c t i o n s have occurred, and these are 10 compared with any p r e c i p i t i n bands which may have formed with unknown st r a i n s . Immunodiffusion is simpler to perform and easier to interpret than agglutination reactions since d i r e c t comparison of unknown and known antigens can be made (Schwinghamer and Dudman, 1980). Since i t is less sensitive than agglutination (Marrack, 1963), higher concentrations of antigen and/or antibody are required for this t e s t . Gel immunodiffusion is most suited to use with preparations of whole Rhizobium c e l l s or of large single root nodules such as those found on soybean roots (Means et a l . , 1964). However, the associated lack of s e n s i t i v i t y in the method precludes the use of small nodules, such as those of Trifolium species, as these do not contain s u f f i c i e n t antigenic material to form p r e c i p i t i n bands in a gel system. In such cases, bacteria must be isolated from the nodule and cultured in broth or on agar u n t i l s u f f i c i e n t c e l l u l a r concentration (10-20 mg/ml c e l l dry matter) (Schwinghamer and Dudman, 1980) i s reached for detection with the assay. If many small nodules are to be studied, the time required for sample preparation quickly becomes p r o h i b i t i v e . Fortunately, two s l i g h t l y more complex serol o g i c a l procedures have been developed which f a c i l i t a t e d i r e c t examination of the microsymbiont without the i s o l a t i o n phase. Immunofluorescent antibody staining (IFAS) has been used for dire c t microscopic examination of nodule squash or s o i l s o l u t i o n p r e p a r a t i o n s ; even the s m a l l e s t root nodules can be d i r e c t l y examined without f i r s t c u l t u r i n g the bacterium ( T r i n i c k , 1969). T h i s technique r e q u i r e s i s o l a t i o n of the immunoglobulin G (IgG) p o r t i o n of the s p e c i f i c antiserum which i s then l a b e l l e d with a f l u o r e s c e n t molecule. There are f i v e c l a s s e s of immunoglobulin molecules of which IgM and IgG are most commonly used in vitro. F l u o r e s c e n t l a b e l s used most o f t e n i n c l u d e f l u o r e s c e i n i s o t h i o c y a n a t e (FITC), l i s s a m i n e rhodamine B, or tetramethylrhodamine i s o t h i o c y a n a t e (Schwinghamer and Dudman, 1980). D e t e c t i o n of the homologous a n t i g e n can be done e i t h e r d i r e c t l y or i n d i r e c t l y . In the d i r e c t procedure, the IgG of the anti-Rhizobium antiserum i s c h e m i c a l l y conjugated to one of the_fluorochromes mentioned above, and the l a b e l l e d a n t i b o d i e s are incubated with a n t i g e n that has been heat f i x e d on a microscope s l i d e . The i n d i r e c t IFAS procedure bypasses the fluorochrome c o n j u g a t i o n step by u s i n g commercially a v a i l a b l e a n t i - a n i m a l fluorochrome-conjugated IgG molecules. Thus, i f the s p e c i f i c anti-Rhi zobi um antiserum was r a i s e d i n a r a b b i t , a n t i - r a b b i t IgG conjugated to FITC would be used. Using e i t h e r technique, homologous b a c t e r i a appear as b r i g h t l y c o l o r e d or glowing c e l l s a g a i n s t a dark background when samples are examined using f l u o r e s c e n c e microscopy. I f a l a r g e number of samples i s to be examined, tedium and f a t i g u e may a f f e c t the q u a l i t y of work i n t h i s type of m i c r o s c o p i c a n a l y s i s . A l s o , data obtained using t h i s technique tends to be q u a l i t a t i v e i n nature, although assessment of the number of f l u o r e s c i n g c e l l s per f i e l d can be made. The ELISA, or enzyme-linked immunosorbent assay ( E n g v a l l and Perlman,1972; V o l l e r et a l . , 1976; C l a r k and Adams, 1977) i s the second technique which i s s e n s i t i v e enough to d e t e c t b a c t e r i a d i r e c t l y i n small root nodules. I t i s based on the same p r i n c i p l e as immunofluorescence microscopy, but assays are u s u a l l y c a r r i e d out i n p o l y s t y r e n e m u l t i w e l l p l a t e s i n s t e a d of on microscope s l i d e s . F l u o r e s c e n t l a b e l s are r e p l a c e d with enzymes capable of c a t a l y z i n g r e a c t i o n s which r e s u l t i n the formation of a c o l o r e d , measurable product. The ELISA technique, which can be completely automated, i s monitored through the change i n o p t i c a l d e n s i t y of the s u b s t r a t e s o l u t i o n with time, thereby q u a n t i f y i n g the r e s u l t s . As with IFAS, d i r e c t and i n d i r e c t ELISA procedures may be employed depending on whether or not the s p e c i f i c antiserum has been enzyme-labelled. The d i r e c t technique was f i r s t a p p l i e d to Rhizobium s t r a i n i d e n t i f i c a t i o n u s i n g the peanut Rhizobium ( K i s h i n e v s k y and Bar-Joseph, 1978). The an t i g e n ( i f present) i s "sandwiched" between l a y e r s of a n t i -Rhizobium antibody; the top l a y e r was conjugated to the enzyme a l k a l i n e phosphatase which may be measured c o l o r i m e t r i c a l l y by the a d d i t i o n of s u b s t r a t e . Conjugation of the enzyme l a b e l to the a n t i b o d i e s can be avoided when the i n d i r e c t ELISA technique i s used. As with IFAS, a commercially a v a i l a b l e a n t i - r a b b i t IgG enzyme-l a b e l l e d conjugate can be purchased. T h i s procedure was f i r s t a p p l i e d to Rhizobium by Berger et a l . (1979) i n i d e n t i f y i n g s t r a i n s of R. I egumi nosarum. In t h i s v a r i a t i o n , a n t i g e n was f i r s t bound to the p o l y s t y r e n e p l a t e , f o l l o w e d by s u c c e s s i v e a d d i t i o n s of anti-Rhi zobi um a n t i b o d i e s and commercial a n t i - r a b b i t enzyme-linked IgG. Q u a n t i f i c a t i o n i s again c a r r i e d out by a d d i t i o n of s u b s t r a t e and c o l o r i m e t r i c e s t i m a t i o n of enzyme a c t i v i t y . While s e r o l o g i c a l assays are the method of c h o i c e f o r Rhizobium s t r a i n i d e n t i f i c a t i o n , there are some disadvantages a s s o c i a t e d with t h e i r use. For example, a necessary p r e r e q u i s i t e to perform these t e s t s i s the a v a i l a b i l i t y of s u i t a b l e a n t i s e r a . T h e r e f o r e , s t u d i e s r e l y i n g on these techniques are r e s t r i c t e d to use of s t r a i n s which are homologues of, or are at l e a s t r e l a t e d t o, the a n t i s e r a that are a v a i l a b l e . As a r e s u l t , l a r g e s c a l e e c o l o g i c a l s t u d i e s to c h a r a c t e r i z e Rhizobium p o p u l a t i o n s at s e v e r a l l o c a t i o n s , f o r example, may not be f e a s i b l e . The a p p r o p r i a t e e x p e r t i s e and f a c i l i t i e s f o r e i t h e r p o l y c l o n a l a n t i s e r a p r o d u c t i o n (see Schwinghamer and Dudman, 1980; V i n c e n t , 1982 f o r d e t a i l s ) or monoclonal a n t i b o d i e s (Wright et a l . , 1986) may a l s o p r o v i d e l o g i s t i c a l o b s t a c l e s to the wide-scale use of s e r o l o g y . Consequently, s t r a i n i d e n t i f i c a t i o n based on non-a n t i g e n i c p r o p e r t i e s of Rhizobium has been i n v e s t i g a t e d . Sodium dodecyl s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS-PAGE) and sodium dodecyl s u l f a t e p o l y a c r y l a m i d e g r a d i e n t g e l e l e c t r o p h o r e s i s (SDS-PAGGE) have been used to separate small numbers of s t r a i n s from one another based on isozyme a n a l y s i s of e s t e r a s e (Murphy and Masterson, 1970) and 3-hydroxybutyrate dehydrogenase ( F o t t r e l l and O'Hora, 1969). Noel and B r i l l (1980), using g e l e l e c t r o p h o r e s i s , demonstrated s u b s t a n t i a l h e t e r o g e n e i t y in the c e l l u l a r p r o t e i n p r o f i l e s of R. j aponi cum i s o l a t e s taken from nodules of f i e l d grown soybeans. These authors showed that eighteen and nineteen d i f f e r e n t p r o t e i n p r o f i l e s were present at two l o c a t i o n s , r e s p e c t i v e l y . In a p p l y i n g s i m i l a r techniques to the study of R. t r i f o l i i , Dughri and Bottomley (1983) showed that twenty-two f i e l d i s o l a t e s of the bacterium were q u i t e heterogeneous f o r s o l u b l e p r o t e i n p r o f i l e s . R e s u l t s of the l a t t e r two s t u d i e s are p a r t i c u l a r l y i n t e r e s t i n g i n view of the f a c t that each i s o l a t e r e a c t e d with at l e a s t one of s e v e r a l a v a i l a b l e a n t i s e r a . Thus, these two s t u d i e s demonstrated that i t was p o s s i b l e to s u b d i v i d e f u r t h e r serogroups of R. japoni cum and R. trifolii using SDS-PAGGE. However, i t has been noted in these i n v e s t i g a t i o n s that e l e c t r o p h o r e t i c a n a l y s i s of p r o t e i n s i n Rhizobium i s u s e f u l only as an a c c e s s o r y method of s t r a i n i d e n t i f i c a t i o n to support r e s u l t s from a primary i d e n t i f i c a t i o n scheme such as g e l immunodiffusion. Immunoelectrophoresis i s a combination of e l e c t r o p h o r e t i c and s e r o l o g i c a l a n a l y s i s i n which samples are r e a c t e d with s p e c i f i c anti-Rhi zobi um antiserum a f t e r e l e c t r o p h o r e t i c s e p a r a t i o n . While u s e f u l i n c h a r a c t e r i z i n g the nature of Rhizobium a n t i g e n s , i t too i s u s u a l l y not c o n s i d e r e d a primary method of s t r a i n i d e n t i f i c a t i o n . R e s i s t a n c e to a n t i m i c r o b i a l substances in i s o l a t e s of Rhizobium has a l s o been i n v e s i g a t e d as a method of s t r a i n i d e n t i f i c a t i o n . While d i f f e r e n t i a l s e n s i t i v i t y to b a c t e r i o c i n s and bacteriophage i n Rhizobium has r e c e i v e d some a t t e n t i o n (Schwinghamer and Dudman, 1980; Br o m f i e l d et a l , 1986), much emphasis has been p l a c e d on the use of a n t i b i o t i c r e s i s t a n c e as a b a s i s f o r s t r a i n i d e n t i f i c a t i o n . Two b a s i c s t r a t e g i e s have been developed based on t h i s p r i n c i p l e : ( i ) use of a n t i b i o t i c r e s i s t a n t mutants to f o l l o w the f a t e of marked i n o c u l a n t s t r a i n s , and ( i i ) s t r a i n i d e n t i f i c a t i o n based on n a t u r a l l y o c c u r r i n g or i n t r i n s i c a n t i b i o t i c r e s i s t a n c e (IAR). Use of the f i r s t s t r a t e g y i s commonly achieved by p l a t i n g out the s t r a i n of i n t e r e s t on i n c r e a s i n g c o n c e n t r a t i o n s of an a n t i b i o t i c , f o l l o w e d by s e l e c t i o n of c o l o n i e s found growing on e l e v a t e d c o n c e n t r a t i o n s of the growth i n h i b i t o r . Streptomycin has been popular with r h i z o b i o l o g i s t s because h i g h l y r e s i s t a n t mutant s t r a i n s s t a b l e to p l a n t passage can be generated e a s i l y . However, the only r e a l advantage t h i s technique o f f e r s i s the cir c u m v e n t i o n of s e r o l o g i c a l procedures; i t i s a l s o of l i m i t e d use i n broad based e c o l o g i c a l s t u d i e s . A major disadvantage a s s o c i a t e d with the technique i s the p o t e n t i a l l o s s of e f f e c t i v e n e s s which has been observed in the s e l e c t e d mutants. While t h i s symbiotic t r a i t i s not a f f e c t e d by mutation to streptomycin r e s i s t a n c e i n many Rhizobium s p e c i e s (Schwinghamer and Dudman, 1980; Gupta and Kleczkowska, 1962; Obaton, 1971; Schwinghamer, 1967), s t r a i n s of R. trifolii have shown a l o s s of e f f e c t i v e n e s s a f t e r mutant s e l e c t i o n (Zelazna-Kowalska, 1971). G e n e r a l l y , r e s i s t a n c e to a n t i b i o t i c s which i n h i b i t c e l l membrane or c e l l w a l l s y n t h e s i s (eg. p e n i c i l l i n ) r e s u l t i n l e s s frequent and d r a s t i c changes i n e f f e c t i v e n e s s , while mutation to r e s i s t a n c e f o r a n t i b i o t i c s that i n h i b i t p r o t e i n s y n t h e s i s (eg. streptomycin) are l e a s t l i k e l y to a f f e c t symbiotic c h a r a c t e r i s t i c s (Schwinghamer, 1977). A r e l a t e d technique that c o u l d allow s t r a i n i d e n t i f i c a t i o n of v i r t u a l l y u n l i m i t e d numbers of i s o l a t e s with no p r e r e q u i s i t e m anipulation (eg. antiserum p r o d u c t i o n or mutant s e l e c t i o n ) i n v o l v e s c h a r a c t e r i z a t i o n of i s o l a t e s based on t h e i r n a t u r a l l y o c c u r r i n g or i n t r i n s i c r e s i s t a n c e to v a r i o u s a n t i b i o t i c s . Rhizobium i s o l a t e s are c u l t u r e d on media c o n t a i n i n g d i f f e r e n t types and amounts of a n t i b i o t i c s . Growth i n h i b i t i o n i s measured q u a l i t a t i v e l y and/or q u a n t i t a t i v e l y r e s u l t i n g in the g e n e r a t i o n of an IAR " f i n g e r p r i n t " of the s t r a i n ; the g r e a t e r the number of a n t i b i o t i c s t e s t e d , the more unique the p a t t e r n can be. IAR has been p o r t r a y e d as a quick and simple method f o r s t r a i n i d e n t i f i c a t i o n w i t h i n the Rhizobiaceae i n comparison with standard s e r o l o g i c a l methods. Heterogeneity i n IAR p a t t e r n s has been demonstrated between s t r a i n s of R. I egumi nosar um (Mahler and Bezdicek, 1978), R. phaseoli (Benyon and Josey, 1980), R. meliloti (Antoun et a l . , 1982), and the cowpea Rhizobium ( S i n c l a i r and Eaglesham, 1984). It has a l s o been a p p l i e d to competition s t u d i e s u s i n g R. meliloti (Marques P i n t o et a l . , 1974; Petersen et a l . , 1983) and mung bean Rhizobium (Gupta et a l . , 1983). However, the u s e f u l n e s s of t h i s method has been questioned due to the v a r i a b i l i t y i n r e s i s t a n c e p a t t e r n s i n Lot ononis Rhizobium ( D i a t l o f f , 1977), Cicer Rhizobium and R. phaseoli ( S t e i n et a l . , 1982). The l a t t e r authors r e p o r t the occurrence of more IAR p a t t e r n s than s t r a i n s t e s t e d which suggests that s t r a i n s may have more than one p a t t e r n or that genetic exchange between c e l l s may f a c i l i t a t e the formation of new p a t t e r n s . I t has long been known that the g e n e t i c i n f o r m a t i o n f o r r e s i s t a n c e to many a n t i b i o t i c s r e s i d e s on plasmid DNA, which i s f r e e l y exchanged between b a c t e r i a l c e l l s of s i m i l a r c o m p a t i b i l i t y groups (Berin g e r , 1974). Thus, i t may be expected that exchange events between c e l l s of s i m i l a r s t r a i n s would s e v e r e l y reduce the u s e f u l n e s s of IAR as a s t r a i n i d e n t i f i c a t i o n method. Data to be presented i n t h i s chapter w i l l support t h i s c o n t e n t i o n when s t r a i n s of R. trifolii are t e s t e d f o r IAR (Chanway and H o l l , 1986). While s e n s i t i v i t y to v a r i o u s a n t i m i c r o b i a l agents has been used i n Rhizobium s t r a i n d i f f e r e n t i a t i n g schemes, m i c r o b i a l p r o d u c t i o n of these agents can a l s o be u s e f u l . Brockwell et a l . (1978) argue that because p r o d u c t i o n of these substances by microbes i s a r a r e r event than s u s c e p t i b i l i t y (as a n t i m i c r o b i a l agents are o f t e n a c t i v e over a broad spectrum of microbes), i d e n t i f i c a t i o n schemes based on p r o d u c t i o n should be more r e l i a b l e . Simple methods f o r assessment of a n t i b i o t i c p r o d u c t i o n are a v a i l a b l e (Neal, 1971),but the p a u c i t y of work i n t h i s area with r e s p e c t to Rhizobium i d e n t i f i c a t i o n p r e c l u d e s examination of the p o t e n t i a l t h i s technique may h o l d . Mayr-Harting et a l . ( l 9 7 2 ) have suggested that s t r a i n t y p i n g on the b a s i s of both p r o d u c t i o n and s u s c e p t i b i l i t y to a n t i m i c r o b i a l agents may r e s u l t i n g r e a t e r r e l i a b i l i t y . Other p o t e n t i a l l y u s e f u l n o n s e r o l o g i c a l s t r a i n i d e n t i f i c a t i o n methods i n c l u d e the use of auxotrophic mutants, which can be used i n much the same way as a n t i b i o t i c r e s i s t a n c e mutants or bi o c h e m i c a l a n a l y s i s of c e l l u l a r metabolism (Schwinghamer and Dudman, 1980)., These types of techniques o f f e r l i t t l e or no advantage over s e r o l o g y and o f t e n r e q u i r e more work than standard antibody t e s t s . Consequently, these too should be c o n s i d e r e d as a c c e s s o r y or secondary methods of Rhizobium s t r a i n i d e n t i f i c a t i o n . F i n a l l y , unique c h a r a c t e r i s t i c s such as the red colony s t r a i n of Rhizobium that nodulates Lot ononis ( N o r r i s , 1958), or the i s o l a t e of R. trifolii which a t y p i c a l l y shows str o n g a b s o r p t i o n of congo red when the dye i s s u p p l i e d i n agar ( B r o m f i e l d and Jones, 1980a) o f f e r obvious s t r a i n i d e n t i f i c a t i o n p r o p e r t i e s . However, unique c h a r a c t e r i s t i c s of t h i s type are very rare and are of l i t t l e use i n most s t u d i e s of Rhizobium b i o l o g y and ecology. Six of the techniques d e s c r i b e d i n t h i s chapter have been a p p l i e d to s t r a i n s of R. trifolii i s o l a t e d from nodules of l o c a l l y growing white c l o v e r p l a n t s . S e r o l o g i c a l procedures i n v e s t i g a t e d i n c l u d e d simple a g g l u t i n a t i o n , g e l immunodiffusion, IFAS, and ELISA while SDS-PAGGE and IAR were i n v e s t i g a t e d as n o n s e r o l o g i c a l procedures. R e s u l t s from and an a p p r a i s a l of these techniques w i l l be presented i n the f o l l o w i n g pages. 20 MATERIALS AND METHODS Mi cr oor gani sms Root nodules were c o l l e c t e d from Trifolium repens and st o r e d d e s i c c a t e d at room temperature. Rhizobium trifolii was i s o l a t e d from nodules of Trifolium repens a c c o r d i n g to Vinc e n t (1970). The i s o l a t i o n procedure i n v o l v e d : (1) washing nodules to remove gross s u r f a c e contamination; (2) momentary immersion in 95% et h a n o l ; (3) immersion i n 0.1% H g C l 2 f o r 3-4 minutes; (4) thorough r i n s i n g i n s i x changes of s t e r i l e water; (5) nodule s l i c e s were str e a k e d onto yeast e x t r a c t mannitol (YEM) agar p l a t e s composed o f : (g/1) K 2HP0 4 0.5; MgS0 4-7H 20 0.2; NaCl 0.1; Mannito l 10; yeast e x t r a c t 0.4; agar 15; (6) i s o l a t e s c u l t u r e d from nodule t i s s u e were re s t r e a k e d on YEM agar to s e l e c t s i n g l e c o l o n i e s ; and (7) were t e s t e d f o r the a b i l i t y to nodulate Trifolium repens i n p l a n t i n f e c t i o n t e s t s . I s o l a t e s were s t o r e d on YEM agar s l a n t s at 4°C. Plant Infection Assay Seeds of Trifolium repens Ladino-type were s u r f a c e s t e r i l i z e d by: (1) soaking in 70% ethanol f o r 60 seconds; (2) a f t e r r i n s i n g in s t e r i l e d i s t i l l e d water, immersion in 20% Chlorox (1% NaOCl) f o r 15 minutes; (3) followed by s e v e r a l r i n s e s i n s t e r i l e water. Seeds were then a s e p t i c a l l y p l a c e d in p l a n t i n f e c t i o n tubes (2.0 cm diameter x 25.0 cm) with c a . 25 ml Jensen's s e e d l i n g agar s o l i d i f i e d in the form of a s l a n t . The s e e d l i n g agar c o n t a i n e d : (g/1) CaHP0 4 1.0; K 2HP0 4 0.2; MgS0 4~7H 20 0.2; NaCl 0.2; F e C l 3 0.1; agar 15. To prepare inoculum, YEM agar s l a n t s were i n o c u l a t e d with R. trifolii and incubated at 30°C fo r 4 days. Growth on the s l a n t s was then suspended i n 1 ml s t e r i l e water and 0.2 ml of the supension was used to i n o c u l a t e seeds i n the p l a n t i n f e c t i o n tubes. Three weeks l a t e r the i d e n t i t y of a l l p u t a t i v e Rhizobium trifolii ' i s o l a t e s was confirmed as nodules were apparent on a l l i n o c u l a t e d c l o v e r s e e d l i n g s . Preparation of Cell Extracts for Electrophoresis I s o l a t e s of R. trifolii were grown i n 40 ml of the d e f i n e d medium (MGA) of Dughri and Bottomley (1983). MGA medium c o n t a i n s : (g/1) mannitol 10; L-glutamic a c i d 1.0; KH 2P0 4 0.5; MgS0 4-7H 20 0.2; NaCl 0.1; c a l c i u m pantothenate 0.01; thiamine h y d r o c h l o r i d e 0.01; b i o t i n 0.3 mg; t r a c e elements 0.1 ml. The t r a c e element s o l u t i o n c o n t a i n e d : (g/1) H 3B0 3 28; MnS0 4-H 20 34; CuS0 4"5H 20 1.0; ZnSO 4-7H 20 2.2; (NH.),Mo0 9.-H 90 1.0). The pH of the medium was a d j u s t e d to 6.8. C e l l s were grown f o r 4 days at 25 C on a r o t a r y shaker, harvested by c e n t r i f u g a t i o n (20,000 x g f o r 20 minutes), and washed three times i n c o l d 10 mM T r i s - H C l b u f f e r (pH 7.6). A f t e r each wash, c e l l s were c e n t r i f u g e d at 10,000 x g f o r 10 minutes. The washed p e l l e t was suspended i n 1 ml of the same b u f f e r and s o n i c a t e d f o r 30 seconds. An equal volume of "sample b u f f e r " (0.08M T r i s - H C l , pH 6.8; 2% (w/v) sodium dodecyl s u l f a t e (SDS); 10% (v/v) g l y c e r o l ; 5% (v/v) 2-mercaptoethanol; and 0.01% (w/v) bromophenol blue) was added to the suspension. C e l l e x t r a c t s were s t o r e d at -20°C u n t i l use. Electrophoresis Gel e l e c t r o p h o r e s i s was performed using s i n g l e c o n c e n t r a t i o n (7.5%) and l i n e a r g r a d i e n t g e l s ( 7 . 5 % - l 5 % ) , with dimensions 180 x 200 x 0.7 mm. A f t e r p o l y m e r i z a t i o n of the s t a c k i n g g e l , 20 to 30 m i c r o l i t e r s of the c e l l e x t r a c t (20 - 30 ug p r o t e i n ) were added to the w e l l s as were p r o t e i n standards. Gels were e l e c t r o p h o r e s e d f o r 8-10 hours at'200 v o l t s and 0.025 amps. Gels were s t a i n e d f o r 1 hour i n 0.1% (w/v) Coomassie b r i l l i a n t blue R-250, 45% (v/v) methanol, and 10% (v/v) g l a c i a l a c e t i c a c i d and then d e s t a i n e d f o r 1 to 2 hours i n 5% (v/v) methanol and 7% (v/v) a c e t i c a c i d . Production of Antiserum Whole Rhizobium c e l l p r e p a r a t i o n s were used as antigens to immunize r a b b i t s , a c c o r d i n g to Dughri and Bottomley (1983). Rhizobium trifolii was grown as p r e v i o u s l y d e s c r i b e d f o r the p r e p a r a t i o n of c e l l e x t r a c t s . A f t e r the c e l l s were washed, the f i n a l p e l l e t was suspended in 0.15M p h y s i o l o g i c a l s a l i n e (g/1: NaCl 8.5; KH 2P0 4 20.4; Na 2HP0 4" Q 7H 20 40.3; pH 7.0) to a c o n c e n t r a t i o n of 5x10 c e l l s per ml. C e l l u l a r d e n s i t y was estimated s p e c t r o p h o t o m e t r i c a l l y using Mcfarland's BaS0 4 standards ( D i f c o ) to generate a r e f e r e n c e curve. C e l l s were then heated at 95°C f o r 60 minutes to denature f l a g e l l a r a n t i g e n s and 1 ml of the heated c e l l p r e p a r a t i o n was thoroughly mixed with 1 ml Freund's complete adjuvant ( D i f c o ) u n t i l a milky suspension was formed. The 2 ml emulsion was i n j e c t e d i n t o the t h i g h muscle of a New Zealand White male r a b b i t . Two r a b b i t s were immunized with each Rhizobium s t r a i n . Four weeks l a t e r , a 1 ml booster c o n t a i n i n g equal p a r t s of a f r e s h l y grown, washed, and heat-q t r e a t e d b a c t e r i a l suspension (5x10 c e l l s ) and Freund's incomplete adjuvant ( D i f c o ) was a d m i n i s t e r e d i n t r a m u s c u l a r l y . Another booster i d e n t i c a l to the four-week i n j e c t i o n was a d m i n i s t e r e d 10 days l a t e r . Two weeks a f t e r the f i n a l booster was given, blood samples (ca. 100 ml) were c o l l e c t e d by c a r d i a c puncture. Blood was l e f t to c l o t o vernight at 4°C. The serum was separated from c e l l u l a r components by c e n t r i f u g a t i o n at 1000 x g f o r 30 minutes and was s t o r e d at -20°C. Isolation of IgG from Antiserum The IgG component of the r a b b i t antiserum was i s o l a t e d using a f f i n i t y chromatography based on the noncovalent b i n d i n g of p r o t e i n A to the Fc region of IgG molecules. Sepharose-Protein A (Pharmacia) was swollen o v e r n i g h t at 4°C in 0.1M potassium phosphate b u f f e r , pH 7.0. The g e l (5 ml) was then poured using a 10 ml s y r i n g e b a r r e l as the column. A s i n t e r e d g l a s s f i l t e r was p l a c e d on top of the g e l to f a c i l i t a t e uniform sample a p p l i c a t i o n . A one ml a l i q u o t of crude antiserum was loaded onto the column and e l u t e d with 0.1M potassium phosphate pH 7.0 at a flow rate of 1 ml per minute to remove unbound serum components. The bound IgG f r a c t i o n of the antiserum was e l u t e d by lowering the pH of the running b u f f e r to pH 2.0 with 0.58% a c e t i c a c i d i n 0.85% NaCl. E l u a t e c o n t a i n i n g the a n t i b o d i e s was c o l l e c t e d i n tubes and was immediately b u f f e r e d to pH 7.0 with a few drops of 10 mM T r i s - H C l pH 8.5 to reduce damage to IgG molecules by the low pH. Each ml of crude antiserum y i e l d e d c a . 10 ml of 1 mg/ml IgG s o l u t i o n . Samples were s t o r e d at -20°C. Cross Adsorption of IgG Cross r e a c t i v i t y of IgG to heterologous a n t i g e n s may be reduced by p r e v i o u s exposure of the antibody molecules to the c r o s s r e a c t i n g a n t i g e n (Vincent, 1982). One ml of the a p p r o p r i a t e IgG s o l u t i o n was thoroughly mixed with a heated, (95°C f o r 1 hour) whole c e l l p r e p a r a t i o n (5.25 X 1 0 1 0 c e l l s ) of the c r o s s r e a c t i n g Rhizobium s t r a i n . The a n t i b o d y / a n t i g e n mixtures were l e f t at room temperature overnight.. Cross adsorbed IgG was separated from the c e l l s by decanting the supernatant a f t e r c e n t r i f u g a t i o n (15,000 x g f o r 10 minutes). Root Nodules with Standard Strains P l a s t i c t r a y s (26 x 7 x 14 cm) were f i l l e d with Turface R and a u t o c l a v e d . Seed of Trifolium repens Ladino-type was s u r f a c e s t e r i l i z e d as p r e v i o u s l y d e s c r i b e d (see Plant I n f e c t i o n Assay) and sown i n the s t e r i l e T u r f a c e R. Seven day o l d c u l t u r e s of the a p p r o p r i a t e R. trifolii s t r a i n growing i n YEM broth were used to i n o c u l a t e p l a n t s by pouring c u l t u r e s (40 ml) d i r e c t l y i n t o the t r a y s c o n t a i n i n g Turface R. P l a n t s were watered as r e q u i r e d with s t e r i l e d i s t i l l e d water. About s i x weeks l a t e r , p l a n t s were harvested and nodules were c o l l e c t e d and s t o r e d i n 10% (v/v) g l y c e r o l at -20°C. Antigen Preparations for Immunoas s ays Root nodules obtained by the procedure d e s c r i b e d above were thawed, b l o t t e d dry, weighed, and p l a c e d i n 1.5 ml m i c r o c e n t r i f u g e tubes (Eppendorf). A f t e r a d d i t i o n of 0.5 ml phosphate b u f f e r e d s a l i n e (PBS) (g/1: Na 9HP0. 4.5; NaH 9P0. 0.44; pH7.0), i n d i v i d u a l nodules were crushed i n the tubes with a s p a t u l a and heated f o r 1 hour at 95°C. Based on nodule f r e s h weight, an a p p r o p r i a t e volume of the heated nodule e x t r a c t was d i l u t e d i n PBS to give the d e s i r e d c o n c e n t r a t i o n ( u s u a l l y 0.3 mg nodule t i s s u e per ml PBS). Whole c e l l p r e p a r a t i o n s of R. trifolii f o r immunoassays were t r e a t e d as p r e v i o u s l y d e s c r i b e d (see Production of A n t i s e r u m ) . Drop Agglutination Procedure Antiserum was d i l u t e d s e r i a l l y from 1:25 to 1:6400 with PBS. Heated whole Rhizobium c e l l p r e p a r a t i o n s were a d j u s t e d q to 5 x 10 c e l l s per ml. A one hundred m i c r o l i t e r drop of antiserum was mixed with an equal volume of the a n t i g e n suspension i n p e t r i d i s h e s and incubated o v e r n i g h t i n a moist chamber. The f o l l o w i n g day drops were examined v i s u a l l y and m i c r o s c o p i c a l l y f o r a g g l u t i n a t i o n or formation of granuoles d i s t i n c t from c o n t r o l t e s t s which r e c e i v e d s a l i n e i n s t e a d of antiserum. Gel Immune-Diffusion Gels composed of 1% (w/v) agar and 0.025% (w/v) sodium a z i d e i n a 0.85% (w/v) sodium c h l o r i d e s o l u t i o n were poured to a t h i c k n e s s of c a . 5 mm i n p e t r i p l a t e s (10 cm d i a m e t e r ) . Hexagonal c o n f i g u r a t i o n s of w e l l s were cut i n the g e l s with a hollow tube (ca. 2 mm diameter) connected to a vacuum system. Each c o n f i g u r a t i o n a l s o i n c l u d e d a c e n t r a l w e l l of the same s i z e . Spacing between the w e l l s was u s u a l l y 0.75 cm w i t h i n a hexagon but was v a r i e d from 0.5 cm to 1.0 cm i n d i f f e r e n t t e s t s . F i v e to ten m i c r o l i t e r s of the h e a t - t r e a t e d a n t i g e n s (whole Rhi zobium c e l l s ) were added to the w e l l s and the a p p r o p r i a t e crude antiserum was p l a c e d i n the c e n t r a l w e l l . Wells d i r e c t l y above and below the c e n t r a l w e l l c o n t a i n e d a n t i g e n s known to be homologous to the antiserum. Antigen 9 1 1 c o n c e n t r a t i o n s that were t e s t e d ranged from c a . 10 to 10 c e l l s per ml PBS. Antiserum c o n c e n t r a t i o n s i n c l u d e d u n d i l u t e d , 1:2, 1:4, and 1:8 (antiserum:PBS). P l a t e s were incubated f o r 1-3 days i n a moist chamber at room temperature before examination f o r p r e c i p i t i n bands. ImmunoJ7 uor e s cent Antibody Staining (IFAS) The i n d i r e c t method of immunofluorescent antibody s t a i n i n g was used to i d e n t i f y r o o t nodule b a c t e r i a in s i t u . Root nodules were crushed i n PBS to a c o n c e n t r a t i o n of 1.25 mg nodule t i s s u e per ml PBS. IgG (1 mg/ml) was d i l u t e d 1:8 or 1:16 (Vincent, 1982) with T r i s - H C l b u f f e r , pH .7.0. Assays were performed as f o l l o w s : (1) a l o o p f u l of antigen was p l a c e d on a microscope s l i d e and a i r d r i e d ; (2) specimens were f i x e d by f l o o d i n g with acetone and r i n s i n g with d i s t i l l e d water f i v e times; (3) s l i d e s were incubated with the anti-Rhizobi um r a b b i t IgG f o r 30 minutes at 37°C; (4) a f t e r r i n s i n g with d i s t i l l e d water, s l i d e s were incubated with f l u o r e s c e i n l a b e l l e d a n t i - r a b b i t IgG (Sigma) d i l u t e d 1:100 with PBS f o r 30 minutes at 37°C; (5) a f t e r washing with d i s t i l l e d water, a drop of mounting f l u i d ( g l y c e r o l 90 ml; PBS 10 ml; p-phenylenediamine 0.1g) was added to the a n t i g e n and a c o v e r s l i p was p l a c e d on the s l i d e . S l i d e s were observed with a Z e i s s U n i v e r s a l microscope equipped with a high pressure mercury lamp (HBO-100 W/Z). E x c i t i a t i o n f i l t e r BG12 and b a r r i e r f i l t e r 50 ( t r a n s m i s s i o n range above 500 nm) were used under e p i - i l l u m i n a t i o n at a m a g n i f i c a t i o n of one thousand. Enzyme-Linked Immunosorbent Assay (ELISA) Enzyme l i n k e d immunosorbent assays were c a r r i e d out as p r e v i o u s l y d e s c r i b e d f o r Rhizobium (Berger et a l . , 1979) with some m o d i f i c a t i o n s . E i t h e r whole c e l l or crushed nodule p r e p a r a t i o n s were used as a n t i g e n s . Undesired c r o s s r e a c t i o n s were minimized by use of c r o s s adsorbed IgG and by o p t i m i z i n g a n t i g e n and antibody c o n c e n t r a t i o n s . The l a t t e r v a l u e s were found to be ca. 10 c e l l s / m l PBS or 0.3 mg nodule t i s s u e / m l PBS f o r a n t i g e n p r e p a r a t i o n s ; and 1:400 (IgG solution:PBS) f o r s t r a i n L1 antiserum and 1:100 (IgG:PBS) f o r s t r a i n s L5 and L6 a n t i s e r a . The assay procedure i n v o l v e d : (1) i n c u b a t i o n of the antigen s o l u t i o n (200 u l / w e l l ) i n w e l l s of covered p o l y s t y r e n e m i c r o t i t e r p l a t e s (Immulon II Dynatech L a b o r a t o r i e s ) o v e r n i g h t at room temperature; (2) p l a t e s were washed f i v e times with 1.9 l i t e r s of tap water d e l i v e r e d over 10 seconds at 12 PSI each time; (3) w e l l s were incubated with 200 u l bovine serum albumin (2 mg/ml) i n PBS at 37°C f o r 90 minutes; (4) a f t e r washing as i n step (2), 200 u l of the ap p r o p r i a t e anti-Rhi zobi um r a b b i t IgG d i l u t e d with PBS was added to w e l l s and p l a t e s were incubated at 37°C f o r 90 minutes; (5) p l a t e s were washed as before, and 200 u l of a l k a l i n e phosphatase l a b e l l e d a n t i - r a b b i t IgG (Sigma) d i l u t e d 1:1000 i n PBS were added to the w e l l s and incubated at 37°C f o r 90 minutes; (6) a f t e r washing, 200 u l of phosphatase s u b s t r a t e , disodium p - n i t r o p h e n y l phosphate hexahydrate (1 mg/ml) i n 10% diethanolamine b u f f e r (diethanolamine 97 ml; water 800 ml; NaN^ 0.2g; MgCl 2"6H 20 0.1g; adjust e d to pH 9.8 with 1M HC1 and made up to 1 l i t e r with d i s t i l l e d water) were added to w e l l s ; and (7) t h i r t y minutes l a t e r the o p t i c a l d e n s i t y at 405 nm was read and recorded u s i n g a m u l t i w e l l spectrophotometer ( T i t r e t e k M u l t i s c a n ) . Antibiotic Disk Assay I n t r i n s i c a n t i b i o t i c r e s i s t a n c e (IAR) p a t t e r n s were determined using an a n t i b i o t i c d i s k assay. S t r a i n s were grown i n MGA medium f o r 4 days and harvested by c e n t r i f u g a t i o n as p r e v i o u s l y d e s c r i b e d (see P r e p a r a t i o n of E x t r a c t s ) . A p p r o p r i a t e d i l u t i o n s of each s t r a i n to g i v e e q u i v a l e n t c u l t u r e d e n s i t i e s were added to 10 ml molten agar h e l d at 45°C. The d i l u t i o n s were poured i n t o p e t r i d i s h e s to give an i n i t i a l b a c t e r i a l c o n c e n t r a t i o n of ca. 10^ c e l l s per ml of agar. A n t i b i o t i c d i s k s (BBL Sensi Disk) were p l a c e d on the s u r f a c e of the agar and became embedded in i t w i t h i n an hour. P l a t e s were incubated at 30°C f o r f i v e days at which time growth i n h i b i t i o n zones were measured. A n t i b i o t i c s used i n the assays were kanamycin, n a l i d i x i c a c i d and c e p h a l o t h i n , each at a c o n c e n t r a t i o n of 30 ug per d i s k . Standard IAR p a t t e r n s presented were determined twice with two r e p l i c a t e s each time. Unknown i s o l a t e s were generated by i n o c u l a t i n g a 40 ml MGA broth with three known s t r a i n s of R. t r i f o l i i . A f t e r four days i n c u b a t i o n at 25°C on a r o t a r y shaker, c u l t u r e s were st r e a k e d onto MGA agar p l a t e s . S i n g l e colony i s o l a t e s were s e l e c t e d and used to i n o c u l a t e separate MGA broths f o r the assay. 31 Antibiotic Gradient Agar Plate Assay IAR p a t t e r n s were a l s o determined using an a n t i b i o t i c g r a d i e n t agar p l a t e assay. S t r a i n s were grown f o r four days in YEM broth and harvested by c e n t r i f u g a t i o n as p r e v i o u s l y d e s c r i b e d (see P r e p a r a t i o n of E x t r a c t s ) . A p p r o p r i a t e d i l u t i o n s of each s t r a i n were made i n YEM broth to y i e l d p f i n a l c e l l c o n c e n t r a t i o n s of 5 x 10 c e l l s / m l . A n t i b i o t i c g r a d i e n t agar p l a t e s ( S z y b a l s k i and Bryson, 1952) were made by: (1) pouring YEM agar i n t o g l a s s p e t r i d i s h e s (10 cm diameter) which were s l a n t e d such that the agar s o l i d i f i e d i n a very t h i n l a y e r at one edge of the p l a t e (ca. 1 mm) and i n a very t h i c k l a y e r at the other edge (ca. 20 mm); (2) the next day an e q u i v a l e n t volume of YEM agar with 2.8 mg/ml of an a n t i b i o t i c d i s s o l v e d i n i t was poured onto the agar s l a n t with p e t r i p l a t e s l y i n g f l a t . T h i s r e s u l t e d i n a r e l a t i v e l y high c o n c e n t r a t i o n of a n t i b i o t i c i n the agar where only a t h i n l a y e r of a n t i b i o t i c -f r e e agar had p r e v i o u s l y s o l i d i f i e d and a r e l a t i v e l y low c o n c e n t r a t i o n at the other end of the p l a t e where most of the a n t i b i o t i c - f r e e agar had accumulated. The p l a t e s were then l e f t at room temperature f o r three days to allow a n t i b i o t i c c o n c e n t r a t i o n s i n the agar to reach e q u i l i b r i u m before use i n the assay. S t e r i l e c o t t o n swabs were i n o c u l a t e d w i t h c e l l s u s p e n s i o n s d e s c r i b e d above and a s i n g l e i n o c u l a t i n g s t r e a k was made f o l l o w i n g t h e g r a d i e n t f r o m one end o f t h e a n t i b i o t i c p l a t e t o t h e o t h e r . A f t e r t h r e e d a y s i n c u b a t i o n a t 30°C, s t r e a k l e n g t h on t h e p l a t e s was meas u r e d . S t a n d a r d s t r e a k l e n g t h s were done i n d u p l i c a t e . A n t i b i o t i c s t e s t e d i n t h i s way i n c l u d e d v a n c o m y c i n , r i f a m p i c i n , n o v o b i o c i n , a m p i c i l l i n , s t r e p t o m y c i n , g e n t a m y c i n , n a l i d i x i c a c i d , p o l y m y x i n B, c a r b e n i c i l l i n , c e p h a l o t h i n , n e o m y c i n , k a n a m y c i n , c h l o r a m p h e n i c o l , and e r y t h r o m y c i n . Of t h e s e , t h e l a t t e r s i x p r o v e d u s e f u l i n s t r a i n i d e n t i f i c a t i o n of R. t r i f o l i i . E i g h t e e n unknown s t r a i n s were g e n e r a t e d f o r t h e s t u d y a s d e s c r i b e d i n t h e p r e v i o u s s e c t i o n w i t h t h e e x c e p t i o n t h a t YEM b r o t h and a g a r was u s e d i n s t e a d o f MGA b r o t h and a g a r . S t r e a k l e n g t h was a s s i g n e d "0" f o r c o m p l e t e i n h i b i t i o n ; " 1 " i f some g r o w t h a l o n g t h e g r a d i e n t was a p p a r e n t ; and "2" i f gr o w t h o c c u r r e d t h e l e n g t h o f t h e g r a d i e n t ( i e . r e s i s t a n c e t o t h e a n t i b i o t i c was o b s e r v e d ) . U s i n g t h i s s c o r i n g s y s t e m , v i r t u a l l y no v a r i a t i o n i n IAR p a t t e r n s w i t h i n t h e a s s a y was o b s e r v e d . RESULTS Examination of p r o t e i n p r o f i l e s of s i x R. trifolii i s o l a t e s using SDS-PAGGE (7.5%-15% l i n e a r g r a d i e n t ) r e v e a l e d s e v e r a l d i f f e r e n c e s between i s o l a t e s (Figure 1-1). I s o l a t e L1 (second from l a s t lane, F i g u r e 1-1) was co n s i d e r e d to be the most d i s t i n g u i s h a b l e of the group on the b a s i s of these d i f f e r e n c e s . Each of the s i x i s o l a t e s possessed some unique c h a r a c t e r i s t i c s i n t h e i r p r o t e i n p r o f i l e s . A f t e r s e v e r a l runs on l i n e a r g r a d i e n t and s i n g l e c o n c e n t r a t i o n g e l s , i s o l a t e s L1, L3, L5, and L6 were chosen f o r serology as these c o n s i s t e n t l y showed unique q u a l i t a t i v e d i f f e r e n c e s i n banding p a t t e r n s . A n t i s e r a r a i s e d a g a i n s t if. trifolii i s o l a t e s L1 and L6 showed the hig h e s t t i t e r when t e s t e d i n the drop a g g l u t i n a t i o n assay while L3 was f o u r - f o l d lower (Table 1-1). S t r a i n L1 was most s p e c i f i c as antiserum r a i s e d a g a i n s t these c e l l s c r o s s - r e a c t e d with i s o l a t e s L5 and L6 only at the 1:50 d i l u t i o n and not at a l l with L3. L1 c e l l s d i d not r e a c t with any of the three heterologous a n t i s e r a . I s o l a t e L5 showed the l e a s t s p e c i f i c response as antiserum r a i s e d a g a i n s t t h i s s t r a i n c r o s s - r e a c t e d with L3 and L6 c e l l s at antiserum d i l u t i o n s of 1:25 and 1:200, r e s p e c t i v e l y , while L5 c e l l s r e a c t e d with a l l a n t i s e r a t e s t e d to v a r y i n g degrees. I s o l a t e s L3 and L6 a l s o showed s u b s t a n t i a l c r o s s - r e a c t i v i t y ; L6 antiserum and c e l l s were p a r t i c u l a r l y r e a c t i v e with c e l l s and antiserum of i s o l a t e L5. C o n s i d e r i n g the f i n d i n g s using the drop a g g l u t i n a t i o n assay, r e s u l t s obtained with g e l immunodiffusion were 34 L6 L5 L4 L3 L2 L l BSA F i g u r e 1-1. S o l u b l e p r o t e i n s of s i x i s o l a t e s of Rhizobium trifolii de termined by sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g r a d i e n t g e l e l e c t r o p h o r e s i s (SDS-PAGGE) G e l s were composed of a l i n e a r g r a d i e n t 7.5%-15% p o l y a c r y l a m i d e . L 1 - L 6 r e p r e s e n t R. trifolii i s o l a t e s 1-6, r e s p e c t i v e l y . BSA - bovine serum albumin (1 mg/ml PBS) Table 1-1. S e n s i t i v i t y of an t i g e n / a n t i b o d y combinations using the drop a g g l u t i n a t i o n procedure with four s t r a i n s of Rhizobium trifolii ANTI SERUM L1 L3 L5 L6 L1 1600 / / / A N T L3 / 400 25 100 I G E L5 50 50 800 200 N L6 50 / 200 1 600 100 u l anti g e n (heat t r e a t e d whole c e l l s ) were mixed with 100 u l of antiserum d i l u t e d with PBS. Values presented are r e c i p r o c a l d i l u t i o n s above which a g g l u t i n a t i o n was not apparent. (/)-no r e a c t i o n somewhat s u r p r i s i n g (Table 1-2). As long as a n t i g e n and antiserum c o n c e n t r a t i o n s were above the l i m i t s of d e t e c t i o n q of the assay (ca. 10 c e l l s / m l and 1:8 antiserum:PBS, r e s p e c t i v e l y ) homologous antigen/antiserum combinations always produced p r e c i p i t i n bands while no unwanted c r o s s r e a c t i o n s were ever observed. T h i s o b s e r v a t i o n was a l s o c o n s i s t e n t with v a r i a b l e spacing between w e l l s i n the hexagon from very c l o s e (0.5 cm) to q u i t e separated (1.0 cm). A l l attempts to take advantage of t h i s s p e c i f i c i t y u s ing antigen p r e p a r a t i o n s from a s i n g l e c l o v e r nodule were u n s u c c e s s f u l , as was expected from the comments of Dudman (1977) r e g a r d i n g immunodiffusion of e x t r a c t s from small root nodules. When suspensions of whole Rhizobium c e l l s ( F i gure 1-2) or nodule t i s s u e e x t r a c t s ( F i g u r e 1-3) of three s e l e c t e d s t r a i n s were used as a n t i g e n s , an obvious q u a n t i t a t i v e r e l a t i o n s h i p between c o n c e n t r a t i o n of a n t i g e n and o p t i c a l 2 d e n s i t y of the s u b s t r a t e s o l u t i o n c o u l d be seen (R >0.95) usi n g the ELISA. R e s u l t s with heated nodule p r e p a r a t i o n s were p a r t i c u l a r l y encouraging as c u l t u r i n g of the microsymbiont c o u l d be avoided i f s u f f i c i e n t s p e c i f i c i t y between homologous antigen/antiserum combinations c o u l d be demonstrated. A s u b s t a n t i a l degree of s p e c i f i c i t y was shown to e x i s t even when a s i n g l e root nodule was crushed i n PBS and assayed without weighing, h e a t i n g , or the use of c r o s s adsorbed IgG p r e p a r a t i o n s . In such ELISA t e s t s , the homologous combinations always showed the h i g h e s t o p t i c a l d e n s i t y . However, the degree of c r o s s r e a c t i o n with heterologous 37 Table 1-2. S p e c i f i c i t y of antigen/antibody combinations using the g e l immunodiffusion procedure with four s t r a i n s of Rhizobium trifolii ANTISERUM L1 L3 L5 L6 L1 A N T L3 I G E L5 N L6 Antigens used were heated whole c e l l p r e p a r a t i o n s . Antiserum used was e i t h e r u n d i l u t e d or d i l u t e d 1:2, 1:4, or 1:8 with PBS. Spacing between w e l l s was 0.5 cm, 0.75 cm, or 1.0 cm. (+) p r e c i p i t i n bands e v i d e n t ; (-) no p r e c i p i t i n bands detected 38 (a) o I 2 3 4 5 6 7 Concenlralion of Cells x 10'' (b) •5. o-M o 2 3 4 5 6 7 Concenlralion of Cells x 10"* (C) >. 0.4 'to <§ "5 u •5. 0 2 o 0.1 I T 1 1 1 r 1 1 r 1 2 3 4 5 6 7 8 Concentration o( Cells x 10 ' F i g u r e 1-2. D e t e c t i o n of homologous s t r a i n s of Rhizobium trifolii whole c e l l s u s i n g the E L I S A S p e c i f i c a n t i s e r u m was d i l u t e d w i t h PBS as f o l l o w s : L1-1/400; L5 and L6-1/100. Means and s t a n d a r d e r r o r s . n=2. (a) o p t i c a l d e n s i t y when R. trifolii s t r a i n L1 c e l l s r e a c t w i t h s t r a i n L1 a n t i s e r u m ; (b) o p t i c a l d e n s i t y when R. trifolii s t r a i n L5 c e l l s r e a c t w i t h s t r a i n L5 a n t i s e r u m ; and ( c ) o p t i c a l d e n s i t y when s t r a i n L6 c e l l s r e a c t w i t h s t r a i n L6 a n t i s e r u m 39 (a) (b) Q o o.io Q "6 0.10-0.01 0.02 0.03 0.04 0.05 Nodule Tissue (mg/ml) 0.01 0.02 0.03 0.04 Nodule Tissue (mg/ml) 0.05 (c) o "B o.io a 0.01 0.02 0.03 0.04 0.05 Nodule Tissue (mg/ml) Figure 1-3 Detection of homologous strains of Rhizobium trifolii in root nodule extracts using the ELISA Specific antiserum was diluted with PBS as follows: L1-1/400; L5 and L6-1/100. Means and standard errors. n=2. (a) optical density when R. trifolii strain L1 extract reacts with strain L1 antiserum; (b) optical density when R. trifolii strain L5 extract reacts with strain L5 antiserum; and (c) optical density when strain L6 extract reacts with strain L6 antiserum antigens was q u i t e v a r i a b l e and l a r g e , sometimes 60%-70% of the homologous r e a c t i o n (data not shown). The a g g l u t i n a t i o n r e a c t i o n (Table 1-1) and i n i t i a l ELISA r e s u l t s before c r o s s a d s o r p t i o n of IgG (Table 1-3) r e v e a l e d a tendency f o r s u b s t a n t i a l c r o s s - r e a c t i o n between s t r a i n s L5 and L6. T h i s o b s e r v a t i o n suggested that a s u b s t a n t i a l degree of homology e x i s t e d between these s t r a i n s as L5 nodule p r e p a r a t i o n s showed > 50% c r o s s - r e a c t i v i t y with L6 antiserum compared with i t s r e a c t i o n to homologous L5 IgG (Table 1-3). A f t e r c r o s s a d s o r p t i o n of each IgG with i t s corresponding heterologous a n t i g e n s (see M a t e r i a l s and Methods), the ELISA was repeated and a s u b s t a n t i a l improvement in s p e c i f i c i t y was observed (Table 1-3, values i n p a r e n t h e s i s ) . The c r o s s -r e a c t i v i t y noted with L5 nodule p r e p a r a t i o n s and L6 antiserum was reduced.to l e s s than one h a l f of the value before c r o s s a d s o r p t i o n (21% vs 52% c r o s s - r e a c t i v i t y ) . The procedure a l s o seemed to remove n o n s p e c i f i c components of c r o s s - r e a c t i v i t y from s t r a i n L6 antiserum as i t c o n c o m i t a n t l y became l e s s r e a c t i v e with nodule e x t r a c t s of s t r a i n L1. V a r i a b i l i t y between ELISA's i n c r o s s r e a c t i v i t y to heterologous a n t i s e r a d i d occur (Table 1-4), but was c o n s i s t e n t and easy to d e f i n e . When the l e v e l of c r o s s -r e a c t i o n exceeded a l i m i t never a t t a i n e d by heterologous antibody/pure a n t i g e n r e a c t i o n s , a m i n o r i t y component c o u l d be concluded to be present i n the a n t i g e n p r e p a r a t i o n . A value of 50% c r o s s - r e a c t i o n was a r b i t r a r i l y s e l e c t e d . Thus, i f the second h i g h e s t o p t i c a l d e n s i t y i n an ELISA was g r e a t e r than 50% of the h i g h e s t , the a n t i g e n p r e p a r a t i o n was Table 1-3. ELISA using unadsorbed IgG r a i s e d a g a i n s t Rhizobium trifolii s t r a i n s L1 , L5, and L6, (values not i n p a r e n t h e s i s ) , compared with ELISA using L5 and L6 IgG c r o s s adsorbed with L6 and L5 c e l l s , r e s p e c t i v e l y (values i n p a r e n t h e s i s ) ANTISERUM % c r o s s r e a c t i o n by heterologous s t r a i n s L1 L5 L6 L1 A N T I G E N L6 L5 .43+.03 (.43+.03) .08+.01 (.08+.01) .09+.01 (.09+.01 ) .05+.01 (.04+.01) .36+.03 (.42+.02) .09+.03 (.09+ .01) .12+.02 (.07+.01) .19+.03 (.09+.01) .51+.02 (.45+.03) 27% (16%) 52% (21%) 18% (21%) Antigens were heated nodule e x t r a c t s (0.3 mg/ml nodule t i s s u e f r e s h weight) i n PBS. IgG d i l u t i o n s (with PBS) were: L1 - 1:400, L5 and L6 - 1:100. % c r o s s r e a c t i o n r e f e r s to the second h i g h e s t OD expressed as a percentage of the hig h e s t OD f o r a given a n t i g e n . Values are means and standard e r r o r s of O D 4 0 5 o b s e r v e d i n ELISA. n=2 42 T a b l e 1-4. P e r c e n t a g e c r o s s r e a c t i o n of r o o t nodule e x t r a c t s i n f e c t e d w i t h s t a n d a r d s t r a i n s of Rhizobium trifolii by h e t e r o l o g o u s a n t i s e r u m A n t i g e n Mean % c r o s s r e a c t i o n o b s e r v e d w i t h h e t e r o l o g o u s a n t i s e r u m L1 20%+2% L5 31%+2% L6 24%+2% V a l u e s p r e s e n t e d a r e the h i g h e s t h e t e r o l o g o u s OD o b s e r v e d f o r an a n t i g e n e x p r e s s e d as a pe r c e n t a g e of the OD a t t a i n e d by the homologous a n t i g e n / a n t i b o d y c o m b i n a t i o n . R e s u l t s p r e s e n t e d a r e a composite of e l e v e n d i f f e r e n t e x p e r i m e n t s . n=2 per ex p e r i m e n t . V a l u e s a re means and s t a n d a r d e r r o r s 43 c o n s i d e r e d t o be a composite of two s t r a i n s . U s i n g t h i s c r i t e r i o n , the m i n o r i t y component i n an a n t i g e n m i x t u r e had t o comprise 23%-43% of the m i x t u r e t o be d e t e c t e d (Table 1-5). T h i s l e v e l of d e t e c t i o n meant t h a t d u a l l y - i n f e c t e d r o o t n odules w i t h r o u g h l y e q u i v a l e n t p r o p o r t i o n s of the s t r a i n s would always be d e t e c t e d , w h i l e mixed i n f e c t i o n s where the m i n o r i t y component c o m p r i s e d a v e r y s m a l l p r o p o r t i o n (< 23%) of the nodule m i c r o s y m b i o n t may be m i s s e d . R e s u l t s of an a n t i b i o t i c d i s k assay a r e summarized i n T a b l e s 1-6 t o 1-8. In t h i s s tudy s t r a i n L3 was s u b s t i t u t e d f o r s t r a i n L1 because i t s growth r a t e i n b r o t h was s i m i l a r t o s t r a i n s L5 and L6. S t r a i n L1 was much sl o w e r t o r e a c h s t a t i o n a r y phase i n b r o t h c u l t u r e than the l a t t e r two s t r a i n s and t h e r e f o r e seemed i n a p p r o p r i a t e f o r an experiment where i s o l a t e s would be competing f o r r e s o u r c e s i n a common b r o t h . An attempt t o i d e n t i f y unknown i s o l a t e s , g e n e r a t e d from a m i x t u r e of known s t r a i n s , by comparing t h e i r response t o a n t i b i o t i c s w i t h " s t a n d a r d " p a t t e r n s of the known s t r a i n s i s p r e s e n t e d i n Table 1-6. A n t i b i o t i c s t e s t e d were kanamycin, neomycin, n a l i d i x i c a c i d , c e p h a l o t h i n , and c h l o r a m p h e n i c o l a l l a t 30 ug of a n t i b i o t i c per d i s k , e r y t h r o m y c i n (15 ug), and a m p i c i l l i n , gentamycin, and s t r e p t o m y c i n a t 10 ug per d i s k . Of the e i g h t a n t i b i o t i c s t e s t e d , kanamycin, n a l i d i x i c a c i d and c e p h a l o t h i n r e s u l t e d i n p o t e n t i a l l y d i s c r i m i n a t i n g growth p a t t e r n s . The i d e n t i t y was a s s i g n e d t o the s t a n d a r d s t r a i n t h a t had i t s c h a r a c t e r i s t i c s a p p e a r i n g most f r e q u e n t l y i n the Table 1-5. Percentage (v/v) of m i n o r i t y a n t i g e n component in a n t i q e n mixtures r e q u i r e d f o r d e t e c t i o n by ELI SA m a j o r i t y m i n o r i t y % (v/v) m i n o r i t y component component component r e q u i r e d for d e t e c t i o n L1 L5 39+4% L1 L6 43 + 2% L5 L1 25+7% L5 L6 23 + 7% L6 L1 24 + 2% L6 L5 27 + 7% Antigens were heated nodule e x t r a c t s c o n t a i n i n g Rhizobium trifolii s t r a i n s L l , L5, or L6 (0.3 mg nodule t i s s u e f r e s h weight per ml PBS heated at 95 C fo r 1 hour). Mixtures were composed of 0%, 10%, 20%, 30%, 40%, and 50% (v/v) of the m i n o r i t y component. M i n o r i t y a n t i g e n components were d e t e c t e d when the second h i g h e s t OD was g r e a t e r than 50% of highest OD. Means and standard e r r o r s of f i v e experiments are presented. n=2 per experiment Table 1-6. Standard s t r a i n s of Rhizobium trifolii most c l o s e l y resembling the response of unknown i s o l a t e s to a n t i b i o t i c s and i d e n t i f i c a t i o n of unknowns using IAR patterns and double d i f f u s i o n (DD) STANDARD STRAIN RESPONSE TO IDENTITY OF UNKNOWN UNKNOWN ANTIBIOTICS ISOLATE USING KAN NA CEP IAR DD 1 L3/L6 L6 NEW L6 L5 2 L3/L6 L6 L3/L5 L3/L6 L5 3 L6 L5 L3 L5 4 L6 L5 L6 L6 L5 5 L3/L6 NEW NEW L3/L6 L3 6 L3/L6 L3 L6 L3/L6 L6 7 L3/L6 L3 L6 L3/L6 L6 8 L3/L6 L6 L6 L6 L6 9 L3/L6 L3L/L6 L6 L6 L6 10 L6 L6 L6 L6 L6 1 1 L3/L6 L5 L6 L6 L5 12 L6 NEW NEW L6 L6 KAN, NA, and CEP are kanamycin, n a l i d i x i c a c i d , and cephalothin, r e s p e c t i v e l y . (NEW) - response not c h a r a c t e r i s t i c of any parental s t r a i n (?) - not i d e n t i f i a b l e Table 1-7. Diameter of growth i n h i b i t i o n zone of Rhizobium trifolii s t r a i n s L3, L5, and L6 in response to a n t i b i o t i c d i s k s (means and standard e r r o r s ) when grown as reference c u l t u r e s in the same experiment as unknown i s o l a t e s , and i d e n t i f i c a t i o n of unknowns using IAR patterns and double d i f f u s i o n STRAIN DIAMETER OF INHIBITION ZONE (mm) IDENTITY USING KAN NA CEP IAR DD L3 23.0±1.0 29.6±0.6 25.0±1 .0 / L3 L5 15.6±0.6 35.0±1.0 19.0±1 .0 / L5 L6 24.0±2.0 25.011.5 12.0±1 .8 / L6 JNKNOWN REFERENCE STRAIN PATTERN 1 L5 L3/L5 L3 L3/L5 L5 2 NEW L3 L5 L3/L5 L5 3 L5 L5 L6 L5 L5 4 L5 L5 L6 L5 L5 5 L3/L6 NEW NEW L3/L6 L3 6 L3/L6 L6 L6 L6 L6 7 L3/L6 L6 NEW L6 L6 8 L3/L6 L3/L6 L6 L6 L6 9 L3/L6 L6 L6 L6 L6 10 L6 L3 L6 L6 L6 11 L3/L6 NEW L3 L3 L5 12 L6 NEW NEW L6 L6 KAN, NA, and CEP are kanamycin, n a l i d i x i c a c i d , and cephalothin, r e s p e c t i v e l y . (NEW) - response not c h a r a c t e r i s t i c of any parental s t r a i n Table 1-8. Diameter of growth i n h i b i t i o n zone in response to a n t i b i o t i c d i s k s (means and standard er r o r s ) and reactions of known (Rhizobium trifolii s t r a i n s L3, L5, and L6) and unknown (progeny) i s o l a t e s with s t r a i n s p e c i f i c a n t i s e r a STRAIN DIAMETER OF INHIBITION ZONE (mm) ANTISERUM KAN NA CEP L3 L5 L6 L3 20.8±2.5 20.215.4 16.015.2 + -L5 13.2±1.4 35.010.6 20.0+0.8 - + -L6 20.014.0 29.612.4 8.611.6 - + UNKNOWN 1 17.0±1.0 32.010.0 28.611.6 + -2 18.610.6 30.6+0.6 21.610.6 + -3 17.6+1.6 37.0+1.0 14.611.6 + -4 16.610.6 33.011.0 11.011.0 + -5 23.011.0 51.610.6 49.013.0 + -6 21.0+1.0 23.610.6 9.011.0 - + 7 23.011.0 24.610.6 5.610.6 - + 8 24.610.6 27.6+0.6 9.0+1.0 - + 9 24.610.6 26.610.6 10.011.0 - + 10 25.6±0.6 29.610.6 11.0+1.0 - + 1 1 1 7.011 . 0 35.01.1 .0 11.610.6 + -12 25.010.0 52.0+0.0 47.011.0 - + KAN, NA, and CEP are kanamycin, n a l i d i x i c a c i d , and cephalothin, r e s p e c t i v e l y r e s i s t a n c e p a t t e r n of the unknown. D e c i s i o n s c o u l d be reached i n seven of the 12 cases, but these were i n agreement with the double d i f f u s i o n r e s u l t s i n only four cases. Only one i s o l a t e , number 10, was p o s i t i v e l y i d e n t i f i e d as i t s IAR p a t t e r n was i d e n t i c a l to that of s t r a i n L6. I s o l a t e s 2, 5, 6, and 7 c o u l d be narrowed down to one of two p a r e n t a l s t r a i n s while unknown i s o l a t e 3 c o u l d not be i d e n t i f i e d because i t s IAR p a t t e r n was a composite of a l l three p a r e n t a l s t r a i n s (Table 1-6). Greater success i n i d e n t i f y i n g unknowns was observed when r e f e r e n c e c u l t u r e s were i n c l u d e d i n the experiment with unknown i s o l a t e s (Table 1-7). The i d e n t i t y of 8 out of 12 unknowns were in agreement with s e r o l o g i c a l r e s u l t s . However, only two i s o l a t e s (numbers 6 and 9) were i d e n t i f i e d unambiguously as a l l o t h e r s s t i l l showed a mixture of p a r e n t a l r e s i s t a n c e p a t t e r n s . A l s o , o n e - t h i r d of the s t r a i n s were e i t h e r u n i d e n t i f i a b l e (numbers 1, 2, and 5) or i d e n t i f i e d i n c o r r e c t l y (number 11) using t h i s "improved" method. In c o n t r a s t to the IAR r e s u l t s , each unknown re a c t e d with only one antiserum u s i n g the double d i f f u s i o n assay a l l o w i n g r a p i d and unambiguous i d e n t i f i c a t i o n s t o be made (Table 1-8). F i n a l l y , r e s u l t s from an a n t i b i o t i c g r a d i e n t agar p l a t e assay u s i n g s t r a i n s of R. trifolii d i f f e r e n t from those used i n the pr e v i o u s study are presented i n Table 1-9. In t h i s experiment only 4 of 18 unknown i s o l a t e s showed IAR p a t t e r n s i d e n t i c a l to a p a r e n t a l p a t t e r n (numbers 3, 11, 13, and 17). Guesses c o u l d be made with 11 of the remaining 14 i s o l a t e s . Table 1-9. Growth response of four s t r a i n s of Rhizobium trifolii and unknown "progeny" i s o l a t e s to a n t i b i o t i c concentration gradients REFERENCE PATTERNS ANTIBIOTIC KNOWN STRAIN ERY KAN NEO CEP CHL CAR IDENTITY A3 2 2 2 2 0 0 A3 A6 0 1 2 2 2 2 A6 A7 0 2 1 2 2 2 A7 C5 0 2 2 0 2 1 C5 UNKNOWN REFERENCE STRAIN PATTERN 1 1 2 2 1 2 2 ? 2 0 1 2 2 2 1 A6 3 0 1 2 2 2 2 A6 4 1 2 2 0 2 1 C5 5 2 2 2 2 1 0 A3 6 1 2 2 2 2 2 ? 7 1 2 2 0 2 1 C5 8 1 2 2 0 2 1 C5 9 1 2 2 0 2 1 C5 10 0 1 2 2 2 1 A6 1 1 2 2 2 2 0 0 A3 1 2 1 2 2 0 2 0 C5 13 0 1 2 2 2 2 A6 14 1 2 2 0 2 1 C5 15 1 2 2 0 2 1 C5 16 1 2 2 1 2 2 C5 17 0 1 2 2 2 2 A6 18 2 2 2 2 2 2 ? Standard patterns were determined i n a previous experiment. A n t i b i o t i c s t e sted were: erythromycin (ERY), kanamycin (KAN), neomycin (NEO), cephalothin (CEP), chloramphenicol (CHL), and c a r b e n i c i l l i n (CAR). Scoring system used was "0" i f no growth was observed, "1" i f some growth along a po r t i o n of the gradient was observed, and "2" i f s o l i d growth along the e n t i r e gradient was observed. A n t i b i o t i c concentrations ranged from near 0 to 2.8 mg/ml. (?) - not i d e n t i f i a b l e while 3 i s o l a t e s (numbers 1, 6, and 18) had t o t a l l y new p a t t e r n s . Three out of four p a r e n t a l s t r a i n s (A6, A7, and C5) were completely i n h i b i t e d by erythromycin c o n c e n t r a t i o n s used i n the assay while the f o u r t h was completely r e s i s t a n t (A3). When unknown i s o l a t e IAR p a t t e r n s were determined, 10 out of 18 s t r a i n s showed " h y b r i d " erythromycin r e s i s t a n c e as they were able to grow on lower c o n c e n t r a t i o n s of the a n t i b i o t i c but not on the h i g h e s t ones. 51 DISCUSSION SDS-PAGGE has been shown to be u s e f u l i n s c r e e n i n g a l a r g e number of R. trifolii i s o l a t e s f o r p o t e n t i a l a n t i g e n i c d i f f e r e n c e s (Dughri and Bottomley, 1983ab). Information based on s o l u b l e p r o t e i n p r o f i l e s of R. trifolii obtained in t h i s study p e r m i t t e d the s e l e c t i o n of four d i s t i n c t i s o l a t e s fo r s e r o l o g i c a l a n a l y s i s . Antiserum subsequently r a i s e d a g a i n s t these i s o l a t e s proved to be completely s p e c i f i c f o r homologous s t r a i n s when t e s t e d i n the immunodiffusion system. Gel e l e c t r o p h o r e s i s may be c o n s i d e r e d u s e f u l f o r t h i s purpose, but p r e d i c t i o n of a n t i g e n i c i t y u s i n g SDS-PAGGE i s not d e f i n a t i v e . The c o r r e l a t i o n between uniqueness of p r o t e i n p r o f i l e and a n t i g e n i c determinants was p e r f e c t i n t h i s study, but i s o l a t e s of R. japonicum (Noel and B r i l l , 1980), R. meliloti (Fuquay et a l . , 1984), and R. trifolii (Dughri and Bottomley, 1983a,1983b) have been shown to possess d i f f e r e n t p r o t e i n p r o f i l e s while belonging to the same serogroup. T h i s phenomenon d e t r a c t s from the u s e f u l n e s s of SDS-PAGGE i n p r e d i c t i n g a n t i g e n i c i t y , but o f f e r s an a c c e s s o r y method of s t r a i n i d e n t i f i c a t i o n beyond the c o n s t r a i n t s of s e r o t y p i n g . Due to the degree of f i n e d e t a i l p r o v i d e d and v a r i a b i l i t y between assays, i t i s not p r a c t i c a l to use SDS-PAGGE and two-dimensional s l a b g e l (Roberts et a l . , 1980) p r o t e i n p r o f i l e s e x c l u s i v e l y i n a s t r a i n i d e n t i f i c a t i o n scheme. However, these techniques p r o v i d e powerful t o o l s i f f u r t h e r c h a r a c t e r i z a t i o n of otherwise c l o s e l y r e l a t e d i s o l a t e s i s d e s i r e d . The other n o n s e r o l o g i c a l technique i n v e s t i g a t e d f o r i d e n t i f y i n g s t r a i n s of R. trifolii appears to be l e s s u s e f u l than SDS-PAGGE. The r e s u l t s presented i n Tables 1-6 to 1-9 suggest that IAR p a t t e r n s are of dubious value i n d i s c r i m i n a t i o n and i d e n t i f i c a t i o n of s t r a i n s belonging to t h i s s p e c i e s . Two major problems with the i n t r i n s i c a n t i b i o t i c r e s i s t a n c e assay are apparent from these data. F i r s t , there i s c o n s i d e r a b l e v a r i a b i l i t y between experiments. C u l t u r e s t r e a t e d i d e n t i c a l l y , but analyzed on separate o c c a s i o n s , showed v a r i a b l e IAR p a t t e r n s as evidenced by the r e l a t i v e l y l a r g e standard e r r o r s of the known s t r a i n data (Table 1-8). T h i s v a r i a b i l i t y i n time i s a l s o r e f l e c t e d i n the o b s e r v a t i o n that more i s o l a t e s c o u l d be c o r r e c t l y i d e n t i f i e d when r e f e r e n c e c u l t u r e s were i n c l u d e d i n the same t e s t s with unknowns (Table 1-7) as opposed to comparing the unknowns to a set of standard data compiled from d i f f e r e n t experiments (Table 1-6). S t r a i n s of R. phaseoli have been shown to possess t h i s c h a r a c t e r i s t i c a l s o (Josey et a l . , 1979). Second, when s t r a i n s are grown together i n mixtures, some degree of g e n e t i c exchange between them may be expected. C h a r a c t e r i s t i c s which are s t a b l e to such " p r o m i s c u i t y " must be used i f s u c c e s s f u l s t r a i n i d e n t i f i c a t i o n from mixtures i s to be accomplished. Such s t a b i l i t y was not the case when IAR p a t t e r n s between s t r a i n s were ana l y z e d . Most of the unknown i s o l a t e s possessed a composite of p a r e n t a l p a t t e r n s , c r e a t i n g a major problem when unknowns were compared to "standard" p a t t e r n s (Table 1-6). For example, unknown i s o l a t e 3, which was a n t i g e n i c a l l y s i m i l a r to s t r a i n L5, possessed kanamycin r e s i s t a n c e c h a r a c t e r i s t i c of s t r a i n L6, n a l i d i x i c a c i d t o l e r a n c e s i m i l a r to s t r a i n L5, and c e p h a l o t h i n r e s i s t a n c e t y p i c a l of s t r a i n L3. In f a c t , only i s o l a t e 10 d i s p l a y e d r e s i s t a n c e r e a c t i o n s to a l l three a n t i b i o t i c s that were c h a r a c t e r i s t i c of a s i n g l e standard s t r a i n (L6). A l l other s t r a i n s t e s t e d showed composite or new IAR p a t t e r n s (Tables 1-6 and 1-8). Use of r e f e r e n c e s t r a i n s i n the experiment reduced t h i s v a r i a b i l i t y but composite IAR p a t t e r n s were s t i l l predominant (Table 1-7, see r e a c t i o n of i s o l a t e s 1, 3, and 4 to c e p h a l o t h i n ) . No improvement was observed when d i f f e r e n t s t r a i n s of R. trifolii were t e s t e d using a n t i b i o t i c c o n c e n t r a t i o n g r a d i e n t s i n agar p l a t e s (Table 1-9). As with the a n t i b i o t i c d i s k assay, recombination appeared to be the r u l e r a t h e r than the e x c e p t i o n s i n c e o n l y 4 out of 18 unknown IAR p a t t e r n s were i d e n t i c a l to a p a r e n t a l one. Perhaps the most obvious example of g e n e t i c reassortment i n t h i s type of system i s i l l u s t r a t e d by the erythromycin r e s i s t a n c e data of the p a r e n t a l and unknown s t r a i n s (Table 1-9). Three of four p a r e n t a l s t r a i n s c o u l d not t o l e r a t e any of the a n t i b i o t i c i n the growth medium while the f o u r t h was completely r e s i s t a n t to the l e v e l s used i n the experiment. Examination of IAR p a t t e r n s generated by t e s t i n g "progeny" s i n g l e colony i s o l a t e s r e v e a l e d that g r e a t e r than 50% (10 out of 18) of the s t r a i n s had obtained p a r t i a l erythromycin r e s i s t a n c e such that they c o u l d not grow the l e n g t h of the a n t i b i o t i c g r a d i e n t but were found part of the way along i t where c o n c e n t r a t i o n s were lower. Thus, a h y b r i d r e s i s t a n c e to erythromycin had developed d u r i n g the four days p a r e n t a l s t r a i n s shared a common b r o t h . Such re c o m b i n a t i o n a l events are not s u r p r i s i n g s i n c e r e s i s t a n c e to many a n t i b i o t i c s i s coded for by plasmid DNA which may be f r e e l y exchanged between b a c t e r i a l c e l l s of s i m i l a r c o m p a t i b i l i t y groups ( B e r i n g e r , 1974). While IAR may be of use i n a manner analogous to SDS-PAGGE ( i e . as an acces s o r y i d e n t i f i c a t i o n technique) (Benyon and Josey, 1980; B r o m f i e l d et a l . , 1982; K i n g s l e y and B o h l o o l , 1983), i t s use as a primary i d e n t i f i c a t i o n scheme f o r s t r a i n s of R. trifolii would r e q u i r e c a r e f u l e v a l u a t i o n i n r e l a t i o n to the problems documented i n t h i s t h e s i s . C l e a r l y , the s i m p l e s t , most s t a b l e , and unambiguous i d e n t i f i c a t i o n scheme was based on a n t i g e n i c determinants i n R. t r i f o l i i . I n i t i a l l y , the drop a g g l u t i n a t i o n assay was t e s t e d with promising r e s u l t s while e x c e l l e n t r e s u l t s were a l s o o b tained with double d i f f u s i o n . The i n t r i n s i c disadvantage of both of these techniques i s t h e i r r e l a t i v e l a c k of s e n s i t i v i t y which r e s t r i c t s t h e i r use to an t i g e n p r e p a r a t i o n s of whole Rhizobium c e l l s or of l a r g e s i n g l e nodules such as i n soybean (Means et a l . , 1964). However, a g g l u t i n a t i o n r e a c t i o n s are c o n s i d e r e d to be up to one thousand times more s e n s i t i v e than those of the double d i f f u s i o n assay (Dughri and Bottomley, 1983a). Thus, Parker and Grove (1970) were ab l e to t e s t e x t r a c t s of nodules from Trifolium repens f o r s t r a i n s of R. trifolii using a m i c r o a g g l u t i n a t i o n technique. U n f o r t u n a t e l y , each crushed nodule c o u l d only be t e s t e d a g a i n s t a s i n g l e antiserum. T h i s r e s t r i c t i o n would s e v e r e l y l i m i t the scope of any study r e l y i n g on t h i s procedure as i t would be of use only i f a s i n g l e inoculum s t r a i n was to be f o l l o w e d through the course of an experiment. C o n s i d e r i n g the t e c h n i c a l problems a s s o c i a t e d with the assays ( i e . n o n - a g g l u t i n a b i l i t y , a u t o a g g l u t i n a t i o n , and d i f f i c u l t y i n d e t e c t i n g t r u e a g g l u t i n a t i o n ) , these procedures are not p a r t i c u l a r l y s u i t a b l e f o r broad spectrum s t r a i n i d e n t i f i c a t i o n i n R. t r i f o l i i . In these s t r a i n a n a l y s e s , i t i s d e s i r a b l e to a v o i d the n e c e s s i t y to c u l t u r e the microsymbiont i s o l a t e d from root nodules to e l i m i n a t e the p o s s i b i l i t y of erroneous r e s u l t s due to contamination and to reduce the t e c h n i c a l workload.. T h e r e f o r e , s t r a i n i d e n t i f i c a t i o n techniques s e n s i t i v e enough to d e t e c t the microsymbiont d i r e c t l y i n i n d i v i d u a l small nodules were i n v e s t i g a t e d . S t r a i n L3 was dropped from these s t u d i e s because there was no corresponding Bacillus s t r a i n i s o l a t e d f o r f u r t h e r e c o l o g i c a l experimentation (see Chapter 3) . Techniques such as immunofluorescent antibody s t a i n i n g and enzyme-linked immunosorbent assays have been a p p l i e d to the study of root nodule b a c t e r i a to f a c i l i t a t e a n a l y s i s of i n d i v i d u a l nodules. IFAS was attempted with s t r a i n s L1, L5, and L6 i n t h i s work (data not shown). While homologous a n t i g e n / antibody combinations r e s u l t e d in d e t e c t a b l e f l u o r e s c e n c e , f u r t h e r work e v a l u a t i n g the s p e c i f i c i t y of IgG p r e p a r a t i o n s was not promising s i n c e at l e a s t some 56 f l u o r e s c i n g c e l l s c o u l d be d e t e c t e d w i t h any a n t i g e n / a n t i b o d y c o m b i n a t i o n t e s t e d . F u r t h e r m o r e , e s t i m a t i o n of t h e number o f f l u o r e s c i n g c e l l s p e r f i e l d d i d not a l w a y s p r o v i d e a c l e a r i n d i c a t i o n of w h i c h a n t i g e n / a n t i b o d y c o m b i n a t i o n s were homologous. T h e s e r e s u l t s were n o t e n t i r e l y u n e x p e c t e d i n v i e w of t h e a g g l u t i n a t i o n t e s t s and i n i t i a l E L I S A d a t a . C e r t a i n c e l l s u r f a c e a n t i g e n i c d e t e m i n a n t s a p p e a r e d t o be common ( e s p e c i a l l y between s t r a i n s L5 and L6) as c r o s s r e a c t i o n s t o v a r i o u s d e g r e e s between a n t i s e r u m and a n t i g e n s of t h e s e i s o l a t e s a l w a y s o c c u r r e d ( T a b l e s 1-1 and 1-3). In c o n t r a s t t o t h e o t h e r immunoassays, t h e r e was a l w a y s c o m p l e t e s p e c i f i c i t y between homologous a n t i g e n / a n t i b o d y c o m b i n a t i o n s when d o u b l e d i f f u s i o n was u s e d . T h e r e a r e two p o s s i b l e f a c t o r s w h i c h may c o n t r i b u t e t o t h e o b s e r v e d d i s c r e p a n c i e s between t h e t e c h n i q u e s . F i r s t i s t h e n a t u r e of t h e a n t i g e n . The O u c h t e r l o n y method depends on t h e d i f f u s i o n of a n t i b o d i e s and s o l u b l e a n t i g e n t o a "zone of e q u i v a l e n c e " i n t h e g e l . Where t h e s e two t y p e s o f m o l e c u l e s meet i n a p p r o x i m a t e l y e q u a l p r o p o r t i o n s , a network of i n t e r l o c k i n g a n t i g e n and a n t i b o d y m o l e c u l e s form a v i s i b l e p r e c i p i t a t e ( i e . p r e c i p i t i n b a n d s ) . Thus, t h e s u r f a c e a n t i g e n s d e t e c t e d by a g g l u t i n a t i o n , IFAS, o r E L I S A do n o t p l a y a m a j o r r o l e i n t h e p r e c i p i t a t i o n r e a c t i o n s d e t e c t e d by i m m u n o d i f f u s i o n . I t i s p o s s i b l e t n a t t h e a n t i g e n i c d e t e r m i n a n t ( s ) of t h e s o l u b l e a n t i g e n ( s ) a r e h i g h l y s p e c i f i c t h e r e b y e l i m i n a t i n g unwanted c r o s s r e a c t i o n s . The s e c o n d f a c t o r w h i c h may c o n t r i b u t e t o t h e s p e c i f i c i t y o b s e r v e d i n g e l i m m u n o d i f f u s i o n i s t h e i n t r i n s i c l a c k of s e n s i t i v i t y i n the assay. I t i s p o s s i b l e that common s o l u b l e a n t i g e n i c determinants capable of ca u s i n g unwanted c r o s s r e a c t i o n s are present i n the system, but at an i n s u f f i c i e n t c o n c e n t r a t i o n to e f f e c t p r e c i p i t i t a t i o n with heterologous antiserum. However, no evidence of t h i s phenomenon was observed when an t i g e n c o n c e n t r a t i o n s one hundred f o l d above the l i m i t s of d e t e c t i o n were t e s t e d a g a i n s t v a r i o u s c o n c e n t r a t i o n s of antiserum. Double d i f f u s i o n i s a l s o p a r t i c u l a r l y i n s e n s i t i v e to r e a c t i o n s i n v o l v i n g the IgM form of antibody (Vincent, 1982). IgM i s thought to be l a r g e l y r e s p o n s i b l e f o r c r o s s r e a c t i o n s i n a g g l u t i n a t i o n t e s t s (Schwinghamer and Dudman, 1980; V i n c e n t , 1982); t h e r e f o r e t h i s source of i n t e r f e r e n c e may a l s o not be det e c t e d i n a g e l system. Assays i n which heterologous c r o s s r e a c t i o n s were observed a l l d e t e c t i n s o l u b l e (eg. c e l l s u r f a c e ) a n t i g e n i c determinants i n v o l v e d i n a g g l u t i n a t i o n - t y p e r e a c t i o n s . The most probable cause of the c r o s s r e a c t i o n s i n these assays i s the occurrence of common a n t i g e n i c s i t e s on the sur f a c e of c e l l s or b a c t e r o i d s t e s t e d . Consequently, i t seems l i k e l y that the di s c r e p a n c y observed i n s p e c i f i c i t y between double d i f f u s i o n and the other assays e v a l u a t e d i s p r i m a r i l y i n the nature of the a n t i g e n , although assay s e n s i t i v i t y may a l s o be a c o n t r i b u t i n g f a c t o r . T h i s hypothesis to e x p l a i n observed d i f f e r e n c e s i s c o n s i s t e n t with the IFAS and ELISA data as both these techniques can detect c e l l s u r f a c e antigens d i r e c t l y . Thus, when IFAS was t e s t e d , some f l u o r e s c e n c e was always observed by c e l l s p o s s e s s i n g common a n t i g e n s . I t may have been p o s s i b l e to improve IFAS such that a b a s e l i n e amount of f l u o r e s c e n c e would be c o n s i d e r e d i n s i g n i f i c a n t , but concurrent work with ELISA suggested that the l a t t e r method was much l e s s s u b j e c t i v e than the f l u o r e s c e n t technique. Using ELISA, i t was p o s s i b l e to " d i l u t e out" the c r o s s r e a c t i n g a n t i g e n s by t e s t i n g s e v e r a l c o n c e n t r a t i o n s of a n t i g e n s and antiserum and s e l e c t i n g the most s p e c i f i c combination. Two c r i t i c a l f a c t o r s were d i s c o v e r e d that allowed c o n t r o l over the v a r i a t i o n i n , and magnitude of, unwanted c r o s s r e a c t i o n s such that t h i s t e s t became u s e f u l and c o n s i s t e n t . F i r s t , was the need to s t a n d a r d i z e the amount of nodule t i s s u e i n the a n t i g e n p r e p a r a t i o n by measuring nodule f r e s h weight before the ELISA was c a r r i e d out. I t was i n i t i a l l y thought that the v a r i a b i l i t y i n root nodule weight would not be important as most Trifolium nodules appear c o n s i s t e n t l y and u n i f o r m l y very small (ca. 1 mg f r e s h weight). While t h i s may be t r u e , the source of e r r o r which r e s u l t s when nodule weights are ignored can be seen i f s l o p e s of the r e l a t i o n s h i p between nodule e x t r a c t c o n c e n t r a t i o n and o p t i c a l d e n s i t y i n ELISA are examined (Fi g u r e 1-3). I t can be seen that the o p t i c a l d e n s i t y i s dependent on antigen c o n c e n t r a t i o n between 0.01 and 0.1 mg nodule t i s s u e per ml as slopes vary between 1.9 and 2.8 f o r the d i f f e r e n t a n t i g e n / a n t i s e r u m combinations. T h e r e f o r e , even small v a r i a t i o n s i n nodule weight w i t h i n t h i s range w i l l g r e a t l y a f f e c t the o p t i c a l d e n s i t y i n ELISA. The degree of s p e c i f i c i t y i n the assay was c o r r e l a t e d to 59 the o p t i c a l d e n s i t y i n that w e l l s overloaded with a n t i g e n r e a c t e d p a r t i a l l y with any antiserum t e s t e d . Under these c o n d i t i o n s , the magnitude of c r o s s r e a c t i o n r e l a t i v e to the homologous combinations was e l e v a t e d to unacceptably high l e v e l s . As a r e s u l t , i t was very important to have an accurate estimate of nodule t i s s u e c o n c e n t r a t i o n to produce r e l i a b l e r e s u l t s . The second important f a c t o r i n r e f i n i n g t h i s t e s t was the need to c r o s s adsorb antiserum with heterologous antigens to improve s p e c i f i c i t y i n the ELISA (Vincent, 1982). Using t h i s procedure, antibody molecules s p e c i f i c to the heterologous antigens or n o n s p e c i f i c s i t e s on otherwise s p e c i f i c antibody molecules should be removed or blocked. Olsen and Rice (1983) s u c c e s s f u l l y used t h i s technique to improve s p e c i f i c i t y of the ELISA i n e v a l u a t i n g R. meliloti s t r a i n s . Consequently, c o n d i t i o n s f o r a r e l i a b l e s e r o l o g i c a l procedure s u i t e d to use with nodule p r e p a r a t i o n s had been e s t a b l i s h e d where c r o s s r e a c t i v i t y between heterologous s t r a i n s was s u b s t a n t i a l l y reduced (3 to 5 f o l d ) compared to homologous r e a c t i o n s (Table 1-3). Repeated ELISA's demonstrated that the assay was c o n s i s t e n t (Table 1-4). A s i n g l e root nodule may be occupied by two a n t i g e n i c a l l y d i s t i n c t s t r a i n s of R. trifolii (Marques P i n t o et a l . , 1974; Brockwell et a l . , 1977; B r o m f i e l d and Jones, 1980b), t h e r e f o r e the l i m i t of d e t e c t i o n of the ELISA used i n these s t u d i e s (see Chapter 3) was i n v e s t i g a t e d to determine the a b i l i t y to d e t e c t mixed i n f e c t i o n s . Using the c r i t e r i o n that a m i n o r i t y component of a d u a l l y - i n f e c t e d nodule should show g r e a t e r than 50% of the o p t i c a l d e n s i t y of the m a j o r i t y component, i t was determined that the assay would d e t e c t the second component i f i t comprised 23%-43% of the mixture depending on the antiserum i n v o l v e d . Highest s e n s i t i v i t y was observed when antig e n combinations i n c l u d e d s t r a i n s L5 or L6 as the m a j o r i t y component. In these cases, the m i n o r i t y component, r e g a r d l e s s of i t s i d e n t i t y , would be d e t e c t e d i n dual i n f e c t i o n s i f i t comprised 20%-25% of nodule occupancy. If L1 was the m a j o r i t y component of a dual i n f e c t i o n , s t r a i n s L5 or L6 would have to comprise about 40% of the c e l l s before being d e t e c t e d . Thus i n a l l p o s s i b l e s c e n a r i o s of dual i n f e c t i o n , i f the m a j o r i t y component d i d not outnumber the m i n o r i t y component by more than 2.5 to 5 times, the double i n f e c t i o n would be d e t e c t e d . The r a t i o of s t r a i n s i n d u a l l y - i n f e c t e d root nodules has been estimated to vary from 2:1 to 10:1 i n soybean (Lindemann et a l . , 1974) and from 1:1 to 20:1 i n Medicago trunculata (Marques P i n t o et a l . , 1974). Brockwell et a l . (1977) estimated that the second s t r a i n may comprise o n l y a small p r o p o r t i o n of the t o t a l b a c t e r o i d p o p u l a t i o n i n a mixed i n f e c t i o n of R. t r i f o l i i . T h e r e f o r e , the ELISA used i n the present work would be expected to d e t e c t some, but not a l l of the dual i n f e c t i o n s t h at might occur. The confounding e f f e c t s of background absorbance and c r o s s r e a c t i v i t y i n the ELISA make IFAS the most s u i t a b l e technique f o r the d e t e c t i o n of mixed i n f e c t i o n s . Since i n d i v i d u a l b a c t e r o i d s are observed when the l a t t e r technique i s used, nodules with an 61 extremely low p r o p o r t i o n of a second s t r a i n c o u l d be i d e n t i f i e d . For such d e t a i l , antiserum of p e r f e c t s p e c i f i c i t y would have to be a v a i l a b l e f o r use. However, f o r the purposes of l a r g e - s c a l e e c o l o g i c a l s t u d i e s , the ELISA i s a h i g h l y s u i t a b l e technique f o r Rhizobium trifolii s t r a i n i d e n t i f i c a t i o n as the only disadvantage a s s o c i a t e d with the assay i s i t s i n a b i l i t y to d e t e c t mixed i n f e c t i o n s when the m i n o r i t y component comprises a very small p r o p o r t i o n of the t o t a l c e l l s . T h i s d i f f i c u l t y does not r e p r e s e n t a s e r i o u s drawback as dual i n f e c t i o n s i n c l o v e r growing i n s o i l are q u i t e r a r e , o c c u r r i n g 1%—3.5% of the time (Brockwell et a l . , 1977; B r o m f i e l d and Jones, 1980b), and the ELISA would miss only some of these. Furthermore, i t may reasonably be argued that the c o n t r i b u t i o n of R. trifolii s t r a i n s comprising l e s s than 20% of the nodule b a c t e r o i d s to p l a n t performance would be s m a l l , thus emphasizing the t r i v i a l consequences of t h i s shortcoming. CHAPTER 2 PLANT GROWTH PROMOTING BACTERIA INTRODUCTION A g r o n o m i c a l l y , p l a n t - b a c t e r i a l a s s o c i a t i o n s may be c a t e g o r i z e d i n t o one of t h r e e g r o u p s : ( i ) a s s o c i a t i o n s where t h e b a c t e r i a l p r e s e n c e i s b e n e f i c i a l i n some way t o p l a n t g r o w t h ; ( i i ) a s s o c i a t i o n s where b a c t e r i a a r e p r e s e n t b ut have no a p p a r e n t e f f e c t on p l a n t g r o w t h ; and ( i i i ) a s s o c i a t i o n s where t h e m i c r o o r g a n i s m has d e l e t e r i o u s e f f e c t s on p l a n t p e r f o r m a n c e ( e g . d i s e a s e ) . The t h i r d c a t e g o r y o f p l a n t / b a c t e r i a i n t e r a c t i o n s f a l l s w i t h i n t h e bounds o f p l a n t p a t h o l o g y and has d e v e l o p e d i n t o an a r e a of s t u d y i n i t s e l f . A s s o c i a t i o n s o f t h e s e c o n d t y p e a r e n o t p e r c e i v e d t o be o f e c o n o m i c i m p o r t a n c e and t h e r e f o r e have n o t r e c e i v e d a g r e a t d e a l o f a t t e n t i o n . However, a s u b s t a n t i a l e f f o r t i s c o n t i n u i n g w h i c h c o n c e n t r a t e s on b e n e f i c i a l p l a n t / b a c t e r i a a s s o c i a t i o n s a s p o t e n t i a l a g r o n o m i c b e n e f i t s a c c r u i n g from s u c h p a r t n e r s h i p s a r e g r e a t . The b e n e f i t s a r e b e s t e x e m p l i f i e d by t h e w e l l known Rhi zobi um-legume s y m b i o s i s , a n a t u r a l l y o c c u r r i n g p l a n t / b a c t e r i a r e l a t i o n s h i p t h a t has been e x p l o i t e d a g r i c u l t u r a l l y f o r d e c a d e s . E x p l o i t a t i o n o f t h i s r e l a t i o n s h i p may r e p l a c e t h e need f o r n i t r o g e n f e r t i l i z e r i n many o f t h e p o o r e r t h i r d w o r l d c o u n t r i e s o r has s u p p l e m e n t e d n i t r o g e n f e r t i l i z e r r e q u i r e m e n t s i n c o u n t r i e s of t h e more a f f l u e n t w e s t e r n w o r l d . E s t i m a t e s of t h e amount of a t m o s p h e r i c n i t r o g e n f i x e d by o r g a n i s m s i n t h i s s y m b i o s i s range from 70 to 100 kg ha f o r peas (Pi sum sativum L.) and beans (Phaseol us sp. ) to over 300 kg ha ^ i n c l o v e r (Trifolium sp.) or a l f a l f a (Medi cago sativa L.) (Postgate, 1982). In the l a s t h a l f century, s e v e r a l r e p o r t s of s t i m u l a t e d p l a n t growth due to i n o c u l a t i o n with v a r i o u s other s p e c i e s of b a c t e r i a have been made (Brown, 1974; Suslow, 1982; Gaskins et a l . , 1985). The taxonomic range of p l a n t s r e p o r t e d to be a f f e c t e d i s broad and i n c l u d e s nionocotyledonous and di c o t y l e d o n o u s angiosperms as w e l l as l e s s advanced p l a n t s such as l i v e r w o r t s and f e r n s (Postgate, 1982). B a c t e r i a r e p o r t e d to have s t i m u l a t o r y e f f e c t s on p l a n t growth represent an e q u a l l y wide range of groups and i n c l u d e a e r o b i c , f a c u l t a t i v e , and anaerobic gram p o s i t i v e and negative s p e c i e s (Mulder and Brotonegoro, 1974; Brown, 1974; Postgate, 1982; Gaskins et a l . , 1985). A survey of l i t e r a t u r e p e r t a i n i n g to the t o p i c of pl a n t growth promoting b a c t e r i a r e v e a l s that there are s e v e r a l p o s s i b l e mechanisms by which r h i z o s p h e r e b a c t e r i a c o u l d s t i m u l a t e p l a n t growth. (The rh i z o s p h e r e i s c o n s i d e r e d to be the zone of s o i l d i r e c t l y i n f l u e n c e d by p l a n t r o o t s ) . Mechanisms that have been e x p e r i m e n t a l l y i n v e s t i g a t e d i n c l u d e : ( i ) i n c r e a s e d a v a i l a b i l i t y of some l i m i t i n g n u t r i e n t , u s u a l l y phosphorus, due to b a c t e r i a l a c t i o n ; ( i i ) s u p pression of pathogenic or d e l e t e r i o u s b a c t e r i a i n the rh i z o s p h e r e of crop p l a n t s by p l a n t growth promoting b a c t e r i a ; ( i i i ) b a c t e r i a l p r o d u c t i o n of p l a n t growth-promoting substances; and ( i v ) r o o t - a s s o c i a t e d f i x a t i o n of atmospheric n i t r o g e n by d i a z o t r o p h i c microbes. It has by no means been r e s o l v e d as to which mechanism(s) are most important in p l a n t responses, as r e s u l t s from s t u d i e s of these systems are o f t e n v a r i a b l e or i n c o n s i s t e n t (Burr and Caesar, 1984). To add to the u n c e r t a i n t y , many of the s p e c i e s of b a c t e r i a s t u d i e d in t h i s regard possess more than one c h a r a c t e r i s t i c that might p o t e n t i a l l y b e n e f i t p l a n t growth. Indeed, some s p e c i e s of p l a n t growth promoting (PGP) b a c t e r i a possess a t t r i b u t e s that are c o n s i s t e n t with a l l of the mechanisms under study. The examples c i t e d i n t h i s review should i l l u s t r a t e the s i g n i f i c a n t p o t e n t i a l that PGP b a c t e r i a h o l d f o r a g r i c u l t u r e while exposing some of the d i f f i c u l t i e s and a m b i g u i t i e s experienced i n determining the exact nature of the observed b e n e f i c i a l e f f e c t s . Phosphorus Solubilization Phosphorus i s o f t e n abundant i n s o i l s , but i n forms l a r g e l y u n a v a i l a b l e to p l a n t s , as part of i n s o l u b l e or p o o r l y s o l u b l e i n o r g a n i c or organic phosphates (Anderson, 1976). Consequently, Gerre t s e n (1948) argued n e a r l y four decades ago that microoganisms capable of i n c r e a s i n g the a v a i l a b i l i t y of phosphorus i n the s o i l c o u l d b e n e f i t p l a n t growth. Mechanisms i n v o l v i n g b a c t e r i a l enzymes (phosphatase or phytase) which c l e a v e phosphate groups from complex molecules, or m i c r o b i a l p r o d u c t i o n of organic a c i d s (eg. 2-ketogluconic a c i d ) (Duff et a l . , 1963) that lower the l o c a l pH and i n c r e a s e the s o l u b i l i t y of phosphorus compounds were envisaged. These e a r l y experiments (Gerretsen, 1948) demonstrated that p l a n t s f e r t i l i z e d with phosphorus and i n o c u l a t e d with s o i l b a c t e r i a show s u p e r i o r growth compared with u n i n o c u l a t e d c o n t r o l s . However, t h i s e f f e c t o c c u r r e d r e g a r d l e s s of whether the a p p l i e d phosphorus was h i g h l y a v a i l a b l e (KH 2P0 4) or p o o r l y s o l u b l e (rock phosphate). Furthermore, p l a n t s grown a s e p t i c a l l y with rock phosphate showed s u b s t a n t i a l i n c r e a s e s i n phosphorus content compared with u n f e r t i l i z e d c o n t r o l s , demonstrating that r o o t s alone c o u l d s o l u b i l i z e phosphorus. T h i s work i n d i c a t e d t hat i n c r e a s e d phosphorus a v a i l a b i l i t y due to m i c r o b i a l a c t i o n can be b e n e f i c i a l to pl a n t growth, but the importance of the b a c t e r i a l c o n t r i b u t i o n was not c l e a r . In what appears t o be a c l a s s i c case of "jumping the gun", the p r a c t i s e of i n o c u l a t i n g crops with p r e p a r a t i o n s of a bacterium capable of in vitro phosphate s o l u b i l i z a t i o n , Bacillus megatherium v a r . phos phat i cum, was endorsed by the So v i e t Union about 30 years ago. I t was claimed that "phosphobacterin", the common name f o r the b a c i l l u s , c o u l d r e s u l t i n y i e l d i n c r e a s e s of the order of 10% i n f i f t y to seventy percent of the crops to which i t was a p p l i e d (Cooper, 1959). Vegetable and glasshouse crops were thought to respond more f a v o r a b l y than f i e l d c r o p s . However, Soviet experiments to determine i n o c u l a t i o n e f f e c t s on cr o p y i e l d s were a p p a r e n t l y not designed and/or analyzed with the usual s t a t i s i c a l r i g o r i n mind. When the inoculum was subsequently t e s t e d i n p r o p e r l y designed experiments, these y i e l d c l a i m s were found to be l a r g e l y u n s u b s t a n t i a t e d as only a small ( M i s h u s t i n and Naumova, 1962) or no (Smith et a l . , 1961; Sundara-Rao et a l . , 1963) b e n e f i t i n y i e l d was observed. Two independent s t u d i e s a s s e s s i n g the o v e r a l l s i g n i f i c a n c e of the S o v i e t inoculum have reached s i m i l a r c o n c l u s i o n s (Cooper, 1959; M i s h u s t i n , 1963). Gaur and Ostwal (1972) observed a negative e f f e c t on y i e l d of wheat (Tri ticum aestivum L.) when t h i s inoculum was t e s t e d but the r e s e a r c h e r s were a b l e to decrease the magnitude of the y i e l d l o s s by the a d d i t i o n of rock phosphate. I t was demonstrated l a t e r t h a t c o m p e t i t i o n f o r phosphorus by r h i z o s p h e r e b a c t e r i a can r e s u l t i n a y i e l d decrease i n b a r l e y (Hordeum vulgare L.) compared with u n i n o c u l a t e d c o n t r o l s (Benians and Barber, 1974). T h i s phenomenon may e x p l a i n the y i e l d l o s s observed with "phosphobacterin" (Gaur and Ostwal, 1972). Barber (1978) argued that the l a c k of evidence f o r y i e l d enhancement in response to i n o c u l a t i o n with phosphorus s o l u b i l i z i n g organisms i s not s u r p r i s i n g f o r two reasons. F i r s t , up to 90% of root and r h i z o s p h e r e microorganisms of common pasture p l a n t s ryegrass (Lolium s p . ) , timothy (Phi eum pratense L . ) , and o r c h a r d grass (Dact yli s glomerata L.) have been shown to produce phosphatases capable of i n c r e a s i n g phosphorus a v a i l a b i l i t y (Greaves and Webley, 1965). Since a c o n s i d e r a b l e f r a c t i o n of the s o i l phosphorus i s i n the organic form, i n o c u l a t i o n with a d d i t i o n a l phosphorus s o l u b i l i z i n g organisms would not be expected to exert a l a r g e e f f e c t on o v e r a l l phosphorus a v a i l a b i l i t y . Second, B. megatherium v a r . phosphaticum i s a spore-forming organism; s p o r u l a t i n g b a c t e r i a have been shown to grow l e s s r e a d i l y i n the r h i z o s p h e r e than other types of b a c t e r i a (Lochhead, 1940). While these are l o g i c a l arguments, i t should be noted t h a t : ( i ) the Greaves and Webley (1965) p r o p o s i t i o n ignored the importance of a l l i n o r g a n i c p ools of phosphorus which c o u l d be made a v a i l a b l e by b a c t e r i a l a c t i o n (see Gaur and Ostwal, 1972), and ( i i ) i n c i t i n g r e s u l t s from Lochhead (1940), Barber g e n e r a l i z e d from the r e s u l t s of o t h e r s , when i t i s c l e a r that p l a n t / b a c t e r i a l i n t e r a c t i o n s are q u i t e v a r i a b l e and dependent on the combination of p l a n t s p e c i e s and b a c t e r i a l s t r a i n t e s t e d (Rennie and Larson, 1979; 1981). In the absence of suppor t i n g data Barbers's (1978) c o n t e n t i o n that B. megatherium var. phosphaticum and other b a c t e r i a capable of in vitro phosphorus s o l u b i l i z a t i o n are poor root c o l o n i z e r s i s unfounded. S e v e r a l genera of s o i l microorganisms have been shown to be capable of phosphate s o l u b i l i z a t i o n and i n c l u d e Bacillus ( e i g h t s p e c i e s ) , Pseudomonas (four s p e c i e s ) , Escherichia (two s p e c i e s ) , Erwinia, Nitrosomonas, Xanthomonas, Fl avobact eri um, Br evi bad er i um, Serratia, Alcaligenes, Ac hr omobact er , Klebsiella, Thi obaci 11 us , Chr omobact er i um, Aci net obact e r , Art hrobact er , Micrococcus, Corynebact eri um, Aeromonas, and Agr obact er i um (Subba Rao, 1982; Barea et a l . , 1976). S t r a i n s of some of these s p e c i e s have been shown to promote p l a n t growth. Subba Rao (1982) c i t e s s e v e r a l p u b l i s h e d r e p o r t s which have t e s t e d the e f f e c t of phosphobacteria on g r a i n and vegetable c r o p s . He concedes that only 10 of 37 f i e l d t r i a l s in I ndia have shown s i g n i f i c a n t y i e l d i n c r e a s e s , most o f t e n with vegetable crops such as potato (Solarium tuberosum L . ) . . However, some of these t r i a l s have r e s u l t e d i n impressive g a i n s . For example, Gaur and Ostwal (1972) demonstrated that Bacillus polymyxa can in c r e a s e g r a i n and straw y i e l d s i n wheat by 38% and 53%, r e s p e c t i v e l y , when rock phosphate i s added to the s o i l . A c o i n c i d e n t i n c r e a s e i n phosphate uptake of 34% was a l s o observed i n these treatments. The authors t e s t e d two other s p e c i e s of phosphate d i s s o l v i n g b a c t e r i a , i n c l u d i n g the S o v i e t "phosphobacterin" and a s t r a i n of Bacillus pul v / / a c i ens. Treatments r e c e i v i n g phosphobacterin and rock phosphate y i e l d e d l e s s than those r e c e i v i n g rock phosphate only, while those r e c e i v i n g B. pulvifaciens y i e l d e d i n t e r m e d i a t e l y between the c o n t r o l s and B. pol ymyxa. However, a l l i n o c u l a t e d treatments r e s u l t e d i n decreased y i e l d s when rock phosphate was omitted. Apparently, the a b i l i t y to s o l u b i l i z e i n o r g a n i c phosphates was c r i t i c a l to the growth response i n these experiments. Kundu and Gaur (1980) have shown g r a i n y i e l d i n c r e a s e s of 59% to 88% in wheat when i n o c u l a t e d with a phosphate s o l u b i l i z i n g s t r a i n of B. pol ymyxa. The e f f e c t was observed with or without added farmyard manure and i n s t e r i l i z e d or u n s t e r i l i z e d s o i l . S i m i l a r l y , Banik and Dey (1985) have shown y i e l d i n c r e a s e s and enhanced phosphorus uptake i n r i c e (Oryza sativa L. ) i n o c u l a t e d with Bacillus firmus. Depending on the study c i t e d , p h o s p h o r u s - s o l u b i l i z i n g b a c t e r i a may have a negative, n e u t r a l , or p o s i t i v e e f f e c t on pl a n t growth. A c r i t i c a l review of the evidence r e v e a l s p o s s i b l e reasons f o r the d i s c r e p a n t c o n c l u s i o n s i n these s t u d i e s . F i r s t , the e f f e c t of d i f f e r e n t s p e c i e s of phosphorus s o l u b i l i z i n g b a c t e r i a must be c o n s i d e r e d . Even w i t h i n the genus Bacillus, Gaur and Ostwal (1972) showed h i g h l y p o s i t i v e e f f e c t s of i n o c u l a t i o n with B. polymyxa while phosphobacterin (B. megatherium var. phosphaticum) has been shown to be l a r g e l y i n e f f e c t i v e (Cooper, 1959; M i s h u s t i n , 1963; Benians and Barber, 1974) or even d e t r i m e n t a l to y i e l d (Gaur and Ostwal, 1972) when used as a crop i n o c u l a n t . Bacillus pul vifaci ens r e s u l t e d i n modest y i e l d i n c r e a s e s of about 20% i n g r a i n and straw y i e l d of wheat. Second, the f e r t i l i t y of the s o i l i n which the t e s t s are ca r r i e d , out can a f f e c t the experimental outcome. In p a r t i c u l a r , the l e v e l of a v a i l a b l e phosphates in the s o i l and the form i n which u n a v a i l a b l e phosphates are found (eg. organic or i n o r g a n i c ) are important f a c t o r s . Kundu and Gaur (1980) found that the y i e l d advantage of wheat due to i n o c u l a t i o n with B. polymyxa was i n c r e a s e d from 59% to 80% when farmyard manure was added. Presumably, the a b i l i t y of the bacterium to s o l u b i l i z e o rganic phosphate was important in the growth response. On the other hand, Gaur and Ostwal (1972) a t t r i b u t e d a 38% y i e l d i n c r e a s e i n wheat y i e l d to i n o c u l a t i o n with the same s p e c i e s of b a c t e r i a , but t h i s was 71 l o s t and a net negative e f f e c t on wheat y i e l d was observed i f i n o c u l a t i o n was done without the a d d i t i o n of rock phosphate. Benians and Barber (1974) observed s i m i l a r y i e l d decreases i n b a r l e y ; i n these experiments, i n o r g a n i c phosphate s o l u b i l i z a t i o n was important. Thus, c a r e f u l s c r e e n i n g of b a c t e r i a l and p l a n t genotypes, c o n t r o l of s o i l f e r t i l i t y l e v e l s with re s p e c t to a v a i l a b l e s o i l phosphorus and knowledge of the form of u n a v a i l a b l e s o i l phosphorus i s r e q u i r e d f o r the maximal b e n e f i t to be r e a l i z e d with these microorganisms. However, Tink e r (1984) noted the lack of evidence i n support of t h i s mechanism of p l a n t growth promoting b a c t e r i a and concluded that the burden of proof f i r m l y r e s t s on those who propose that phosphorus s o l u b i l i z a t i o n i s important i n m i c r o b i a l l y - s t i m u l a t e d p l a n t growth. Disease Suppression A second mechanism by which PGP b a c t e r i a may s t i m u l a t e p l a n t growth i s through suppression of d i s e a s e organisms i n the r h i z o s p h e r e of crop p l a n t s (Suslow, 1982; Linderman, 1986; Schroth and Weinhold, 1986). T h i s h y p othesis d i f f e r e n t i a t e s between major p l a n t pathogenic b a c t e r i a , microorganisms known to cause p l a n t d i s e a s e s with c l a s s i c symptoms and r e s u l t a n t y i e l d l o s s , and d e l e t e r i o u s r h i z o b a c t e r i a (DRB) or minor pathogens. The l a t t e r group of b a c t e r i a are n a t u r a l l y - o c c u r r i n g s o i l microorganisms that c o l o n i z e root systems and r h i z o s p h e r e s of host p l a n t s and cause y i e l d r e d u c t i o n s while the p l a n t remains symptom-free (Suslow and Schroth, 1982). Suppression of one microbe by another i s a w e l l known phenomenon. B i o l o g i c a l c o n t r o l of pathogenic micoorganisms i n root systems and r h i z o s p h e r e s of crop p l a n t s i s j u s t beginning to b e n e f i t a g r i c u l t u r a l systems (Schroth and Hancock, 1981; Suslow, 1982). Phenomena such as b a c t e r i o c i n and phage p r o d u c t i o n by the c o n t r o l microbe (Vidaver, 1976) and p r e d a t i o n of pathogenic fu n g i and b a c t e r i a by mycophageous i n s e c t s , protozoa, predaceous nematodes, or amoebae (Alexander, 1964; C u r l , 1979; Old and P a t r i c k , 1979; Schroth and Hancock, 1981) are demonstrable under c o n t r o l l e d c o n d i t i o n s and are probably important i n c e r t a i n ecosystems. However, a n t i b i o s i s and c o m p e t i t i o n f o r l i m i t e d resources are thought to be the most important mechanisms by which p o p u l a t i o n s of microorganisms a f f e c t one another (Suslow, 1982) . A n t i b i o s i s i s d e f i n e d as the p r o d u c t i o n of a metabolic agent by an organism that i s harmful to another organism (Schroth and Hancock, 1981). Such products may i n c l u d e w e l l known a n t i b i o t i c s such as p e n i c i l l i n or r e c e n t l y d i s c o v e r e d a n t i m e t a b o l i t e s such as siderophores,' which complex i r o n and decrease i t s a v a i l a b i l i t y to other organisms (Kloepper et a l . , 1980). Examples of b i o l o g i c a l c o n t r o l i n c l u d e the use of Agrobact eri um radi obact er s t r a i n K-84 to suppress Agrobact eri um tumefaciens i n p l a n t r h i z o s p h e r e s (Kerr, 1980; Moore, 1979; Moore and Warren, 1979), i n o c u l a t i o n of sugarbeet {Beta vulgaris L.) with Bacillus subtilis to c o n t r o l damping-off (Dunlevy, 1952), or the use of Bacillus pol ymyxa to reduce the in c i d e n c e of b a c t e r i a l w i l t i n tomato {Lycopersicon esculentum M i l l . ) ( C e l i n o and G o t t l i e b , 1952). B i o l o g i c a l c o n t r o l of such major pathogens does not d i r e c t l y concern the i s s u e of PGP b a c t e r i a , as the l a t t e r group of microorganisms e l i c i t e f f e c t s on p l a n t s not o b v i o u s l y i n f e c t e d by pathogens. N e v e r t h e l e s s , i f one accepts the concept of symptomless d i s e a s e , then work with the major pathogens re p r e s e n t s a model f o r the p o t e n t i a l mechanisms of pl a n t growth promotion by b a c t e r i a . So c a l l e d d e l e t e r i o u s r h i z o b a c t e r i a (DRB) have been i s o l a t e d from the roo t s and r h i z o s p h e r e s of sugarbeet (Suslow and Schroth, 1982), potato (Howie and Echandi, 1982), z i n n i a (.Zinnia elegans Jacq.) (Yuen and Schroth, 1986), and from feeder r o o t s of rough lemon {Citrus jambhiri Lush.) (Gardner et a l . , 1984). In these s t u d i e s , the b a c t e r i a l f l o r a from ro o t s and rhi z o s p h e r e s o i l samples were i s o l a t e d and separated. Each of the i s o l a t e s was then i d e n t i f i e d (at l e a s t t e n t a t i v e l y ) and used as a s i n g l e s t r a i n inoculum on t e s t p l a n t s grown under b a c t e r i o l o g i c a l l y c o n t r o l l e d c o n d i t i o n s . I n o c u l a t i o n of p l a n t s with some of these s t r a i n s r e s u l t e d i n y i e l d decreases of 21% to 41% i n sugarbeet (Suslow and Schroth, 1982) and up to 50% i n c i t r u s s p e c i e s (Gardner et a l . , 1984) compared with u n i n o c u l a t e d c o n t r o l s . S i m i l a r l y , Bowen and Ro v i r a (1961) found that the r o o t s of tomato, subterranean c l o v e r [Trifolium subterraneum L . ) , reed canarygrass (Phalaris arundi naceae L . ) , and Monterey pine (Pi nus radiata D. Don) were stunted by the presence of s o i l microbes when compared with growth under s t e r i l e c o n d i t i o n s . In c o n t r a s t , i n o c u l a t i o n with PGP b a c t e r i a reduced c o l o n i z a t i o n of p l a n t root systems by DRB and r e s u l t e d i n concurrent y i e l d i n c r e a s e s of 13% to 150% i n t e s t p l a n t s (Kloepper and Schroth, 1981; Suslow and Schroth, 1982). I t was a l s o demonstrated in r a d i s h (Raphanus sativus L.) that PGP pseudomonads had l i t t l e or no e f f e c t when p l a n t s were grown under s t e r i l e c o n d i t i o n s (Kloepper and Schroth, 1981). R e s u l t s from these s t u d i e s s t r o n g l y suggest that the mechanism of growth promotion by these s t r a i n s i s dependent on the presence of minor pathogens which can be suppressed by the PGP bacterium. I t i s worth n o t i n g that i n both of these s t u d i e s (Kloepper and Schroth, 1981; Suslow and Schroth, 1982) c e r t a i n PGP b a c t e r i a l s t r a i n s caused anatomical changes to root systems t y p i c a l of hormonal e f f e c t s (eg. an i n c r e a s e i n the number of l a t e r a l r o o t s or i n the l e n g t h of the primary r o o t ) , but the authors e i t h e r f a i l t o mention such e f f e c t s (Suslow and Schroth, 1982) or conclude that they were unimportant to the growth response (Kloepper and Schroth, 1981). I t seems l i k e l y t h a t c e r t a i n PGP b a c t e r i a are capable of suppressing d e l e t e r i o u s microbes t h a t normally c o l o n i z e r o o t s systems. Two b a c t e r i a l s t r a i n s belonging to the Ent erobact eri aceae have been r e p o r t e d to a c t i n t h i s way (Loper and Schroth, 1986) but most of these PGP b a c t e r i a belong to the genus Pseudomonas. Members of t h i s genus account for only a small p r o p o r t i o n of b a c t e r i a r e p o r t e d to promote p l a n t growth upon i n o c u l a t i o n (Gaskins et a l . , 1985; Vose and Ruschel, 1981). Furthermore, p l a n t growth promoting pseudomonads are c o n s i d e r e d to be v i g o r o u s root c o l o n i z e r s t hat can s u c c e s s f u l l y outcompete indigenous d e l e t e r i o u s b a c t e r i a f o r s i t e s on the root or i n the rhizosphere (Kloepper et a l . , 1980). T h i s c h a r a c t e r i s t i c has yet to be demonstrated i n other genera of PGP b a c t e r i a (eg. Azotobacter, Azospi ri 11 um, or Bacillus). Indeed, as s t a t e d e a r l i e r , b a c t e r i a belonging to the genus Bacillus are not noted f o r t h i s c h a r a c t e r i s t i c (Lochhead, 1940). T h e r e f o r e , where a response to a PGP b a c t e r i a l s t r a i n i s observed, three c o n d i t i o n s must be met f o r the d i s e a s e suppression hypothesis to gain g e n e r a l acceptance as the mechanism by which PGP b a c t e r i a a c t . I t must be demonstrated t h a t : ( i ) DRB p r o l i f e r a t e i n the absence of the b e n e f i c i a l bacterium; ( i i ) DRB c o l o n i z a t i o n of r o o t s and r h i z o s p h e r e s o i l i s s i g n i f i c a n t l y reduced by the PGP bacterium; and ( i i i ) other a t t r i b u t e s the PGP s t r a i n may possess such' as p r o d u c t i o n of p l a n t growth promoting substances are unimportant to the growth response of the p l a n t s . The l a t t e r argument ( i i i ) seems u n l i k e l y i n view of r e p o r t s that p l a n t growth promotion i n p e a r l m i l l e t (Pennisetum americanum L.) by Azospirillum brasilense (Venkateswarlu and Rao, 1983) and wheat by Bacillus polymyxa (Kundu and Gaur, 1980) occurs under s t e r i l e or u n s t e r i l e c o n d i t i o n s . These two b a c t e r i a l s p e c i e s are both d i a z o t r o p h i c and produce phytohormones or phosphorus s o l u b i l i z i r t g compounds. S i m i l a r l y , Barber and M a r t i n (1976) have demonstrated l a r g e decreases i n wheat shoot and root weights when growth i n s t e r i l e s o i l was compared with that i n u n s t e r i l i z e d s o i l . Plant Growth Substances The t h i r d p o s s i b l e mechanism by which p l a n t growth promoting b a c t e r i a may act i s through the p r o d u c t i o n of p l a n t growth promoting substances (Brown, 1974). S e v e r a l genera of s o i l b a c t e r i a can produce compounds with phytohormone a c t i v i t y s i m i l a r to auxins, g i b b e r e l l i n s , c y t o k i n i n s , or ethlyene (Pegg, 1985). These i n c l u d e Aci net obact er, Agrobact eri um, Aeromonas, Art hrobact er, Azospi ri I I um, Azot obact er, Bacillus, Chromobact eri um, Corynebacteri um, Fl avobact eri um, Klebsiella, Micrococcus, Pseudomonas, Rhizobium, Xant homonas, and members of the Ent erobacteriaceae (Katznelson and Cole, 1965; Brown and Burlingham, 1968; Brown and Walker, 1970; P h i l l i p s and Torrey, 1972; Barea et a l . , 1976; Azcon and Barea, 1975; Brown, 1976; T i e n et a l . , 1979; Loper and Schroth, 1986). A l s o , Azot obact er has been shown to produce v i t a m i n s such as n i a c i n , b i o t i n , cyanocobalamine, thiamine, r i b o f l a v i n , and pantothenic a c i d (Gonzolez-Lopez et a l . , 1983; El-Essawy et a l . , 1984) as w e l l as the amino a c i d s tryptophan, methionine, l y s i n e , and glutamic a c i d (Gonzolez-Lopez et a l . , 1983). Demonstration of the presence of growth promoting substances i n c u l t u r e e x t r a c t s of v a r i o u s microbes i s a r e l a t i v e l y simple procedure. R e l a t i n g the c a p a c i t y to produce growth substances in vitro to p l a n t response when i n o c u l a t e d with PGP b a c t e r i a i s c o n s i d e r a b l y more d i f f i c u l t . Most s t u d i e s attempt to mimic the response to these b a c t e r i a with exogenously a p p l i e d hormone (Brown and Burlingham, 1968; Brown et a l . , 1968; T i e n et a l . , 1979) while some re s e a r c h e r s ( L i b b e r t and M a n t e u f f e l , 1970) have been able to measure higher l e v e l s of growth substances i n p l a n t t i s s u e s when e p i p h y t i c b a c t e r i a were pr e s e n t . Responses of p l a n t s to phytohormones are e a s i l y r e c o g n i z a b l e , o f t e n a f f e c t i n g stem internode l e n g t h , the degree of branching of shoots or r o o t s , number of f l o w e r s , and other r e a d i l y measured c h a r a c t e r i s t i c s . Brown and Burlingham (1968) and Brown et a l . (1968) showed s i m i l a r i t i e s between the response of tomato p l a n t s t r e a t e d with a s t r a i n of Azotobacter chr oococcum that produces g i b b e r e l l i n - l i k e substances and exogenously a p p l i e d g i b b e r e l l i n (GA^). T r e a t e d p l a n t s showed e l o n g a t i o n of stem internodes and expansion of leaves while the number of flower buds and the time of f r u i t formation was a l s o a l t e r e d from c o n t r o l p l a n t s . Brown (1976) has s i m i l a r l y demonstrated that the n i t r o g e n f i x i n g bacterium Azotobacter pas pal i probably a f f e c t s growth of i t s host p l a n t t r o p i c a l Bahia grass (Paspal um not atum F l i g g e ) by s e c r e t i n g phytohormones and not by root a s s o c i a t e d n i t r o g e n f i x a t i o n as was p r e v i o u s l y suggested (Dobereiner et a l . , 1972). I n o c u l a t i o n of the t r o p i c a l grass with A. paspali r e s u l t e d i n dry weight gains of 168% compared with u n i n o c u l a t e d c o n t r o l s . These e f f e c t s were a l s o observed when p l a n t s were t r e a t e d with c e l l - f r e e e x t r a c t s of A. pas pali which had been shown to c o n t a i n i n d o l e a c e t i c a c i d (IAA), GA^, and c y t o k i n i n a c t i v i t y . N i t r o g e n f i x a t i o n ( a c e t y l e n e r e d u c t i o n ) was d e t e c t a b l e at low l e v e l s but was not always c o r r e l a t e d with the presence of A. pas pal i i n the root system. Ti e n et a l . (1979) s t u d i e d the growth response of p e a r l m i l l e t {Pennisetum americanum L.) to i n o c u l a t i o n with another n i t r o g e n f i x i n g bacterium, Azospirillum brasilense. P l a n t responses to i n o c u l a t i o n i n c l u d e d a 41% i n c r e a s e i n shoot weight; a 47% i n c r e a s e i n the number of l a t e r a l r o o t s ; and a 24% i n c r e a s e i n the t o t a l l e n g t h of the l a t e r a l root system. A l s o , r o o t s of i n o c u l a t e d p l a n t s had a dense c o v e r i n g of root h a i r s i n c o n t r a s t to c o n t r o l p l a n t r o o t s which were almost bare. No n i t r o g e n f i x a t i o n c o u l d be d e t e c t e d i n any of the i n o c u l a t e d treatments and i t appears that p l a n t s were grown under s t e r i l e c o n d i t i o n s which would e l i m i n a t e the s u p p r e s s i o n of DRB as a p o s s i b l e mechanism of the growth response. P l a n t responses to i n o c u l a t i o n c o u l d be approximated by a d d i t i o n of exogenous phytohormones l e a d i n g the authors to conclude that m i c r o b i a l p r o d u c t i o n of these growth substances was a l i k e l y cause of the observed e f f e c t s . Loper and Schroth (1986) measured the l e v e l of IAA i n c u l t u r e supernatants of s e v e r a l s t r a i n s of Ps eudomonas s p e c i e s , the Enterobact eri aceae, and i n one Flavobacteri um s t r a i n . T h i s c h a r a c t e r i s t i c was then c o r r e l a t e d to the e f f e c t of b a c t e r i a l i n o c u l a t i o n on primary root l e n g t h i n sugarbeet. A s i g n i f i c a n t n e g a t i v e r e l a t i o n s h i p was observed between the c o n c e n t r a t i o n of IAA produced in vitro and the e f f e c t of i n o c u l a t i o n on root l e n g t h . Highest IAA p r o d u c t i o n was observed in s t r a i n s of b a c t e r i a that had d e l e t e r i o u s e f f e c t s on root l e n g t h , while low IAA producers were shown to possess p l a n t growth promoting a c t i v i t y . The authors concluded that m i c r o b i a l p r o d u c t i o n of IAA can be s i g n i f i c a n t , but that t h i s phenomenon i s important i n e x p l a i n i n g the e f f e c t s of DRB, not PGP b a c t e r i a . In c o n t r a s t to these r e s u l t s , Venkateswarlu and Rao (1983) performed a s i m i l a r study using f i v e s t r a i n s of Azospirillum brasilense and p e a r l m i l l e t as the t e s t p l a n t . Data obtained from t h e i r experiments l e a d to c o n c l u s i o n s e x a c t l y opposite to those of Loper and Schroth (1986). Regression of the amount of IAA produced in vitro by b a c t e r i a l c u l t u r e s on root l e n g t h of i n o c u l a t e d p e a r l m i l l e t showed a p o s i t i v e r e l a t i o n s h i p . Furthermore, t h i s t r a i t 2 showed a strong p o s i t i v e c o r r e l a t i o n to root (R =0.81) and 2 shoot (R =0.92) y i e l d s of i n o c u l a t e d p l a n t s . Thus, there i s c o n v i n c i n g evidence that m i c r o b i a l l y produced phytohormones can p l a y a r o l e i n the response of c e r t a i n p l a n t s to PGP b a c t e r i a (Brown, 1974; T i e n et a l . , 1979; Venkateswarlu and Rao, 1983). The c o n f l i c t i n g c o n c l u s i o n s of the two s t u d i e s that have t r i e d to r e l a t e in vitro IAA p r o d u c t i o n to the nature and magnitude of p l a n t growth responses demonstrate the s p e c i f i c i t y that can e x i s t between b a c t e r i a l s t r a i n and p l a n t genotype. Indeed, the amount of IAA produced in vitro by the PGP s t r a i n s of A. brasilense s t u d i e d by Venkateswarlu and Rao (1983) were 80 s i m i l a r to the the l e v e l s of the phytohomone produced by d e l e t e r i o u s r h i z o b a c t e r i a i n the sugarbeet study of Loper and Schroth (1986). I t may simply be that sugarbeet i s more s e n s i t i v e to m i c r o b i a l sources of IAA, thus showing root growth i n h i b i t i o n at lower c o n c e n t r a t i o n s than does p e a r l m i l l e t . An a l t e r n a t i v e e x p l a n a t i o n i s that IAA i s not i n v o l v e d i n the PGP responses observed t h e r e f o r e trends in / n vitro p r o d u c t i o n of t h i s phytohormone are not r e l a t e d to the degree of p l a n t growth s t i m u l a t i o n . Associative Nitrogen Fixation A f i n a l proposed mechanism f o r the a c t i o n of p l a n t growth promoting b a c t e r i a i s that of root a s s o c i a t e d n i t r o g e n f i x a t i o n by f r e e l i v i n g d i a z o t r o p h i c b a c t e r i a (Vose and Ruschel, 1981). At the same time that "phosphobacterin" was being used i n the S o v i e t Union to i n c r e a s e a v a i l a b i l i t y of s o i l phosphorus, an i n o c u l a n t that was reputed to i n c r e a s e s o i l n i t r o g e n content was a l s o a p p l i e d r e g u l a r l y . The inoculum, known as " a z o t o b a c t e r i n " , c o n s i s t e d of s e v e r a l s p e c i e s of the genus Azotobacter which have long been known to be able to f i x atmospheric n i t r o g e n . Claims s i m i l a r to those made for "phosphobacterin" were made f o r the n i t r o g e n f i x i n g product, i n that over h a l f of the crops t e s t e d with the b a c t e r i a l p r e p a r a t i o n were repo r t e d to show an average 10% y i e l d i n c r e a s e (Cooper, 1959). C e r e a l y i e l d i n c r e a s e s i n the Ukraine of 15% to 20% were a t t r i b u t e d to " a z o t o b a c t e r i n " , while sugar beet was to shown to have y i e l d i n c r e a s e s of up to 7%. However, upon r i g o r o u s s c i e n t i f i c i n v e s t i g a t i o n , the b e n e f i t s that c o u l d be a t t r i b u t e d to t h i s inoculum were again found to be i n s i g n i f i c a n t (Cooper, 1959; M i s h u s t i n , 1963). In a d d i t i o n , there was l i t t l e evidence that n i t r o g e n f i x a t i o n c o n t r i b u t e d to any of the observed responses (Brown, 1982). The d i s c o v e r y that the nitrogenase enzyme c o u l d reduce the CpN bond as w e l l as N 2 (Dilworth, 1966) l e d to the development of the w e l l known ac e t y l e n e r e d u c t i o n assay (Hardy et a l . , 1968). T h i s t e s t f a c i l i t a t e d the wide s c a l e e v a l u a t i o n of n i t r o g e n f i x i n g a c t i v i t y i n the f i e l d . Subsequently, Dobereiner (1961) reported that c e r t a i n c u l t i v a r s of Pas pal um notatum had rh i z o s p h e r e p o p u l a t i o n s of Azotobacter s p e c i e s i n s o i l that commonly supported l a r g e p o p u l a t i o n s of Derxia gummosa. A d d i t i o n a l work confirmed t h i s a s s o c i a t i o n and i t was determined that the bacterium was a new s p e c i e s of Azotobacter capable of f i x i n g n i t r o g e n i n a s s o c i a t i o n with the grass rhizosphere (Dobereiner and Campelo, 1971; Dobereiner et a l . , 1972). The bacterium was a p p r o p r i a t e l y named Azotobacter pas pal i (Neyra and Dobereiner, 1977). The process of n i t r o g e n f i x a t i o n ( a c e t y l e n e reduction) was shown to be h i g h l y s e n s i t i v e to oxygen which was expected i n view of the known s e n s i t i v i t y of the n i t r o g e n a s e complex to t h i s molecule (Postgate, 1982). E a r l y r e p o r t s suggested that up to 100 kg n i t r o g e n ha 1 yr 1 c o u l d be f i x e d by t h i s p l a n t / b a c t e r i a a s s o c i a t i o n , but these were l a t e r c o r r e c t e d to more r e a l i s t i c estimates of 20 kg h a ~ 1 y r _ 1 (Postgate, 1982). While there i s l i t t l e doubt as to the p o t e n t i a l growth s t i m u l a t i o n that may accrue from t h i s p a r t n e r s h i p (Brown, 1976; Postgate, 1982), subsequent experimentation, which was d i s c u s s e d i n the p r e v i o u s s e c t i o n , has produced c o n v i n c i n g evidence that the growth response i s p r i m a r i l y caused by phytohormones of b a c t e r i a l o r i g i n , with n i t r o g e n f i x a t i o n p l a y i n g a secondary and o f t e n undetermined r o l e (Brown, 1976). In a s i m i l a r d i s c o v e r y , Dobereiner and Day (1976) re p o r t e d a new a s s o c i a t i o n between a s p i r a l bacterium and d i g i t g r a s s {Digit aria decumbens S t e n t . ) . The bacterium was i n i t i a l l y i d e n t i f i e d as Spirillum lipoferum but the genus has s i n c e been renamed Azospi rilium with two s p e c i e s , lipoferum and brasilense (Tarrand et a l . , 1978). The authors were able to show that the b a c t e r i a invaded the root c o r t e x of the grass and, using t e t r a z o l i u m , a v i t a l s t a i n , c o u l d be demonstrated i n s i d e c o r t i c a l c e l l s . Strong c o r r e l a t i o n s between root p i e c e n i t r o g e n a s e a c t i v i t y and t e t r a z o l i u m -reducing b a c t e r i a i n the cortex l e a d to the c o n c l u s i o n that A. lipoferum was the major organism r e s p o n s i b l e f o r the a c e t y l e n e reducing a c t i v i t y . Subsequent work has shown that i n o c u l a t i o n of p l a n t s with Azospirillum s p e c i e s r e s u l t s i n s u b s t a n t i a l y i e l d i n c r e a s e s i n wheat (Rennie and Larson, 1979; 1981; Reynders and Vlassak, 1982; Kapulnik et a l . , 1983; M i l l e t et a l . , 1985) and other crop p l a n t s (Vose and Ruschel, 1981). Perhaps the most s i g n i f i c a n t of a l l demonstrations of a s s o c i a t i v e n i t r o g e n f i x a t i o n has been made by Rennie and Larson (1979; 1981) u s i n g disomic chromosome s u b s t i t u t i o n l i n e s o f wheat. These l i n e s c o n t a i n 20 p a i r s of indigenous chromosomes p l u s one p a i r from a donor l i n e , a l l o w i n g f o r the study of e f f e c t s of the "donated" p a i r i n an otherwise constant g e n e t i c background. They were able to i d e n t i f y chromosomes 2 and 5 of the common wheat c u l t i v a r s "Cadet" and "Rescue" as the sources of s p e c i f i c i t y i n e f f e c t i v e wheat/Azospi riI I um or wheat/Baci I I us a s s o c i a t i o n s . Chromosome 5B i s i n v o l v e d i n root r o t r e s i s t a n c e while 5D a f f e c t s the number of n i t r a t e reducing b a c t e r i a i n the r h i z o s p h e r e (Rennie and Larson, 1979). A h i g h l y s p e c i f i c response to b a c t e r i a l s p e c i e s was a l s o observed using t h i s type of a n a l y s i s . For example, i n o c u l a t i o n of wheat c u l t i v a r Rescue R-C2D ("Rescue" genotype with chromosome 2D from cv Cadet) with a b a c i l l u s l a t e r i d e n t i f i e d as B. pol ymyxa, r e s u l t e d i n no net gain i n p l a n t weight, while treatment with A. brasilense r e s u l t e d in a 41% y i e l d i n c r e a s e . However, when the same wheat c u l t i v a r was t e s t e d with the C5B chromosome from "Cadet" s u b s t i t u t e d f o r chromosome C2D, i n o c u l a t i o n with B. pol ymyxa more than t r i p l e d p l a n t dry weight while the response to A. brasilense d i d not change. S i m i l a r l y impressive gains i n p l a n t n i t r o g e n content compared with u n i n o c u l a t e d c o n t r o l s and a n a l y s i s of n i t r o g e n balance data l e d the authors to conclude that the mechanism of the growth response was r e l a t e d to the a b i l i t y of each bacterium to f i x n i t r o g e n . L e t h b r i d g e and Davidson (1983) s t u d i e d the n i t r o g e n balance i n the same two s p r i n g wheat c u l t i v a r s ("Rescue" and "Cadet") and t h e i r r e c i p r o c a l chromosome s u b s t i t u t i o n l i n e s C-R5D and R-C5D when i n o c u l a t e d with d i a z o t r o p h i c b a c t e r i a . Using a c e t y l e n e r e d u c t i o n and N i s o t o p e d i l u t i o n techniques (Rennie et a l . , 1977; Rennie and Rennie, 1983), these authors were not able to demonstrate r o o t - a s s o c i a t e d n i t r o g e n f i x a t i o n without the a d d i t i o n of exogenous carbohydrates. I t was concluded that without such a d d i t i o n s , the i n c o r p o r a t i o n of m i c r o b i a l l y - f i x e d n i t r o g e n by wheat was n e g l i g i b l e . S e v e r a l p o i n t s of c o n t e n t i o n are apparent from t h i s work. F i r s t , L e t h bridge and Davidson (1983) were working with a system i n which l i t t l e or no growth response to i n o c u l a t i o n was observed (eg. 7%). T h i s r e l a t i o n s h i p i s i n c o n t r a s t to the 300% i n c r e a s e i n p l a n t weight that Rennie and Larson (1979) c o u l d a t t r i b u t e to b a c t e r i a l i n o c u l a t i o n . If the former authors c o u l d not d u p l i c a t e the i n o c u l a t i o n response r e p o r t e d by Rennie and Larson (1979), i t i s d i f f i c u l t to see how t h e i r c o n c l u s i o n s may be a p p l i e d to p l a n t growth responses to d i a z o t r o p h i c organisms. 1 5 Second, the N study u t i l i z e d d i f f e r e n t b a c t e r i a l s t r a i n s than those used i n the o r i g i n a l chromosome s u b s t i t u t i o n work of Rennie and Larson (1979). Although s t r a i n s of A. brasilense and B. pol ymyxa were used, L e t h b r i d g e and Davidson (1983) themselves p o i n t out that i t was not known to what extent B. pol ymyxa from S c o t t i s h s o i l s d i f f e r s from i s o l a t e s of the same s p e c i e s used i n the A l b e r t a work (Rennie and Larson, 1979; 1981). S e v e r a l s t u d i e s have suggested that the matchup between p l a n t and bacterium i s c r i t i c a l i n determining the nature and magnitude of the growth response (Gaur and Ostwal, 1972; Neyra and Dobereiner, 1977; Rennie and Larson, 1979; 1981) and s t r a i n to s t r a i n 85 v a r i a t i o n w i t h i n a s p e c i e s can be s u b s t a n t i a l (Venkateswarlu and Rao, 1983; Loper and Schroth, 1986). T h i r d , the nature of the c o n t r o l used in the L e t h b r i d g e and Davidson (1983) experiment i s q u i t e d i f f e r e n t from that normally used, and appears to c o n t r i b u t e s u b s t a n t i a l l y to the d i s c r e p a n t growth responses. H e a t - k i l l e d b a c t e r i a l c u l t u r e s were used as c o n t r o l s and, upon o b s e r v i n g an apparent growth response when these were used as inoculum, the authors s t a t e that the autoclaved b a c t e r i a l c e l l s appear to be a good source of n i t r o g e n f o r the p l a n t s . In the one set of data where u n i n o c u l a t e d p l a n t s are compared with those r e c e i v i n g l i v e inoculum, a 43% i n c r e a s e i n p l a n t dry weight was observed. However, the response to b a c t e r i a l i n o c u l a t i o n becomes an 18% decrease i f h e a t - k i l l e d c e l l s are c o n s i d e r e d to be the c o n t r o l . C l e a r l y , c u l t u r e s of h e a t - k i l l e d c e l l s have a major e f f e c t on p l a n t growth and should be c o n s i d e r e d a treatment, not a c o n t r o l . F i n a l l y , the authors showed that i n c o r p o r a t i o n of b a c t e r i a l l y - f i x e d n i t r o g e n d i d not occur i n the absence of exogenously a p p l i e d carbohydrate. These r e s u l t s were obtained using p l a n t s growing i n sand c u l t u r e under h i g h l y a r t i f i c i a l c o n d i t i o n s . E x t r a p o l a t i o n of r e s u l t s from p l a n t s growing under these c o n d i t i o n s to p l a n t s growing i n the f i e l d i s tenuous and u s u a l l y not j u s t i f i e d . To d i s p u t e the e f f e c t claimed by Rennie and Larson (1979), p l a n t s would have to have been grown i n the f i e l d and should have shown a l a r g e e f f e c t due to i n o c u l a t i o n with no evidence of i n c o r p o r a t i o n of b a c t e r i a l l y f i x e d n i t r o g e n by the p l a n t s . 86 Many s t u d i e s of d i a z o t r o p h i c r h i z o c o e n o s i s have r e l i e d on the a c e t y l e n e r e d u c t i o n t e s t (Hardy et a l . , 1968) to estimate nitrogenase a c t i v i t y a s s o c i a t e d with root systems (Vose and Ruschel, 1981). However, d i f f i c u l t i e s with the technique have r e s u l t e d i n i n a c c u r a c i e s i n the e s t i m a t i o n of the c o n t r i b u t i o n of a s s o c i a t i v e f i x a t i o n to the n i t r o g e n economy of an ecosystem (Witty and Day, 1977, Brown, 1982; Witty 1983); f i x a t i o n l e v e l s i n such a s s o c i a t i o n s have u s u a l l y been overestimated. A s u b s t a n t i a l improvement in measuring n i t r o g e n f i x a t i o n o c c u r r e d with the development of 1 5 N i s o t o p e techniques (Bergersen and Turner, 1983; Rennie and Rennie, 1983). The assay i s based on the o b s e r v a t i o n 15 1 4 . that the r a t i o of N: N i n s o i l n i t r o g e n i s much higher than that of n i t r o g e n i n the atmosphere. P l a n t s r e c e i v i n g n i t r o g e n i nputs from atmospheric sources w i l l t h e r e f o r e tend 15 14 to have a lower N: N r a t i o than those u t i l i z i n g s o i l n i t r o g e n . Using a mass spectrometer, the d i f f e r e n c e i n the r a t i o can be d e t e c t e d as i t occurs i n nature, or a f t e r 1 5 supplemental N has been added to the s o i l as f e r t i l i z e r to exaggerate the ' e f f e c t . While t h i s technique i s not without i t s l i m i t a t i o n s (Chalk, 1985), i t y i e l d s an estimate of n i t r o g e n f i x e d that i s i n t e g r a t e d over the e n t i r e growing season and i s not p a r t i c u l a r l y a f f e c t e d by l o c a l d a i l y or seasonal f l u c t u a t i o n s i n n i t r o g e n a s e a c t i v i t y . As data from these types of s t u d i e s accumulate, i t i s becoming apparent that a s s o c i a t i v e n i t r o g e n f i x a t i o n does occur, but that i t s importance i n a g r i c u l t u r a l systems may be questioned (Kapulnik, 1985). However, under c o n d i t i o n s of p l e n t i f u l carbon s u b s t r a t e , low amounts of a v a i l a b l e n i t r o g e n , low oxygen t e n s i o n s , and an adequate supply of moisture, a p o s i t i v e c o n t r i b u t i o n to the n i t r o g e n economy of an ecosystem can be made (Brown, 1982). Such a c o n t r i b u t i o n may be observed e a s i l y i n a g r i c u l t u r a l systems that t r a d i t i o n a l l y ignore n i t r o g e n f e r t i l i z a t i o n yet harvest crops year a f t e r year with no apparent d e f i c i e n c i e s i n p l a n t n i t r o g e n . For example, sugar cane {Saccharum officinarum L. ) has been grown i n monoculture f o r more than 100 years i n B r a z i l without n i t r o g e n f e r t i l i z e r (Neyra and Dobereiner, 1977). The l a t t e r authors c i t e the work of Verdade (1967) who determined that only h a l f of these f i e l d s responded to f e r t i l i z a t i o n with n i t r o g e n even when phosphorus and potassium were a p p l i e d c o n c u r r e n t l y . Dobereiner (1961) has shown that the d i a z o t r o p h Beijerinckia sp. i s s e l e c t i v e l y s t i m u l a t e d under sugar cane with r e s u l t a n t n i t r o g e n f i x a t i o n a c t i v i t y . The same type of s i t u a t i o n can be seen i n t r o p i c a l r i c e c u l t u r e where the AzolIa/Anabaena a s s o c i a t i o n can f i x up to 600 kg ha 1 i n r i c e paddies and i s b e l i e v e d to have maintained the f e r t i l i t y of P h i l i p p i n e r i c e t e r r a c e s f o r c e n t u r i e s (Postgate, .1982). It i s safe to conclude that the general area of p l a n t growth promoting b a c t e r i a i s i n a s t a t e of f l u x . Growth responses, some s p e c t a c u l a r (Rennie and Larson, 1979; 1981; Suslow and Schroth, 1982), are o f t e n observed but can be c o r r e l a t e d to s e v e r a l p o t e n t i a l l y b e n e f i c i a l b a c t e r i a l c h a r a c t e r i s t i c s . For example, s p e c i e s of both Azotobacter and Azospi ri 11 um i n i t i a l l y appeared to cause growth responses i n host p l a n t s by f i x i n g atmospheric n i t r o g e n i n the host r h i z o s p h e r e (Neyra and Dobereiner, 1977; Dobereiner and Day, 1976). Since then, Azospirillum has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n as a p l a n t i n o c u l a n t as i t has been shown to i n c r e a s e y i e l d in wheat (Rennie and Larson, 1979; 1981; Subba Rao et a l . , 1979; Dobereiner and D e P o l l i , 1980; Okon et a l . , 1980; 1981; Subba Rao, 1980; Hegazi et a l . , 1981; Kapulnik et a l . , 1981; Vlassak and Reynders, 1981; Kapulnik et a l . , 1985; Bashan, 1986a; 1986b; Boddey et a l . , 1986), p e a r l m i l l e t , Pani cum sp., (Subba Rao et a l . , 1985), sorghum (Sorghum hi col or L . ) , f i n g e r m i l l e t (Eleusine coracana L . ) , (Subba Rao et a l . , 1985), maize (Zea mays L . ) , and ryegrass (Barea et a l . , 1983; Vose and Ruschel, 1981). However, as with Azot obact er pas pali and Paspalum notatum, evidence has accumulated that n i t r o g e n f i x a t i o n i s not the s o l e , or primary mechanism by which these b a c t e r i a i n f l u e n c e p l a n t growth (Kapulnik et a l . , 1985; K i p e - N o l t et a l . , 1985). For example, growth s t i m u l a t i o n has been observed at temperatures below which n i t r o g e n a s e a c t i v i t y i n these a s s o c i a t i o n s ceases ( A v i v i and Feldman, 1982), at r a t e s of n i t r o g e n f e r t i l i z a t i o n that should i n h i b i t n i t r o g e n f i x a t i o n ( M i l l e t and Feldman, 1984), and by mutants of the bacterium unable to f i x n i t r o g e n (O'Hara et a l . , 1981). The p r o d u c t i o n of p l a n t growth substances by the bacterium appears to be the most l i k e l y cause of the growth s t i m u l a t i o n at t h i s time (Tien et a l . , 1979; Vlassak and Reynders, 1981) a l t h o u g h t h i s c o n t e n t i o n remains to be proven. Bacillus polymyxa has been shown to have a p o s i t i v e e f f e c t on the growth of Penni set um s p e c i e s (Dart and Subba Rao, 1981) and common wheat (Gaur and Ostwal, 1972; Neal et a l . , 1973; Neal and Larson, 1976; Rennie and Larson, 1979; 1981; Kundu and Gaur, 1980). Due to i t s a b i l i t y to s p o r u l a t e , PGP s t r a i n s of Bacillus pol ymyxa c o u l d prove to be u s e f u l a g r i c u l t u r a l i n o c u l a n t s . I t would be h e l p f u l to determine the mechanism of the growth promoting a c t i v i t y so that p o t e n t i a l l y u s e f u l s t r a i n s c o u l d be d e t e c t e d using l a b o r a t o r y s c r e e n i n g procedures. However, s t r a i n s of B. pol ymyxa have been shown to possess a v a r i e t y of a t t r i b u t e s , any or a l l of which c o u l d c o n t r i b u t e to p l a n t growth. These c h a r a c t e r i s t i c s i n c l u d e the a b i l i t y to s o l u b i l i z e i n o r g a n i c and organic sources of phosphorus (Gaur and Ostwal, 1972; Kundu and Gaur, 1980; H o l l et a l . , 1987), the suppression of pathogenic microorganisms ( C e l i n o and G o t t l i e b , 1952), the p r o d u c t i o n of p l a n t growth substances i n c l u d i n g g i b b e r e l l i n and IAA (Katznelson and Cole, 1965; H o l l et a l . , 1987), and the a b i l i t y to f i x n i t r o g e n in vitro and i n a s s o c i a t i o n with p l a n t roots (Neal and Larson, 1976; Witty, 1979; Rennie and Larson, 1979; 1981)). .Strains of t h i s s p e c i e s are not r e s t r i c t e d to b e n e f i c i a l e f f e c t s on p l a n t growth as they have a l s o been shown to ex e r t negative e f f e c t s on crop growth f o l l o w i n g i n o c u l a t i o n , perhaps through c o m p e t i t i o n f o r phosphorus (Gaur and Ostwal, 1972), and to be pathogenic (Dowson, 1949). While members of the genus Bacillus may not be encountered as f r e q u e n t l y as other genera i n the rhi z o s p h e r e (Lochhead, 1940), B. pol ymyxa has been r e p o r t e d to comprise 90 an important f r a c t i o n of r h i z o p l a n e Bacillus s p e c i e s ( C l a r k , 1949). W u l l s t e i n et a l . (1979) have i s o l a t e d a d i a z o t r o p h i c B. polymyxa-like organism from the r h i z o s h e a t h s of x e r i c g r a s s e s , but e f f e c t s of the i s o l a t e on p l a n t performance were not i n v e s t i g a t e d . Mechanisms r e p o r t e d to be i n v o l v e d i n the observed s t i m u l a t o r y growth responses a t t r i b u t e d to B. polymyxa i n c l u d e phosphate s o l u b i l i z a t i o n (Gaur and Ostwal, 1972; Kundu and Gaur, 1980) and n i t r o g e n f i x a t i o n (Neal and Larson, 1976; Rennie and Larson, 1979; 1981). Advanced p l a n t development in wheat, s i m i l a r to the phytohormonal e f f e c t s c i t e d by Brown (1974), has a l s o been a t t r i b u t e d to i n o c u l a t i o n with t h i s bacterium ( R o v i r a , 1963; R o v i r a and Davey, 1974). A study of the e f f e c t s of B. pol ymyxa i n o c u l a t i o n on Lol i um perenne L., Trifolium repens L., and Agropyron cristatum L. i s d e s c r i b e d i n the f o l l o w i n g pages. To a v o i d the confounding e f f e c t s of genotype-genotype i n t e r a c t i o n s i n c l o v e r - r y e g r a s s mixtures, the e v a l u a t i o n of the growth response to B. pol ymyxa i n o c u l a t i o n was focussed on c r e s t e d wheatgrass. P r e l i m i n a r y experiments i n t h i s l a b o r a t o r y (F.B. H o l l , p e r s o n a l communication) i n d i c a t e d that t h i s s p e c i e s showed a c o n s i s t e n t and r e p r o d u c i b l e response to i n o c u l a t i o n . Evidence w i l l be presented which l i n k s in vitro p r o d u c t i o n of i n d o l e a c e t i c a c i d to p l a n t growth promoting a c t i v i t y of the b a c i l l u s . MATERIALS AND METHODS Bacterial Culture and Evaluation Bacillus pol ymyxa was i s o l a t e d from s o i l from a 45 year o l d permanent pasture with a b o t a n i c a l composition i n c l u d i n g p e r e n n i a l ryegrass {Lolium perenne L . ) , orchardgrass (Dactyl is glomerata L . ) , v e l v e t g r a s s (Hoicus I anal us L . ) , and white c l o v e r (Trifolium repens L . ) . S o i l was d i l u t e d 1:10 (w/v) with 0.2M phosphate-buffered s a l i n e (g/1: Na 2HP0 4 4.5; NaH 2P0 4 0.4; pH 7.2) and streaked onto p l a t e s of combined carbon medium (Rennie, 1981). The combined carbon medium was prepared i n two s o l u t i o n s as f o l l o w s : S o l u t i o n I: (g) K 2HP0 4 8.0; KH 2P0 4 0.2; NaCL 0.1; Na 2FeEDTA 0.028; Na 2Mo0 4-2H 20 0.025; yeast e x t r a c t 0.1; mannitol 5.0; sucrose 5.0; sodium l a c t a t e 0.5 ml (60% v / v ) ; d i s t i l l e d H 20 900 ml. S o l u t i o n I I : (g) MgS0 4~7H 20 0.2; C a C l 2 0.6; d i s t i l l e d H 20 100 ml. The s o l u t i o n s were a u t o c l a v e d s e p a r a t e l y , c o o l e d , and mixed; f i l t e r - s t e r i l i z e d b i o t i n (5 ug/1) and p-aminobenzoic a c i d (PABA) (10 ug/1) were added; and the f i n a l pH was a d j u s t e d to 7.0. A t y p i c a l Bacillus (Buchanan and Gibbons, 1974) was i s o l a t e d a f t e r 5 days growth i n anaerobic j a r s (Baltimore B i o l o g i c a l L a b o r a t o r i e s ) at 30°C. C o l o n i e s were then p u r i f i e d by r e s t r e a k i n g on the same medium and t e s t e d f o r a c e t y l e n e reducing c a p a b i l i t y ( D i l w o r t h , 1966). Assays i n v o l v e d : 92 ( i ) i s o l a t i n g medium (2 ml), i n a s t e r i l e 6 ml tube f i t t e d with a t e f l o n s e a l , was i n o c u l a t e d with b a c t e r i a ; ( i i ) a f t e r 24 h growth at 30°C, a c e t y l e n e was added to a f i n a l c o n c e n t r a t i o n of 10%; ( i i i ) 1 day a f t e r a d d i t i o n of the a c e t y l e n e , 1 ml samples of gas i n the v i a l were analyzed f o r ethylene by flame i o n i z a t i o n gas chromatography f o l l o w i n g s e p a r a t i o n i n a s t a i n l e s s s t e e l column (0.3 x 180 cm) c o n t a i n i n g Porapak N (80-100 mesh) at 50°C with n i t r o g e n c a r r i e r gas at a flow r a t e of 40 ml per minute. I d e n t i t y of i s o l a t e s was confirmed using micoscopy and standard b i o c h e m i c a l t e s t s i n the API 20E system (Buchanan and Gibbons, 1974; Rennie, 1980). Bacillus pol ymyxa was maintained on n u t r i e n t agar s l a n t s and s t o r e d at 4°C. Plant Growth and Inoculation C r e s t e d wheatgrass cv Fairway was sown, 15 seeds per 7.5 cm diameter pot, i n t o a 50:50 (v/v) mixture of p o t t i n g s o i l and T u r f a c e R. The s o i l mix c o n t a i n e d 580 kg ha 1 t o t a l n i t r o g e n , 28 kg ha 1 a v a i l a b l e phosphorus, and 79 kg ha 1 a v a i l a b l e potassium. S o i l i n each pot was i n o c u l a t e d g immediately with 25 ml (ca. 10 c e l l s ) of e i t h e r v i a b l e c u l t u r e s d i l u t e d with n u t r i e n t broth or f i l t e r - s t e r i l i z e d medium from b a c t e r i a l c u l t u r e s grown f o r one week i n n u t r i e n t b r o t h . The s t e r i l e c u l t u r e f i l t r a t e s were d i l u t e d i n the same manner as the l i v e c u l t u r e s . A d d i t i o n a l c o n t r o l pots were t r e a t e d with s t e r i l e n u t r i e n t b r o t h alone. No d i f f e r e n c e was observed between s t e r i l e c u l t u r e f i l t r a t e s and unused n u t r i e n t broth c o n t r o l s . Upon emergence, s e e d l i n g number was recorded d a i l y . S e e d l i n g s were thinned to f i v e per pot a f t e r emergence was complete, 17 days a f t e r p l a n t i n g . P l a n t s were watered with tap water as r e q u i r e d and were grown fo r 8 weeks before a s s a y i n g f o r n i t r o g e n f i x a t i o n ( a c e t y l e n e r e d u c t i o n ) and h a r v e s t . Experiments with p e r e n n i a l ryegrass and white c l o v e r were conducted using wooden f l a t s c o n t a i n i n g the same s o i l -Turf ace ®> mix. M a t e r i a l f o r these experiments was c o l l e c t e d from a permanent pasture which was seeded 45 years ago. P a i r s of p l a n t s (one white c l o v e r and one p e r e n n i a l r yegrass) growing s i d e - b y - s i d e were c o l l e c t e d and microorganisms were i s o l a t e d from a s s o c i a t e d r h i z o s p h e r e s o i l . P l a n t s were then progagated i n the greenhouse and c l o n e s of those genotypes were used i n the experiments d e s c r i b e d . Whenever p l a n t and/or b a c t e r i a l genotypes which were o r i g i n a l l y c o l l e c t e d from the same l o c a t i o n i n the f i e l d were grown together e x p e r i m e n t a l l y , the term "homologous" has been used to i d e n t i f y t h i s treatment combination. F l a t s were a u t o c l a v e d f o r 90 minutes p r i o r to use i n these experiments. To reduce v a r i a b i l i t y , c l o n e s of t h r e e ryegrass genotypes and three white c l o v e r genotypes were used. I n d i v i d u a l t i l l e r s ( r y e g r a s s ) or s t o l o n t i p s ( c l o v e r ) were c l i p p e d from stock p l a n t s , r i n s e d f o r 5 seconds i n 80% ethanol and then i n s t e r i l e d i s t i l l e d water to reduce random b a c t e r i a l contamination, and p l a n t e d i n rows c o n t a i n i n g a l t e r n a t i n g grass and legume p l a n t s . Intra-row and inter-row s p a c i n g was ca. 18 mm and 75 mm, r e p e c t i v e l y . F l a t s were then d i v i d e d i n t o 16 compartments of equal s i z e (75 mm x 110 mm) which were arranged i n 4 rows and 4 columns u s i n g p l a s t i c p a r t i t i o n s . These b a r r i e r s separated below ground compartments completely and extended 100 mm above the s o i l s u r f a c e to discourage above ground competition between p l a n t s i n d i f f e r e n t c e l l s . To i n v e s t i g a t e the e f f e c t s of B. pol ymyxa on the y i e l d of ryegrass and c l o v e r , f l a t s r e c e i v e d dual i n o c u l a t i o n of R. trifolii and B. polymyxa. Each f l a t r e c e i v e d 3 x 10 1 (^ R. trifolii c e l l s ( 1 0 1 ^ c e l l s of each of the three homologous s t r a i n s ) i n 200 ml MGA medium (Dughri and Bottomley, 1983a) and 10 1^ c e l l s of B. polymyxa in n u t r i e n t b r o t h . Inoculum was a d m i n i s t e r e d by pouring 50 ml a l i q u o t s evenly along each row of p l a n t s i n the f l a t f o l l o w e d by e x t e n s i v e watering. C o n t r o l f l a t s r e c e i v e d R. trifolii but no B. polymyxa i n o c u l a t i o n . P l a n t s were grown under n a t u r a l l i g h t with a day/night temperature regime of 30°/20°C i n a greenhouse and were c l i p p e d back to the height of the p l a s t i c p a r t i t i o n twice d u r i n g the growth p e r i o d . A f t e r 2 months growth, a d e s t r u c t i v e harvest was c a r r i e d out. In each c e l l , the outer two p l a n t s were d i s c a r d e d as these may have experienced s u b s t a n t i a l border e f f e c t s with the p l a s t i c p a r t i t i o n s . The inner four p l a n t s were separated from each ot h e r , d i v i d e d i n t o r o o t s and shoots, d r i e d at 55°C f o r 4 days, and weighed. 95 Nitrogen Fixation P l a n t s to be e v a l u a t e d f o r n i t r o g e n f i x a t i o n a c t i v i t y u s i ng 1^N/ 1 4N r a t i o s were f e r t i l i z e d with 10 kg n i t r o g e n ha 1 ( 5 % - 1 5 N l a b e l as C a ( 1 5 N 0 3 ) 2 ) at the beginning of the experiment. At h a r v e s t , biomass was d r i e d at 55°C f o r 2 days, ground to 2 mm and analyzed f o r 1^N/ 1 4N us i n g a s t a b l e i s o t o p e mass spectrometer (VG Is o g a s ) . For the a c e t y l e n e r e d u c t i o n assay, i n d i v i d u a l pots were p l a c e d i n 1 l i t r e p l a s t i c screw-cap c o n t a i n e r s (Nalgene), s e a l e d , and ac e t y l e n e was added to a f i n a l c o n c e n t r a t i o n of 10% ( v / v ) . A f t e r 4 h at room temperature, a 5 ml gas sample was removed and 1 ml of the sample was a n a l y z e d ' f o r ethylene p r o d u c t i o n by gas chromatography as d e s c r i b e d f o r the b a c t e r i a l c u l t u r e s . Supplemental Phosphorus and Growth Regulators Treatments r e c e i v i n g a d d i t i o n a l s o l u b l e phosphorus (P) were given a t o t a l of 28 kg ha 1 P as NaH2PC>4 (pH 7.0) a p p l i e d i n four equal a l i q u o t s ; at p l a n t i n g and then at three subsequent two week i n t e r v a l s . These a d d i t i o n s doubled the a v a i l a b l e P i n the s o i l to 56 kg ha 1 d u r i n g the course of the experiment. Treatments r e c e i v i n g growth r e g u l a t o r s were given a t o t a l of e i t h e r 0.12 ug or 1.2 ug i n d o l e a c e t i c a c i d (IAA) pot 1 with or without i s o p e n t y l adenine (IPA) (0.01 ug pot 1 ) , a l s o a p p l i e d i n 4 equal a l i q u o t s . In treatments r e c e i v i n g e i t h e r a d d i t i o n a l P or growth r e g u l a t o r s , the f i r s t a p p l i c a t i o n was made i n n u t r i e n t b r o t h while water was used f o r subsequent a p p l i c a t i o n s . To t e s t f o r P s o l u b i l i z a t i o n , B. polymyxa was str e a k e d onto p l a t e s of m o d i f i e d Pikovskaya's medium (Parry et a l . , 1983) c o n t a i n i n g (g/1): glucose 10; ( N H 4 ) 2 S 0 4 0.5; KC1 0.2; MgS0 4~7H 20 0.1; MnS0 4 0.005; FeS0 4 0.005; yeast e x t r a c t 0.5; agar 15; and d i s t i l l e d water to 1 l i t e r . Calcium phytate (2 g/1) was i n c l u d e d i n those p l a t e s used to t e s t f o r organic P s o l u b i l i z a t i o n t e s t s , while CaHPC>4 (1 g/1) was i n c l u d e d i n the i n o r g a n i c P s o l u b i l i z a t i o n t e s t s . P l a t e s were incubated f o r 2 weeks at 30°C and then examined v i s u a l l y f o r c l e a r i n g of the agar. Isolation of Plant Growth Promoting Substances B. polymyxa s t r a i n L6 was grown f o r 6 days i n n u t r i e n t medium c o n t a i n i n g (g/1): (NH 4) 2HPC> 4 1.0; KC1 0.2; MgS0 4"7H 20 0.2; yeast e x t r a c t 0.2; and sucrose 10. The c u l t u r e (300 ml) was h a r v e s t e d by c e n t r i f u g a t i o n at 12,000 x g f o r 30 minutes; the supernatant was decanted and reduced to 100 ml by r o t a r y e v a p o r a t i o n at l e s s than 35°C. The con c e n t r a t e d supernatant was a c i d i f i e d to pH 2.5 with 1N HCl and p a r t i t i o n e d three times a g a i n s t equal volumes of g l a s s - d i s t i l l e d e t h y l a c e t a t e . The combined e t h y l a c e t a t e phase was evaporated to dryness, r e - d i s s o l v e d i n acetone and chromatographed on K i e s e l g e l 60-F254 TLC p l a t e s (Merck) using an isobutanol:ammonia:water (10:1:1) (v/v) s o l v e n t . The chromatograph was cut i n 10 equal s t r i p s (10 mm), each of which was scraped i n t o i n d i v i d u a l t e s t tubes. For g i b b e r e l l i n b i o a s s a y s , the s i l i c a g e l s c r a p i n g s were e l u t e d with 2 ml acetone. For i n d o l e a c e t i c a c i d (IAA) b i o a s s a y s , the s c r a p i n g s were e l u t e d overnight at 4°C i n 6 ml of assay b u f f e r (0.1M phosphate, pH 6.4). The aqueous phase f o l l o w i n g e t h y l a c e t a t e p a r t i t i o n i n g was evaporated b r i e f l y to remove r e s i d u a l e t h y l a c e t a t e p r i o r to a d j u s t i n g the pH to 7.5 with 5N KOH. The s o l u t i o n was p a r t i t i o n e d f i v e times with 1/5 volume g l a s s - d i s t i l l e d n-butanol. The b u t a n o l i c phase was evaporated and the r e s i d u e taken up i n a minimum volume of methanol. The methanolic f r a c t i o n was d i v i d e d i n t o three samples to p r o v i d e r e p l i c a t i o n f o r the b i o a s s a y . E x t r a c t s were st r e a k e d onto chromatography paper (Whatman 3MM); descending chromatography were run using a chloroformrmethanol (17:3)(v/v) s o l v e n t . The chromatograms were a i r - d r i e d and cut i n t o ten equal s e c t i o n s (1 cm) f o r i n c l u s i o n i n the bioassay medium. Bi oas s ays C y t o k i n i n was bioassayed u s i n g soybean c a l l u s ( M i l l e r , 1965) with three r e p l i c a t e f l a s k s (125 ml) each c o n t a i n i n g 20 ml medium and three p i e c e s of c a l l u s f o r each sample to be analyzed. A f t e r 2 weeks at 30°C the c a l l u s e s were b l o t t e d and weighed. G i b b e r e l l i n s were bioassayed as f o l l o w s (Murakami, 1968). Seeds of r i c e , Oryza sativa L. cv Tan Ginbozu were sur f a c e s t e r i l i z e d i n 1% (w/v) sodium h y p o c h l o r i t e f o r 20 minutes, r i n s e d r e p e a t e d l y i n s t e r i l e d i s t i l l e d water then germinated at 30°C f o r 72 h. Uniformly sprouted s e e d l i n g s were t r a n s f e r r e d to P e t r i d i s h e s (10 cm diameter) c o n t a i n i n g 1% (w/v) agar and d i v i d e d i n t o quadrats. Four s e e d l i n g s per quadrat were used f o r each of three r e p l i c a t e s to provide 12 p l a n t s per treatment. Dishes were exposed f o r 48 h to continuous i l l u m i n a t i o n (200 uEm ^s 1) at 30°C. A microdrop (1 u l ) of t e s t s o l u t i o n or g i b b e r e l l i c a c i d (GA^) standard was a p p l i e d to the base of each c o l e o p t i l e . T r e a t e d p l a n t s were grown f o r an a d d i t i o n a l 72 h under the same c o n d i t i o n s before the len g t h of the second l e a f sheath was measured. A u x i n - l i k e a c t i v i t y was determined using the Avena c o l e o p t i l e s t r a i g h t growth assay ( N i t s c h and N i t s c h , 1956). Seeds of Avena sativa L. cv V i c t o r y were germinated i n the dark i n moistened v e r m i c u l i t e f o r 72-96 h. A l l subsequent manipulation of the m a t e r i a l was c a r r i e d out under dim red l i g h t (CBS Red 650 s a f e l i g h t ) . The a p i c a l 3 mm of the shoot were e x c i s e d with a razor blade and d i s c a r d e d ; the s u b - a p i c a l 6 mm s e c t i o n was removed and used i n the bi o a s s a y . F i v e segments i n each of three P e t r i d i s h e s (2.8 cm diameter) c o n t a i n i n g 0.1M phosphate b u f f e r , pH 6.4 supplemented with 1% (w/v) sucrose were incubated on a r o t a r y shaker at room temperature f o r 20 h and were then measured with an o p t i c a l micrometer. S e c t i o n s incubated i n b u f f e r alone were used as c o n t r o l s to evaluate the e f f e c t of the i s o l a t e d f r a c t i o n s . Gas Chr omat ogr aphy Samples showing p u t a t i v e growth promoting substances i n bio a s s a y s were d e r i v a t i z e d with B i s - t r i m e t h y l s i l y l t r i f l u o r a c e t a m i d e (Sigma) and analyzed by gas l i q u i d chromatography (Kingman and Moore, 1982). Sample i d e n t i t i e s were subsequently checked using c o - e l u t i o n chromatography with known commercial standards. Experimental De s i gn and St at i s i c a I Analysis The e f f e c t of B. pol ymyxa on growth of a white c l o v e r -p e r e n n i a l ryegrass mixture was s t u d i e d using a s p l i t - s p l i t -s p l i t p l o t design with inoculum a s s i g n e d to main p l o t s ( f l a t s ) , r y egrass c l o n e s to s u b - p l o t s (rows w i t h i n f l a t s ) and c l o v e r c l o n e s to sub-sub-plots (columns w i t h i n f l a t s ) . Main p l o t s were randomized w i t h i n 3 complete b l o c k s . The e n t i r e experiment was repeated and the combined data analyzed as a s i n g l e experiment with blocks as main p l o t s and experiments as the r e p l i c a t e s . The e f f e c t of B. pol ymyxa i n o c u l a t i o n , a d d i t i o n a l s o l u b l e phosphorus, and growth r e g u l a t o r s on emergence and growth of c r e s t e d wheatgrass was e v a l u a t e d using a l a t i n square d e s i g n . The experiment c o n s i s t e d of two 8 x 8 l a t i n squares (n=16). A l l treatments were r e p l i c a t e d at l e a s t once in other experiments. Mean s e p a r a t i o n and t e s t s of s i g n i f i c a n c e were performed using ANOVA, with s i n g l e degree of freedom orthogonal c o n t r a s t s . Homogeneity of v a r i a n c e t e s t s f o r p e r e n n i a l ryegrass shoot, r o o t , and p l a n t dry weight and f o r white c l o v e r shoot and p l a n t dry weight were s i g n i f i c a n t . R e c i p r o c a l data of these y i e l d v a r i a b l e s were r e - t e s t e d f o r e q u a l i t y of v a r i a n c e and F - t e s t s were not s i g n i f i c a n t . T h e r e f o r e , p r o b a b i l i t y l e v e l s a s c r i b e d i n t e s t s of these y i e l d v a r i a b l e s r e f e r to a n a l y s i s of transformed data. 100 RESULTS I d e n t i f i c a t i o n of Bacillus polymyxa s t r a i n L6 was achieved by studying f e a t u r e s of i t s p h y s i o l o g y and bio c h e m i s t r y (Table 2-1) with the API 20E system which has been adapted f o r use i n i d e n t i f i c a t i o n of d i a z o t r o p h i c b a c t e r i a (Rennie, 1980). The system was o r i g i n a l l y developed for the r a p i d i d e n t i f i c a t i o n of members of the Ent erobact eri aceae. C h a r a c t e r i s t i c s l i s t e d (Table 2-1) are c o n s i s t e n t with p u b l i s h e d accounts of the p r o p e r t i e s of B. pol ymyxa (Buchanan and Gibbons, 1974; Rennie, 1980). I n o c u l a t i o n of pure and mixed stands of p e r e n n i a l ryegrass and white c l o v e r with B. pol ymyxa r e s u l t e d i n the d i s c o v e r y of complex i n t e r a c t i o n s between neighbours and genotypes of the pasture p l a n t s and the bacterium (Tables 2-2 to 2-8). Pure stands of p l a n t s c o n s i s t e d of c l o n e s of three genotypes of the a p p r o p r i a t e s p e c i e s . I n o c u l a t i o n of ryegrass or c l o v e r when grown i n pure stands had no s i g n i f i c a n t e f f e c t on any of the parameters measured (Table 2-2), but there was a n o n s i g n i f i c a n t tendency f o r p l a n t weight to decrease in response to i n o c u l a t i o n . However, when B. polymyxa was used to i n o c u l a t e pure stands of ryeg r a s s or c l o v e r p l a n t s homologous to i t , these tendencies were rev e r s e d . Though not s t a t i s t i c a l l y s i g n i f i c a n t , r yegrass p l a n t weight showed a s l i g h t gain due to i n o c u l a t i o n (2%) mainly because of a l a r g e e f f e c t on root weight (39%) (Table 2-3). The same s t a t i s t i c a l l y i n s i g n i f i c a n t trends were evident i n pure stands of c l o v e r where a 4% decrease i n p l a n t 101 Table 2 - 1 . P h y s i o l o g i c a l and b i o c h e m i c a l c h a r a c t e r i s t i c s of Bacillus polymyxa s t r a i n L 6 C h a r a c t e r i s t i c Reaction a e r o b i c growth + anaerobic growth + growth on b i l e s a l t s gram s t a i n + spores + f l a g e l l a + a c e t y l e n e r e d u c t i o n + n i t r a t e r e d u c t i o n + n i t r i t e r e d u c t i o n c a s e i n h y d r o l y s i s + c a t a l a s e a c t i v i t y g e l l i q u i f a c t i o n + i n d o l e p r o d u c t i o n B - g a l a c t o s i d a s e a c t i v i t y + a c e t o i n p r o d u c t i o n + phytase a c t i v i t y + 102 T a b l e 2-2 . E f f e c t of Bacillus pol ymyxa i n o c u l a t i o n on p l a n t d r y weight and r o o t / s h o o t r a t i o i n pure s tands of g e n o t y p i c m i x t u r e s of p e r e n n i a l r y e g r a s s or white c l o v e r P e r e n n i a l r y e g r a s s White c l o v e r C h a r a c t e r I n o c u l a t i o n I n o c u l a t i o n %change - + %change Shoot weight (mg) 328 275 -16 766 706 - 8 Root weight (mg) 167 141 -16 202 227 + 12 T o t a l weight (mg) 495 416 -16 968 933 - 4 Root /Shoot r a t i o 0.51 0.51 0 0.26 0.32 + 23 P l a n t s were grown i n s o i l under greenhouse c o n d i t i o n s . n=!8 103 T a b l e 2-3. E f f e c t of Bacillus pol ymyxa i n o c u l a t i o n on p l a n t d r y weight and r o o t / s h o o t r a t i o i n pure s t a n d s of p e r e n n i a l r y e g r a s s or w h i t e c l o v e r genotypes homologous t o B. pol ymyxa P e r e n n i a l r y e g r a s s White c l o v e r C h a r a c t e r I n o c u l a t i o n I n o c u l a t i o n — + %change + %change Shoot weight (mg) 373 308 -17 654 820 + 25 Root weight (mg) 191 266 + 39 1 93 220 + 14 T o t a l weight (mg) 564 574 + 2 847 1 040 + 23 Root/Shoot r a t i o 0.51 0.86 + 69 0.30 0.27 -1 1 P l a n t s were grown i n s o i l under greenhouse c o n d i t i o n s . n=6 104 Table 2-4. Eff e c t of Bacillus pol ymyxa inoculation on plant dry weight and root/shoot r a t i o in a 50:50 (by numbers) genotypic and species mixture of perennial ryegrass and white clover Perennial ryegrass White clover Character Inoculation Inoculation + %change - + %change Shoot weight (mg) 1087 1 056 - 3 560 672 + 20* Root weight (mg) 544 446 -22 1 28 1 73 + 35* Total weight (mg) 1631 1 502 - 9 688 845 + 23* Root/Shoot r a t i o 0.50 0.42 - 1 9 0.23 0.26 + 12 Plants were grown in s o i l under greenhouse conditions. n=l8 * P < 0.05 105 Table 2-5. E f f e c t of Bacillus pol ymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o i n a 50:50 (by numbers) mixture of p e r e n n i a l ryegrass and white c l o v e r P e r e n n i a l r y e g r a s s White c l o v e r Character Inoculat ion %change In o c u l a t ion %change + + Shoot weight (mg) 1210 1 1 36 - 6 573 813 + 42* Root weight (mg) 646 684 + 6 1 48 212 + 43* T o t a l weight (mg) 1856 1820 - 2 721 1025 + 42* Root/Shoot r a t i o 0.53 0.60 + 13 0.26 0.26 0 P e r e n n i a l ryegrass c l o n e s homologous to B. pol ymyxa were grown with a genotypic mixture of a l l c l o v e r c l o n e s . White c l o v e r c l o n e s homologous t o B. pol ymyxa were grown with a genotypic mixture of a l l p e r e n n i a l ryegrass c l o n e s . P l a n t s were grown i n s o i l under greenhouse c o n d i t i o n s . n=18-24 * P < 0.05 106 Table 2-6. E f f e c t of Bacillus pol ymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o i n a 50:50 (by numbers) mixture of p e r e n n i a l ryegrass and white c l o v e r c l o n e s homologous to each other and B. pol ymyxa P e r e n n i a l ryegrass. White c l o v e r Character I n o c u l a t ion I n o c u l a t i o n + %change — + %change Shoot weight (mg) 1 063 1 1 25 + 6 473 753 + 59 Root weight (mg) 561 751 + 34 146 232 + 59 T o t a l weight (mg) 1 624 1 876 + 16 619 985 + 59 Root/Shoot r a t i o 0.53 0.67 + 26 0.31 0.31 0 P l a n t s were grown in s o i l under greenhouse condi i.tions. n = 6 107 Table 2-7. E f f e c t of Bacillus polymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o i n a 50:50 (by numbers) mixture of p e r e n n i a l ryegrass and white c l o v e r P e r e n n i a l ryegrass White c l o v e r Character I n o c u l a t ion %change I n o c u l a t i o n %change + + Shoot weight (mg) 1 023 1099 + 7 619 571 - 8 Root weight (mg) 529 392 -25 1 57 171 + 9 T o t a l weight (mg) 1 552 1 491 - 4 776 742 - 4 Root/Shoot r a t i o 0.52 0.36 -31* 0.30 0.25 + 20* P e r e n n i a l ryegrass comprised a s i n g l e genotype not homologous to B. polymyxa grown with a genotypic mixture of c l o v e r c l o n e s . White c l o v e r comprised a s i n g l e genotype not homologous to B. pol ymyxa grown with a genotypic mixture of p e r e n n i a l r y e g r a s s c l o n e s . P l a n t s were grown i n s o i l under greenhouse c o n d i t i o n s . n=l8-24 * P < 0.05 108 Table 2-8. E f f e c t of Bacillus polymyxa i n o c u l a t i o n on p l a n t dry weight and root/shoot r a t i o i n a 50:50 (by numbers) mixture of p e r e n n i a l ryegrass and white c l o v e r P e r e n n i a l ryegrass White c l o v e r Character I n o c u l a t ion %change I n o c u l a t i o n %change + + Shoot weight (mg) 1311 1 283 - 2 430 524 -18 Root weight (mg) 626 577 - 8 1 37 168 -18 T o t a l weight (mg) 1937 1860 - 4 567 692 -18 Root/Shoot r a t i o 0.48 0.45 - 6 0.32 0.32 0 P e r e n n i a l r y e g r a s s comprised a s i n g l e genotype not homologous t o B. polymyxa grown with a c l o n e s of the c l o v e r genotype homologous to B. pol ymyxa. White c l o v e r comprised a s i n g l e genotype not homologous to B. polymyxa grown with c l o n e s of the p e r e n n i a l ryegrass genotype homologous to B. polymyxa. P l a n t s were grown i n s o i l under greenhouse c o n d i t i o n s . n=6 weight of mixed genotypes (Table 2-2) became a 23% i n c r e a s e when the homologous genotype comprised the e n t i r e pure stand (Table 2-3). Neighbour e f f e c t s became evident when i n o c u l a t i o n of mixed stands of the pasture p l a n t s was c a r r i e d out (Table 2-4). If ryegrass c l o n e s were a composite of a l l three genotypes c o l l e c t e d , the n o n s i g n i f i c a n t tendency to r e a c t n e g a t i v e l y to i n o c u l a t i o n with B. polymyxa was observed when ryegrass p l a n t y i e l d was measured. However, c l o v e r c l o n e s c o n s i s t i n g of the three genotypes c o l l e c t e d showed s i g n i f i c a n t y i e l d i n c r e a s e s when i n o c u l a t e d i n mixture with r y e g r a s s . Root weight was most respo n s i v e to i n o c u l a t i o n (35%) but shoot weight was s i g n i f i c a n t l y higher as w e l l (20%). These changes r e s u l t e d i n i n o c u l a t e d p l a n t s y i e l d i n g 23% more biomass than c o n t r o l s . As was suggested by work with pure stands, the y i e l d response due to i n o c u l a t i o n c o u l d be i n c r e a s e d , sometimes s i g n i f i c a n t l y , by u s i n g ryegrass and/or c l o v e r c l o n e s homologous to the s t r a i n of B. polymyxa used fo r i n o c u l a t i o n . Ryegrass was the l e a s t responsive but y i e l d s i n c r e a s e d from 9% below c o n t r o l s (Table 2-4) to 2% below c o n t r o l p l a n t weight when homologous ryegrass c l o n e s were grown with a composite of a l l three c l o v e r genotypes (Table 2-5). T h i s s l i g h t y i e l d disadvantage became a 16% i n c r e a s e i f the homologous c l o v e r genotype was used as the neighbour i n s t e a d of the genotypic mixture (Table 2-6). However, a l l responses in p e r e n n i a l ryegrass y i e l d were not s t a t i s t i c a l l y s i g n i f i c a n t . 110 White c l o v e r showed responses s i m i l a r t o ry e g r a s s when grown i n a mixture of the two s p e c i e s , but the magnitude of the y i e l d responses was more impr e s s i v e . The 23% (P < 0.05) y i e l d advantage observed when i n o c u l a t e d c l o v e r p l a n t s were grown i n a mixture with clones of a l l ryegrass genotypes almost doubled to 42% (P < 0.05) when the y i e l d of the c l o v e r genotype homologous to B. pol ymyxa was measured (Table 2-5). A f u r t h e r i n c r e a s e i n y i e l d to 59% above c o n t r o l p l a n t s was observed when the homologous r y e g r a s s genotype e x c l u s i v e l y comprised the grass neighbour p o p u l a t i o n (Table 2-6). The 59% i n c r e a s e i n y i e l d was not s t a t i s t i c a l l y s i g n i f i c a n t due to the decrease i n r e p l i c a t i o n of t h i s f a c t o r i a l combination. The y i e l d advantage a c c r u i n g from homologous p l a n t / b a c t e r i a a s s o c i a t i o n s compared with y i e l d s i n stands of mixed genotypes suggests that c e r t a i n nonhomologous genotype combinations do not r e a c t p o s i t i v e l y to i n o c u l a t i o n . R e s u l t s p r esented i n Tables 2-7 and 2-8 c o n f i r m t h i s i d e a . When ryegr a s s homologous to the bacterium was grown i n mixture with c l o n e s of a l l the c l o v e r genotypes, no s i g n i f i c a n t e f f e c t on p l a n t performance was observed (Table 2-5). However, i n o c u l a t i o n of a nonhomologous ryegrass genotype under these c o n d i t i o n s r e s u l t e d i n a r e l a t i v e l y l a r g e but i n s i g n i f i c a n t 25% decrease i n root weight (Table 2-7). T h i s t r e n d was a l s o evident i n white c l o v e r y i e l d where a 42% (P < 0.05) y i e l d advantage i n the homologous genotype (Table 2-5) was l o s t , as no e f f e c t i n p l a n t weight was observed i n the y i e l d of a nonhomologous c l o v e r genotype (Table 2-7). S i m i l a r l y , the l a r g e but s t a t i s t i c a l l y i n s i g n i f i c a n t y i e l d advantage i n ryegrass and c l o v e r p l a n t weight which occurred when a l l three organisms were homologous (Table 2-6) was completely r e v e r s e d when the y i e l d of a nonhomologous p l a n t genotype was s t u d i e d (Table 2-8). For example, i n o c u l a t e d white c l o v e r showed a 59% y i e l d i n c r e a s e when a l l organisms were homologous (Table 2-6), but the y i e l d of a nonhomologous i n o c u l a t e d c l o v e r genotype grown under the same c o n d i t i o n s was 18% below c o n t r o l p l a n t y i e l d s (Table 2-8). To a v o i d the c o m p l e x i t i e s i n t r o d u c e d by the r y e g r a s s / c l o v e r neighbour and genotype e f f e c t s , work to e l u c i d a t e the mechanism of the p l a n t growth promoting a c t i v i t y of B. pol ymyxa s t r a i n L6 was continued using pure stands of c r e s t e d wheatgrass (Agropyron cri st at um L . ) . S o i l i n o c u l a t i o n with the bacterium at sowing r e s u l t e d i n a g r e a t e r number of s e e d l i n g s which emerged compared with c o n t r o l s e e d l i n g s (Table 2-9). T h i s e f f e c t became s i g n i f i c a n t when a l l p l a n t s had emerged (day 17, Table 2-9), r e s u l t i n g i n 23% (P < 0.05) more p l a n t s i n i n o c u l a t e d pots than i n c o n t r o l s t r e a t e d with f i l t e r - s t e r i l i z e d c u l t u r e supernatant. Treatments f e r t i l i z e d with supplemental s o l u b l e phosphorus showed a s i m i l a r t r e n d toward enhanced emergence by day 17, although t h i s e f f e c t was not s t a t i s t i c a l l y s i g n i f i c a n t . A d d i t i o n of IAA, with or without IPA, i n i t i a l l y tended to r e t a r d emergence, but these treatments 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 the u n i n o c u l a t e d c o n t r o l s by day 17. A f t e r e i g h t weeks growth, a s i g n i f i c a n t y i e l d i n c r e a s e in c r e s t e d wheatgrass has c o n s i s t e n t l y been observed Table 2-9. E f f e c t of p l a n t growth r e g u l a t o r s , s o l u b l e phosphorus and i n o c u l a t i o n with Bacillus polymyxa on emergence of c r e s t e d wheatgrass 5 to 17 days a f t e r sowing. n=8-16 Mean Number of Seedlings Emerged Days N u t r i e n t . broth 1 S t e r i l e c u l t u r e » I n o c u l a t e d Phosphorus f i l t r a t e (B. polymyxa) (28 kg/ha) IAA 4 IAA+IPA 5 5.0 4.5 5.6 ( 2 4 ) 3 5.0 (10) 4.3 (- 16) 4.8 (-4) 8 8.8 8.4 9.5 (13) 9.0 ( 2) 8.1 ( -8) 8.4 (-4) 10 9.0 9.1 10.1 (11) 10.1 (12) 8.8 ( -2) 9.0 ( 0) 1 2 9.3 9.1 10.8 (13) 10.8 (16) 9.2 ( -1 ) 9.3 ( 0) 17 9.7 9.2 11.3 (23)* 11.3 (16) 9.7 ( 0) 9.8 ( 1) 1 N u t r i e n t b r o t h was used as the c o n t r o l f o r comparisons i n v o l v i n g supplemental phosphorus and growth r e g u l a t o r s . 2 F i l t e r - s t e r l l i z e d c u l t u r e supernatant was used as the c o n t r o l f o r comparisons i n v o l v i n g i n o c u l a t e d versus u n i n o c u l a t e d treatments. 3 Values i n p a r e n t h e s i s represent percent change i n treatment compared to the a p p r o p r i a t e c o n t r o l . 4 Values f o r IAA treatments are the pooled data from 0.12 and 1.2 ug per pot. * P < 0.05 f o l l o w i n g i n o c u l a t i o n . T y p i c a l l y , a minor e f f e c t on shoot growth i s a s s o c i a t e d with a s u b s t a n t i a l i n c r e a s e i n root (28%) and t o t a l p l a n t (17%) dry weights; higher root/shoot r a t i o s are, t h e r e f o r e , c h a r a c t e r i s t i c of the i n o c u l a t e d p l a n t s (Table 2-10). Biological Nitrogen Fixation Acetylene r e d u c t i o n assays of broth c u l t u r e s of B. pol ymyxa were c a r r i e d out to ensure that the s t r a i n used in these experiments c o u l d produce an a c t i v e nitrogenase in vitro. The p r o v i s i o n of n i t r o g e n through b i o l o g i c a l f i x a t i o n by B. polymyxa was an obvious h y p o t h e s i s f o r enhanced p l a n t performance, but these data do not support t h i s premise (Table 2-11). No d e t e c t a b l e in vivo a c e t y l e n e r e d u c t i o n a c t i v i t y was a s s o c i a t e d with i n o c u l a t e d c r e s t e d wheatgrass assayed a f t e r 8 weeks growth. Subsequent experimentation . . . 1 5 . u t i l i z i n g N enrichment a l s o y i e l d e d no evidence of in vivo n i t r o g e n f i x i n g a c t i v i t y . Phosphate Solubilization B. polymyxa L6 was t e s t e d f o r the a b i l i t y to s o l u b i l i z e phosphate using in vitro t e s t s . C o l o n i e s of t h i s s t r a i n were able to c l e a r agar p l a t e s c o n t a i n i n g organic phosphate (c a l c i u m p h y t a t e ) , but had no e f f e c t on medium s a t u r a t e d with an i n o r g a n i c phosphate source (CaHP0 4) (Table 2-11). To t e s t the h y p o t h e s i s that s o l u b i l i z a t i o n of organic phosphate compounds was r e s p o n s i b l e f o r the observed growth response, c r e s t e d wheatgrass p l a n t s were supplemented with twice the Table 2-10. E f f e c t of p l a n t growth r e g u l a t o r s , s o l u b l e phosphorus and i n o c u l a t i o n with Bacillus polymyxa on p l a n t dry weight and root/shoot r a t i o of c r e s t e d wheatgrass grown i n s o i l under greenhouse c o n d i t i o n s . n=8-l6 S t e r i l e . Character N u t r i e n t . c u l t u r e _ I n o c u l a t e d Phosphorus IAA b r o t h f i l t r a t e (B. polymyxa) (28 kg/ha) Shoot wt (mg) 539 585 625 ( 7 ) 3 599 (11) 576 ( 7) Root wt (mg) 570 589 751 (28)** 618 ( 8) 658 (15)* P l a n t wt (mg) 1109 1174 1376 (17)* 1217 (10) 1234 (11)* Root/Shoot 1.07 1.02 1.20 (18)* 1.05 (-2) 1.14 ( 7) r a t i o N u t r i e n t broth was used as the c o n t r o l f o r comparisons i n v o l v i n g supplemental phosphorus and growth r e g u l a t o r s . 2 F i l t e r - s t e r i l i z e d c u l t u r e supernatant was used as the c o n t r o l f o r comparisons i n v o l v i n g i n o c u l a t e d versus un i n o c u l a t e d treatments. ^Values i n p a r e n t h e s i s represent percent change i n treatment compared to the a p p r o p r i a t e c o n t r o l . 4 Values f o r IAA treatments are the pooled data from 0.12 and 1.2 ug per pot. * P < 0.05 ** P < 0.01 Table 2-11. Survey of c h a r a c t e r i s t i c s of Bacillus polymyxa s t r a i n L6 used i n these experiments which may account f o r the observed p l a n t growth response Character Presence (+) or Absence (-) Nitrogenase a c t i v i t y (in vitro acetylene r e d u c t i o n ) + (1.3 nmole ethylene /ml/h) Root a s s o c i a t e d N_ f i x a t i o n (in vivo a c e t y l e n e r e d u c t i o n ) S o l u b i l i z a t i o n of organic + phosphorus S o l u b i l i z a t i o n of i n o r g a n i c phosphorus A n t i b i o t i c a c t i v i t y C y t o k i n i n production (i n vil ro) G i b b e r e l l i n p r oduction (in vitro) IAA p r o d u c t i o n (in vitro ) + (38 ug l " 1 ) 1Calcium phytate (2 g 1~ 1) 2 -1 Calcium phosphate, d i b a s i c (1 g 1 ) ^Bioassay d e t e c t i o n l i m i t was 2.5 ng 1 1 IPA e q u i v a l e n t s 4 -1 Bioassay d e t e c t i o n l i m i t was 60 ug 1 GA., e q u i v a l e n t s a v a i l a b l e p h o s p h a t e and compared w i t h i n o c u l a t e d t r e a t m e n t s ( T a b l e 2 -10 ) . W h i l e r o o t and s h o o t w e i g h t s were i n c r e a s e d by t h e a d d i t i o n a l p h o s p h a t e , t h e m a g n i t u d e of t h e r o o t r e s p o n s e was c o n s i d e r a b l y l o w e r t h a n t h a t o b s e r v e d i n t h e i n o c u l a t e d t r e a t m e n t s . F u r t h e r m o r e , i n c r e a s e s i n r o o t and s h o o t w e i g h t s were c o m p a r a b l e when p h o s p h a t e was added, t h u s p r o d u c i n g a s u b s t a n t i a l l y l o w e r (20%) r o o t / s h o o t r a t i o t h a n i n o c u l a t e d t r e a t m e n t s . Growth Promoting Activity B i o a s s a y o f c e l l - f r e e e x t r a c t s of B. pol ymyxa b r o t h c u l t u r e s f o r p l a n t g r o w t h p r o m o t i n g s u b s t a n c e s r e v e a l e d s u b s t a n t i a l IAA a c t i v i t y ( T a b l e 2 -11 ) . The i d e n t i f i c a t i o n o f IAA a s t h e a c t i v e component was c o n f i r m e d by c o - e l u t i o n o f t h e a c t i v e f r a c t i o n w i t h a known IAA s t a n d a r d i n t h e gas c h r o m a t o g r a p h i c a n a l y s i s ( d a t a n ot shown). C y t o k i n i n and g i b b e r e l l i n - l i k e a c t i v i t y were not d e t e c t e d i n t h e e x t r a c t s . The o b v i o u s s t i m u l a t i o n of r o o t g r o w t h r e l a t i v e t o s h o o t g r o w t h i n t h e c r e s t e d w h e a t g r a s s r e s p o n s e t o i n o c u l a t i o n may r e s u l t f r o m c h a n g e s i n t h e amounts o f phyto h o r m o n e s i n t h e p l a n t r h i z o s p h e r e . The e f f e c t of i n o c u l a t i o n w i t h B. polymyxa on gro w t h of c r e s t e d w h e a t g r a s s was m i m i c k e d by exogenous a p p l i c a t i o n o f IAA. The ma g n i t u d e o f t h e r e s p o n s e t o IAA a p p e a r e d t o be l e s s t h a n t h a t t o i n o c u l a t i o n , b u t t r e n d s i n a l l c h a r a c t e r s m e a s u r e d were s i m i l a r f o r b o t h t r e a t m e n t s ( T a b l e 2 -10 ) . B e c a u s e c y t o k i n i n c a n a l s o e x e r t r o o t g r o w t h e f f e c t s , some t r e a t m e n t s i n c l u d e d , i n c o m b i n a t i o n w i t h IAA, a low c o n c e n t r a t i o n of c y t o k i n i n ( 0 . 0 1 ug pot , i s o p e n t e n y l a d e n i n e ) . The response to these t rea tments was not s t a t i s i c a l l y d i f f e r e n t from the e f f e c t s of IAA a l o n e , a l t h o u g h r o o t / s h o o t r a t i o s tended to be l ower . 118 DISCUSSION Plant Growth Promotion in Mixed Forage Stands The r e s u l t s d i s p l a y e d i n Tables 2-2 to 2-8 r e f l e c t the complex nature of p l a n t growth promotion by Bacillus polymyxa s t r a i n L6. Two f a c t o r s which are very important i n the response can be i d e n t i f i e d from these d a t a . F i r s t i s the e f f e c t of neighbours. The y i e l d of white c l o v e r (composite genotypes) i n o c u l a t e d i n a pure stand was not a f f e c t e d by the bacterium. However, when c l o v e r y i e l d was measured i n mixture with r y e g r a s s , shoot, root, and t h e r e f o r e p l a n t y i e l d i n c r e a s e s were noted. The i n o c u l a t e d p l a n t s had higher root/shoot r a t i o s . Ryegrass p l a n t s were not s i g n i f i c a n t l y a f f e c t e d by i n o c u l a t i o n , but tended to r e a c t n e g a t i v e l y whether grown i n pure or mixed stands. The e x p l a n a t i o n f o r the y i e l d enhancement observed i n c l o v e r when grown in mixture with ryegrass may be r e l a t e d to a change i n r e l a t i v e c o m p e t i t i v e a b i l i t i e s below ground of the two s p e c i e s f o l l o w i n g i n o c u l a t i o n . In pure stands, the presence of the bacterium decreased ryegrass root weight by 16% while i n c r e a s i n g c l o v e r root weight by 12%. However, both t r e n d s were not s t a t i s t i c a l l y s i g n i f i c a n t . S i m i l a r t e n dencies but of g r e a t e r magnitude were observed when p l a n t s were grown i n mixture. Under these circumstances, ryegrass root weight decreased by 22% compared with u n i n o c u l a t e d mixtures, again not s i g n i f i c a n t l y , but c l o v e r root weight was i n c r e a s e d by 35% (P < 0.05). These trends were a l s o e vident when above ground p l a n t y i e l d was measured but to a s m a l l e r 119 d e g r e e . Thus, p l a n t g r o w t h p r o m o t i o n of c l o v e r i n m i x t u r e w i t h r y e g r a s s may be a t t r i b u t a b l e i n p a r t t o i n c r e a s e d a v a i l a b i l i t y o f r e s o u r c e s as a c o n s e q u e n c e o f l e s s v i g o r o u s r y e g r a s s r o o t g r o w t h . However, y i e l d i n c r e a s e s ( s t a t i s i c a l l y i n s i g n i f i c a n t ) were o b s e r v e d i n p u r e s t a n d s of t h e p a s t u r e p l a n t s ( T a b l e 2 - 3 ) , t h e r e f o r e i t i s p o s s i b l e t h a t o t h e r f a c t o r s s u c h as m i c r o b i a l IAA p r o d u c t i o n a l s o c o n t r i b u t e t o t h i s r e s p o n s e . The s e c o n d f a c t o r w h i c h a p p e a r s t o be i m p o r t a n t t o t h e d i r e c t i o n and m a g n i t u d e of t h e g r o w t h r e s p o n s e i s t h e g e n o t y p i c c o r r e l a t i o n between p l a n t s and b a c t e r i a . I n o c u l a t i o n of p l a n t s homologous t o t h e b a c t e r i u m i n p u r e s t a n d s may r e s u l t i n a r e v e r s a l o f t h e n e g a t i v e y i e l d e f f e c t s o b s e r v e d when g e n o t y p i c m i x t u r e s were s t u d i e d . A d e f i n a t i v e c o n c l u s i o n i s not p o s s i b l e b e c a u s e t h e s e e f f e c t s were no t s t a t i s i c a l l y s i g n i f i c a n t (P < 0 . 0 5 ) . However, t h i s t r e n d was most a p p a r e n t when r y e g r a s s r o o t w e i g h t s o r c l o v e r s h o o t w e i g h t s a r e compared. The g e n o t y p i c s p e c i f i c i t y between p l a n t s and b a c t e r i a was p a r t i c u l a r l y e v i d e n t i n t h e y i e l d o f w h i t e c l o v e r (P < 0.05) when m i x e d s t a n d s o f p e r e n n i a l r y e g r a s s , w h i t e c l o v e r , and B. pol ymyxa were t e s t e d . Yield Response using Homologous Organisms D a t a f o r p l a n t y i e l d a r e a v a i l a b l e f o r c o m p a r i s o n s w h i c h p r o g r e s s f r o m l i t t l e homology between p l a n t s and b a c t e r i a t h r o u g h t h o s e w i t h homologous p l a n t / b a c t e r i a , but not p l a n t n e i g h b o u r c o m b i n a t i o n s , t o t o t a l homology between a l l o r g a n i s m s t e s t e d . As o r g a n i s m s a r e grown i n more " f a m i l i a r " 120 b i o t i c surroundings, p l a n t y i e l d s i n c r e a s e a c c o r d i n g l y , sometimes s i g n i f i c a n t l y . T h e r e f o r e , i n o c u l a t e d r y e g r a s s p l a n t s showed a 9% decrease i n p l a n t weight when a composite of genotypes was grown i n mixture with a l l three types of c l o v e r c l o n e s . T h i s response was reduced to a 2% y i e l d decrease when the ryegrass genotype homologous to B. polymyxa was t e s t e d with the same nonhomologous c l o v e r neighbours, while a y i e l d i n c r e a s e of 16% was noted when the homologous c l o v e r genotype comprised the neighbouring p l a n t p o p u l a t i o n . However, a l l these d i f f e r e n c e s i n p e r e n n i a l ryegrass performance were not s t a t i s t i c a l l y s i g n i f i c a n t . More impressive gains i n the y i e l d of white c l o v e r were apparent under c o n d i t i o n s of i n c r e a s i n g b i o t i c f a m i l i a r i t y . White c l o v e r p l a n t y i e l d i n c r e a s e d by 23% (P < 0.05) when sp e c i e s mixtures of a l l c l o v e r and ryegrass genotypes were i n o c u l a t e d . The y i e l d advantage almost doubled (42%) (P < 0.05) when the homologous c l o v e r / b a c t e r i u m comination was t e s t e d with a composite of a l l r y e g r a s s genotypes. I f homologous ryegrass genotypes were used as the neighbouring p l a n t s i n these t e s t s , a f u r t h e r y i e l d i n c r e a s e of near 50% r e s u l t e d such that c l o v e r p l a n t s o u t y i e l d e d u n i n o c u l a t e d c o n t r o l s by 59%. The decrease i n r e p l i c a t i o n of the l a t t e r treatment c o n t r i b u t e d to the y i e l d response not being s t a t i s i c a l l y s i g n f i c a n t . The importance of these b i o t i c e f f e c t s i s emphasized when corres p o n d i n g y i e l d s of nonhomologous p l a n t genotypes are s t u d i e d . T h i s comparison i s demonstrated e f f e c t i v e l y when the y i e l d of a nonhomologous p l a n t genotype i s compared 121 to p l a n t y i e l d s under c o n d i t i o n s of some homology. The 42% (P < 0.05) y i e l d advantage observed when white c l o v e r was homologous to B. pol ymyxa i s l o s t i f the y i e l d of a nonhomologous c l o v e r genotype i s measured. Under the l a t t e r c o n d i t i o n s , i n o c u l a t i o n had no e f f e c t on performance of white c l o v e r . S p e c i f i c i t y between p l a n t and bacterium has been p r e v i o u s l y r e p o r t e d to occur between B. pol ymyxa and wheat (Rennie and Larson, 1979; 1981). In those s t u d i e s , the authors were able to a s s i g n s p e c i f i c i t y to a s i n g l e chromosome i n the wheat genome. The bacterium used in t h e i r work was i s o l a t e d from s o i l i n which wheat had been c u l t i v a t e d f o r over 60 years (Neal and Larson, 1976). It i s p o s s i b l e that p l a n t and b a c t e r i a l genotypes had coadapted in that time because wheat i s a s e l f - p o l l i n a t i n g c r o p and v a r i e t i e s adapted to the area would c o n s i s t e n t l y be used. S i m i l a r l y , Dobereiner and Campelo (1971) observed a very s p e c i f i c response between Azot obact er pas pal i and Pas pal um not alum i n n a t u r a l ecosystems. The s p e c i f i c i t y observed i n the present work may a l s o have a r i s e n from c o a d a p t i v e mechanisms between p l a n t s and microorganisms as the pasture from which c o l l e c t i o n s were made has been u n d i s t u r b e d f o r 45 ye a r s . Mechansims of Plant Growth Promotion The mechanism by which B. polymyxa can promote growth was i n v e s t i g a t e d u s i n g pure stands of c r e s t e d wheatgrass which has been shown to c o n s i s t e n t l y i n c r e a s e root weight r e l a t i v e to shoot weight when i n o c u l a t e d . These e f f e c t s are s i m i l a r to those observed in pure or mixed stands of ryegr a s s and c l o v e r when s t i m u l a t i o n of p l a n t growth r e s u l t e d from b a c t e r i a l i n o c u l a t i o n . The s t r a i n of B. pol ymyxa used in these experiments possesses s e v e r a l c h a r a c t e r i s t i c s which c o u l d p o t e n t i a l l y a f f e c t p l a n t growth and a syst e m a t i c i n v e s t i g a t i o n of some of these p o s s i b i l i t i e s was undertaken. Because the bacterium possesses an a c t i v e n i t r o g e n a s e in vitro, the p o s s i b i l i t y that the growth response r e s u l t e d from a s s o c i a t i v e n i t r o g e n f i x a t i o n i n the p l a n t r h i z o s p h e r e was t e s t e d . No evidence i n support of t h i s idea was found when p l a n t / b a c t e r i a a s s o c i a t i o n s were analyzed f o r a c e t y l e n e 1 5 r e d u c t i o n or N d i l u t i o n . Furthermore, there i s str o n g evidence to r e j e c t t h i s e x p l a n a t i o n i n the d i f f e r e n t i a l r oot/shoot response. I f B. pol ymyxa were p r o v i d i n g supplemental n i t r o g e n to the p l a n t , i t i s l i k e l y that the growth response would a l s o be r e f l e c t e d i n enhanced shoot p r o d u c t i o n . In a l l experiments with c r e s t e d wheatgrass, shoot dry weights showed a marginal response to i n o c u l a t i o n , while the t o t a l dry weight i n c r e a s e was i n e v i t a b l y a consequence of the s u b s t a n t i a l gains in root biomass. S e v e r a l r e s e a r c h e r s have concluded that a s s o c i a t i v e n i t r o g e n f i x a t i o n i s unimportant i n p l a n t responses to p l a n t growth promoting b a c t e r i a (Brown, 1976; Let h b r i d g e and Davidson, 1983; Kapulnik et a l . , 1985). The growth response of c l o v e r , however, d i d i n c l u d e s u b s t a n t i a l gains i n shoot weight, e s p e c i a l l y when homologous B. pol ymyxa/white c l o v e r combinations were present (Table 123 2-5). A l s o , nodule weight of c l o v e r p l a n t s was shown to be up to 30% higher when the legume was grown with homologous bacterium/ryegrass p a r t n e r s (see Chapter 3). Larger root nodules are c o n s i d e r e d to be more e f f i c i e n t i n n i t r o g e n f i x a t i o n than smaller ones (Mytton and Jones, 1971). Th e r e f o r e , i t i s p o s s i b l e that the growth response i n c l o v e r was p a r t i a l l y due to enhanced n i t r o g e n n u t r i t i o n which was i n d i r e c t l y a f f e c t e d by neighbouring organisms. Burns et a l . (1981) s i m i l a r l y demonstrated that Azotobacter vinelandii can in c r e a s e n o d u l a t i o n when used as a c o - i n o c u l a n t f o r v a r i o u s legumes i n c l u d i n g Trifolium repens L. The observed growth response may a l s o be f a c i l i t a t e d by the s uppression of pathogenic organisms i n the p l a n t r h i z o s p h e r e by B. polymyxa ( C e l i n o and G o t t l i e b , 1952). However, d u r i n g the course of the experiments r e p o r t e d here, no v i s u a l evidence of d i s e a s e was observed i n any of the treatments; t h e r e f o r e , suppression of major pathogens was c o n s i d e r e d to be an u n l i k e l y mechanism. Rhizosphere s o i l was not t e s t e d f o r the presence of d e l e t e r i o u s r h i z o b a c t e r i a (DRB) (Suslow and Schroth, 1982; Gardiner et a l . , 1984). The use of steam s t e r i l i z e d s o i l reduces but does not e l i m i n a t e the p o s s i b i l i t y that such microorganisms were pres e n t . Furthermore, Rhizobium s t r a i n s s e n s i t i v e to s e v e r a l a n t i b i o t i c s (Chanway and H o l l , 1986) showed no negative response to in vitro t e s t s of a n t i m i c r o b i a l a c t i v i t y by the s t r a i n of B. polymyxa used i n these experiments (data not shown). Bacillus s p e c i e s are c o n s i d e r e d to be poor r h i z o s p h e r e c o l o n i z e r s (Lochhead, 1950) and most DRB s u p p r e s s i n g microbes are vigorous root c o l o n i z i n g pseudomonads (Kloepper et a l . , 1980). Because B. pol ymyxa s t r a i n L6 does not possess obvious a n t i m i c r o b i a l a c t i v i t y and belongs t o a genus of b a c t e r i a not noted f o r v i g o r o u s c o m p e t i t i o n i n the rh i z o s p h e r e , i t seems u n l i k e l y that p l a n t growth promotion r e s u l t e d from the suppression of DRB. S o l u b i l i z a t i o n of u n a v a i l a b l e s o i l phosphorus was a l s o i n v e s i g a t e d as a p o s s i b l e source of p l a n t growth s t i m u l a t i o n by B. pol ymyxa. I n o c u l a t i o n of wheat with a s t r a i n of t h i s bacterium r e s u l t e d i n s u b s t a n t i a l y i e l d gains when rock phosphate was added to the s o i l (Gaur and Ostwal, 1972). However, B. pol ymyxa s t r a i n L6 was unable to s o l u b i l i z e i n o r g a n i c phosphate, although phytase a c t i v i t y was shown to be p r e s e n t . Doubling the a v a i l a b l e s o i l phosphorus d i d not s i g n i f i c a n t l y a f f e c t emergence and growth of c r e s t e d wheatgrass, but more s e e d l i n g s emerged and these developed i n t o h e a v i e r p l a n t s than those i n c o n t r o l p o t s . Because t h i s treatment mimicked the e f f e c t of B. polymyxa i n o c u l a t i o n on emergence of c r e s t e d wheatgrass, i n c r e a s e d a v a i l a b l i l i t y of t h i s n u t r i e n t may be important i n the s e e d l i n g response. However, i n response to a d d i t i o n a l phosphorus root and shoot dry weight i n c r e a s e s were only marginal and not s t a t i s t i c a l l y s i g n i f i c a n t , r e s u l t i n g i n a 10% p l a n t y i e l d advantage over c o n t r o l s . The root/shoot r a t i o decreased s l i g h t l y i n response to i n o c u l a t i o n . These e f f e c t s are not s i m i l a r to those t y p i c a l l y seen when p l a n t s are i n o c u l a t e d with B. polymyxa, as a d d i t i o n of the bacterium r e s u l t e d i n s u b s t a n t i a l and s i g n i f i c a n t root weight ga ins (28%) r e l a t i v e to shoot weight and h i g h e r (17%) r o o t / s h o o t r a t i o s . T h e r e f o r e , wh i l e phosphate s o l u b i l i z a t i o n may c o n t r i b u t e to the e f f e c t of i n o c u l a t i o n on s e e d l i n g emergence of c r e s t e d wheatgrass , i t does not appear to a f f e c t subsequent p l a n t growth i n the same manner as t h i s s t r a i n of B. pol ymyxa. T h i s c o n c l u s i o n has been reached by o t h e r s ( B a r b e r , 1978; T i n k e r , 1984) . Bacillus pol ymyxa s t r a i n L6 was shown to produce IAA in vitro. R e s u l t s from exper iments comparing the e f f e c t of exogenous ly a p p l i e d IAA w i t h i n o c u l a t i o n of c r e s t e d wheatgrass support the idea tha t m i c r o b i a l p r o d u c t i o n of the growth r e g u l a t o r i s i n v o l v e d i n the response to i n o c u l a t i o n . Whi le exogenous IAA w i t h or wi thout IPA d i d not approximate the e f f e c t s of i n o c u l a t i o n on s e e d l i n g emergence, subsequent p l a n t growth was c l o s e l y mimicked by a p p l i c a t i o n of the phytohormone. Increase s i n root and p l a n t weight were observed upon a d d i t i o n of the p l a n t growth s u b s t a n c e . As w i t h B. pol ymyxa i n o c u l a t i o n , shoot weight was l e s s r e s p o n s i v e , t h e r e f o r e r o o t / s h o o t r a t i o s were a l s o i n c r e a s e d . The magnitude of e f f e c t s when IAA was exogenous ly a p p l i e d was l e s s than e f f e c t s which were a t t r i b u t e d to i n o c u l a t i o n . T h i s i s not s u r p r i s i n g because i t i s o n l y p o s s i b l e to e s t imate the amount of growth r e g u l a t o r tha t the p l a n t may be exposed to through i n o c u l a t i o n . F u r t h e r m o r e , i n o c u l a t e d p l a n t s may be exposed to a slow but c o n t i n u o u s source of IAA as opposed to the p l a n t s t r e a t e d w i t h exogenous phytohormone which r e c i e v e d four e q u a l doses a d m i n i s t e r e d 126 over e i g h t weeks. C o n s i d e r i n g these p o t e n t i a l sources of v a r i a b i l i t y , the data do provide c o n v i n c i n g evidence f o r the p r o d u c t i o n of IAA as the mechanism by which p l a n t growth i s s t i m u l a t e d by t h i s i s o l a t e of B. pol ymyxa. I t i s a l s o p o s s i b l e to e x p l a i n the r e s u l t s observed with i n o c u l a t e d ryegrass and c l o v e r i n terms of m i c r o b i a l IAA p r o d u c t i o n . The tendency f o r b a c t e r i a l i n o c u l a t i o n to n e g a t i v e l y a f f e c t p e r e n n i a l r y e g r a s s root y i e l d may r e s u l t from the b u i l d up of i n h i b i t o r y c o n c e n t r a t i o n s of IAA i n the r h i z o s p h e r e . Loper and Schroth (1986) have demonstrated t h i s e f f e c t with sugarbeet when i n o c u l a t e d with IAA-producing s t r a i n s of b a c t e r i a . Those s t r a i n s producing the most IAA in vitro were shown to cause the g r e a t e s t i n h i b i t i o n of root growth. However, the amount of IAA exposed to c l o v e r r o o t s may be s t i m u l a t o r y , as was found with p e a r l m i l l e t i n o c u l a t e d with IAA producing s t r a i n s of Azospirilium brasilense (Venkateswarlu and Rao, 1983). They showed that high IAA producing s t r a i n s s t i m u l a t e d root growth. T h e r e f o r e , the nature of the growth response depends on the s e n s i t i v i t y of the p l a n t to the phytohormone, as w e l l as the r a t e and d i s t r i b u t i o n of p r o d u c t i o n by r h i z o s p h e r e microbes. Some s t r a i n s of phytohormone-producing b a c t e r i a r e q u i r e p r e c u r s o r s f o r the p r o d u c t i o n of p l a n t growth substances. For example, Primrose (1976) has demonstrated that methionine i s r e q u i r e d f o r the m i c r o b i a l p r o d u c t i o n of ethylene, while T i e n et a l . (1979) showed that tryptophan had to be present in the medium f o r PGP s t r a i n s of Azos pi ri Hum to produce IAA. Rhizosphere microbes are l a r g e l y dependent on root exudates f o r t h e i r supply of o r g a n i c n u t r i e n t s (Rovira, 1969; 1971) and genotypic v a r i a b i l i t y has been shown to occur i n the q u a l i t y of s o l u b l e root exudates (Kipe-Nolt et a l . , 1985). In an undisturbed permanent pasture the r h i z o s p h e r e m i c r o f l o r a would have ample o p p o r t u n i t y to adapt to the q u a l i t y and q u a n t i t y of p l a n t root exudation. These exudates may not only a f f e c t the p o p u l a t i o n s i z e and c o n s t i t u t i o n of r h i z o s p h e r e b a c t e r i a , but a l s o the nature and amount of b a c t e r i a l metabolic products, some of which may a f f e c t p l a n t growth. I t i s tempting to s p e c u l a t e that the genotypic s p e c i f i c i t y observed when p l a n t s were grown under c o n d i t i o n s of i n c r e a s i n g b i o t i c f a m i l i a r i t y may be a t t r i b u t a b l e to the e f f e c t of c o a d a p t a t i o n of the Bacillus polymyxa r h i z o s p h e r e p o p u l a t i o n to p l a n t root exudation of the homologous ryegrass and c l o v e r genotypes. CHAPTER 3 GENOTYPE SPECIFICITY BETWEEN PLANTS AND MICROORGANISMS 129 INTRODUCTION Fi n e s c a l e b i o t i c s p e c i a l i z a t i o n has been demonstrated i n p l a n t communities by s e v e r a l i n v e s t i g a t o r s . A l l a r d and Adams (1969) showed that l i n e s of b a r l e y , which had a pre v i o u s h i s t o r y of being grown i n mixture, y i e l d e d more when grown together than when c u l t i v a t e d i n pure stands. L i n e s which had no h i s t o r y of i n t e r a c t i o n d i d not show t h i s response. Joy and L a i t i n e n (1980) observed s i m i l a r tendencies when mixture and pure stand y i e l d s of Phi eum pratense L. and Trifolium pratense L. were compared between c u l t i v a r s t h a t had p r e v i o u s l y c o e x i s t e d and those which had not. C h a r l e s (1968) demonstrated that grass p l a n t s can a f f e c t the g e n e t i c makeup of the neighbouring white c l o v e r or red c l o v e r p o p u l a t i o n . Turkington and Harper (1979) s t u d i e d the s e l e c t i v e p r e s s u r e s exerted by v a r i o u s s p e c i e s of grass on po p u l a t i o n s of Trifolium repens i n a 65-70 year o l d permanent g r a s s l a n d s i t e i n Wales. In t h e i r study, white c l o v e r p l a n t s were c o l l e c t e d from four s i t e s i n the f i e l d , each s i t e dominated by a d i f f e r e n t grass s p e c i e s (Agrostis capillar is syn. A. tenuis S i b t h . , Cynosurus cristatus L., Hoi cus lanatus L., or Lol i um perenne L . ) . C u t t i n g s from the c l o v e r p l a n t s were t r a n s p l a n t e d e i t h e r back i n t o each of the four g r a s s -dominated areas of the f i e l d , or i n t o f l a t s sown with seeds of each grass s p e c i e s in a greenhouse experiment. G e n e r a l l y , c l o v e r p l a n t s y i e l d e d h i g h e s t when grown with the grass neighbour with which they had c o e x i s t e d i n the f i e l d . These DO r e l a t i o n s h i p s were evident i n both f i e l d and greenhouse experiments. Aarssen and Turkington (1985) f u r t h e r r e f i n e d the degree to which t h i s f i n e - s c a l e b i o t i c s p e c i a l i z a t i o n may occur by studying genets of Trifolium repens and Lol i um perenne. P l a n t s were c o l l e c t e d from a permanent pasture i n southwestern B r i t i s h Columbia which had been sown 39 years p r e v i o u s l y . Four p a i r s of p l a n t s (one white c l o v e r and one p e r e n n i a l ryegrass) growing s i d e - b y - s i d e i n the f i e l d were c o l l e c t e d and p e r e n n i a l r y e g r a s s t i l l e r s or white c l o v e r ramets of these genotypes were produced. Clones of the p l a n t genets were then grown in a l l p o s s i b l e genotypic combinations in s p e c i e s mixtures i n a greenhouse. P l a n t s were c l i p p e d r e g u l a r l y and were harvested a f t e r one year. When t o t a l biomass was measured, c l o v e r y i e l d again was found to be s u p e r i o r when grown with the r y e g r a s s genotype i t had p r e v i o u s l y "known" i n the f i e l d . These r e s u l t s demonstrate that b i o t i c s p e c i a l i z a t i o n occurs between neighbouring genotypes i n a n a t u r a l community. Evans et a l . (1985) have a l s o demonstrated s p e c i f i c i t y in Swiss, French, and I t a l i a n p o p u l a t i o n s of white c l o v e r and p e r e n n i a l r y e g r a s s . However, t h e i r experiments were not designed to study r e l a t i o n s h i p s between p l a n t s at such a f i n e - s c a l e as those of Aarssen and Turkington (1985). T h e r e f o r e , these authors have demonstrated that b i o t i c s p e c i a l i z a t i o n between p l a n t s may occur, but at a d i f f e r e n t l e v e l than was observed by the l a t t e r r e s e a r c h e r s . A n a l y s i s of the above ground behaviour of these p l a n t s o f f e r e d no obvious e x p l a n a t i o n f o r such c o m p a t i b i l i t y . T h e r e f o r e , the mechanism by which genotypic s p e c i f i c i t y occurs may l o g i c a l l y be p r e d i c t e d to i n v o l v e r h i z o s p h e r e e f f e c t s . P l a n t r o o t s can have d i r e c t e f f e c t s on each other r e s u l t i n g i n e i t h e r p o s i t i v e or negative growth responses (Rovira and Davey, 1974; C u r l and T r u e l o v e , 1986). Examples of the s t i m u l a t o r y e f f e c t s t h a t r o o t s can exert upon the performance of other p l a n t s may be found i n seed germination s t u d i e s of p l a n t - p a r a s i t i c phanerograms. For example, seeds of broomrape (Orobanche ramosa L . ) , which i s a root p a r a s i t e of solanaceous and leguminous cro p s , germinate when exposed to root exudates of the host p l a n t ( C u r l and T r u e l o v e , 1986) S i m i l a r l y , the angiosperm p a r a s i t e , Striga lutea Lour., shows d e c l i n i n g germination as d i s t a n c e from i t s hosts r o o t s i s i n c r e a s e d (Brown and Edwards, 1944). Newman and R o v i r a (1975) grew four s p e c i e s each of grasses and herbaceous d i c o t s i n separate c o n t a i n e r s of sand Leachates were c o l l e c t e d from these s p e c i e s and were a p p l i e d s e p a r a t e l y to d i f f e r e n t pots c o n t a i n i n g t e s t p l a n t s of the same s p e c i e s . I t was found that l e a c h a t e s from Hoi cus I anat us, Trifolium repens, and Hypochoeris radicata L. reduced growth of a l l other s p e c i e s with no v i s i b l e symptoms of i n j u r y . Lol i um perenne, PI ant ago I anceolat a L., Hypochoeris radicata L., and Trifolium repens were i n h i b i t e d more by t h e i r own l e a c h a t e than by that of other s p e c i e s while Ant hoxant hum odoratum L., Cynosurus cri status, and Hoi cus I anat us grew b e t t e r when t h e i r own l e a c h a t e was a p p l i e d . The authors concluded that p l a n t root l e a c h a t e s 132 c o u l d have p o s i t i v e or negative e f f e c t s on p l a n t growth. R e s u l t s from t h i s work and other s t u d i e s on a l l e l o p a t h y i n d i c a t e the p o t e n t i a l e f f e c t s p l a n t s may have on themselves and each other, but d e f i n i t e proof of such mechanisms of i n t e r a c t i o n i s l a c k i n g . Often a c r i t i c a l c o n t r o l i s absent or an a l t e r n a t i v e e x p l a n a t i o n of the r e s u l t s i s p o s s i b l e (Harper, 1977). In the study of Newman and Ro v i r a (1975), microorganisms present i n the le a c h a t e s may have been r e s p o n s i b l e f o r the observed e f f e c t s as the "donor" p l a n t pots were i n o c u l a t e d with 100 g of f i e l d s o i l to introduce the normal s o i l m i c r o f l o r a to the experimental p l a n t s . Y i e l d - r e d u c i n g DRB which can i n f l u e n c e p l a n t growth without i n d u c i n g symptoms of d i s e a s e , or PGP microbes are o f t e n found i n p l a n t r h i z o s p h e r e s (see Chapter 2). Ambiguous experimental r e s u l t s do not exclude the p o s s i b i l i t y t h a t d i r e c t e f f e c t s between p l a n t r o o t s are important. For example, the underground t r a n s f e r of n u t r i e n t s between neighbouring p l a n t s has been demonstrated 3 2 u s i n g P (Woods and Brock, 1964). In a d d i t i o n , the roots and l e a v e s of walnut (Juglans sp.) are known to cause i n j u r y to p l a n t s growing nearby ( C u r l and Truelov e , 1986). The i n h i b i t o r y juglone, 5-hydroxy-napthoquinone, has been i s o l a t e d from leaves of t h i s s p e c i e s . T h e r e f o r e , the p o t e n t i a l t r a n s f e r of n u t r i e n t s or other compounds between root systems and the s t i m u l a t i o n or i n h i b i t i o n of p l a n t s by other p l a n t s suggest that r h i z o s p h e r i c i n t e r a c t i o n s c o u l d be very important i n above ground p l a n t performance. Even where p l a n t r o o t s can d i r e c t l y a f f e c t one another, 133 the l i k e l i h o o d that s o i l m i c r o f l o r a i n f l u e n c e such processes or a f f e c t p l a n t growth themselves i s c o n s i d e r a b l e . S u b s t a n t i a l numbers of microorganisms are present i n f i e l d s o i l , of which b a c t e r i a are the most abundant ( C u r l and 6 8 T r u e l o v e , 1986). Estimates of between 10 and 10 c e l l s per gram dry weight or per c u b i c centimeter of s o i l have been made ( G r i f f i n , 1972). R u s s e l l (1973) c a l c u l a t e d the l i v e weight of b a c t e r i a i n the top 15 cm of Rothamsted f i e l d s o i l to be between 1500 and 3750 kg ha 1 . Rhizosphere s o i l i s 9 even more populous as up to 3 x 10 b a c t e r i a l c e l l s per gram (dry weight) of s o i l have been recorded (Rouatt and Katznelson, 1961). Furthermore, b a c t e r i a are more abundant than fungi i n r h i z o s p h e r e s o i l ( R o v i r a and Davey, 1974). Because a l l n u t r i e n t s obtained by the p l a n t root must pass through the r h i z o s p h e r e , m i c r o b i a l a l t e r a t i o n of these compounds c o u l d a f f e c t p l a n t growth. The e f f e c t s that b a c t e r i a can e x e r t on p l a n t growth have been w e l l documented (see Chapter 2). Nonsymbiotic a s s o c i a t i o n s can r e s u l t i n y i e l d i n c r e a s e s of over 300% (Rennie and Larson, 1979) while the Rhi zobi wm-clover symbiosis can i n c r e a s e s o i l n i t r o g e n by 300 kg ha 1 per year (Postgate, 1982). I f m i c r o b i o l o g i c a l phenomena c o n t r i b u t e to the observed genotype s p e c i f i c i t y between white c l o v e r and p e r e n n i a l r y e g r a s s (Aarssen and T u r k i n g t o n , 1985), the most i n f l u e n t i a l process may be symbiotic n i t r o g e n f i x a t i o n . T h i s c o n t e n t i o n i s supported by the o b s e r v a t i o n that a f t e r 1 year of growth i n mixture, only the c l o v e r component showed a y i e l d advantage when matched with i t s " c o r r e c t " grass neighbour. However, Evans et a l . (1985) have demonstrated that s i m i l a r b e n e f i c i a l e f f e c t s have occurred f o r the rye g r a s s component i n the second year of growth. The r e l a t i v e l y immediate y i e l d advantage seen i n white c l o v e r may r e f l e c t enhanced symbiotic n i t r o g e n f i x a t i o n . B e n e f i t s to the grass neighbour from t h i s p r ocess would not be immediately apparent as n i t r o g e n t r a n s f e r from the symbiosis occurs s l o w l y , when root t i s s u e and nodules senesce and decay (Walton, 1983). If symbiotic n i t r o g e n f i x a t i o n was an important mechanism in the observed s p e c i f i c i t y , then c e r t a i n Rhizobium i s o l a t e / c l o v e r genotype combinations should be expected to o u t y i e l d other microsymbiont/legume p a r t n e r s h i p s . Heterogeneity f o r a range of b i o c h e m i c a l , m o r p h o l o g i c a l , and p h e n o l o g i c a l c h a r a c t e r i s t i c s i n c l u d i n g n i t r o g e n f i x a t i o n between white c l o v e r genotypes has been demonstrated (Mytton, 1975; Burdon 1980). S i m i l a r l y , v a r i a b i l i l t y i n IAR (Hagedorn, 1979; Chanway and H o l l , 1986), a n t i g e n i c determinants ( T r i n i c k , 1969; Hagedorn and C a l d w e l l , 1981; Dughri and Bottomley, 1983; Chanway and H o l l , 1986), and n i t r o g e n f i x a t i o n (Mytton, 1975; Mytton and de F e l i c e , 1977; Hagedorn and C a l d w e l l , 1981; Smith et a l . , 1982; Materon and Hagedorn, 1983) has been demonstrated between i s o l a t e s of Rhi zobi um trifolii . V a r i a b i l i t y i n symbiotic e f f e c t i v e n e s s between s p e c i f i c lequme/Rhi zobi um combinations i s a l s o w e l l documented (Mytton, 1975; Mytton and de F e l i c e , 1977; Mytton et a l . , 1977; Hobbs and Mahon, 1983) and i s not unexpected. The complex i n t e r a c t i o n between the host and microsymbiont ensures that there are many f a c t o r s i n the symbiosis which c o u l d a f f e c t n i t r o g e n f i x a t i o n q u a l i t a t i v e l y and q u a n t i t a t i v e l y . For example, the enzyme nit r o g e n a s e i s extremely o x y g e n - l a b i l e but the process of n i t r o g e n f i x a t i o n i s endergonic; t h e r e f o r e s u b s t a n t i a l a e r o b i c r e s p i r a t i o n i s r e q u i r e d f o r g e n e r a t i o n of ATP. An oxygen c a r r y i n g p r o t e i n , leghaemoglobin, r e g u l a t e s the d e l i c a t e balance between oxygen t o x i c i t y and a e r o b i c energy requirements. The p r o t e i n , analogous to human myoglobin in s t r u c t u r e , i s thought to f u n c t i o n by p r o t e c t i n g n i t r o g e n a s e from excess C>2 while s u p p l y i n g the element to a c t i v e l y r e s p i r i n g root c e l l s . I t i s easy to see how d i f f e r e n c e s i n the q u a l i t y or q u a n t i t y of t h i s p r o t e i n between symbiotic systems c o u l d d r a s t i c a l l y a f f e c t n i t r o g e n f i x a t i o n . The p o t e n t i a l f o r v a r i a t i o n i n t h i s t r a i t i s great as the haem p o r t i o n of the molecule i s coded f o r by b a c t e r i a l genes while the p r o t e i n o r i g i n a t e s i n the p l a n t ( M e i j e r , 1982). D i f f e r e n c e s i n g e n e t i c c o m p a t i b i l i t y between host and microsymbiont c o u l d cause v a r i a b i l i t y i n the e f f e c t i v e n e s s of leghaemoglobin and hence i n the amount of n i t r o g e n f i x e d . Other processes such as the export e f f i c i e n c y of the end product (NH^) from the nodule c o u l d a l s o a f f e c t the system as accumulation of ammonia can be t o x i c to the root nodule. Mytton (1975) i n v e s t i g a t e d the p o s s i b i l i t y that homologous white c l o v e r genotype/if. trifolii i s o l a t e combinations might r e s u l t i n s u p e r i o r y i e l d s of the legume D6 when compared with the average performance of a l l c l o v e r and Rhizobium genotypes. The term homologous was used as i t has been d e f i n e d i n Chapter 2; r e f l e c t i n g a common plant/Rhi zobi um source of experimental organisms. S t r a i n s of R. trifolii were i s o l a t e d from the root nodules of e i g h t d i f f e r e n t white c l o v e r p l a n t s . E i g h t homologous s t r a i n s of R. trifolii were combined i n a l l p o s s i b l e p a i r w i s e combinations with the e i g h t c o r r e s p o n d i n g genotypes of white c l o v e r . Homologous Rhi zobi um/'clover combinations were shown to o u t y i e l d heterologous microbe/legume combinations by 25% when p l a n t dry weight was measured. Rhizobium p o p u l a t i o n x p l a n t v a r i e t y i n t e r a c t i o n s had the l a r g e s t e f f e c t on c l o v e r y i e l d when s e v e r a l p l a n t v a r i e t i e s and m i c r o b i a l s t r a i n s were t e s t e d at d i f f e r e n t l o c a t i o n s with v a r y i n g s o i l types (Mytton and L i v e s l y , 1983). S i m i l a r l y , s u b s t a n t i a l gains i n c l o v e r y i e l d have been observed when s p e c i f i c c l o v e r v a r i e t y / i ? . trifolii s t r a i n combinations were used (Sherwood and Masterson, 1974; Young and Mytton, 1983; Mytton and Hughes, 1984). For example, when c l o v e r v a r i e t i e s S184 and Hui a were i n o c u l a t e d with R. trifolii s t r a i n "B", v a r i e t y S184 showed a y i e l d advantage i n the f i e l d (Young and Mytton, 1983). When R. trifolii s t r a i n "A" was used as the i n o c u l a n t , t h i s trend was reversed and Hui a showed the s u p e r i o r y i e l d . Mytton (1978; 1981) has d i s c u s s e d the importance of the s p e c i f i c i n t e r a c t i o n between host and microsymbiont with respect to c u r r e n t legume breeding programs and concluded that the Rhizobium s t r a i n should be c a r e f u l l y matched with the legume v a r i e t y to be t e s t e d . Microbe/legume s p e c i f i c i t y has been demonstrated i n other symbiotic systems as w e l l . The i n t e r a c t i o n between R. meliloti and a l f a l f a (Medi cago sativa L.) genotypes has been shown to comprise a s i g n i f i c a n t component of the v a r i a b i l i t y i n y i e l d of the forage s p e c i e s (Mytton et a l . , 1984). Experimentation with p r e v i o u s l y u n r e l a t e d Rhizobium Ieguminosarum s t r a i n s and f i e l d bean {Vicia faba L.) v a r i e t i e s demonstrated that the s p e c i f i c i n t e r a c t i o n between microbe and legume genotypes accounted f o r a s t r i k i n g 74% of the v a r i a t i o n i n p l a n t dry weight (Mytton et a l . , 1977). S i m i l a r e f f e c t s have been observed with peas (Pi sum sativum L.) (Hobbs and Mahon, 1982; 1983) and crimson c l o v e r (Trifolium incarnatum L. ) (Smith et a l . , 1982). However, the environment p l a y s an important r o l e i n these i n t e r a c t i o n s as Dean et a l . (1980) found no s t r a i n x c u l t i v a r i n t e r a c t i o n between genotypes of f i e l d bean and R. Iegumi nosarum i n t e s t s c a r r i e d out i n Manitoba. Large environmental e f f e c t s have a l s o been noted i n work with c l o v e r and R. trifolii (Mytton, 1981; Mytton and Hughes, 1984). A s i g n i f i c a n t b a r r i e r to a g r i c u l t u r a l e x p l o i t a t i o n of p l a n t qenotype/Rhi zobi um s t r a i n i n t e r a c t i o n s i n v o l v e s the r e l a t i v e a b i l i t y of the microbe to i n f e c t the host p l a n t root h a i r s when i n c o m p e t i t i o n with other s t r a i n s of i n f e c t i v e Rhizobium. The r e l a t i o n s h i p between i n f e c t i v e n e s s (the c a p a c i t y t o form root nodules) and e f f e c t i v e n e s s (the a b i l i t y to f i x atmospheric n i t r o g e n a f t e r root nodules have been formed) has been s t u d i e d using R. trifolii and v a r i o u s 138 s p e c i e s of c l o v e r . Apparent host s e l e c t i o n of R. trifolii s t r a i n s has been observed i n white c l o v e r (Masterson and Sherwood, 1974; Marques P i n t o et a l . , 1974; R u s s e l l and Jones, 1975; Mytton and de F e l i c e , 1977; Jones and Hardarson, 1979; Hardarson and Jones, 1979) and i n red c l o v e r (Robinson, 1969; R u s s e l l and Jones, 1975; Materon and Hagedorn, 1983). In some cases, the most c o m p e t i t i v e R. trifolii s t r a i n s have r e s u l t e d i n the most e f f e c t i v e n i t r o g e n f i x i n g a s s o c i a t i o n s (Robinson, 1969; Masterson and Sherwood, 1974; Marques P i n t o et a l . , 1974; Materon and Hagedorn, 1983). However, l e s s e f f e c t i v e or completely i n e f f e c t i v e s t r a i n s have a l s o been shown to form the m a j o r i t y of nodules i n d i f f e r e n t s p e c i e s of c l o v e r (Vincent and Waters, 1953; Mytton and de F e l i c e , 1977). Thus, i n f e c t i v e n e s s and e f f e c t i v e n e s s can be c o r r e l a t e d under c e r t a i n c o n d i t i o n s but host s e l e c t i o n of the most e f f e c t i v e R. trifolii i s o l a t e from a s t r a i n mixture i s by no means assured. Rhizobium s t r a i n c o m p e t i t i o n f o r nodule formation depends on s e v e r a l f a c t o r s i n c l u d i n g the i n f l u e n c e of the host p l a n t , the s t r a i n of Rhizobium, and the v a r i o u s environmental i n t e r a c t i o n s between them (Date and Br o c k w e l l , 1978). The l a t t e r group of f a c t o r s can in c l u d e b i o t i c . i n t e r a c t i o n s such as a n t i b i o s i s , c o mpetition, and b a c t e r i o c i n or phage p r o d u c t i o n by other s o i l microbes i n c l u d i n g competing s t r a i n s of Rhizobium ( T r i n i c k , 1982). P h y s i c a l and edaphic f a c t o r s are a l s o important i n sap r o p h y t i c c o m p e t i t i o n and s u r v i v a l of Rhizobium s t r a i n s (Bushby, 1982). These i n c l u d e s o i l t e x t u r e , s t r u c t u r e , moisture, temperature, pH, organic matter content, and nutrient levels. However, the broad specif ic i ty observed between legumes and Rhizobium of various cross inoculation groups (Postgate, 1982) and the much more defined legume genotype/Rhi zobi um isolate specif ic i ty demonstrated within different cross inoculation groups (Marques Pinto et a l . , 1974; Mytton and de Fel ice , 1977) suggests that biotic interactions between the host and microsymbiont are very important in strain selection. Marques Pinto et a l . (1974) studied the effect of inoculum and root surface ratios on nodule formation using several strains of Rhizobium and different species of Trifolium and Medicago. Generally, they found that root surface ratios were more closely correlated to nodule occupancy than was the ratio of strains in the inoculum. Because strains differed in the ab i l i ty to colonize root surfaces, the ratio of strains in the the inoculum was not closely related to the root surface representation of each strain. In some cases, however, the host plant influence was strong enough to override the effect of root surface ratio in nodule formation, as the minority component of the inoculum formed most of the nodules. Other workers have reached the same conclusion that relative nodulation success is not always related to inoculum or root surface ratios (Means et a l . , 1961; Robinson, 1969; Skrdleta and Karimova, 1969; Labandera and Vincent, 1975; Franco and Vincent, 1976). Furthermore, Hardarson and Jones (1979) have demonstrated that the strain preference of parental white clover genotypes can be inherited by F progeny. The demonstration of such host-microsymbiont specif icity suggests that some type of molecular signal(s) may exist between plant roots and the bacterium. The observation that root surface ratios are more relevant than inoculum ratios in determining nodulation success of Rhizobium strains suggests that microbial interactions in the rhizosphere are important. Factors such as motility (El-Haloui et a l , 1986) or the secretion of root exudates such as homoserine, b iot in , or thiamine which may selectively stimulate Rhizobium strains in the rhizosphere (van Egeraat, 1975; Date and Brockwell, 1978) could be central to the mechanism involved in early recognition of the host and microsymbiont. Physical attachment of Rhizobium ce l ls to root hairs precedes root hair curl ing, development of the infection thread, and subsequent nodule formation (Dart, 1977). Host specif ic i ty is expressed at about the same time as attachment, before penetration of the root-hair c e l l wall (Li and Hubbell, 1969) or the formation of the infection thread (Napoli and Hubbell, 1975). The discovery that in vitro binding of soybean (Glycine max L. ) seed lect in and the a b i l i t y of these strains to infect soybean roots were signif icantly correlated (Bohlool and Schmidt, 1974) led to the formation of the lect in hypothesis to explain host-microsymbiont spec i f ic i ty . Lectins are carbohydrate-binding proteins or glycoproteins. This hypothesis states that recognition at infection sites involves the binding of highly specific plant lectins to unique carbohydrates found only on the bacterial c e l l surface (Dazzo and Hubbell, 1982). The reaction envisaged is not different from that occurring between a homologous antibody/antigen combination where the lect in molecule would act as a bridge linking the bacterial c e l l and plant root hair surfaces. Lectin-meditated attachment has also been implicated in the clover/Rhi zobi um symbiosis as perfect correlations between in vitro lect in binding and strain specif ic i ty have been described (Dazzo and Hubbell, 1975). Binding in these studies was shown to be inhibited by the hapten 2-deoxyglucose. Haptens are low molecular weight molecules which bind to reactive sites on the antibody thereby blocking further immunological reaction of the molecules. The seed lec t in , t r i f o l i i n , which was orig inal ly used to demonstrate the speci f ic i ty , has been shown to be present on root hair surfaces where attachment of the bacterial c e l l occurs before infection (Dazzo et a l . , 1978). Some earlier studies with soybean and R. japonicum showed no correlation between infect iv i ty with in vitro lect in binding (Bohlool and Schmidt, 1974; Bhuvaneswari et a l . , 1977), but i t was later discovered that strains used in those experiments would bind lect in i f preincubated in soybean root exudate (Bhuvaneswari and Bauer, 1978). However, there are s t i l l several reports demonstrating that attachment of Rhizobium to various surfaces, including soybean and clover roots, is nonspecific and does not involve host lectins (Law et a l . , 1982; Badenoch-Jones et a l . , 1984; Mi l l s and Bauer, 1985; Vesper and Bauer, 1986). The latter authors present evidence to support the hypothesis that 142 m i c r o b i a l p i l i , and not p l a n t l e c t i n s , a r e i m p o r t a n t i n the s p e c i f i c attachment of R. j aponi cum t o hos t r o o t s . F a c t o r s such as the n i t r a t e or ammonium i o n c o n c e n t r a t i o n i n the growth medium or the age of the b a c t e r i a l c u l t u r e used f o r i n o c u l a t i o n may a l s o a f f e c t l e c t i n b i n d i n g between p l a n t r o o t s and b a c t e r i a l c e l l s . For example, i n c r e a s i n g the c o n c e n t r a t i o n of e i t h e r NO^ or N H 4 + r e s u l t s i n a p a r a l l e l d e c r e a s e i n l e v e l s of d e t e c t a b l e t r i f o l i i n and the attachment of R. trifolii t o r o o t s h a i r s (Dazzo and B r i l l , 1978). A l s o , c u l t u r e s of R. trifolii a r e more l i k e l y t o b i n d s p e c i f i c a l l y t o r o o t h a i r s when i n the e a r l y s t a t i o n a r y r a t h e r than the e x p o n e n t i a l phase of growth (Hrabak e t a l . , 1981). T h i s v a r i a b l e and t r a n s i e n t n a t u r e of l e c t i n b i n d i n g a c t i v i t y would c o n t r i b u t e t o t h e c o n f l i c t i n g e v i d e n c e p r e s e n t e d above. Dazzo and H u b b e l l (1982) suggest t h a t the b r i e f appearance of l e c t i n b i n d i n g a c t i v i t y by R. j aponi cum between l a g and e x p o n e n t i a l phases of growth (Bhuvaneswari et a l . , 1977) c o u l d be of e c o l o g i c a l s i g n i f i c a n c e . They suggest t h a t the p h y s i o l o g i c a l s t a t e of the b a c t e r i a i n l a t e l a g phase may resemble t h a t of micr o b e s when q u i e s c e n t Rhizobium c e l l s f i r s t e n counter the n u t r i e n t -r i c h legume r h i z o s p h e r e . T u r n i n g on a s p e c i f i c attachment mechanism a t t h a t p o i n t i n the b a c t e r i a l l i f e c y c l e would h e l p t o ensure t h a t the i n f e c t i o n p r o c e s s was i n i t i a t e d . In summary, i t appears t h a t p l a n t l e c t i n s a t l e a s t c o n t r i b u t e t o h o s t / m i c r o s y m b i o n t s p e c i f i c i t y . Whether or not t h i s phenomenon e l i c i t s a major response i n the s p e c i f i c b i n d i n g of c e l l s t o r o o t h a i r s c l e a r l y r e q u i r e s f u r t h e r i n v e s t i g a t i o n . Another potentially important factor in the lequme/Rhi zobium symbiosis is the effect of other so i l microbes on the infect iv i ty and effect ivity of Rhizobium strains and on the process of nodulation. Burns et a l . (1981) demonstrated that coinoculation of various legume species including white clover and soybean with Azotobacter vinelandii and root nodule bacteria resulted in substantial increases in root nodule number. Increases in root nodule weight were also observed but these responses were less consistent. El-Bahrawry (1983) confirmed these trends by studying the effect of Azot obact er sp. on the soybean symbiosis. Singh and Subba Rao (1979) observed increases in nodule number and weight, and in shoot and grain yields when soybean was coinoculated with Azospirillum brasilense and R. j aponi cum. Iruthayathas et a l . (1983) discovered a Rhizobium x Azospirillum genotype-genotype interaction when studying the effect of dual inoculation on the yield of winged bean (Psophocarpus t et r agonolobus DC.) and soybean. Certain combinations of Rhizobium and Azospirillum resulted in substantial increases in nodule weight, nodule number, acetylene reduction, and shoot weight of test plants while others did not. Thus, neighbouring microflora may have direct effects on plant growth (see Chapter 2) or indirect effects through changes in legume nodulation. Legumes are reported to induce greater rhizospheric populations of bacteria than nonleguminous plants (Rovira and Davey, 1974; Curl and Truelove, 1986), but this observation does not mean that the latter group supports t r i v i a l root-a s s o c i a t e d m i c r o b i a l p o p u l a t i o n s . For example, Rouatt and Katznelson (1961) showed that while b a c t e r i a l p o p u l a t i o n s i n the red c l o v e r r h i z o s p h e r e were over 3 b i l l i o n per gram (dry weight) of s o i l , oats c o u l d support over 1 b i l l i o n b a c t e r i a l c e l l s per gram of s o i l . R o vira and Stern (1961) examined the s o i l m i c r o f l o r a i n sown swards of Lol i um rigidum Gaud, and Trifolium subterraneum L. and found that c l o v e r o f t e n had higher r h i z o s p h e r e p o p u l a t i o n s of b a c t e r i a per gram dry weight of s o i l than neighbouring grass p l a n t s , but g d i f f e r e n c e s were not great (eg. 1.4 x 10 f o r c l o v e r vs 1.9 x 10 f o r g r a s s ) . Depending on the n i t r o g e n n u t r i t i o n (which a f f e c t e d the b o t a n i c a l dominance in the mixture) and the stage of growth, ryegrass was shown to support l a r g e r Q r h i z o s p h e r e p o p u l a t i o n s of b a c t e r i a than c l o v e r (eg 1.2 x 10 Q f o r c l o v e r vs 1.9 x 10 c e l l s f o r g r a s s ) . Work by C h r i s t i e et a l . (1974; 1978) and Newman et a l . (1979) has demonstrated that the presence of p l a n t neighbours does a f f e c t r h i z o p l a n e ( r o o t - s u r f a c e ) m i c r o f l o r a . Rhizosphere m i c r o f l o r a was not examined. These experiments i n c l u d e d treatments where Lol i um perenne and Trifolium repens were grown e i t h e r alone or i n mixture. When p l a n t performance and r o o t - s u r f a c e m i c r o f l o r a were examined, a h i g h l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between the r a t i o s shoot weight in mixture:shoot weight in monoculture and m i c r o b i a l abundance i n m i x t u r e : m i c r o b i a l abundance i n monoculture was observed. A s i m i l a r r e l a t i o n s h i p was observed when %N content was s u b s t i t u t e d f o r shoot weight. S i m i l a r l y , c o r r e l a t i o n s between b a c t e r i a l abundance and shoot N content were h i g h l y s i g n i f i c a n t (r=.902) but t h i s was not the case when shoot P or K content was t e s t e d . Root s u r f a c e fungi and endomycorrhizae were, however, s i g n i f i c a n t l y c o r r e l a t e d to both shoot N and P content. F u r t h e r work by t h i s group (Newman et a l . , 1979), showed that above ground co m p e t i t i o n c o u l d i n f l u e n c e root m i c r o f l o r a q u a n t i t a t i v e l y and q u a n t i t a t i v e l y . When Lolium perenne and Tri folium repens were separated below but not above ground, and compared with t h e i r r e s p e c t i v e monocultures, i t was found that the l e n g t h of f u n g a l mycelium on r o o t s of L. perenne was reduced to h a l f of the monoculture value when above ground com p e t i t i o n o c c u r r e d . R e s u l t s from t h i s work have l e d t h i s group to conclude that p l a n t root exuduates can be a f f e c t e d by i n t e r s p e c i f i c p l a n t c o m p e t i t i o n and that p l a n t s able to d e r i v e more n i t r o g e n i n mixture exude more amino a c i d s than in monoculture. Since many rh i z o s p h e r e b a c t e r i a are amino, a c i d users (Gray and W i l l i a m s , 1971), i n c r e a s e d n i t r o g e n exudation as amino a c i d s c o u l d account f o r the l a r g e r r h i z o s p h e r e p o p u l a t i o n s and e x p l a i n the p o s i t i v e c o r r e l a t i o n observed between m i c r o b i a l abundance and N s t a t u s of the p l a n t s (Newman et a l . , 1979). However, Turner and Newman (1984) found that d e c r e a s i n g n i t r o g e n s u p p l i e s , which decreased p l a n t y i e l d , had no e f f e c t on s u r v i v o r s h i p of b a c t e r i a when r o o t s of pasture grasses i n c l u d i n g L. perenne were i n o c u l a t e d . Instead, a response to phosphorus was observed such t h a t b a c t e r i a l abundance i n c r e a s e d i f P was d e f i c i e n t . D i s c r e p a n c i e s i n c o n c l u s i o n s r e g a r d i n g N s t a t u s and m i c r o b i a l growth i n the r h i z o s p h e r e are probably r e l a t e d to the f a c t t h a t many of these s t u d i e s make l i t t l e e f f o r t to i d e n t i f y the b a c t e r i a l s t r a i n s t h a t are observed. While gross g e n e r a l i z a t i o n s with r e s p e c t to gram r e a c t i o n , spore formation (Lochhead and Chase, 1940), or n u t r i t i o n a l requirements (Lochhead, 1943) of r h i z o s p h e r e b a c t e r i a are of some use, comparisons between s t u d i e s are tenuous at best and i n f o r m a t i o n on p a r t i c u l a r s p e c i e s of b a c t e r i a i s not a v a i l a b l e . Some of the most i n f o r m a t i v e work on r h i z o s p h e r e p o p u l a t i o n s t u d i e s has been done with s p e c i e s of Rhizobium (Parker et a l . , 1977; Schmidt, 1978). While the legume r h i z o s p h e r e supports l a r g e r p o p u l a t i o n s of microbes i n g e n e r a l , s e v e r a l r e s e a r c h e r s have i n v e s t i g a t e d the s p e c i f i c p o p u l a t i o n dynamics of Rhizobium i n homologous and heterologous p l a n t r h i z o s p h e r e s . There i s some evidence to suggest that a legume s e l e c t i v e l y s t i m u l a t e s Rhizobium s t r a i n s of the proper c r o s s - i n o c u l a t i o n group when other root nodule b a c t e r i a are p r e s e n t , but o p i n i o n s d i f f e r as to whether t h i s phenomenon occurs c o n s i s t e n t l y (Parker et a l . , 1977). R o v i r a (1961) and Tuzimura and Watanabe (1961; 1962) have demonstrated the s t i m u l a t i o n of Rhizobium i n the host r h i z o s p h e r e r e l a t i v e to other a e r o b i c microbes, but most s t u d i e s have j u s t f o l l o w e d Rhizobium p o p u l a t i o n s and ignored a l l other types of microorganisms (Schmidt, 1978). U n f o r t u n a t e l y , t h i s approach obscures the t r u e r h i z o s p h e r e e f f e c t as Rhizobium may comprise only a small p o r t i o n of the t o t a l m i c r o f l o r a , but s t i m u l a t i o n of that component may be very important i n subsequent n o d u l a t i o n and n i t r o g e n f ixat ion. Robinson (1967) found that in a mixed subterranean c lover /a l fa l fa sward, R. meliloti could not be detected in subterranean clover rhizospheres, but up to 5 mil l ion ce l l s of the bacterium per gram of root were found in the a l fa l fa rhizosphere. R. trifolii was apparently less selective as i t was found in either plant rhizosphere in large numbers with a slight tendancy to associate preferentially with the clover component of the mixture. Krasil 'nikov (1958) similarly demonstrated that legumes selectively stimulate infective Rhizobium strains tenfold over noninfective isolates when he tested clover and a l fa l fa with homologous and heterologous microsymbionts in s ter i le nutrient solution. In contrast, Tuzimura and Watanabe (1962) found that R. trifolii populations were only s l ight ly enhanced in the rhizosphere of i ts host ladino clover, but populations were substantially stimulated in the non-host legume rhizospheres of a l f a l f a , soybean, and peanut (Arachis hypogaea L . ) . Evidence for qualitative and quantitative differences in plant rhizospheres and associated microbial populations can be found from the work of Foster and Rovira (1976) discussed by Rovira (1978). Transmission electron microscopy of the root systems of Trifolium subterraneum, Pas pal um di I at ut um P o i r . , and wheat revealed substantial differences in the organization and structure of the rhizoplane and rhizosphere. The rhizosphere of T. subt erraneum was shown to possess -several dist inct types of bacteria based on ce l lu lar morphology which form a layer (10-20 um) between the root and the s o i l . The P. dilatutum rhizosphere was shown to consist of a thick layer of organic material with microcolonies scattered throughout, while the wheat rhizosphere did not extend outward from the root surface and was comprised solely of the rhizoplane and invaded cort ica l and epidermal ce l l s . Scanning electron microscopy of Trifolium repens roots (Campbell and Rovira, 1973) revealed that mucilage was confined mainly to the root surface while L. perenne appeared to spread its mucigel layer throughout sand and clay particles in the rhizosphere. As pointed out by Rovira (1978), these studies demonstrate that numerous ecological niches are available for microbial growth on roots and in rhizospheres. The differences in physical characteristics of plant rhizospheres discussed above i l lustrate the diversity that exists between plant root systems. Even within a root system, the presence of microcolonies of bacteria along root surfaces suggests that d i f ferent ia l colonization occurs within a rhizosphere thus providing further var iab i l i ty for specific sites of colonization. Whether these differences are characteristic of the plant genotype and therefore influence the composition of the associated microflora, or are the result of previous microbial colonization, the potential for species and strain specific niches clearly exists between and within root systems of different plants. The biot ic specif ic i ty observed between genotypes of white clover and perennial ryegrass (Aarssen and Turkington, 1985) could, therefore, be mediated by influences of the neighbouring ryegrass root system on s o i l m i c r o f l o r a which may subsequently a f f e c t c l o v e r p l a n t growth. For example, i f the grass root system p r o v i d e d f a v o r a b l e growth c o n d i t i o n s f o r a bacterium that c o n t r i b u t e s to i n c r e a s e d legume nodule s i z e or number (Burns et a l , 1981; El-Bahrawry, 1983), a grass-mediated p o s i t i v e e f f e c t on i t s c l o v e r neighbour would r e s u l t . Rhizosphere m i c r o f l o r a of t h i s type c o u l d e i t h e r ( i e x i s t i n the grass r h i z o s p h e r e but a f f e c t c l o v e r performance d i r e c t l y or through n o d u l a t i o n , or ( i i ) s t i m u l a t e the c l o v e r or grass to increase root exudation thereby i n c r e a s i n g the r h i z o s p h e r e p o p u l a t i o n i n c l u d i n g Rhizobium. The p o s s i b i l i t y of a f f e c t i n g or s t i m u l a t i n g root exudation was advanced by the work of Norman (1955; 1961), who found t h a t c e r t a i n a n t i b i o t i c s such as polymyxin, which i s formed by some s t r a i n s of Bacillus polymyxa, a l t e r s c e l l p e r m e a b i l i t y and i n c r e a s e s leakage from r o o t s . Even though a root nodule can a r i s e from a s i n g l e i n f e c t i o n , l a r g e p o p u l a t i o n s of Rhizobium i n the s o i l are r e q u i r e d to e s t a b l i s h e f f e c t i v e symbiosis (Brown et a l . , 1968). In t h i s regard, host r h i z o s p h e r e p o p u l a t i o n s of the a p p r o p r i a t e Rhizobium s p e c i e s are important. However, i n a pasture where v a r i o u s non-legume s p e c i e s may s i g n i f i c a n t l y outnumber the legume component, s u r v i v a l of Rhizobium i n non legume r h i z o s p h e r e s c o u l d be very important. R o v i r a (1961) noted that c e r t a i n Rhizobium s p e c i e s do so w e l l i n the r h i z o s p h e r e s of non-legumes that neighbouring p l a n t s p e c i e s c o u l d be an i n f l u e n t i a l f a c t o r i n s u r v i v a l of the microbe in the absence of i t s host. T h i s phenomenon would a l s o be 150 important during the saprophytic stages of the l i fecycle of root nodule bacteria when legume infection and nodulation does not occur. Rovira (1961) compared rhizosphere stimulation of R. trifolii between pure stands of red clover (Trifolium pratense) and the grass Paspalum diI at at um. The clover rhizosphere was shown to support higher numbers of bacteria, including Rhizobium (32 mill ion per gram of root), but the grass rhizosphere had a substantial Rhizobium population, 20 mil l ion ce l l s per gram of root, as well. The pH of the so i l used in this experiment was low (pH 4.8) therefore the effect of liming was investigated. Addition of calcium oxide, which increased the pH to 7.0, resulted in spectacular gains in rhizospheric populations of Rhizobium in both species. After so i l amendment, the grass was shown to support 142 mill ion root nodule bacteria per gram of root while clover root populations of Rhizobium increased to 680 mil l ion per gram of root tissue. Krasil 'nikov (1958) also showed substantial prol i feration of clover and a l fa l fa microsymbionts in the non-host rhizospheres of cotton (Gossypium sp.) and wheat when grown in nutrient medium. Robinson's (1967) examination of subterranean clover and a l fa l fa Rhizobium populations included a site comprised of Poa austral is R.Br, tussocks. Both R. trifolii and R. meliloti were found in substantial numbers in the rhizosphere of the Australian grass. Al fa l fa Rhizobium was not detected in the subterranean clover rhizosphere in this study therefore Robinson (1967) demonstrated the preference of root nodule bacteria for the rhizosphere of a grass over one of a non-host legume. Others have shown that R. trifolii can proliferate in the rhizosphere of Brassica sp., tomato, Urtica sp. , and Gnaphal i um sp. (Tazimura and Watanabe, 1962; Brown et a l . , 1968; Chatel and Greenwood, 1973). The latter group has further demonstrated strain differences within R. trifolii in their capacity to colonize root systems of three pasture weeds in Western Australia (Arctotheca calendula L . , Vulpi a myuros L . , and Hypochoer i s glabra L . ) . Rhizosphere studies of the microflora in mixed stands of Wimmera ryegrass (Lolium rigidum Gaud.) and subterranean clover {Trifolium subt erraneum) have demonstrated that branched or pleomorphic forms of bacteria such as Ar t hr obact er sp. and Nocardia sp. predominate on roots of both plant species (Sperber and Rovira, 1959; Rovira and Stern, 1961). Generally, plant roots have been shown to favor gram negative, non-sporing, rod-shaped bacteria; often pseudomonads (Curl and Truelove, 1986). Katznelson (1965) suggested that Art hrobact er can be a common rhizosphere inhabitant but i t is often suppressed by vigorous members of the genus Pseudomonas. It is of particular interest in the clover/ryegrass studies (Sperber and Rovira, 1959; Rovira and Stern, 1961) that of 305 clover and 286 ryegrass root isolates, only 1 (from clover) was identified as Rhizobium even though abundant nodules were evident on test plants. This observation suggests that a neighbouring grass plant which supported R. trifolii in i ts root system could be of benefit to subsequent clover and pasture productivity as a source of inoculum of root nodule bacteria. Diatloff (1969) tested such an idea by inoculating seeds of cereals and non-host legumes with R. japoni cum in an novel attempt to introduce strains of this bacterium to the s o i l . His greenhouse results showed that Rhizobium was stimulated suff iciently in the non-host rhizosphere to provide an adequate inoculum for a subsequent crop of soybeans, but f ie ld tests were not part icularly successful as only small populations of the root nodule bacterium were established. Neighbouring grass species can have negative effects on the lequme/Rhi zobi um symbiosis as well. In an area which had a history of nodulation problems, Parle (1964), cited in Parker et a l . (1977), showed that roots of the grass Notodanthonia sp. completely inhibited growth of R. trifolii and R. meliloti. Vir tual ly no root nodule bacteria were detected in host rhizospheres in the New Zealand pasture examined, but when grass roots were k i l l ed or removed, 4 rhizosphere populations of Rhizobium rose to 10 per gram of s o i l . If Rhizobium is selectively stimulated in non-legume rhizospheres, i t may be possible that the microbe benefits growth of the non-host in some way (see Chapter 2). Rhizobium is capable of fixing nitrogen while in a free l iv ing state (Kurz and LaRue, 1975) and also produces phytohormones (Meijer, 1982). A positive y ie ld response has been observed in perennial ryegrass inoculated with R. trifolii (J . Thompson, University of Liverpool, personal 153 communication). Similarly, Kavimandan (1985; 1986) has observed a growth response when wheat was inoculated with various species of Rhizobium. The author speculates that the growth response results.from rhizospheric nitrogen fixation, but this is unlikely because careful manipulation of growth conditions is necessary to induce nitrogenase act iv i ty in culture (Trinick, 1982). Smith and Rice (1986) agree with the conclusion that nonlegume nitrogen fixation by Rhizobium is unlikely. In view of increasing evidence for the role of plant growth substances in PGP bacteria (Brown, 1974), i t seems l ike ly that the wheat response is phytohormonal in nature. Specific associations between plant genotypes and pathogenic strains of microbes are well known (Flor, 1955; Person, 1959). Similarly, specif ic i ty between plant and bacterium in nonpathogenic associations has been demonstrated (Dobereiner and Campelo, 1971; Neyra and Dobereiner, 1977; Rennie and Larson, 1979; Baldani and Dobereiner, 1980). The association of Azot obact er pas pal i with Paspalum notatum in so i l that normally supports large populations of Derxia gummosa as the free l iv ing diazotroph (Dobereiner and Campelo, 1971) is a good example of such an association at the species leve l . However, of 33 ecotypes of the grass studied, only 5 tetraploid types stimulated A. pas pali in the rhizosphere (Neyra and Dobereiner, 1977) which suggests that genotypes within a species may exert di f ferent ia l effects on root bacteria populations. Host plant-microbe specif ic i ty of this type has also been observed with maize, wheat, and rice inoculated with Azospirillum species (Baldani and Dobereiner, 1980). The work of Neal et a l . (1970; 1973) and Rennie and Larson (1979; 1981) with disomic chromosome substitution lines of wheat demonstrated that a single chromosome can qualitat ively change rhizosphere microflora and subsequently affect plant growth. Similarly, Johnson and Mattern (1977) and Klucas and Pedersen (1980) found that group 5 chromosomes of Cheyenne and Atlas wheat were important in establishment of effective nitrogen fixing associations. Thus, specif ic i ty in plant/microbe interactions can be attributed to small changes in plant genotype as i t is possible that only one or a few genes on the substituted chromosome were responsible for the observed effects. Holl (1983) cites several examples of genetic var iab i l i ty in nonlegume nitrogen fixation between plant and bacterial species. The spectacular gains conferred by some PGP bacteria, such as the 300% increase in plant weight of wheat (Rennie and Larson, 1979), could have a large effect on plant fitness. While i t is not alway true that "bigger is better" when fitness is considered (Harper, 1977), i t is easy to see. that under certain environmental circumstances, a large plant would be more successful than a smaller one in reproductive output. For example, under conditions of limited so i l phosphorus, a large root system can result in a substantial competitive advantage as this element is very immobile in so i l (Nye, 1966). Enhanced phosphorus ava i lab i l i ty could aid either parental survival or production of reproductive p r o p a g u l e s w h i c h a r e t h e two e l e m e n t s i m p o r t a n t i n f i t n e s s . T h e r e f o r e , w i t h t h e p o t e n t i a l f o r r e l a t i v e l y s m a l l c h a n g e s i n p l a n t g e n e t i c makeup t o r e s u l t i n a l a r g e a d v a n t a g e i n p l a n t f i t n e s s ( and t h e n e c e s s a r y v a r i a b i l i t y ) , t h e p r o c e s s o f n a t u r a l s e l e c t i o n i n p l a n t / b a c t e r i a l i n t e r a c t i o n s s h o u l d be i m p o r t a n t . Some e v i d e n c e f o r t h i s phenomenon has been p r e s e n t e d i n C h a p t e r 2, where an i s o l a t e o f Bacillus pol ymyxa was d e m o n s t r a t e d t o i n d u c e a l a r g e r s t i m u l a t o r y e f f e c t on homologous g e n o t y p e s of p e r e n n i a l r y e g r a s s and w h i t e c l o v e r t h a n on h e t e r o l o g o u s ones u s i n g e x p e r i m e n t a l m a t e r i a l c o l l e c t e d f r o m a 45 y e a r o l d p a s t u r e . The f o l l o w i n g s t u d y examines y i e l d r e l a t i o n s h i p s between p e r e n n i a l r y e g r a s s and w h i t e c l o v e r grown i n m i x t u r e when c o m p o s i t i o n o f t h e s o i l m i c r o f l o r a was q u a l i t a t i v e l y c o n t r o l l e d . The f i r s t e x p e r i m e n t i n v e s t i g a t e d t h e p r o d u c t i v i t y o f p a s t u r e p l a n t s when R. t r i f o l i i i s o l a t e s homologous t o t h e c l o v e r a n d / o r r y e g r a s s g e n o t y p e s c o m p r i s e d t h e r o o t n o d u l e b a c t e r i a p o p u l a t i o n compared w i t h y i e l d s when h e t e r o l o g o u s Rhizobium i s o l a t e s were u s e d a s i n o c u l a . I n f e c t i v i t y of homologous i s o l a t e s o f R. t r i f o l i i as a f f e c t e d by t h e c l o v e r and r y e g r a s s g e n o t y p e s was a l s o i n v e s t i g a t e d . The s e c o n d e x p e r i m e n t e xamined s i m i l a r y i e l d r e l a t i o n s h i p s a s a f f e c t e d by homologous o r h e t e r o l o g o u s i s o l a t e s o f d i a z o t r o p h i c Bacillus s p e c i e s . In t h i s i n v e s t i g a t i o n , a l l p l a n t s r e c e i v e d a c o m p o s i t e i n o c u l u m o f R. t r i f o l i i i s o l a t e s , e a c h o f w h i c h was homologous t o one o f t h e c l o v e r g e n o t y p e s u s e d i n t h e e x p e r i m e n t s . T h e r e f o r e , t h e e f f e c t of the a s s o c i a t e d Bacillus i s o l a t e on R. trifolii s t r a i n s e l e c t i o n by c l o v e r genets was a l s o examined. E v i d e n c e i s p r e s e n t e d which suggests tha t p l a n t s and b a c t e r i a have undergone c o a d a p t a t i o n or c o e v o l u t i o n i n the 45 year o l d p a s t u r e . The e f f e c t of s o i l b a c t e r i a on the genotype s p e c i f i c i t y observed between p e r e n n i a l r y e g r a s s and white c l o v e r genotypes (Aarssen and T u r k i n g t o n , 1985) was shown to be s u b s t a n t i a l and the i m p l i c a t i o n s of t h i s f i n d i n g are d i s c u s s e d . MATERIALS AND METHODS Plants and Microorganisms Pairs of plants (one Loli um perenne and one Trifolium repens) growing side-by-side were collected from perennial ryegrass-dominated areas of a 45 year old pasture. The sward had a botanical composition which included perennial ryegrass (Lolium perenne L . ) , orchardgrass (Dactyl i s glomerata L . ) , velvetgrass (Hoi cus lanatus L . ) , and white clover (Trifolium repens L . ) . Rhizobium trifolii was isolated from root nodules of T. repens as outlined earl ier (see Microorganisms, Chapter 1). Diazotrophic Bacillus spp. were isolated from rhizosphere so i l of the pasture plant pairs also as previously described (see Bacterial Culture and Evaluation, Chapter 2). These collection and isolation procedures resulted in the procurement of three sets of experimental organisms. Each group consisted of a perennial ryegrass plant, a white clover plant, an isolate of R. trifolii, and an isolate of a diazotrophic Bacillus species, similar to Bacillus pol ymyxa. These experimental combinations were designated as follows: Rhizobium (R1 , R2, R3) ; Lol i um (L1 , L2, L3) ; Bacillus (B1, B2, B3); Trifolium (T1, T2, T3). Where the numerical designation is the same (eg. R1T1), the organisms were isolated from the same original association in the pasture and represent a homologous combination. For comparative purposes to the identif ication studies described in Chapter 1, i t should be noted that the Rhizobium strains R1, R2, and R3 were original ly isolated and 158 characterized as L1, L5, and L6, respectively. Plants were vegetatively propagated in the greenhouse so that clones of the plant genotypes and subcultures of the microorganisms could be used for subsequent experimentation. Plant Growth and Inoculation Experiments were conducted using wooden flats (45 x 30 x 6.5 cm) containing a 50:50 (v/v) mixture of potting so i l and Turface ® . The so i l mix contained 580 kg ha 1 total nitrogen, 28 kg ha 1 available phosphorus, and 79 kg ha 1 available potassium. Flats were autoclaved for 90 minutes prior to use in experiments. Individual t i l l e r s (ryegrass) or stolon tips (clover) were clipped from stock plants, rinsed for 5 seconds in 80% ethanol and then in s ter i le d i s t i l l e d water to reduce random bacterial contamination and were planted in 4 rows containing alternating grass and legume plants. Intra-row and inter-row spacing was ca. 18 mm and 75 mm, respectively. Flats were then divided into 16 compartments of equal size (75 x 110 mm) which were arranged in 4 rows and 4 columns using plastic partit ions. Partitions were surface, s ter i l i zed in 95% ethanol before use. These barriers separated below ground compartments completely and extended 100 mm above the so i l surface to discourage above ground competition between plants in different ce l l s . Two types of experiments were established depending on the inoculum combination used. The f i r s t experiment was designed to investigate the effectiveness of R. trifolii isolates over a long term. Flats received 101^ cel l s of the appropriate R. trifolii strain (ca 7x10 m ) in 200 ml MGA medium. Inoculum was administered by pouring 50 ml aliquots evenly along each of the four rows of plants followed by extensive watering of the f lats . To ensure that root nodules were formed by the desired strain of R. t r i f o l i i , inoculation was repeated three months later. Plants were grown under natural l ight in the greenhouse with a day/night temperature regime of 30°C/20°C. Plants were clipped back to the height of the plastic partit ion 9 times during the year-long growth period, about every 5 weeks. Clippings were separated into species components and were dried at 55°C for 4 days and weighed. After one year's growth, plants were destructively harvested. In each c e l l , the outer two plants were discarded as these reflected substantial border effects with the plastic part i t ions . The inner four plants (2 ryegrass and 2 clover) were separated from each other and divided into roots and shoots, dried, and weighed as before. Several root nodules from each clover plant were collected and stored frozen in 10% glycerol (v/v) for subsequent identif ication of the nodule inhabitants. The second experiment was designed to investigate the effect of Bacillus isolates on the yie ld of perennial ryegrass and white clover and on the relative infectiveness of R. trifolii strains in root nodule formation. Therefore, flats received dual inoculation of R. trifolii and Bacillus sp. Each flat received 3X10 1^ R. trifolii ce l l s (101(^ cel ls of each homologous R. trifolii strain) in 200 ml MGA medium and a further 101^ ce l l s of the appropriate Bacillus isolate 160 in 200 ml of nutrient broth. Inoculum was administered 50 ml row 1 as in the previous experiment. Control flats were inoculated with R. trifolii in MGA medium and also received 200 ml of steri le nutrient broth. Plants were grown in the greenhouse under similar conditions to the f irs t experiment. Plants were clipped back to the height of the plastic partit ion twice, about every three weeks, and after 8 weeks growth were destructively harvested as in the long term exper iment. Identification of the Microsymbiont in Root Nodules Individual root nodules were thawed, weighed, and analyzed using the ELISA procedure described in Chapter 1 (see Enzyme-Linked Immunosorbent Assay). Three nodules per plant were assayed; therefore, 36 nodules per experimental treatment were analyzed (3 nodules/plant x 2 plants/replicate x 3 replicates/experiment x 2 experiments). Experimental Design and Analysis The effectiveness of three R. trifolii isolates as indicated by growth of genotypic and species mixtures of perennial ryegrass and white clover was studied using a s p l i t - s p l i t plot design with inoculum assigned to main plots ( f lats) , ryegrass clones to sub-plots (rows within f la t s ) , and clover clones to sub-sub-plots (columns within f la t s ) . Main plots were randomized with 5 complete blocks on a bench in the greenhouse. Homogeneity of variance tests were not rejected therefore analysis was performed on raw data. The 161 design and analysis of experiments which investigated the effect of Bacillus isolates on plant yield have been previously described (see Experimental Design and Stat i s ica l Analysis, Chapter 2). In both experiments, a single degree of freedom (orthogonal contrast) was used to test the hypothesis that homologous genotype combinations outyielded heterologus combinations for the yield variables of interest. The associated probability level for this test is recorded at the bottom of each graph (Figures 3-1 to 3-43). Separation of individual treatment means was accomplished using Tukey's procedure. Means which have not been assigned lower case letters or those with the same letter are not s ignif icantly different (P < 0.05). In experiments to evaluate R. trifolii infectiveness, the design and analysis was identical to that of the latter experiment except that the yield variable was the number nodules containing the various R. trifolii strains. Homogeneity of variance tests for these yield variables were also not significant therefore untransformed data were used in the analysis. Analysis of variance tables and orthogonal contrasts for the experiments presented in this Chapter are lodged with Dr. F .B . Ho l l , Department of Plant Science and may be obtained upon request. 162 RESULTS One of the major goals of this research was to determine the effect R. trifolii had on genotype specif ic i ty between clones of white clover and perennial ryegrass. The effect of perennial ryegrass neighbours on f inal clover root, shoot, and plant weights and on total clover biomass accumulation over the year-long experiment is presented (Figure 3-1). A very small advantage (5%; not significant) was observed when the average yield of white clover plants grown with homologous ryegrass neighbours was compared to the yield of plants growing with heterologous neighbours (Figure 3-2c, harvest 10). The effect was more pronounced for root weights (Figure 3—1b) where two of the three homologous combinations tended to outyield heterologous ones. These small and s ta t i s t i ca l ly nonsignificant yie ld advantages were evident by the second harvest and were consistent throughout the experiment (Figure 3-2) . However, when the matched T. repens/L. perenne clones were inoculated with homologous R. t r i f o l i i , a substantial clover yie ld advantage was observed (Figures 3-2 to 3-4). Under these conditions, white clover root weights were 50% higher (Figure 3-3b) while f inal clover plant weight showed an average increase of 28% (Figure 3-4a). When total clover biomass was analyzed, plants growing with homologous organisms were shown to outyield the average heterologous combination by 27% (P < 0.005) (Figure 3-2c, harvest 10 and 3-4b). This yie ld advantage also appeared relatively early in Trifolium Shoot Weight Trifolium Root Weight 3500 3000 1300 *-' 2000 I * BOO 300-L1 TI T2 13 L2 ab ab 11 T2 T3 13 TI T2 T3 fj> 600 ^ 400 11 ab ab 1 L2 I L3 A t ab I TI T2 T3 T1 T2 T3 TI T2 T3 N.S. N.S. 4000 3500-3000-f 2300 "fe 1000-I woo -500 Trifolium Plant Weight 13 Total Trifolium Biomass L2 T3 TI T2 T3 TI T2 T3 WO00 8000 JL 6000-I 4000-NS. 0-N.S. L1 I TI T2 T3 L2 TI T2 T3 L3 T1 T2 T3 F i g u r e 3-1. E f f e c t of L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of T. repens The p r o b a b i l i t y l e v e l (bottom l e f t ) r e f e r s to the c o n t r a s t : homologous genotype c o m b i n a t i o n s (hatched bars ) o u t y i e l d h e t e r o l o g o u s c o m b i n a t i o n s (open b a r s ) . h=Lolium, T= Trifolium. N . S . - not s i g n i f i c a n t . I n d i v i d u a l means a s s i g n e d no l e t t e r or the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (Tukey ' s t e s t ; P < 0 . 0 5 ) . n=15. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t r o o t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated biomass 164 "frifolium Yield Cumulative Harvest 2 3 4 5 6 7 8 9 10 Cumulative Harvest Trifolium Yield Cumulative Harvest 4 5 6 7 8 Cumulative Harvest Legend o R«L O RxT 10 Figure 3 -2 . Advantage observed in growth of T. repens when the y i e l d of homologous genotype combinations i s expressed as a percentage of the y i e l d observed in heterologous combinations (a) and (b) d e p i c t % y i e l d advantage at each of 10 h a r v e s t s taken over 12 months; (c) and (d) d e p i c t c u m u l a t i v e y i e l d advantage a t each h a r v e s t . LxT - homologous L. perenne/T. repens c o m b i n a t i o n s , n=15; RxL - homologous R. trifolii/L. perenne c o m b i n a t i o n s , n=15; RxT -homologous R. trifolii/T. repens c o m b i n a t i o n s , n=15; RxLxT - homologous R. trifolii/L. perenne/T. repens c o m b i n a t i o n s , n=5. Open symbols (P > 0 . 0 5 ) ; C l o s e d symbols (P < 0.05) Trifolium Shoot Weight 4000-| 3S00 3000 If 2500-"S> 2000 I 1500-Q 1000 50n-R2 / / / / / / / / <> / / / R1 / / / / / / / / / / R3 p<0.025 f p f p p f c c ; sssaaa 33: PPCPPPPPP ppppPFPPP 555 333333 Trifolium Root Weight 1600- R2 cn 1400-1200-1000-"§> 800-I 600-o 400-200-R1 R3 p<0.005 p p p p p p p p e 555333333 PRPPPPPPP 555333333 PPPPPPPPP 555333333 F i g u r e 3 - 3 . E f f e c t of R. t r i f o l i i / L . perenne/T. repens genotype combinat ions on the y i e l d of T. repens S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=5. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t w e i g h t . W=Rhi zobi um, L=Lolium, ?=Tr i f oli um Trifolium Plant Weight 166 SOOO-i 4500 4000-350)-15) 3000-"Si 2500-^ 2000-Q 1500 1000 500 0 p<0 R1 R2 R3 025 ^ § § § § ^ 5 i S s s ^ i l i Total Trifolium Biomass 10000 9000-8000 7000 JE, 6000-O) 5000 4000 H Q 3000 2000 1000 0 p<0. R1 / ' H R2 /-I / / / / R3 005 aaaaaasss PC! F i g u r e 3 -4. E f f e c t of R. trifolii/L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of T. repens S t a t i s t i c s are d e s c r i b e d in F i g u r e 3 - 1 . n=5. (a) f i n a l h a r v e s t p l a n t weight (b) t o t a l accumulated b iomass . R=Rhizobi um, L=Lolium, T=Trifolium the year ( F i g u r e s 3-2a and c , h a r v e s t 3) and peaked by h a r v e s t 6, when homologous p a r t n e r s h i p s o u t y i e l d e d h e t e r o l o g o u s ones by an average of 47% ( F i g u r e 3 -2a , h a r v e s t 6 ) . To i l l u s t r a t e f u r t h e r the white c l o v e r y i e l d advantage , t o t a l legume biomass a c c u m u l a t i o n may be e v a l u a t e d when R. trifolii was not r e l a t e d to e i t h e r the white c l o v e r or the p e r e n n i a l r y e g r a s s genotypes t e s t e d ( F i g u r e 3~5a) . In F i g u r e 3-5b the c l o v e r y i e l d observed when on ly p l a n t s were homologous has been r e p l a c e d by the mean y i e l d of the legume when a l l organ i sms , i n c l u d i n g R. t r i f o l i i , were r e l a t e d . C l e a r l y , the genotype s p e c i f i c i t y of the type d i s c o v e r e d by Aars sen and T u r k i n g t o n (1985) i s e v i d e n t in the l a t t e r c a s e . A t e s t of s i g n i f i c a n c e was not p o s s i b l e u s i n g these data due to r e s t r i c t i o n s of o r t h o g o n a l i t y ( F i g u r e 3 - 5 ) . The y i e l d of r y e g r a s s when grown in m i x t u r e wi th c l o v e r was not s i g n i f i c a n t l y a f f e c t e d by d i f f e r e n c e s i n the n e i g h b o u r i n g legume genotype , but a tendency f o r lower y i e l d s to occur w i t h p r e v i o u s l y "known" c l o v e r p a r t n e r s was observed ( F i g u r e s 3-6d and 3 - 7 c ) . F i n a l weight of such p l a n t s was 13% lower than g r a s s e s growing w i t h h e t e r o l o g o u s c l o v e r ne ighbours ( F i g u r e s 3-6c and 3 -7a , h a r v e s t 10) . An 8% y i e l d decrease was observed when t o t a l Lolium biomass was a n a l y z e d ( F i g u r e s 3-6d and 3-7c , h a r v e s t 10) . S i m i l a r s t a t i s t i c a l l y i n s i g n i f i c a n t t r e n d s were observed when p e r e n n i a l r y e g r a s s was grown w i t h homologous white c l o v e r and R. trifolii genotypes ( F i g u r e s 3-7 to 3 - 9 ) , except that r o o t weight was s l i g h t l y i n c r e a s e d (8%) ( F i g u r e 3-8) compared w i t h a s l i g h t 168 Total Trifolium Biomass 8000 7000 6000-5000 "B> 4000-I C" 3000 O 2000-1000 Ll TI T2 T3 L2 1 TI T2 T3 L3 TI T2 T3 Total Trifolium Biomass 10000 9000 8000 7000 'cn ,E, 6000 H "g> 5000 3S >^  4000-o 3000-2000-1000 0 L2 Ll T I T 2 T 3 TI T2 T3 L3 A I T1 T2 T3 F i g u r e 3 - 5 . E f f e c t of L. per enne/T. repens genotype c o m b i n a t i o n s on the y i e l d of T. repens (a) R. trifolii was h e t e r o l o g o u s to both p l a n t genotypes ; (b) R. trifolii was matched to homologous p l a n t genotype c o m b i n a t i o n s . L=Loiium, T=7> i f ol i um Lolium Shoot Weight Lolium Root Weight »oo-800-•00-L1 L2 L3 Tt T2 T3 TI T2 T3 T1 T2 T3 J , 600 % I 400 <5 L2 L3 TI T2 T3 TI T2 T3 Tt T2 T3 N.S. N.S. BOO-i 0-N.S. Lolium Plant Weight L l L2 L3 TI T2 T3 TI T2 T3 TI T2 T3 5000 2S00 ;§> BOO £ woo Total Lolium Biomass L3 L l L2 TI T2 T3 TI T2 T3 Tt T2 T3 N.S. F i g u r e 3 -6 . E f f e c t of L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of L. perenne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=l5 . (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t r o o t , w e i g h t (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . "L-Lolium, T=Tr i folium 170 Lolium Yield individual Harvest Lolium Yield Individual Harvest 3 4 5 6 7 8 9 10 Individual Harvest 4 5 6 7 Individual Harvest Lolium Yield Cumulative Harvest 4 5 6 7 8 Cumulative Harvest Lolium Yield Cumulative Harvest - Legend - O R«L - 0 R»T - D K n— p. .41 O B----J,-' H , - , f l , - B-4 5 6 7 8 Cumulative Harvest 10 F i g u r e 3 - 7 . Advantage observed i n growth of L. perenne when the y i e l d of homologous genotype c o m b i n a t i o n s i s expressed as a percentage of the y i e l d observed i n h e t e r o l o g o u s c o m b i n a t i o n s (a) and (b) d e p i c t % y i e l d advantage a t each of 10 h a r v e s t s taken over 12 months; (c) and (d) d e p i c t c u m u l a t i v e y i e l d advantage a t each h a r v e s t . Symbols a r e e x p l a i n e d i n F i g u r e 3-2 171 1000 800 cn !c cn I Q 600 400 200 Lolium Shoot Weight R1 n R2 R3 N.S. m ^ m m & m $ n m n ® cn 1200-1 1000 800-"§> 600 Q 400 200 N.S. Lolium Root Weight R2 RI R3 r.B pp Epcg p saaaaasss F i g u r e 3 -8 . E f f e c t of R. trifolii/L. perenne/T. repens genotype combinat ions on the y i e l d of L. perenne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=5, (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t root we ight . R=Rhizobium, L=Loli um, T=7>/ folium Lolium Plant Weight 172 2000-, R1 R2 R3 1500-o) woo-I L. o 500-0-N.S. I—PI / / aaaaaass: pepP B E t c p Total Lolium Biomass 3500- R1 R2 R3 3000-2500-E ^ 2000-JZ O) 3t 1500->~ Q 1000-500-/ / / / / / / / / / / N.S. PC sa B P f f C f . B P fcCPPpPPBP ssaaa 3 3 3 5 5 5 a a a 3 3 3 P P C E R P C B P F i g u r e 3-9. E f f e c t of 1?. trifolii/L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of L. per e nne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=5. (a) f i n a l h a r v e s t p l a n t weight (b) t o t a l accumulated b iomass . R=Rhizobium, L=Lolium, T=Tr i folium decrease (9%) when R. trifolii was not homologous (Figure 3-6b) . Because white clover formed most of the biomass in mixture, the yie ld trends observed when the legume component of the stand was studied were also seen when total mixture biomass was analyzed (Figures 3-10 to 3-13). No yield advantage was apparent when Trifolium and Lolium genotypes were homologous (Figures 3-10 and 3-11) but inclusion of the "correct" R. trifolii strain resulted in significant gains. Final combined plant weight was 20% higher (P < 0.025) (Figure 3-11a, harvest 10), while total forage biomass which had accumulated during the year long growth period was increased by 18% (P < 0.025) (Figure 3-11c, harvest 10). It can be seen from Figures 3-3 and 3-4 that the white clover yie ld advantage which occurred when a l l three organisms were related was not necessarily maximized when heterologous clover genotypes were excluded. If the strain of R. trifolii was homologous to the genotype of L. perenne higher yields resulted, often without regard to the clover genotype (see homologous Rhi zobi um/Lol i um (R/L) combinations Figures 3-3 and 3-4). Therefore, when various two-factor interactions were studied, the R. trifolii/L. perenne relationship was seen to affect yie ld positively and to be highly significant (Figures 3-2, and 3-14 to 3-16). A l l parameters measured showed this trend when white clover genotypes were pooled and dry weights were assessed. The yield advantage began early in the experiment and continued throughout the period of study Combined Shoot Weight Ll v L2 „ L3 Combined Root Weight T I « ' T 3 TI T2T3 TI T2 T3 oi J. I, I WOO N.S. fl-its. Ll L3 I TI T2 T3 TI T2 13 TI T2 13 Combined Plant Weight Ll TI T2 T3 L2 TI T2 T3 L3 TI T2 T3 10000 sooo aooo 7000 6000 i& 5000 I >. 4000-o 3000-2000 woo Total Combined Ll L2 Biomass L3 i TI Ti T3 TI T2 T3 TI T2 T3 N.S. N.S. F i g u r e 3-10 . E f f e c t of L. perenne/T. repens genotype combinat ions on combined forage y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=15. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t root weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . h=Lolium, T=Trifolium 175 I > < Combined Yield Individual Harvest 50-40-30-20-10-0 --w-- 2 0 -Legend • LxT - 3 0 - O RxLxT - 4 0 -- 3 0 -1 2 3 4 5 6 7 8 9 10 Combined Yield individual Harvest Individual Harvest 1 2 3 4 5 6 7 8 9 10 Individual Harvest Combined Yield Cumulative Harvest 4 5 6 7 8 Cumulative Harvest Combined Yield Cumulative Harvest .» — • --» e-Legend a r « l O R«T 2 3 4 5 6 7 8 9 10 Cumulative Harvest Figure 3-11. Advantage observed in combined forage growth when the y i e l d of homologous genotype combinations i s expressed as a percentage of the y i e l d observed in heterologous combinations (a) and (b) d e p i c t % y i e l d advantage a t each of 10 h a r v e s t s taken over 12 months; (c) and (d) d e p i c t c u m u l a t i v e y i e l d advantage a t each h a r v e s t . Symbols a r e e x p l a i n e d i n F i g u r e 3-2 176 Combined Shoot Weight 5000-1 4500-4000-3500-.E, 3000-"5> 2500 I >. 2000 o 1500-1000 500-0 RI R2 R3 p<0.1 c b f p p p p p p aaaaaasss ppppppppp fpppppppp aaaaaaas:? aaaaaassa Combined Root Weight 2500-1 R2 2000-JE, 1500 -I ^ 1000 -o 500-r-i RI 7 tpppppcp; aaaaaass: 7\ R3 a a a a 3 3ddj ^^j33j4 JPBR p<0.025 F i g u r e 3 -12 . E f f e c t of R. trifolii/L. perenne/T. repens genotype c o m b i n a t i o n s on combined f o r a g e y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=5. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t root w e i g h t . W=Rhi zobium, L=Lolium, T=Trifolium 177 6OOO-1 5000 4000 g> 3000 I Q 2000 1000 . E . 12000 10000 8000 "g> 6000 I Q 4000 2000-Combined Plant Weight R1 R2 R3 P<o.o25 gggiSSSSS Total Combined Biomass Rl R2 R3 P<o.o25 ggSSSSSSS £§£§££§£§ ££§£££5S§ F i g u r e 3 -13 . E f f e c t of R. trifolii/L. perenne/T. repens genotype c o m b i n a t i o n s on combined f o r a g e y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=5. (a) f i n a l h a r v e s t p l a n t weight (b) t o t a l accumulated b iomass . R=Rhizobium, h-Lolium, T=Trifolium 178 Trifolium Shoot Weight Trifolium Root Weight 2500-2000-•§> 1500 1000-500-R1 Lt L2 L 3 L T s 1 L2 L3 "ft, Ll L2 L3 I200-, WOO « S 100-J , "§> 600-I <^ 400 200 RI i R2 1 Ll L2 L3 Ll L2 L3 R3 Ll L2 L3 p<0.0l p<0.0l 3500-3000-2500-2000-1500-W00-Trifolium Plant R2 Weight Total Trifolium Biomass RI Ll R3 / / L2 L3 Ll L2 L3 Ll L2 L3 W000 9000 8000-7000-*— cn 6000-•& 5000-I 4000 3000 2000-W00-Rl Ll L2 L3 R2 Ll L2 L3 R3 ! L I L 2 L 3 p<0.005 p<0.005 F i g u r e 3-14 . E f f e c t of R. t r i f o l i i / L . perenne genotype combinat ions on the y i e l d of T. repens S t a t i s t i c s are d e s c r i b e d in F i g u r e 3 - 1 . n=l5 . (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t root weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . R=Rhi zobi um, L=Lolium 179 Lolium Shoot Weight Lolium Root Weight J , 600 I 200 Rl R2 R3 I LI L2 L3 LI L2 L3 LI L2 L3 J, 600 I 200-R1 R2 R3 LI L2 L3 LI L2 L3 LI L2 L3 N:S. p<0.01 Lolium Plant Weight Total Lolium Biomass 2000-1800-1600-1400 X 1200 •§> tooo £ _, >. 800-Q 600-400-200-0 R1 I R2 LI L2 L3 L1 L2 L3 LI L2 L3 R3 I 2300-•& 1500 I O 1000 L1L2L3 R2 LI L2L3 R3p^ LI L2 Li p<0.05 N.S. F i g u r e 3 -15 . E f f e c t of R. trifolii/L. perenne genotype c o m b i n a t i o n s on the y i e l d of L. perenne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=l5 . (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . R=Rhizobi um, L=Lolium 4000 -i 3500-3000-2500-"§) 2000-£ 1500 -1000-Combined Shoot Weight R2 R3 Combined Root Weight RI Ll L2.L3 I Ll L2L3 U L 2 L 3 "§> 1000 'I 500-R1 R2 R3 Ll L2 L3 U 12 L3 Ll L2 L3 p<0.005 p<0.005 Combined Plant Weight Total Combined Biomass 6000 5000-•& 3000 I Q 2000 -Rt 1 I Lt L2 L3 R2 Ll 12 L3 R3 Z Lt L2 L3 12000-1 10000 "S> 6000 a 4000 p<0.005 o-p<0.005 RI Ll 12 L3 R2 —f<^— Ll 12 L3 R3 1 J Ll L2 L3 F i g u r e 3-16 . E f f e c t of R. c o m b i n a t i o n s t ri fol i i /L. on combined perenne genotype forage y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=l5 . (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . R=Rhizobi um, L=Lolium (Figures 3-14; 2b and d, harvest 3). Lolium yields were less responsive (Figures 3-7 and 3-15), but f inal plant weight conformed to this trend (Figure 3~15c) (P < 0.05) mainly due to an unambiguous effect on root weight (Figure 3-15b) (P < 0.01). Because white clover dominated the mixture, the effect of homologous Rhi zobi um/Lol i um combinations was apparent in total accumulated forage biomass as well (Figures 3-11 to 3-16). There was a tendency for homologous R. trifolii/T. repens combinations to outyield the average heterologous partnerships when the legume yie ld was examined (Figures 3-2 and 3-17). These trends were rarely significant (P < 0.05), and decreased to minor proportions (eg. 2% - 7%) by the end of the experiment (Figures 3-2b and 3-2d). However, a highly significant 28% yield advantage (P < 0.01) was observed midway through the experiment (Figure 3-2b, harvest 6). Yields of L. perenne were not s ignif icantly affected by the homology between T. repens and ii!. trifolii (Figures 3-7 and 3-18), but there was a tendency to react negatively to such combinations. As a consequence of the small magnitude of the effect on white clover y ie ld , total forage biomass was only s l ight ly enhanced by the presence of homologous white clover//?, trifolii combinations (Figures 3-11 and 3-19). The ecological combining ab i l i ty of various genotype combinations was investigated by analyzing the quotient: minority component of the mixture (usually ryegrass) f majority component of the mixture (Figures 3-20 and 3-21). No advantage (indicated by a quotient approaching unity) was 182 2500 - 2000 J, "6l 1500 °S S >-£> (000 Trifolium Shoot Weight Rl R2 R3 Trifolium Root Weight I ab TI T2 T3 T? ab T2 T3 T1T2 T3 tooo BOO >§. 600 400-R1 R2 R3 TI T2 T3 TI T2 T3 TI T2 T3 N.S. N.S. 4000-3500-3000 2500 ,g> 2000 1500 -Trifolium Plant Weight Rl R2 R3 Total Tri folium Biomass •D TI T2 T3 TI TF 1U T3 Tt T2 T3 10000 9000 6000-7000-cn j i , 6000 5000 4000 3000 2000-1000 N.S. 0-N.S. Rl i TI T2 T3 R2 Ri .ah. TI T2 T3 TI T2 T3 F i g u r e 3 -17 . E f f e c t of R. trifolii/T. repens genotype c o m b i n a t i o n s on the y i e l d of T. repens S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=15. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t root weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . B.=Rhi zobi um, T=Tri f ol i um Lolium Shoot Weight Lolium Root Weight R1 R2 R3 Tt T2 T3 Tt T2 T3 Tt T2 T3 800 «oo N.S. 0-p<0.1 Rt R2 R3 31 Tt T2 T3 Tt T2 T3 Tt T2 T3 0-p<O.K> Lolium Plant Weight Total Lolium Biomass RI R2 R3 Tt T2 T3 Tt T2 T3 TI T2 T3 3000-1 2500 "6> 1500 I 1000 Rt Tt T2 T3 R2 TI T2 T3 R3 J TI T2 Ti N.S. F i g u r e 3-18 . E f f e c t of R. trifolii/T. repens genotype c o m b i n a t i o n s on the y i e l d of L. perenne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=15. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . R=Rhizobi um, T=Tri f olium 184 I 2500-"§> 2000-I £• BOO -o 1000-Combined Shoot Weight RI TI T2 T3 R2 TI T 2 T 3 R3 J TI T2 T3 2000 cn J . "Si 1000 I 500-N.S. 0-N.S. Combined Root Weight RI TI T2 T3 R2 R3 TI T2 T3 TI T2 T3 4000-Oi ,E, 3000 2000 Combined Plant Weight RI R2 R3 Total Combined Biomass 0-N.S. M T3 TI T 2 T 3 T I T ? T3 M>000-i 9000-8000 7000-6000 •§> 5000 ^ 4000-o 3000-2000 1000 RI TI T2 T3 TI T2 T3 R3 TI T2 T3 N.S. F i g u r e 3-19 . E f f e c t of R. trifolii/T. repens genotype c o m b i n a t i o n s on combined forage y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n= l5 . (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t root weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . R=Rhi zobi um, T=Trifolium Combining Ability Combining Ability Rl 1 LI L2 Li R2 1 LI L2 L3 R3 1 LI L2 L3 o £ 0.8 O O o 3 o a E o u .2" 0.2 *c o c a Rt ab . I i t R2 ab ab. R3 I TI T2 T3 TI T2 TS TI T2 TJ N.S. p<0.1 Combining Ability c F i g u r e 3 -20 . E f f e c t of genotype combinat ions on e c o l o g i c a l combin ing a b i l i t y between L. perenne and T. repens (a) R. trifolii/L. perenne genotypes ; (b) R. trifolii/T. repens genotypes; (c) L. per enne/T. repens g enotypes . n=15. S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . R=Rhizobium, L=Lolium T=Tri folium Combining Ability 1-1 0.8-R1 R2 R3 0.6-0.4 0.2-p<0.05 f »- « N W KMOtO ^ J , J J , j J .J .J HP KHt<E<f Bt< F i g u r e 3 -21 . E f f e c t of R. trifolii/L. perenne/T. repens genotype c o m b i n a t i o n s on e c o l o g i c a l combinin a b i l i t y between L. perenne and T. repens S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 - 1 . n=5 R=Rhi zobi um, L=Loli um T=Trifolium 187 observed when homologous combinations were tested. Indeed, when a l l three homologous combinations were grown together, the lowest values were obtained (Figure 3-21). The results of the R. trifolii strain competition experiments are presented in Figures 3-22 to 3-24. Mixed infections in these experiments were found to be rare, comprising 3.3% of the 1728 nodules examined. This finding is in agreement with previous estimates of the frequency of dual infections in Trifolium sp. (Brockwell et a l . , 1977; Bromfield and Jones, 1980) and was not altered by inoculation with Bacillus isolates. Most of the mixed infections (2.7%) contained R. trifolii isolates R2 and R3 while the combination of strains R1 and R2 formed the fewest dual infections (0.2%). Strains R1 and R3 were found in the remaining 0.4%. When mixed infections were detected, the presence of each microsymbiont was assigned to the treatment combination being evaluated. Only one s ta t i s t i ca l ly significant observation was apparent from these data. When white clover genotypes were grown in pure stand and inoculated with equal numbers of the three strains of R. t r i f o l i i , each plant harbored heterologous microsymbiont strains in preference to homologous ones (Figure 3-24a). This response was apparent when plants were grown in species mixture, but it was not s ta t i s t i ca l ly significant (Figure 3-22a). L. perenne genotypes had no effect on this phenomenon when data for a l l clover genotypes were pooled or examined separately (Figures 3-22 and 23). 188 Nodule Occupancy 2.5- R2 in o 3 O z E 3 0.5-R1 i T1 T2 T3 T1 T2 T3 R3 T1 T2 T3 N.S. 2.5-1 JK D O '-^  0) E ' z 0.5-N.S. Nodule Occupancy R2 RI 1 R3 L1 L2 L3 L1 L2 L3 L1 L2 L3 F i g u r e 3 -22 . E f f e c t of p l a n t genotypes on R. trifolii s t r a i n s e l e c t i o n by T. repens The yie ld variable is the mean number of root nodules per plant that contained the isolate of R. trifolii (R1-R3). (a) T. repens genotype effect; (b) L. perenne genotype effect. Stat is t ics are described in Figure 3-1. n=l8. R1, R2, and R3 are R. trifolii isolates 1, 2, and 3 f respectively. h=Lolium, T=Trifolium 189 N o d u l e O c c u p a n c y 2.5-i 03 13 TD O OJ 2-1.5 0.5 R1 0 R2 R3 N.S. C\J K\ T- f\| K> T- f\| K\ E-"HE-»tr<EHlHe-<l^  IX 5^ ^ ,) J ,4 j j ^ a a ass: '— r\j CNJ OJ WA KA «\ J ^ ,j J .-3 »J .3 . J F i g u r e 3 -23 . E f f e c t of L. perenne/T. repens genotype c o m b i n a t i o n s on R. trifolii s t r a i n s e l e c t i o n by T. repens The y i e l d variable i s the mean number of root nodules per plant that contained the isolate of R. trifolii (R1-R3). S t a t i s t i c s are described in Figure 3-1. n=l8. R1, R2, and R3 are R. trifolii i s o l a t e s 1, 2, and 3, respectively. L=Lolium, T=Trifolium 190 Nodule Occupancy 2 5 - R2 in -e t-3 Z 0.5-p<0.05 RI JZ2L TI T2 T3 J TI T2 T3 R3 3 I T1 T2 T3 Pure Stand Trifolium Yield RxT 50-a> cn _o c D > < >-40-30-20-10-- 2 0 -- 3 0 --40 --50 -Legend • Individual Harvests O Cumulative Harvests 1 2 3 4 5 6 7 8 9 10 Harvest Number Figure 3-24. Relationship between R. trifolii isolates and T. repens genotypes when grown without L. perenne (a) R. trifolii strain selection by T. repens genotypes (T1-T3); R1, R2, and R3 are the yie ld variables. (b) y ie ld advantage of T, repens when inoculated with homologous R. trifolii isolates and compared to performance with heterologous isolates. Stat is t ics and symbols are described in Figures 3-1, 3-2, and 3-22. n=6 191 To test the hypothesis that homologous R. trifolii/T. repens combinations underyield when compared with heterologous ones, pure stands of white clover were grown concurrently in the species mixture experiment and inoculated with single R. trifolii strains (Figure 3-24b). No such tendencies were apparent, especially at the beginning of the study which coincided with the length of the strain competition experiment. In fact, y ie ld advantages of 20% -25% were seen early in the study (Figure 3-24b, harvest 2) when the yie ld of T. repens inoculated with homologous R. trifolii was compared to that of legumes inoculated with heterologous bacterial isolates. The strain competition experiments were also designed to investigate the effect of homologous diazotropthic Bacillus isolates on plant y ie ld . Two of the three Bacillus isolates (B2 and B3) were identified as Bacillus pol ymyxa while the third (B1) reacted ambiguously in biochemical tests and was not positively identif ied. However, i t was very similar in overall behavior to B. pol ymyxa and was considered to be a B. polymyxa-like microorganism. Therefore, a l l f lats were inoculated with a single Bacillus sp. isolate, similar to Bacillus polymyxa, in addition to a composite inoculum of root nodule bacteria which consisted of a l l three R. trifolii strains used in the previous experiment (Figures 3-1 to 3-24) . Analysis of the white clover component of these mixtures revealed l i t t l e effect of Bacillus inoculation on plant performance. Homologous Bacillus/T. repens or Bacillus/T. r e pe ns/L. perenne combinations showed no yield advantage when compared with the average performance of heterologous combinations (Figures 3-25 to 3-27). Again, the specific interaction between white clover and perennial ryegrass genotypes noted by Aarssen and Turkington (1985) was not evident (Figure 3-28). However, when clover root weight and total clover accumulated biomass were measured, a significant yie ld advantage was seen in the average performance of homologous Bacillus/L. perenne combinations compared with heterologous ones (Figure 3-29). Root weight was 32% higher (P < 0.05) and total clover biomass was increased by 16% (P < 0.05) mainly due to the large response of the B2L2 combination (Figure 3-29d). Root nodule weight of white clover plants was also found to be highest when homologous Bacillus/ perennial ryegrass combinations were present (Figure 3-30a). However, this yie ld parameter showed a further increase when the homologous white clover genotype was included in a three-way combination (Figure 3-31). No significant trends in nodule weight were observed when homologous Bacillus/T. repens or Lolium/T, repens partnerships were tested (Figure 3-30b and c) . Analysis of the L. perenne component of the mixture revealed similar trends to those seen in white clover, but of greater magnitude (Figures 3-32 to 3-34). Bacillus/T. repens or L. perenne/T. repens homologous combinations conferred no yield advantage to perennial ryegrass (Figures 3-33 and 3-34), but a l l ryegrass parameters measured were Trifolium Shoot Weight Trifolium Root Weight 1000-200-0 J N.S. Bl B3 B2 I TI T2 T3 Tt T2 TS TI T2 T3 J , 300 5. I 0-N.S. Bl B2 B3 »b »b 1 All I TI T2 T3 TI T2 T3 Tt T2 T3 Trifolium Plant Weight Total Trifolium Biomass Bl B2 T1 T2 T3 TI T2 T3 TI T2 T3 Co N.S. 0-N.S. Bl B2 B3 TI T2 T3 TI T2 T3 Tt T2 T3 Figure 3-25. Effect of B. polymyxa/T. repens genotype combinations on the yield of T. repens The probability level (bottom left) refers to the contrast: homologous genotype combinations (hatched bars) outyield heterologous combinations (open bars). B=Baci11 us , T=7> / fol i um. N .S . - not significant. Individual means assigned no letter or the same letter are not s ignif icantly different (Tukey's test; P < 0.05). n=l8. (a) f inal harvest shoot weight (b) f inal harvest root weight (c) f inal harvest plant weight (d) total accumulated biomass 194 Trifolium Shoot Weight o> E 1200 n 1000-B00 "§) 600 I O 400-200 Bl B2 n B3 N.S. 500 400 Cn 300 a 200-100-ggsssssss ggsssssss sssssssss Trifolium Root Weight B1 B3 B2 r N.S. F i g u r e 3-26 . E f f e c t of B. pol ymyxa/L. perenne/T. repens genotype combinat ions on the y i e l d of T. repens S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 -25 . n=6. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t w e i g h t . B-Bacillus, L=Lolium, T-Trifolium. 195 1200 1000 800-"Cft .600 I >-Q 400-200-Trifolium Plant Weight B1 B2 B3 N.S. §!§§§S53s §§§§££§g§ Total Trifolium Biomass 2000-1 1500 CD *§> 1000 I >» »_ o 500-B1 B2 B3 N.S. P B F P P P P C P C B P P R P p p p P B P P P P C P P 5 5 5 3 3 3 3 3 3 5 5 5 3 3 3 3 3 3 5 3 5 3 3 3 3 3 3 F i g u r e 3 -27 . E f f e c t of B. polymyxa/L. perenne/T. repens genotype combina t ions on the y i e l d of T. repens S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 -25 . n=6. (a) f i n a l h a r v e s t p l a n t weight (b) t o t a l accumulated b iomass . B=Bacillus, L=Lolium, T=Trifoli um. 196 Trifolium Shoot Weight Tri folium Root Weight I 2 0 0 -12 Lt L5 TI T2 T3 TI T2 T3 TI T2 T3 250 200 » § , ISO-wo SO-LI L2 L3 I 1 TI T2 T3 TI T2 T3 TI T2 T3 N .S . p<0.1 ,E, «oo-0 -N.S. Trifolium Plant Weight Total Trifolium Biomass L2 I L3 TI T2 T3 TI T2 T3 TI T2 T3 1500 500 0 -N .S . LI L2 I L3 TI T2 T3 TI T2 T3 TI T2 T3 F i g u r e 3-28. E f f e c t of L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of T. repens S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 -25 . n=24. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t r o o t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . h=Lolium, T=Tr i f ol i um. 197 Trifolium Shoot Weight Trifolium Root Weight 800-.£ , «00-1 200-0-N.S. Bl B2 B3 Ll L2 L3 L1 L2 L3 Ll L2 L3 cn J . 300 t o Bl B2 B3 Ll L2 L3 Ll L2 L3 Lt L2 L3 1 p<0.05 Trifolium Plant Weight Total Trifolium Biomass 800-C7> £ 800-I o B1 B2 B3 L1 L2 L3 Ll L2 L3 Lt L2 L3 1500 - ~ - WOO •6. i N.S. 0-p<0.05 Bl B2 B3 Ll L2 L3 Lt L2 L3 Lt L2 L3 F i g u r e 3 -29 . E f f e c t of B. pol ymyxa/L. perenne genotype c o m b i n a t i o n s on the y i e l d of T. repens S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25 . n=l8 . (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t root weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . B=Bacillus, li-Lol i um 198 Nodule Weight 2-I 0.5-B l 62 I B3 LI L2 L3 LI L2 L3 LI L2 L3 p<0.01 2.5 cn J . I 0.5-0 J N.S. Nodule Weight T2 I Bl B2 B3 81 B2 B3 Bl B2 B3 T3 I 2-Nodule Weight TI T2 is-LI L2 L3 LI L2 L3 LI L2 L3 N.S. F i g u r e 3 -30 . E f f e c t of genotype combinat ions on roo t nodule weight of T. repens S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25. (a) B. pol ymyxa/L. perenne (BxL) genotype c o m b i n a t i o n s , n=l8; (b) B. polymyxas/T. repens (BxT) genotype c o m b i n a t i o n s , n=l8; (c) L. perenne/ T. repens (LxT) genotype c o m b i n a t i o n s , n=24 Nodule Weight 199 T2 T3 Ic CJ) 3-2-T1 m S3 oa CQ £4 P3 m 81 IP 5 5 S DI DI Di 132 2 to M m n n n n n n _) J _1 J J J J J J S ^ S DI DI DI322 p<0.05 F i g u r e 3 -31 . E f f e c t of B. pol ymyxa/L. perenne/T. repens genotype combinat ions on r o o t nodule weight of T. repens S t a t i s t i c s are d e s c r i b e d in F i g u r e 3-25 . n=6. B=Bacillus, h=Loli um, T=Trifolium Lolium Shoot Weight 1500 I r O 500 LI 0-p<0.0l L2 LJ Bl B2 B3 Bl B2 83 Bl B2 B3 vi, soo Lolium Root Weight L2 1 L3 Bl B2 B3 Bl B2 B3 Bl B2 B3 p<0.05 Lolium Plant Weight Total Lolium Biomass 2000 cn J . "Si 1000 I 300-L3 2500 L 2 p<0.005 Bl B2 B3 Bl B2 B3 Bl B2 B3 cn .E, 1500 LI L2 1 L3 B 1 B 2 B 3 Bl B2 B3 1 Bl 82 B3 p<0.005 F i g u r e 3-32. E f f e c t of B. pol ymyxa/L. perenne genotype combinat ions on the y i e l d of L. perenne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25. n=l8 . (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . B=Bacillus, L=Lolium 201 uua-1000-(mg) 800-(mg) fc 600-Dry 400-200-Lolium Shoot Weight ^ Bl B2 B3 CD ,£, 300-I » 2C O Lolium Root Weight 61 B2 B3 TI T2 T3 TI T2 T3 TI T2 T3 TI T2 T3 TI T2 T3 TI T2 T3 N.S. N.S. Lolium Plant Weight 2000-1 cn J. fc 1000-I 300 0-N.S. 61 62 63 TI T2 T3 TI T2 T3 TI T2 T3 fc woo I a Total Lolium Biomass B3 ^ 8 1 B2 TI T2 T3 TI T2 T3 TI T2 T3 N.S. F i g u r e 3-33 . E f f e c t of B. pol ymyxa/T. repens genotype combinat ions on the y i e l d of L. perenne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25 . n=l8 . (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . B=Bacillus, T=Trifoli um 202 1200-•&> 6 0 0 -I £ 4 0 0 -2 0 0 -Lolium Shoot Weight Ll L2 | | L3 J00 a 200 Lolium Root Weight Ll L3 I L2 TI T2 T3 TI T2 T3 TI T2 T3 TI T2 T3 TI 12 T3 TI T2 T3 N . S . N.S . Lolium Plant Weight Total Lolium Biomass 1500-O l J , • f i l 1000-§ 500-0 -N.S. L2 L3 TI T2 T3 TI T2 T3 TI T2 T3 2500 2000-,E, 1500 I, 500 0 -N.S. I TI T2 T3 L2 TI T2 T3 L3 TI T2 13 F i g u r e 3-34. E f f e c t of L. per enne/T. repens genotype combinat ions on the y i e l d of L. perenne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25. n=24. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . L=Lolium, T=Trifol i um significantly higher when the Bacillus isolate matched the Lolium genotype (Figure 3-32). Root weight was the most responsive as a 46% increase was noted (P < 0.05). Shoot (20%), plant (27%), and total perennial ryegrass biomass (25%) were also substantially increased (P < 0.01). When a l l three organisms were homologous the average yield of perennial ryegrass was signif icantly higher than the mean of heterologous three-way combinations (Figures 3-35 and 3-36), but the association of B2L2T2 did not conform to this trend. In these experiments, where soi l nitrogen was probably not l imit ing due to the relat ively short growth period, L. perenne dominated white clover in terms of dry matter y ie ld . Therefore, combined forage yield showed similar trends to those observed when the grass component of the mixture was analyzed (Figures 3-37 to 3-41). Homologous Bacillus/T. repens or L. perenne/T. repens combinations did not outyield heterologous ones (Figures 3-37 and 3-38), but shoot (16%), root (42%), plant (22%) and total biomass (21%) weights were highest when the Bacillus isolate and the L. perenne genotype were homologous (P < 0.05). When a l l three organisms were related, significant yie ld increases were observed in root, plant, and total biomass weights (Figures 3-40 and 3-41), but these specific combinations were not always the highest. No s ta t i s t i ca l ly significant results were observed when the effect of Bacillus isolates on R. trifolii strain occupancy of clover root nodules was analyzed (Figures 3-42 and 3-43). However, certain trends were noted and are of potential importance to these y ie ld relationships. F i r s t , 204 Lolium Shoot Weight 1500-1250-O 1000-J , "B> 750-I >» Q 500-250-B1 B2 B3 P P P C P P P P P S C P P P C P c c P R P P P P P P E 5 5 5 3 3 3 3 3 3 assaaasss 5 5 5 3 3 3 3 3 3 p<0 .05 800-Lolium Root Weight 700-600-|> 500-"S> 400-Q 200-100-B1 B2 B3 PI / / / * * 4 p < 0 . 0 5 P P P P P P P B P p c p p p p c p p p p p p p p c p p 3 3 5 3 3 3 2 3 3 3 5 5 3 3 3 3 3 3 5 5 5 3 3 3 3 3 3 F i g u r e 3 -35 . E f f e c t of B. polymyxa/L. perenne/T. repens genotype combinat ions on the y i e l d of L. pe r e nne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25 . n=6. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t r o o t w e i g h t . B=Baci11 us , L=Lolium, T=Trifolium 205 Lolium Plant Weight 2000- B1 B2 B3 1500-g> looo-I o 500-aaaaaa:;:;: fcPPPPP aaaaaa pee §§§§§ 3 ~ i p<0.05 Total Lolium Biomass 2500-2000-rj) ^E, 1500-cn 'I 1000-500-81 B2 B3 H ' / / / / / / / / / / / / / / / ppcrcpt a aaass: S P P P P E aaaaaa: :0S CPPPPP aaaaaa: p<0.05 F i g u r e 3-36 . E f f e c t of B. polymyxa/L. perenne/T. repens genotype c o m b i n a t i o n s on the y i e l d of L. perenne S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3 -25 . n=6. (a) f i n a l h a r v e s t p l a n t weight (b) t o t a l accumulated b iomass . B=Bacillus, L=Lolium, T=Trifolium 206 2000 -i 1 lb WOO-i lis 500-0-N.S. Combined Shoot Weight Bl B2 B3 Combined Root Weight TI T2 T3 TI T2 T3 TI T2 T3 WOO-i oi , £ , 600 I ->. «oo-200 61 B2 B3 TI T2 T3 TI T2 T3 TI T2 T3 N.S. 2500--3 2 0 0 0 -J. •§> 1500 -I £ woo-Combined Plant Weight Total Combined Biomass Bl TI T2 T3 B2 TI T2 T3 B3 TI T2 T3 J500- , 2500-2000-oi J. Ii S 1500 Bl TI T2 T3 B2 TI T2 T3 B3 1 TI T2 T3 N.S. N.S. F i g u r e 3-37. E f f e c t of B. polymyxa/T. repens genotype combinations on combined forage y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25. n=l8. (a) f i n a l harvest shoot weight (b) f i n a l harvest root weight (c) f i n a l harvest p l a n t weight (d) t o t a l accumulated biomass. B=Baci1I us , T=7>if oli um 207 Combined Shoot Weight Combined Root Weight 2000 1800-teoo MOO J ! 1200-1 "§> 1000 I 4 >. MO-600-400 200 0 Lt 12 Tt T2 T3 L3 TI T2 T 3 T t T2 T3 800 J ! 600 I >. 400 200 Lt ab L2 L3 1 TI T2 T3 TI T2 T3 TI T2 Ti N.S. N.S. Combined Plant Weight 2300-1 2000 .fc, 1500-I J «oooH 500 L2 L3 TI T2 T3 Tt T2 T3 Tt T2 T3 SOOO-i 2500 2000 •& BOO-<^  woo Total Combined Biomass r j ] Lt | , L2 L3 TI T2 T3 TI T2 T3 TI T2 T3 N.S. N .S. Figure 3-38. Effect of L. perenne/T. repens genotype combinations on combined forage y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25 . n=24. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t r o o t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . h=Lolium, T=Trifolium 208 2000-0 1 1230-"6> woo-I £ 7 5 °-250-Combined Shoot Weight L2 L I r73 L3 \7\ 1 Bl B2 63 BIB2B3 Bl B2 BJ J . 600 I 400 C o Combined Root Weight L3 iftl L2 Bl B2 B3 Bl B2 BJ Bl B2 BJ p<0.05 p<0.005 Combined Plant Weight 3 0 0 0 -2500-L1 Bl B2 63 L2 Bl B2 B3 L3 I J B1.B2 B3 350O-. 3000-2500 cn E W 2000 * 1500 Total Combined Ll L2 Biomass L3 Bl 62 63 Bl 82 BJ Bl 62 BJ p<0.01 p<0.005 F i g u r e 3-39 . E f f e c t of B. polymyxa/L. perenne genotype c o m b i n a t i o n s on combined forage y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25 . n=18. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t weight (c) f i n a l h a r v e s t p l a n t weight (d) t o t a l accumulated b iomass . B=Baci11 us, L=Lolium 209 Combined Shoot Weight 2500 -i 2000-B2 CD E cn I O 1500-1000-500-B1 B3 PI rr' / / / / / / / / / / / / / / aa- P P P H C E 3 3 3 332 PBEf. a aa a BPS PP 33333 r.p; N.S. Combined Root Weight 1000-800-83 v§, 600-JZ cn I >-o 400-200-B1 B2 p<0.05 P B P P P P P C ; aaa33333" P P P C P P P P E P P P P P C P B P aaaaaa333 aaaaaa335 F i g u r e 3 -40 . E f f e c t of B. pol ymyxa/L. perenne/T. repens genotype combinat ions on combined forage y i e l d S t a t i s t i c s are d e s c r i b e d in F i g u r e 3 -25 . n=6. (a) f i n a l h a r v e s t shoot weight (b) f i n a l h a r v e s t roo t w e i g h t . B=Bacillus, h=Lolium, T=Trifoli um 210 Combined Plant Weight .E. 3000-2500-2000-B1 D> 1500-I O 1000-500-0-p<0.1 B2 r f / / / / 8 3 n / / P C P C R P P C P 5 5 5 3 3 3 3 3 3 KSPPBPfcP 5 5 5 3 3 3 3 3 3 {.PPPCPPPP 5 5 5 3 3 3 3 3 3 Total Combined Biomass 4000 3500 3000 |* 2500-D) 2000 I 1500 O 1000-500 B1 B2 r f / B3 p<0.05 3 3 3 3 3 3 555§SS§S§ 5 5 5 3 3 3 »4 -> Figure 3-41. Effect of .5. pol ymyxa/L. per enne/T. repens genotype combinations on combined forage y i e l d S t a t i s t i c s are d e s c r i b e d i n F i g u r e 3-25. n=6. (a) f i n a l h arvest p l a n t weight (b) t o t a l accumulated biomass. B=Bacillus, L=Lolium, T-Trifolium 211 18-16-14-12-0.8-0.6-0.4-0.2-0-N.S. Nodule Occupancy R2 1 R3 Bl B2 B3 Bl B2 BJ Bt B2 BJ l l il H V z "5 & o. 0. 0. 0.2-Nodule Occupancy R2 Rt I R3 N.S. Ll L2 L3 Ll L2 L3 Ll L2 L3 1.1 Ll « I 1 t. z "5 k. f 0 2 o, 0. 0. 0-N.S. Nodule Occupancy R2 RI I TI T2 T3 TI T2 T3 R3 TI T2 T3 F i g u r e 3-42 . E f f e c t of p l a n t and m i c r o o r g a n i s m genotypes on R. trifolii s t r a i n s e l e c t i o n by T. repens S t a t i s t i c s and symbols are d e s c r i b e d i n F i g u r e s 3-22 and 3-25. (a) B. polymyxa genotype e f f e c t , n=54; (b) L. perenne genotype e f f e c t , n=72; (c) T. repens genotype e f f e c t , n=72. R1, R2, and R3 are R. trifolii i s o l a t e s 1, 2, and 3, r e s p e c t i v e l y . B=Baci11 us, L=Lolium, T^Tri folium .212 Nodule Occupancy 2.5 -i R2 O -Q E 1.5 1-0.5-R1 N.S. 3 2i S S S S S 3 S en ro tn m w w 3 3 S 3 ! J J 312 R3 »- OJ KA r- OJ KA «— OJ KN J J ^ J J J J J >-l I KA KA KA F i g u r e 3-43. E f f e c t of B. pol ymyxa/L. perenne genotype combinat ions on R. trifolii s t r a i n s e l e c t i o n by T. repens S t a t i s t i c s and symbols are d e s c r i b e d i n F i g u r e s 3-22 and 3-25 . n=6. R1 , R2, and R3 are R. trifolii i s o l a t e s 1, 2, and 3, r e s p e c t i v e l y . B=Bacillusf L-Lol i um 2D the repulsion of homologous strains by white clover genotypes noted in the treatments without Bacillus was evident when these isolates were included in the experiments. On average, clover genotypes harbored nonhomologous R. trifolii strains in preference to homologous ones (Figure 3-42c). Second, L. perenne genotypes had l i t t l e effect on strain selection (Figure 3-42b). While microsymbionts homologous to perennial ryegrass genotypes L2 and L3 were selected most often, those homologous to L1 were found least often when clones of the appropriate grass genotype was present. In a l l cases, the differences between treatments was small. Third, in a l l three cases R. trifolii formed most of the root nodules when the homologous Bacillus isolate/perennial ryegrass genotype combination was present (Figure 3-43). However, this effect was equalled in magnitude when the Lolium component was ignored and individual Bacillus effects were tested (Figure 3-42a). In the latter case, R. trifolii strain 1 formed 32% more of the nodules when Bacillus isolate 1 was present. Similarly, R. trifolii strains 2 and 3 formed 9% and 15% more nodules respectively, when their matching Bacillus isolates were present. Other specific interactions (eg. Bacillus/T. repens or Bacillus/T. repens/L. perenne) did not affect the selection of homologous R. trifolii strains by genotypes of white clover. 214 DISCUSSION The data presented in this chapter support the hypothesis that root-associated microorganisms are important in genotype specif ic i ty between white clover and perennial ryegrass (Aarssen and Turkington, 1985). L i t t l e evidence of this compatibility was apparent when the effect of R. trifolii strains was ignored or when only heterologous R. trifolii isolates were tested. However, when homologous R. trifolii was used to inoculate matched T. repens/L. perenne genotypes, significant yield advantages were observed. It is of interest that in the present study genotype specifity betweem L. per enne/T. repens was not reflected in white clover yields when the influence of homologous R. trifolii isolates was disregarded. There are at least two possible reasons why the "Aarssen effect" was not detected under those conditions. The f i r s t is related to recent experiments which have revealed that certain genotype-specific white clover characteristics are not stable when plants are removed from their natural habitat and are stored in a common garden (eg. a greenhouse) (Evans, 1986). If the genotype specif ic i ty between white clover and perennial ryegrass is phenotypically p las t ic , then plants may lose the characteristics which allow this phenomenon to occur as time passes since the material was collected. Plant material used in these experiments had been propagated in the greenhouse for c irca 15 months before use; therefore, these clones may have lost some or a l l of the attributes necessary to show this fine-scale biotic special ization. Such attributes could include the effect of naturally-occurring root-associated microbes that have adapted to the plant genotypes. A second explanation assumes that root-associated microbes are central to observed genotype specif ic i ty between pasture plants. In the experiments described in this thesis, so i l was autoclaved before use and clover stolon tips or ryegrass t i l l e r s were immersed f irs t in 80% ethanol and then in s teri le water before planting. Immediately after planting, a heavy inoculum of the appropriate R. trifolii strain was administered followed by a s imil iar inoculation three months later. Use of these procedures ensured that root nodules were formed by the "appropriate" strain of R. t r i f o l i i . This intention was confirmed with ELISA analysis of the root nodule microsymbionts at the conclusion of the one year growth period. In contrast, Aarssen and Turkington (1985) did not attempt to control any aspect of the so i l microbiology and planted clones direct ly into standard potting mix without Rhizobium inoculation. It is l ike ly that the grass t i l l e r s used in their experiments were carrying so i l microbes, possibly Rhi zobi um, on the root or crown tissue. The grass plants may therefore have provided neighbouring clover ramets with a source of bacterial inoculum, possibly R. t r i f o l i i , which had adapted to the grass genotype. S imi l iar ly , the potting mix may have contained sufficient microbial inoculum for effective strains to form root nodules. The disparity in findings between the results of Aarssen and Turkington (1985) and the ones presently described may, therefore, result from the fact that R. trifolii was "forced" on plants in the latter case while in the former study R. trifolii may have either been transferred with plant tissue or have been present in some diversity in the potting s o i l . Results from another recently completed longterm experiment support the idea that genotype specif ic i ty between white clover and associated grass species disappears when the natural so i l microflora are manipulated (J. Thompson, University of Liverpool, personal communication). In this study, which was simil iar to that of Turkington and Harper (1979), white clover genotypes did not show biotic specialization when the response to different species of neighbouring grasses was tested. Plants in these experiments were also surface s ter i l i zed with ethanol and grown in autoclaved s o i l . However, differences in application of treatments, experimental design, and management not withstanding, the magnitude of the growth response in the experiment of Aarssen and Turkington (1985) and the present work is remarkably s imi l iar . When total white clover biomass accumulation was measured, Aarssen and Turkington (1985) found that homologous combinations outyielded heterologous ones by 49%. In the present work, a 27% yield advantage was seen when clover was grown with homologous L. perenne/R. trifolii combinations, but a 47% yield advantage was seen midway through the experiment. Clover root weight also showed a 50% yield advantage under these conditions. Because the L. perenne/T. repens genotype interaction c o n t r i b u t e d o n l y a s m a l l p o r t i o n of t h e R. t r i f o l i i / L . perenne/T. repens y i e l d a d v a n t a g e , t h e e f f e c t s o f t h e o t h e r two-way i n t e r a c t i o n s were s t u d i e d . The y i e l d a d v a n t a g e c o n f e r r e d t o w h i t e c l o v e r when i n o c u l a t e d w i t h i t s homologous m i c r o s y m b i o n t was s i g n i f i c a n t a t c e r t a i n t i m e s of t h e y e a r , but was s m a l l (7%) and n o t s t a t i s t i c a l l y s i g n i f i c a n t when t o t a l w h i t e c l o v e r b i o m a s s was a n a l y z e d . T h i s a n a l y s i s s u g g e s t s t h a t t h e s p e c i f i c c o m b i n a t i o n o f homologous w h i t e c l o v e r g e n o t y p e s and R. t r i f o l i i i s o l a t e s c o u l d be i m p o r t a n t i n o v e r a l l p r o d u c t i v i t y b u t t h a t i t does n o t r e p r e s e n t a l a r g e component of t h e t h r e e - w a y i n t e r a c t i o n . F u r t h e r d o u b t i s c a s t on t h e s i g n i f i c a n c e o f t h e R. t r i f o l i i / T . repens homologous i n t e r a c t i o n when t h e u n e x p e c t e d r e s u l t s o f t h e s t r a i n s e l e c t i o n e x p e r i m e n t a r e e v a l u a t e d . I t was i n i t i a l l y h y p o t h e s i z e d t h a t homologous w h i t e c l o v e r a nd R. t r i f o l i i g e n o t y p e s would " f i n d e a c h o t h e r " when t h e Rhizobium i s o l a t e s were i n c o m p e t i t i o n w i t h o t h e r m i c r o s y m b i o n t s t r a i n s , b u t l i t t l e e v i d e n c e o f t h i s was d i s c o v e r e d . In f a c t , when t h e i n f l u e n c e o f c l o v e r g e n o t y p e s on R. t r i f o l i i s t r a i n s e l e c t i o n was examined, t h e major t r e n d was f o r t h e e x a c t o p p o s i t e t o o c c u r . W h i t e c l o v e r p l a n t s t e n d e d t o h a r b o u r h e t e r o l o g o u s , n ot homologous, m i c r o s y m b i o n t s i n t h e i r r o o t n o d u l e s . Lolium g e n o t y p e s g e n e r a l l y d i d not a f f e c t t h i s p r o c e s s but R. t r i f o l i i s t r a i n R1 formed more r o o t n o d u l e s when t h e s p e c i f i c c o m b i n a t i o n o f L1T1 ( p e r e n n i a l r y e g r a s s and w h i t e c l o v e r g e n o t y p e s 1) was p r e s e n t . T h i s was t h e o n l y o b s e r v a t i o n w h i c h s u g g e s t e d t h a t c l o v e r g e n o t y p e s c o u l d contribute to the selection of homologous R. trifolii strains by white clover. However, when yields of homologous R. trifolii/L. perenne combinations were analyzed, a 24% (P<0.005) increase in total white clover biomass accumulation was discovered. The magnitude of this effect compares favorably with that seen when homologous R. trifolii/L. per enne/T. repens combinations were tested and accounts for almost a l l of the 27% yield advantage observed in the latter case. Similar trends were evident in f inal shoot, root, and plant y ie ld of T. r epens . Therefore, regardless of the white clover genotype, homologous R. trifolii/L. perenne combinations were the major determinants in genotype specif ic i ty between the pasture plants in this experiment. This effect is further i l lustrated by comparing white clover genotype yields within homologous R. trifolii/L. perenne combinations. Final plant weight for the yields of the three clover genotypes in each of the homologous Rhi zobi um/qrass combinations revealed that the homologous white clover genotype was the highest yielding clone in only one case (R1L1T1), while i t was intermediate (R2L2T2) or lowest (R3L3T3) in the other two cases. When total white clover biomass was measured, homologous white clover genotypes yielded highest in two of the three cases (R1L1T1 and R2L2T2) but yields of the other genotypes were not s ignif icantly or substantially different (compare R2L2T1 with R2L2T2 in Figure 3-4a). In the case of R3L3, the homologous white clover genotype was intermediate in yie ld compared with the other two clover genotypes. Aarssen and Turkington (1985) found that L. perenne yields tended to be lower when grown with homologous white clover genotypes. In the present set of experiments, L. perenne y ie ld was shown to be s ta t i s t i ca l ly unaffected by the presence of clover neighbours but tended to show a general negative trend. Because perennial ryegrass dominated white clover in the experiments of Aarssen and Turkington (1985), combined forage yield was not positively affected by this genotype speci f ic i ty . These authors also showed that the ecological combining a b i l i t y of homologous plant genotype combinations was higher than that of heterologous ones (ie. grass - clover quotients were highest in the homologous combinations). This relationship occurred because the yie ld of the dominant component (perennial ryegrass) decreased in these partnerships while the minimum component (white clover) showed corresponding increased yie lds . However, in the data from this thesis, white clover dominated the grass component in species mixture. Therefore, the combined forage yield did show a positive y ie ld response when homologous R x L x T combinations were tested. When ecological combining ab i l i ty was calculated, homologous R x L x T combinations were lowest, in contrast to the findings of Aarssen and Turkington (1985). The reason for the discrepant trends in ecological combining a b i l i t y may be explained by the reversal of dominance between the two species when grown in mixture. In 220 both exper iments (Aarssen and T u r k i n g t o n , 1985, and the p r e s e n t work) , homologous genotype combinat ions r e s u l t e d i n white c l o v e r y i e l d i n c r e a s e s but c o n c u r r e n t d e c r e a s e s in L. perenne dry we ight . However, i n t h i s t h e s i s , the minimal component ( r y e g r a s s ) decreased i n y i e l d whi le the dominant component (white c l o v e r ) i n c r e a s e d , t h e r e f o r e homologous q u o t i e n t s (minimium component - maximum component) were s m a l l e s t , not l a r g e s t (P<0.05) . T h i s f i n d i n g c a s t s doubt on the c o n c l u s i o n of Aarssen and T u r k i n g t o n (1985) t h a t p l a n t s whose genotypes are matched show enhanced e c o l o g i c a l combining a b i l i t y . An a l t e r n a t e e x p l a n a t i o n i s t h a t T. repens r e a c t s p o s i t i v e l y wh i l e L. perenne r e a c t s n e g a t i v e l y in these s p e c i f i c combinat ions r e g a r d l e s s of which component dominates the m i x t u r e . R e s u l t s from exper iments d e s i g n e d to examine the e f f e c t of homologous Bacillus i s o l a t e s on p l a n t performance showed s i m i l a r i t i e s to the R. trifolii s t u d y . A g a i n , the L. perenne/T. repens genotype s p e c i f i c i t y was not a p p a r e n t . When homologous Bacillus i s o l a t e s were used as inocu lum, L2T2 and L3T3 c o m b i n a t i o n s tended to have h igher white c l o v e r y i e l d s but these d i f f e r e n c e s were not s t a t i s t i c a l l y s i g n i f i c a n t . However, L. perenne d i d show s i g n i f i c a n t g a i n s in y i e l d when a l l the organims were homologous, but the c o m b i n a t i o n B2L2T2 was not r e s p o n s i v e . When a l l p o s s i b l e two-way i n t e r a c t i o n s were examined, i t was d i s c o v e r e d tha t the Bacillus i s o l a t e x L. perenne genotype a s s o c i a t i o n was the most i m p o r t a n t . No c o n s i s t e n t t r e n d s i n the y i e l d of e i t h e r m i x t u r e component were seen 221 when homologous Bacillus/T. repens or L. perenne/T. repens combinations were analyzed. However, homologous grass/bacteria combinations resulted in superior white clover root, root nodule, and total accumulated biomass weight. Similarly, a l l L. perenne parameters were signif icantly and substantially increased when homologous combinations of the latter type were studied. Because the Bacillus/L. perenne/T. repens combination contained one association (B2L2T2) that reacted negatively when yields were compared with heterologous associations, the average yie ld advantage was only 18%. In contrast, a l l homologous Bacillus x Lolium combinations, including B2L2, showed yield increases. The average homologous Baci 11 us/Loli um combination yielded 25% more than the mean of the heterologous partnerships, regardless of the white clover genotype. Therefore, it was concluded that the specific grass/bacteria partnership again was the most important component of the three-way bacteria/ryegrass/clover association. Since L. perenne dominated the mixture in these experiments and T. repens tended to react positively to homologous Bacillus/L. perenne combinations, combined forage yie ld of the two species was also enhanced by this interaction. The grass/bacteria relationship contributed to nonsignificant but potentially important trends for homologous strains of R. trifolii to form more clover root nodules regardless of the white clover genotype. Further examination of the data revealed that i t was the Bacillus 222 component which specif ical ly contributed to this effect. Therefore, the grass may be important as a source of inoculum of B. polymyxa-like organisms and may contribute in this way to the numerous effects attributed to this specific plant/microoganism interaction. The only s ta t i s t i ca l ly significant trend in the strain competition experiments was for white clover genotypes to select heterologous microsymbiont strains when grown in pure stands. This repulsion was not correlated to a negative yield response as homologous plant/microbe combinations yielded up to 25% more than heterologous ones (P<0.05). Mytton ( 1975) also found that homologous white clover/i?. trifolii combinations outyielded heterologous ones by 25%. While infect ivi ty and effect iv i ty can be correlated (Robinson, 1969; Masterson and Sherwood, 1974), i t has been shown that the many competitive strains do not necesarily form the most effective symbioses (Vincent and Waters, 1953; Mytton and De Fel ice , 1977). The present experimental evidence supports the contention that these two symbiotic characteristics are unrelated as strains repulsed by white clover genotypes did result in y ie ld increases when tested as single strain inocula with homologous white clover genotypes. The transient and variable nature of lect in binding sites (Dazzo and Hubbell, 1982) may contribute to the observed strain selection effects because cultures in the stationary phase of growth were used exclusively as inoculum. It is possible that under different environmental conditions and with cultures of different ages a strong selection effect 223 f o r homologous s t r a i n s may have been observed. These r e s u l t s do i n d i c a t e , however, that genotype communication between p l a n t root and bacterium does occur s i n c e homologous s t r a i n s were o b v i o u s l y d i s c r i m i n a t e d a g a i n s t in root nodule format i o n . C l e a r l y , the common component i n a l l of t h i s work i s the i n f l u e n c e of the g r a s s / b a c t e r i a i n t e r a c t i o n on the performance of both forage s p e c i e s when grown i n mixture. In work with R. trifolii or Bacillus i s o l a t e s , the g r e a t e s t e f f e c t on y i e l d was observed when the L. perenne genotype was homologous to the b a c t e r i a l s t r a i n t e s t e d . I t i s p a r t i c u l a r l y i n t e r e s t i n g to see that two organisms ( p e r e n n i a l ryegrass and b a c t e r i a ) can combine to a f f e c t the performance of a t h i r d (white c l o v e r ) . T h i s i n t e r a c t i o n i s e x e m p l i f i e d by the y i e l d of white c l o v e r when grown with homologous L. perenne/R. trifolii genotypes. S i m i l a r l y , the presence of homologous p e r e n n i a l ryegrass/Baci11 us sp. combinations r e s u l t e d i n s u p e r i o r c l o v e r r o o t , root nodule, and t o t a l biomass weights. Normally, t h i s kind of i n t e r a c t i o n may be a n t i c i p a t e d to i n v o l v e a Rhizobi um/legume combination p o s i t i v e l y a f f e c t i n g the subsequent growth of a nonlegume through n i t r o g e n f i x a t i o n . However, the e f f e c t s d e s c r i b e d here seem to be t r u l y novel i n an a g r i c u l t u r a l c o n t e x t . The mechanism by which such e f f e c t s occur cannot be determined from these data, but informed s p e c u l a t i o n i s p o s s i b l e . Increases of up to 50% were noted i n white c l o v e r root weight when homologous R. trifolii/L. perenne combinations were p r e s e n t . Homologous Bacillus x Lolium 224 associations resulted in an average 46% increase in root weight of the grass. L. perenne f inal shoot weight was only marginally responsive to matched perennial ryegrass/./?. trifolii combinations while root weight showed unambiguous and substantial gains. S imi l iar ly , the Bacillus/L. perenne interaction had a clear effect on white clover root weight, but shoot weight was not affected in as specific a manner. Therefore, when growth stimulation was correlated to homologous grass/bacteria associations, root weight was affected more clearly and to a greater degree than was the above ground biomass accumulation. Consequently, i t seems reasonable to assume that enhanced root growth due to microbial influences may affect subsequent shoot growth in these cases and result in the observed above ground effects. If this is so, the beneficial bacterial strains may be di f ferent ia l ly stimulated in the perennial ryegrass root system such that larger rhizosphere populations are present. Examples of the di f ferent ia l stimulation of R. trifolii in host and nonhost legume rhizospheres and in nonlegume rhizospheres are numerous (Krasil 'nikov, 1958; Rovira, 1961; Tuzimura and Watanabe, 1962; Robinson, 1967; Chatel and Greenwood, 1973). Bacillus species are thought to be poorer rhizoplane inhabitants than other bacteria, but B. pol ymyxa was shown to comprise a substantial portion of the rhizoplane microflora (Clark, 1949). If the flourishing bacterial population produces compounds that stimulate growth of either or both of the pasture plants, the observed effects may result from the increased bacterial presence. Another potential mechanism by which plants may stimulate growth promoting microbes is through the effect of d i f ferent ia l root exudation on the production of microbial exudates. For example, Tien et a l . (1979) found that Azospirillum brasilense could produce IAA but only if tryptophan was present in the growth medium. S imi l iar ly , Primrose (1976) showed that many so i l microbes are capable of ethylene production but only i f an adequate supply of methionine is available. Therefore, i t is possible that root microbial populations do not differ greatly between genotypes of the pasture plants, but that qualitative differences in exudation influence microbial production of substances which may subsequently affect plant growth. Differences in root exudation have been shown to occur between plant species (Rovira and Davey, 1974; Curl and Truelove, 1986) and between cult ivars and genotypes of the same species (Kraft, 1974; Keeling, 1974; Baldani and Dobereiner, 1980). Shading has also been shown to affect root exudation (Rovira, 1959; Hale et a l . , 1978) and to influence the composition of root microflora (Christie et a l . , 1974; 1978; Newman et a l . , 1979). Several other factors, some of which could be influenced by neighbouring plants, may similarly alter this process. These include plant developmental stage, plant nutri t ion, plant injury, l ight , temperature, moisture, and fo l iar sprays. It is of interest that Rhizobium spp. and Bacillus pol ymyxa are known to have growth requirements for thiamine and biot in, both of which can be present in plant root exudates (Curl and Truelove, 1986). Van Egeraat (1975) has demonstrated that homoserine may be involved in the di f ferent ia l stimulation of Rhizobium in pea root systems. Therefore, diversity in rhizosphere populations may also be attributed to very specific qualitative differences between plant root exudates. The complexity of these effects is further i l lustrated by reports that root-associated microorganisms can affect the process of plant root exudation direct ly (Rovira and Davey, 1974; Hale et a l . , 1978; Curl and Truelove, 1986). The former authors l i s t four ways by which microbes may accomplish this: (i) by affecting the permeability of root ce l l s , ( i i ) by affecting metabolism of roots, ( i i i ) by absorption of certain exuded compounds, and (iv) by altering nutrient ava i lab i l i ty to the plant. Hale and Moore (1979) have demonstrated that inoculation of legumes with root nodule bacteria qualitat ively changes root exudation. Addition of Rhizobium to a l fa l fa was shown to increase levels of nonreducing sugars and amino nitrogen in exudates while decreasing the amount of reducing sugars and total phenols (Curl and Truelove, 1986). Such changes may have substantial effects on rhizosphere microorganisms resulting in larger bacterial populations (R. trifolii or Bacillus polymyxa-like organisms) or altered production of microbial metabolites. Antibiot ics , such as polymyxin, have been shown to affect membrane permeability thereby increasing leakage from roots (Norman, 1955). Because some strains of B. polymyxa are known to produce this antimicrobial agent, i t is possible that the Bacillus isolates used in these experiments influenced the production of root exudates through a similar mechanism. The specific interaction between perennial ryegrass and Bacillus sp. was shown to affect nodulation of white clover. The effect of free l iv ing diazotrophic microbes on nodulation of soybean and clover has been previously demonstrated (Singh and Subba Rao, 1979; Burns et a l . , 1981; El-Bahrawry, 1983). In the current work larger root nodules (indicated by increased fresh weight) were formed when homologous Bacillus and Lolium genotypes were present. Plants with fewer large root nodules are considered to be more efficient in nitrogen fixation than those with many small ones because less energy is wasted on processes associated with nodule formation. Nodule number was not assessed in the present experiments, but it may be reasonably assumed that larger nodule size confers an advantage to plant performance regardless of nodule number. Furthermore, the homologous R. trifolii strain was found more often in white clover root nodules when the matched grass/bacteria association was present. A yield advantage of 20% - 25% was observed when clover was inoculated with its homologous R. trifolii strain even though these combinations were not favored in the strain selection experiments. 228 T h e r e f o r e , the grass/Bacillus i n t e r a c t i o n may exert a dual p o s i t i v e e f f e c t on legume n i t r o g e n f i x a t i o n by c a u s i n g the formation of l a r g e r nodules which c o n t a i n homologous microsymbionts more f r e q u e n t l y . Increased legume n i t r o g e n f i x a t i o n does not e x p l a i n the enhanced p e r e n n i a l ryegrass performance because the study was of r e l a t i v e l y short d u r a t i o n and n i t r o g e n was abundant i n the s o i l m ixture. However, even i f n i t r o g e n were l i m i t i n g growth, i t i s u n l i k e l y that t r a n s f e r of t h i s element from legumes to grasses would occur only two months a f t e r p l a n t i n g . Such e f f e c t s c o u l d , however, be important i n long-term pasture p r o d u c t i v i t y . The Bacillus i s o l a t e s may a l s o have d i r e c t p l a n t growth promoting a c t i v i t y on white c l o v e r performance (Chapter 2 ) . If PGP s t r a i n s of B. polymyxa are s t i m u l a t e d i n the p e r e n n i a l ryegrass root system, a t h i r d b e n e f i t would accrue to l o c a l white c l o v e r p l a n t s due to the g r a s s / b a c t e r i a i n t e r a c t i o n . The concept of r y e g r a s s - s t i m u l a t e d b a c t e r i a l p o p u l a t i o n s can be used to e x p l a i n the r e s u l t s of the R. trifolii/plant genotype s p e c i f i c i t y experiment. The most c o n s i s t e n t t r e n d in that study was the y i e l d advantage, mainly i n white c l o v e r , c o n f e r r e d by homologous R. trifolii/L. perenne combinations. Under those c o n d i t i o n s , L. perenne may serve as a source of inoculum f o r white c l o v e r by a i d i n g i n the s a p r o p h y t i c s u r v i v a l of R. trifolii before nodules are formed. T h i s idea i s supported by the o b s e r v a t i o n that white c l o v e r responded to a g r e a t e r degree than p e r e n n i a l r y e g r a s s when these combinations were t e s t e d . Performance of L. perenne was unaffected unt i l f inal plant weight was measured, one year after planting. This late yie ld advantage for the grass component may have resulted from enhanced levels of nitrogen becoming available as white clover roots and nodules decayed. Evans et a l . (1985) also demonstrated that the perennial ryegrass component of a similar mixture did not show enhanced yield unt i l the second year of growth with "familiar" white clover genotypes. However, i t should not be forgotten that Rhizobium sp. can produce significant amounts of phytohormones (Meijer, 1982). Some species have been shown to promote growth of nonlegumes (Kavimandan, 1985; 1986), therefore, direct PGP effects of R. trifolii on both species of pasture plant may occur. If perennial ryegrass aided the survival of the microbe or supplied a c r i t i c a l growth factor which affected microbial exudation of plant growth promoting substances, the type of grass/bacteria interaction noted here could result . In both types of experiments (Bacillus and Rhizobium) significant effects were found when the homologous three-way partnerships (bacteria x ryegrass x clover) were tested, but often the white clover genotype contributed l i t t l e or performed worse than heterologous genotypes. This response suggests that the clover genotype effect is weak and unimportant relative to the specific effects of the grass/bacterium association. Therefore, the latter partnership may strongly influence the performance of the local white clover population in natural systems. Such e f f e c t s may be i m p o r t a n t i n t h e e a r l y s t a g e s of c o a d a p t a t i v e or c o e v o l u t i o n a r y r e l a t i o n s h i p s between p a s t u r e p l a n t s and m i c r o o r g a n i s m s . F o r example, t h e g r a s s / b a c t e r i a r e l a t i o n s h i p may d e v e l o p s h o r t l y a f t e r s o w i n g of t h e p a s t u r e w i t h t h e n e i g h b o u r i n g c l o v e r p l a n t s becoming an i n t e g r a l p a r t of t h e t r i o a s t i m e p a s s e s . W h i t e c l o v e r g e n o t y p e s d i d n o t have l a r g e s p e c i f i c e f f e c t s on f o r a g e y i e l d . However, e v i d e n c e f o r c l o v e r g e n o t y p e s p e c i f i c i t y w i t h i n t h e t h r e e - w a y c o m b i n a t i o n i s e x e m p l i f i e d by t h e a p p a r e n t c o m m u n i c a t i o n ( i . e . r e p u l s i o n ) between homologous legume c l o n e s and R. t r i f o l i i i s o l a t e s . I n t h i s s c e n a r i o , t h e m i c r o s y m b i o n t would be p r i m a r i l y homologous t o t h e L. perenne g e n o t y p e ( o r t h e grass/Bacillus c o m b i n a t i o n ) , and n o t t h e c l o v e r g e n o t y p e from w h i c h i t was o r i g i n a l l y i s o l a t e d . The g r a s s / b a c t e r i a i n t e r a c t i o n does n o t p r o v i d e t h e d e f i n a t i v e e x p l a n a t i o n f o r .these r e s u l t s , i n a s much as t h e a d d i t i o n o f homologous Bacillus i s o l a t e s d i d n o t r e v e r s e t h e r e p u l s i o n o f homologous R. t r i f o l i i by w h i t e c l o v e r g e n o t y p e s ( d a t a n ot shown). The e c o l o g i c a l s i g n i f i c a n c e of s e l e c t i o n a g a i n s t homologous m i c r o s y m b i o n t s t r a i n s r e m a i n s a m y s t e r y , but i t s h o u l d be t a k e n a s a f u r t h e r example o f t h e l e v e l a t w h i c h c o m m u n i c a t i o n between p l a n t s and m i c r o b e s may o c c u r . T h e r e i s a n o t h e r c o n s i d e r a t i o n w h i c h s h o u l d be made i n t h e i n t e r p r e t a t i o n o f t h e s e d a t a . I t i s n o t p o s s i b l e t o d e t e r m i n e whether or n o t t h e documented e f f e c t s a r e c r i t i c a l t o y i e l d r e l a t i o n s h i p s between t h e p a s t u r e p l a n t s , o r i f t h e y s i m p l y r e p r e s e n t a few p o i n t s on t h e s c a l e o f b i o t i c f a m i l i a r i t y n o t e d i n C h a p t e r 2. F o r example, homologous L. perenne/T. repens genotypes were shown to have a very small and s ta t i s i ca l ly insignificant positive effect on yield of white clover (5%), while homologous T. repens/R. trifolii combinations were s l ightly more influential (15%). L. per enne/R. trifolii or L. per enne/R. trifolii/T. repens showed the greatest yield advantage, up to 50%. Similarly, Bacillus x T. repens interactions generally affected yie ld in a positive way but Bacillus/Lolium or Baci 11 us/Lol i um/Trifol i um were best. Does this indicate that testing any rhizosphere microorganism would result in the same type of trend or are R. trifolii and B. pol ymyxa particularly important to pasture productivity? There is no doubt about the potential influence root nodule bacteria may exert in this regard, but the importance of the Bacillus isolates used in these experiments may be more ambiguous. Was i t simply fortunate or coincidental to isolate this particular bacterium from so i l samples? No definative answer to this question may be derived from these data, but certain clues are available. F i r s t , the Bacillus strains isolated represented a minority component of aerobic isolates of so i l bacteria but a majority of the anaerobic ones. The isolating medium was composed of several carbon substrates and growth factors peculiar to diazotrophic bacteria, but most groups of bacteria would be able to proliferate under these conditions. Since there were many more aerobic isolates than anaerobic ones, these Bacillus strains represented a minority class of microorganisms present in this s o i l . Second, the growth s t i m u l a t i o n a f f o r d e d by the Baci 11 us/Lol i um i n t e r a c t i o n was measured r e l a t i v e to h e t e r o l o g o u s g r a s s / b a c t e r i a combinat ions and was not always compared wi th u n i n o c u l a t e d c o n t r o l s . When y i e l d of L. perenne was c o n s i d e r e d i n m i x t u r e , the average e f f e c t of i n o c u l a t i o n wi th Bacillus was a s l i g h t d e p r e s s i o n of y i e l d . The presence of homologous g r a s s / b a c t e r i a a s s o c i a t i o n s would o f t e n i n c r e a s e y i e l d from s l i g h t l y below to s l i g h t l y above u n i n o c u l a t e d c o n t r o l y i e l d s . T h i s r e s u l t e d i n a s i g n i f i c a n t e f f e c t r e l a t i v e to h e t e r o l o g o u s c o m b i n a t i o n s but not compared w i t h u n i n o c u l a t e d c o n t r o l s . T h e r e f o r e i t appears tha t these exper iments have uncovered f i n e s c a l e b i o t i c s p e c i a l i z a t i o n w i t h i n s p e c i e s whose a b s o l u t e e f f e c t s are s m a l l , but whose r e l a t i v e e f f e c t s are s i g n i f i c a n t . I t i s p o s s i b l e tha t o ther microorgan i sms i s o l a t e d from an o l d f i e l d such as t h i s one and t e s t e d i n a s i m i l i a r way might show the same s m a l l but d e t e c t a b l e e f f e c t s . On the o ther hand, one of the Bacillus s t r a i n s t e s t e d i n these exper iments was shown to possess s i g n i f i c a n t p l a n t growth promot ing a c t i v i t y , o f t en m a n i f e s t e d by i n c r e a s e s i n r o o t weight (Chapter 2 ) . The other two s t r a i n s showed s i m i l i a r t r e n d s but were not examined i n such d e t a i l . T h e r e f o r e , from a " p l a n t ' s - e y e - p o i n t of view", these i s o l a t e s may r e p r e s e n t a s p e c i a l component of the r h i z o s p h e r e m i c r o f l o r a . S o i l was e x t e n s i v e l y t e s t e d f o r the presence of o t h e r w e l l known d i a z o t r o p h i c PGP b a c t e r i a b e l o n g i n g to the genera Azospirillum and Azotobacter. However, i s o l a t e s b e l o n g i n g to these groups were not d e t e c t e d i n any of the 233 samples c o l l e c t e d from r h i z o s p h e r e s o i l of these p a s t u r e p l a n t s . In t h i s regard i t i s i n t e r e s t i n g to note that Sperber and R o v i r a (1959) i s o l a t e d three Bacillus s t r a i n s from subterranean c l o v e r r o o t s but none from r o o t s of Wimmera ryegrass when the pasture p l a n t s grew in mixture. In the present study, i t was not p o s s i b l e to a s c r i b e the Bacillus i s o l a t e s to e i t h e r the white c l o v e r or p e r e n n i a l ryegrass r h i z o s p h e r e as the bacterium was i s o l a t e d from s o i l samples in which both root systems were i n t e r m i n g l e d . The importance of the f i n d i n g that Bacillus i s o l a t e s comprised the m a j o r i t y of the anaerobic b a c t e r i a l s t r a i n s i s unknown. The d i a z o t r o p h i c s t r i c t anaerobe Clostridium pasteurianum i s common i n g r a s s l a n d s o i l s (Warembourg and M o r r a l , 1978) and has been shown to f i x n i t r o g e n i n the r h i z o s p h e r e (Dobe r e i n e r , 1974; Knowles, 1977) e s p e c i a l l y at the root s u r f a c e at lower depths (Ogan, 1979). S i m i l a r l y , B. polymyxa i s a common s o i l microbe whose p o t e n t i a l p l a n t growth s t i m u l a t i o n has been w e l l documented (Chapter 2). T h e r e f o r e i t c o u l d be argued that f a c u l t a t i v e Bacillus s p e c i e s c o n t r i b u t e more to y i e l d r e l a t i o n s h i p s between pasture p l a n t s than other f r e e - l i v i n g s o i l microorganisms. Regardless of the exact mechanisms at work, the r e s u l t s of these s t u d i e s i n d i c a t e t h a t r h i z o s p h e r i c i n t e r a c t i o n s between p l a n t and b a c t e r i a l genotypes are important i n above ground p l a n t performance. In p a r t i c u l a r , the s i g n i f i c a n c e of the Rhi zobi um component to the system has been u n e q u i v o c a l l y demonstrated. Turkington (1985) p r e d i c t e d t h a t the R. .234 trifolii genotype may be adapted to the grass and legume components of the mixture. In the present work both of these r e l a t i o n s h i p s were shown to e x i s t but the Rhi zobi um/Lol i um i n t e r a c t i o n appears to be the major determinant of b i o l o g i c a l accommodation between genotypes of the pasture p l a n t s . I t i s not s u r p r i s i n g that i n t e r a c t i o n s i n v o l v i n g the microsymbiont are important because of the p o t e n t i a l e f f e c t s b i o l o g i c a l n i t r o g e n f i x a t i o n may have on pl a n t performance. However, phytohormonal i n f l u e n c e s of the microbe may a l s o be important and r e q u i r e f u r t h e r study. R e s u l t s of the Bacillus experiments are s i m i l a r l y impressive and f u r t h e r demonstrate the i n f l u e n c e that r h i z o s p h e r i c b a c t e r i a may exert on p l a n t growth and the complex c o e v o l u t i o n a r y processes that occur between p l a n t s and microorganisms. Perhaps the most important .demonstration of these r e l a t i o n s h i p s i n an a g r i c u l t u r a l context i s the b i o t i c f a m i l i a r i t y noted i n Chapter 2. As b i o t i c surroundings became l e s s f o r e i g n with r e s p e c t to i n t r a s p e c i f i c and i n t e r s p e c i f i c neighbouring genotypes, forage y i e l d was shown to i n c r e a s e . When p l a n t and b a c t e r i a l genotypes were homologous, a y i e l d advantage of 42% was observed. While b a c t e r i a comprise the m a j o r i t y of r h i z o s p h e r e m i c r o f l o r a ( C u r l and Truelo v e , 1986), the i n f l u e n c e of the fun g a l component should a l s o be c o n s i d e r e d i n plant/microbe r e l a t i o n s h i p s . M y c o r r h i z a l fungi are a group of symbiotic microorganisms that invade p l a n t root systems and are thought to enhance n u t r i e n t uptake. V e s i c u l a r - a r b u s c u l a r mycorrhizae (VAM) comprise one of several types of these microbes. Members of this group of fungi l ive between ce l l s of the root cortex and form temporary hyphal projections that penetrate cort ica l c e l l s . The projections may take the form of swollen vesicles or they may consist of finely branched masses called arbuscules. VAM fungi are considered to be the most important group of mycorrhizal organisms because they occur in most cultivated crops and in most plants in natural ecosystems (Hayman, 1978). Inoculation with spores of these microbes has been shown to result in y ie ld increases in a number of crop species probably through enhanced uptake of phosphorus. Beneficial effects on symbiotic nitrogen f ixation, similar to those noted with so i l bacteria (Singh and Subba Rao, 1979; Burns et a l . , 1981), have also been observed when legumes were coinoculated with VAM fungi (Subba Rao et a l . , 1986). Furthermore, specif ic i ty between VAM fungal species and T. subterranean has been reported (Green et a l . , 1983) which suggests that genotype specif ic i ty between plants and mycorrhizae may occur. Consequently, only continuing experimentation with qualitative and quantitative control and assessment of microbial growth, including mycorrhizal assocations, can provide further insights into the role of microorganisms in above ground compatibility between Lolium perenne and Trifolium repens. Summary V a r i a b i l i t y i n i n t r i n s i c a n t i b i o t i c r e s i s t a n c e was no t a s u i t a b l e b a s i s f o r s t r a i n i d e n t i f i c a t i o n w i t h i n Rhizobium t r i f o l i i . H y b r i d IAR p a t t e r n s were common and r e s u l t e d i n ambiguous o r i n c o r r e c t i d e n t i f i c a t i o n s . S e r o l o g i c a l a s s a y s b a s e d on p o l y c l o n a l a n t i s e r u m a l l o w e d R. t r i f o l i i s t r a i n s t o be r e l i a b l y s e p a r a t e d on t h e b a s i s o f a n t i g e n i c d e t e r m i n a n t s . Of t h e s e p r o c e d u r e s , a g g l u t i n a t i o n and i m m u n o f l u o r e s c e n c e were t h e l e a s t s a t i s f a c t o r y , b u t i m m u n o d i f f u s i o n and EL I S A were h i g h l y s u i t a b l e . By u s i n g t h e l a t t e r t e c h n i q u e , R. t r i f o l i i c o u l d be i d e n t i f i e d d i r e c t l y i n i n d i v i d u a l r o o t n o d u l e s . A l o c a l l y - i s o l a t e d s t r a i n o f Bacillus polymyxa was shown t o promote g r o w t h of c r e s t e d w h e a t g r a s s (Agropyron cristatum) and w h i t e c l o v e r (Trifolium repens). T y p i c a l l y , t h e e f f e c t i n c l u d e d i n c r e a s e s i n r o o t w e i g h t b u t g a i n s i n w h i t e c l o v e r s h o o t w e i g h t were a l s o o b s e r v e d . P e r e n n i a l r y e g r a s s was no t s i g n i f i c a n t l y a f f e c t e d by i n o c u l a t i o n . When T. repens and B. pol ymyxa were homologous, t h e r e s u l t a n t y i e l d i n c r e a s e s were above t h o s e s een when g e n o t y p i c m i x t u r e s of t h e legume were t e s t e d w i t h t h e b a c t e r i u m . T h i s t r e n d was s t a t i s i c a l l y s i g n i f i c a n t o n l y i n s p e c i e s m i x t u r e . A n a l y s i s of t h e p l a n t g r o w t h r e s p o n s e i n c r e s t e d w h e a t g r a s s r e v e a l e d t h a t b a c t e r i a l p r o d u c t i o n o f IAA was t h e most l i k e l y c a u s e of g r o w t h s t i m u l a t i o n . O t h e r p o t e n t i a l l y i m p o r t a n t a t t r i b u t e s of B. pol ymyxa, s u c h a s t h e a b i l i t y t o f i x a t m o s p h e r i c n i t r o g e n o r t o s o l u b i l i z e o r g a n i c p h o s p h o r u s , were shown t o be u n r e l a t e d t o t h e gro w t h r e s p o n s e . C o m p a t i b i l i t y between g e n o t y p e s o f T. repens and L. perenne was n o t d e t e c t e d when t h e e f f e c t o f R. t r i f o l i i was i g n o r e d . However, t h i s b i o t i c s p e c i a l i z a t i o n w h i c h was c h a r a c t e r i z e d by y i e l d i n c r e a s e s i n t h e w h i t e c l o v e r component o f t h e s p e c i e s m i x t u r e , was e v i d e n t when homologous R. t r i f o l i i i s o l a t e s were u s e d t o i n o c u l a t e s p e c i e s m i x t u r e s . Most o f t h e y i e l d a d v a n t a g e o b s e r v e d when homologous p l a n t s were i n o c u l a t e d w i t h m a t c h i n g R. t r i f o l i i s t r a i n s was a c c o u n t e d f o r by t h e s p e c i f i c i t y of t h e L. perenne/R. t r i f o l i i c o m b i n a t i o n . Homologous g r a s s / b a c t e r i a c o m b i n a t i o n s c o n f e r r e d a y i e l d a d v a n t a g e t o n e i g h b o u r i n g w h i t e c l o v e r p l a n t s r e g a r d l e s s of t h e legume g e n o t y p e . B e c a u se T. repens was t h e dominant component o f t h e s p e c i e s m i x t u r e , t o t a l f o r a g e y i e l d showed s i m i l a r t r e n d s t o t h o s e seen i n t h e legume. However, t h i s r e s p o n s e r e s u l t e d i n homologous c o m b i n a t i o n s showing t h e l o w e s t e c o l o g i a l c o m b i n i n g a b i l i t y . When T. repens was i n o c u l a t e d w i t h a m i x t u r e o f R. t r i f o l i i s t r a i n s , h e t e r o l o g o u s i s o l a t e s f ormed more r o o t n o d u l e s t h a n d i d homologous o n e s . The p r e s e n c e o f L. perenne d i d not m i t i g a t e t h i s e f f e c t . However, y i e l d a d v a n t a g e s were o b s e r v e d when T. repens was i n o c u l a t e d w i t h homologous R. trifolii . S u p e r i o r y i e l d s were o b s e r v e d i n r o o t w e i g h t , r o o t nodule we ight , and t o t a l accumulated biomass of T. repens when homologous B. pol ymyxa/L. perenne combinat ions were p r e s e n t . A l l v a r i a b l e s measured i n L. perenne and in combined forage y i e l d a l s o showed t h i s t r e n d . 240 LITERATURE CITED Aarssen, L.W. 1983. I n t e r a c t i o n s and c o e x i s t e n c e of s p e c i e s in p asture community e v o l u t i o n . Ph.D. T h e s i s . U n i v e r s i t y of B r i t i s h Columbia. Vancouver, B r i t i s h Columbia. 249 pp. Aarssen, L.W., and R. Tu r k i n g t o n . 1985. B i o t i c s p e c i a l i z a t i o n at the genotype l e v e l in Lolium perenne and Trifolium repens from a permanent p a s t u r e . J . E c o l . 73:605-614. Alexander, M. 1964. I n t r o d u c t i o n to S o i l M i c r o b i o l o g y . Wiley, New York/London. 472 pp. A l l a r d , R.W., and J . Adams. 1969. Po p u l a t i o n s t u d i e s i n predominantly s e l f - p o l l i n a t i n g s p e c i e s . X I I I . I n t e r g e n o t y p i c c o m p e t i t i o n and p o p u l a t i o n s t r u c t u r e i n ba r l e y and wheat. Amer. Natur. 103:621-645. Anderson, G. 1976. Other or g a n i c phosphorus compounds. In: Organic S o i l Components. J . E. G e i s e k i n g (Ed.). S p r i n g e r - V e r l a g , B e r l i n , pp. 305-331. Antoun, H., L.M. Bordeleau, and D. Prevost. 1982. S t r a i n i d e n t i f i c a t i o n i n Rhizobium meliloti using the a n t i b i o t i c d i s k s u s c e p t i b i l i t y t e s t . P l a n t and S o i l 66:45-50. A v i v i , Y. , and M." Feldman. 1982. The response of wheat to b a c t e r i a of the genus Azospirillum. I s r a e l J . Bot. 31:237-245. Azcon, R., and J.M. Barea. 1975. Synth e s i s of auxins, g i b b e r e l l i n s , and c y t o k i n i n s by Azotobacter vinelandii and Azotobacter beijerinckii r e l a t e d to e f f e c t caused on tomato p l a n t s . P l a n t and S o i l 43:609-619. Badenoch-Jones, J . , D.J. F l a n d e r s , and B.G. R o l f e . 1984. A s s o c i a t i o n of Rhizobium s t r a i n s with r o o t s of Trifolium repens. Appl. Env. Mi c r o . 49:1511-1520. B a l d a n i , V.L.D., and J . Dobereiner. 1980. Host p l a n t s p e c i f i c i t y i n the i n f e c t i o n of c e r e a l s with Azospirillum ssp. S o i l B i o l . Biochem. 12:433-439. Banik, S., and B.K. Dey. 1985. E f f e c t of i n o c u l a t i o n with n a t i v e phosphate s o l u b i l i z i n g microorganisms on the a v a i l a b l e phosphorus content i n the rh i z o s p h e r e and uptake of phosphorus by r i c e , grown i n an Indian a l l u v i a l s o i l . Z b l . M i k r o b i o l . 140:455-464. Barber, D.A. 1978. N u t r i e n t uptake. In: I n t e r a c t i o n s Between Non-Pathogenic S o i l Microorganisms and P l a n t s . Y.R. Dommergues and S.V. Krupa (Eds.). E l s e v i e r S c i e n t i f i c , Amsterdam/Oxford/New York. pp. 131-162. 241 Barber, D.A. , and J . K . Martin. 1976. The release of organic substances by cereal roots into s o i l . New Phytol. 76:69-80. Barea,- J . M . , E . Navarro, and E. Montoya. 1976. Production of plant growth regulators by rhizosphere phosphate-solubil iz ing bacteria. J . Appl. Bact. 40:129-134. Barea, J . M . , A . F . Bonis, and J . Olivares. 1983. Interactions between Azospirillum and VA mycorrhizae and their effects on growth and nutrition of maize and ryegrass. Soi l B i o l . Biochem. 15:705-709. Bashan, Y. 1986a. Significance of timing and level of inoculation with rhizosphere bacteria on wheat plants. Soi l B io l . Biochem. 18:297-301. Bashan, Y. 1986b. Enhancement of wheat root colonization and plant development by Azospirillum brasilense Cd. following temporary depression of rhizosphere microflora. Appl. Env. Micro. 51:1067-1071. Benians, G . J . , and D.A. Barber. 1974. The uptake of phosphate by barley plants from so i l under aseptic and non-sterile conditions. Soil B i o l . Biochem. 6:195-200. Benyon, J . L . , and D.P. Josey. 1980. Demonstration of heterogeneity in a natural population of Rhizobium phasedi using variation in intr ins ic antibiotic resistance. J . Gen. Micro. 118:437-442. Berger, J . A . , S.N. May, L.R. Berger, and B.B. Bohlool. 1979. Colorimetric enzyme-linked immunosorbent assay for the identif ication of strains of Rhizobium in culture and in the nodules of l e n t i l s . Appl. Env. Micro. 37:642-646. Bergegsen, F . J . , and G.L. Turner. 1983. An evaluation of N methods for estimating nitrogen fixation in a subterranean clover-perennial ryegrass sward. Aust. J . Agr. Res. 34:391-401. Beringer, J . E . 1974. R Factor transfer in Rhizobium Ieguminosarum. J . Gen. Micro. 84:188-198. Bhuvaneswari, T . V . , S.G. Pueppke, and W.D. Bauer. 1977. The role of lectins in plant-microorganism interactions. I . Binding of soybean lect in to rhizobia. Plant Physiol. 60:486-491. Bhuvaneswari, T . V . , and W.D. Bauer. 1978. The role, of lectins in plant-microorganism interactions. III. Binding of soybean lect in to root cultured rhizobia. Plant Physiol. 62:71-74. 242 Boddey, R .M. , V . I . D . Baldani, J . I . Baldani, and J . Dobereiner. 1986. Effect of inoculation of Azospirillum spp. on nitrogen accumulation by f ie ld grown wheat. Plant and Soi l 95:109-121. Bohlool, B .B . , and E . L . Schmidt. 1974. Lectins: a possible basis for specif ic i ty in the Rhi zobi um I egumi nosarum symbiosis. Science 185:269-271. Bowen, G.D. , and A.D. Rovira. 1961. Effects of micro-organisms on plant growth. I. Development of roots and root hairs in sand and agar. Plant and Soi l 15:166-188. Brockwell, J . , E .A. Schwinghamer, and R.R. Gault. 1977. Ecological studies of root-nodule bacteria introduced into f ie ld environments - V. A c r i t i c a l examination of the s tabi l i ty of antigenic and streptomycin-resistance markers for identif ication of strains of Rhizobium trifolii. Soi l B i o l . Biochem. 9:19-24. Brockwell, J . , A. Diatloff , and E.A. Schwinghamer. 1978. An appraisal of methods for distinguishing between strains of Rhizobium and their application to the identif ication of isolates from f ie ld environments. In: Microbial Ecology. M.W. Loutit and J . A . R . Miles (Eds.). Springer-Verlag, Berlin/Heidelberg, pp. 390-397. Bromfield, E . S . P . , and D.G. Jones. 1980a. A strain marker in Rhizobium trifolii based on the absorption of congo-red. Zbl. Bakt. II, Abt. 135:290-295. Bromfield, E . S . P . , and D.G. Jones. 1980b. Studies on double strain occupancy of nodules and the competitive ab i l i ty of Rhizobium trifolii on red and white clover grown in so i l and agar. Ann. Appl. B i o l . 94:51-59. Bromfield, E . S . P . , M. Stein, and R.P. White. 1982. Identification of Rhizobium strains on antibiot ic concentration gradients. Ann. Appl. B i o l . 101:269-277. Bromfield, E . S . P . , I .B. Sinha, and M.S. Wolynetz. 1986. Influence of location, host cul t ivar , and inoculation on the composition of naturalized populations of Rhizobium meliloti in Medicago sativa nodules. Appl. Env. Micro. 51:1077-1084. Brown, M.E. 1974. Seed and root bacterization. Ann. Rev. Phytopath. 12:181-197. Brown, M.E. 1 976. Role of Azotobacter pas pali in association with Paspalum notatum. J . Appl. Bact. 40:341-348. 243 Brown, M.E. 1982. Nitrogen f i x a t i o n by f r e e - l i v i n g bacteria associated with plants - fact or f i c t i o n ? In: Bacteria and Plants. M.E. Rhodes-Roberts and F.A. Skinner (Eds.). Academic Press, New York. pp. 25-41. Brown, M.E., and S.K. Burlingham. 1968. Production of plant growth substances by Azotobacter chroococcum. J . Gen. Micro. 53:135-144. Brown, M.E., R.M. Jackson, and S.K. Burlingham. 1968. Growth and effects of bacteria introduced into s o i l . In: The Ecology of S o i l Bacteria. T.R.G. Gray and D. Parkinson (Eds.). Liverpool University Press, Liverpool. pp. 531-551. Brown, M.E., R.M. Jackson, and S.K. Burlingham. 1968. E f f e c t s produced on tomato plants, Lycopersi cum esculentum, by seed or root treatment with g i b b e r e l l i c acid and indolyl-3-acetic acid. J . Exp. Bot. 19:544-552. Brown, M.E., and N. Walker. 1970. Indolyl-3-acetic acid formation by Azotobacter chr oococcum. Plant and S o i l 32:250-253. Brown, R., and M. Edwards. 1944. The germination of the seed of Striga lutea. I. Host influence and the progress of germination. Ann. Bot. 8:131-148. Buchanan, R.E., and N.E. Gibbons (Eds.). 1974. Bergey's Manual of Determinative Bacteriology, 8th e d i t i o n . Williams and Wilkins, Baltimore. 1094 pp. Burdon, J . J . 1980. I n t r a - s p e c i f i c d i v e r s i t y in a natural population of Trifolium repens. J. Ecol. 68:717-735. Burns, T.A., J r . , P.E. Bishop, and D.W. I s r a e l . 1981. Enhanced nodulation of leguminous plant roots by mixed cultures of Azotobacter vinelandii and Rhizobium. Plant and S o i l 62:399-412. Burr, T., and A. Caesar. 1984. B e n e f i c i a l plant bacteria. CRC C r i t i c a l Reviews in Plant Science 2:1-20. Bushby, H.V.A. 1982. Ecology. In: Nitrogen Fixation Volume 2: Rhizobium. W.J. Broughton (Ed.). Clarendon Press, Oxford, pp. 312-331. Campbell, R., and A.D. Rovira. 1973. The study of the rhizosphere by scanning electron microscopy. S o i l B i o l . Biochem. 5:747-752. Celino, M.S., and D. G o t t l i e b . 1952. Control of b a c t e r i a l w i l t of tomatoes by Bacillus polymyxa. Phytopath. 42:4. 244 Chalk, P.M. 1985. Estimation of N 2 fixation by isotope di lut ion: an appraisal of techniques involving N enrichment and their application. Soil B i o l . Biochem. 17:389-410. Chanway, C P . , and F .B . Ho l l . 1986. Suitabi l i ty of intr ins ic antibiotic resistance as a method of strain identification in Rhizobium t r i f o l i i . Plant and Soi l 93:287-291. Charles, A.H. 1968. Some selective effects operating upon white and red clover swards. J . Br. Grassl . Soc. 23:20-25. Chatel, D . L . , and R.M. Greenwood. 1973. The location and distribution in so i l of rhizobia under senesced annual legume pastures. Soil B io l . Biochem. 5:809-813. Chris t ie , P . , E . I . Newman, and R. Campbell. 1974. Grassland species can influence the abundance of microbes on each other's roots. Nature 250:570-571. Chris t ie , P . , E . I . Newman, and R. Campbell. 1978. The influence of neighbouring grassland plants on each other's endomycorrhize and root-surface microorganisms. Soil B i o l . Biochem. 10:521-527. Clark, F . E . 1949. Soi l microorganisms and plant roots. Adv. Agron. 1:242-288. Clark, M . F . , and A.N. Adams. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J . Gen. Virology 34:475-483. Cooper, R. 1959. Bacterial f er t i l i z er s and their effectiveness. Mikrobiologiya 32:911-917. Cur l , E .A. 1979. Effects of mycophageous collembola on Rhizoctonia solani and cotton-seedling disease. In: Soil-Borne Plant Pathogens. B. Schippers and W. Gams (Eds.). Academic Press, London/New York/San Fransisco. pp. 253-269. Cur l , E . A . , and. B. Truelove. 1986. The Rhizosphere. Springer-Verlag, Berlin/Heidelberg. 289 pp. Dart, P . J . 1977. Infection and development of leguminous nodules. In: A Treatise on Dinitrogen Fixation. Section III. Biology. R.W.F. Hardy and W.S. Silver (Eds.). John Wiley and Sons, New York. pp. 367-472. 245 Dart, P.J., and R.V. Subba Rao. 1981. Nitrogen f i x a t i o n a s s o c i a t e d with sorghum and m i l l e t . In: A s s o c i a t i v e N„-F i x a t i o n . V o l . 1. P.B. Vose and A.P. Ruschel (Eds.) . CRC Press, Boca Raton, F l o r i d a , pp. 169-177. Date, R.A., and J . Br o c k w e l l . 1978. Rhizobium s t r a i n c o m p e t i t i o n and host i n t e r a c t i o n f o r n o d u l a t i o n . In: P l a n t R e l a t i o n s in Pastur e s . J.R. Wilson (Ed.). Commonwealth S c i e n t i f i c and I n d u s t r i a l Research O r g a n i s a t i o n , East Melbourne. pp. 202-216. Dazzo, F.B., and W.J. B r i l l . 1978. Regulat i o n by f i x e d n i t r o g e n of host-symbiont r e c o g n i t i o n i n the Rhizobi urn-clover symbiosis. P l a n t P h y s i o l . 62:18-21. Dazzo, F.B., W.E. Yanke, and W.J. B r i l l . 1978. T r i f o l i i n , a Rhizobium r e c o g n i t i o n p r o t e i n from white c l o v e r . Biochem. Biophys. Acta 539:276-286. Dean, J.R., B. Toomsan, and K.W. C l a r k . 1980. Rhizobium s t r a i n s e l e c t i o n f o r fababeans. Can. J . Pl a n t S c i . 60:385-397. D i a t l o f f , A. 1969. The i n t r o d u c t i o n of Rhizobium j aponi cum to s o i l by seed i n o c u l a t i o n of non-host legumes and c e r e a l s . Aust. J . Exp. A g r i c . Anim. Husb. 9:357-360. D i a t l o f f , A. 1977. E c o l o g i c a l s t u d i e s of root-nodule b a c t e r i a i n t r o d u c e d i n t o f i e l d environments. 6. A n t i g e n i c and symbiotic s t a b i l i t y i n Lot ononis Rhizobia over a 12-year p e r i o d . S o i l B i o l . Biochem. 9:85-88. D i l w o r t h , M.J. 1966. Acetylene r e d u c t i o n by n i t r o g e n -f i x i n g p r e p a r a t i o n s from CI ost r i di um past euri anum. Biochem. Biophys. A c t a . 127:285-294. Dobereiner, L. 1961. N i t r o g e n - f i x i n g b a c t e r i a of the genus Beijerinckia Derx i n the rh i z o s p h e r e of sugar cane. Pla n t and S o i l 15:211-217. Dobereiner, J . 1974. N i t r o g e n - f i x i n g b a c t e r i a i n the rh i z o s p h e r e . In: The Bi o l o g y of Nitrogen F i x a t i o n . A. Q u i s p e l (Ed.). North-Holland, Amsterdam, pp. 86-120. Dobereiner, J . , and A.B. Campelo. 1971. Non-symbiotic n i t r o g e n f i x i n g b a c t e r i a i n t r o p i c a l s o i l s . P l a n t and S o i l Spec. V o l . pp. 457-470. Dobereiner, J . , J.M. Day, and P.J. Dart. 1972. Nitrogenase a c t i v i t y and oxygen s e n s i t i v i t y of the Pas palum notatum-Azotobacter paspali a s s o c i a t i o n . J . Gen. M i c r o . 71:103-116. 246 Dobereiner, J . , and J .M. Day. 1976. Associative symbiosis in tropical grasses: Characterization of microorganisms and dinitrogen fixing sites . In: Proceedings of the F irs t International Symposium on Nitrogen Fixation. Vo l . 2. W.E. Newton and C . J . Nyman (Eds.). Washington State University Press, Pullman. pp. 518-538. Dobereiner, J . , and De-Pol l i , H. 1980. Diazotrophic rhizocoenosis. In: Nitrogen Fixation 18th Annual Proceedings Phytochemical Society of Europe. W.D.P. Stewart and J.R. Gallon (Eds.). Academic Press, London, pp. 301-333. Dowson, W.J. 1949. Manual of Bacterial Plant Diseases. Adam and Charles Black, London. 183 pp. Dudman, W.F. 1977. Serological methods and their application to dinitrogen fixing organisms. In: A Treatise on Dinitrogen Fixation. Section IV. Agronomy and Ecology. R.W.F. Hardy and W.S. Silver (Eds.). John Wiley and Sons, New York. pp. 487-508. Duff, R . B . , D.M. Webley, and R.P. Scott. 1963. Solubil ization of minerals and related materials by 2-ketogluconic acid producing bacteria. Soi l Sc i . 95:105-114. Dughri, M.H. , and P . J . Bottomley. 1983a. Complementary methodologies to delineate the composition of Rhizobium trifolii populations in root nodules. Soil Sc i . Soc. Am. J . 47:939-945. Dughri, M.H. , and P . J . Bottomley. 1983b. Effect of acidity on the composition of an indigenous so i l population of Rhi zobi um trifolii found in nodules of Trifolium subterraneum L . Appl. Env. Micro. 46:1207-1213. Dunlevy, J .M. 1952. Control of damping-off of sugarbeet by Bacillus s u b t i l i s . Phytopath. 42:465. El-Bahrawy, S.A. 1983. Associative effect of mixed cultures of Azotobacter and different rhizosphere fungi with Rhizobium japonicum on nodulation and symbiotic fixation of soybean. Zbl . Mikrobiol. 138:443-449. El-Essawy, A . A . , M.A. El-Sayed, Y.A.H. Mohamed, and A. E l -Shanshoury. 1984. Effect of combined nitrogen in the production of plant-growth regulators by Azotobacter chr oococcum. Zbl . Mikrobiol . 139:327-333. El-Haloui , N . E . , D, Ochin, and R. T a i l l i e z . 1986. Infection competitivity between Rhizobium meliloti strains: role of moti l i ty . Plant and Soi l 95:337-344. 247 Engvall, E . , and P. Perlmann. 1972. Enzyme-linked immunosorbent assay, ELISA. III . Quantitation of specific antibodies by enzyme-labelled ant i -immunoglobulins in antigen-coated tubes. J . Immunol. 109:129-1235. Evans, D.R. , J . H i l l , T.A. Williams, and I. Rhodes. 1985. Effects of coexistence on the performance of white clover-perennial ryegrass mixtures. Oecologia 66:536-539. Evans, R. 1986. Morphological variation in a b iot ica l ly patchy environment: Evidence from a pasture population of Trifolium repens. M.Sc. Thesis. , University of Br i t i sh Columbia. Vancouver, Bri t i sh Columbia. 80 pp. F lor , H.H. 1955. Host-parasite interactions in flax rust-its genetics and other implications. Phytopath. 45:680-685. Foster, R . C . , and A.D. Rovira. 1976. Ultrastructure of wheat rhizosphere. New Phytol. 76:343-352. F o t t r e l l , P . F . , and A. O'Hora. 1969. Multiple forms of (D)3-hydroxybutyrate dehydrogenase in Rhizobium. J . Gen. Micro. 57:287-292. Franco, A . A . , and J .M. Vincent. 1976. Competition amongst Rhizobial strains for the colonization and nodulation of two tropical legumes. Plant and Soil 45:27-48. Fuquay, J . I . , P . J . Bottomley, and M.B. Jenkins. 1984. Complementary methods for the differentiation of Rhi zobi um meliloti isolates. Appl. Env. Micro. 47:663-669. Gardner, J . M . , J . L . Chandler, and A.W. Feldman. 1984. Growth promotion and inhibition by antibiotic-producing fluorescent pseudomonads on citrus roots. Plant and Soi l 77: 103-113. Gaskins, M.H. , S .L. Albrecht, and D.H. Hubbell. 1985. Rhizosphere bacteria and their use to increase productivity: a review. Agriculture, Ecosystems, and Environment 12:99-116. Gaur, A . C . , and K.P. Ostwal. 1972. Influence of phosphate dissolving b a c i l l i on yield and phosphate uptake by wheat crop. Ind. J . Exp. B i o l . 10:393-394. Gerretsen, F . C . 1948. The influence of micro-organisms on the phosphate intake by the plant. Plant and Soi l 1:51-85. 248 Gonzalez-Lopez, J . , V. Salmeron, M.V. Martinez-Toledo, F. Ballasteros, and A. Ramos-Cormenzana. 1986. Production of auxins, gibberellins and cytokinins by Azotobacter vinelandii ATCC 12837 in chemically-defined media and dialysed soi l media. Soi l B i o l . Biochem. 18:119-120. Gray, T . R . G . , and S.T. Williams. 1971. Soi l Micro-organisms. Oliver and Boyd, Edinburgh. 240 pp. Greaves, M.P. , and D.M. Webley. 1965. A study of the breakdown of organic phosphates by micro-organisms from the root region of certain pasture grasses. J . Appl. Bact. 28:454-465. Green, N . E . , M.D. Smith, W.D. Beavis, and E . F . Aldon. 1983. Influence of vesicular-arbuscular mycorrhizal fungi on the nodulation and growth of subclover. J . Range Manage. 36:576-578. G r i f f i n , D.M. 1972. Ecology of Soil Fungi. Chapman and H a l l , London. 193 pp. Gupta, R . P . , M.S. Kaira, S.C. Bhandari, and A.S. Khurana. 1983. Intrinsic multiple antibiotic resistance markers for competitive and effectiveness studies with various strains of mung bean rhizobia. J . Bioscience 5:253-260. Gupta, B .M. , and J . Kleczkowska. 1962. A study of some mutations in a strain of Rhizobium t r i f o l i i . J . Gen. Micro. 27:473-476. Hagedorn, C. 1979. Relationship of antibiotic resistance to effectiveness in Rhizobium trifolii populations. Soi l Sc i . Soc. Am. J . 43:921-925. Hagedorn, C , and B.A. Caldwell. 1981. Chracterization of diverse Rhizobium trifolii isolates. Soi l Sc i . Soc. Am. J . 45:513-516. Hale, M.G. , L.D. Moore, and G . J . G r i f f i n . 1978. Root exudates and exudation. In: Interactions Between Non-Pathogenic Soi l Microorganisms and Plants. Y.R. Dommergues and S.V. Krupa (Eds.). Elsevier Sc ient i f ic , Amsterdam/Oxford/New York. pp. 163-203. Hale, M . G . , and L.D. Moore. 1979. Factors affecting root exudation II. 1970-1978. Adv. Agron. 31:93-124. Hardarson, G . , and Jones, D.G. 1979. The inheritance of preference for strains of Rhizobium trifolii by white clover {Trifoli um repens). Ann. Appl. B i o l . 92:329-333. 249 Hardy, R.W.F., R.D. Holsten, E .K. Jackson, and R.C. Burns. 1968. The acetylene-ethylene assay for ^ fixation: labratory and f ie ld evaluation. Plant Physiol. 43:1185-1207. Harper, J . L . 1977. Population Biology of Plants. Academic Press, London. 892 pp. Hayman, D.S. 1978. Endomycorrhizae. In: Interactions Between Non-Pathogenic Soi l Microorganisms and Plants. Y.R. Dommergues and S.V. Krupa (Eds.). Elsevier Scient i f ic , Amsterdam/Oxford/New York. pp. 401-442. Hegazi, N .A. , H. Khawas, and M. Monib. 1981. Inoculation of wheat with Azospirillum under Egyptian conditions. In: Current Perspectives in Nitrogen Fixation. A.H. Gibson and W.E. Israel (Eds.). Australian Academy of Sciences, Canberra. p 493. Hobbs, S . L . A . , and J .D. Mahon. 1982. Effects of pea (Pi sum sativum) genotypes and Rhi zobi um I egumi nosarum strains on N 9 ( C H 9 ) fixation and growth. Can. J . Bot. 60:2594-26007 ^ * Hobbs, S . L . A . , and J .D. Mahon. 1983. Var iab i l i ty and interaction in the Pi sum sativum L . - R h i zobi um I egumi nosarum symbiosis. Can. J . Plant Sc i . 63:591-599. Hol l , F .B . 1983. Plant genetics: manipulation of the host. Can. J . Micro. 29:945-953. Hol l , F . B . , C P . Chanway, R. Turkington, and R.A. Radley. 1987. Response of crested wheatgrass (Agropyron cristatum L . ) , perennial ryegrass (Lolium perenne L . ) , and white clover (Trifolium repens L . ) to inoculation with Bacillus polymyxa. Soi l B i o l . Biochem.: in press. Howie, W . J . , and E . Echandi. 1983. Rhizobacteria: influence of cult ivar and so i l type on plant growth and yie ld of potato. Soi l B i o l . Biochem. 15:127-132. Hrabak, E . , M. Urbano, and F.B. Dazzo. 1981. Growth-phase-dependent immunodeterminants of Rhizobium trifolii lipopolysaccharide which bind t r i f o l i i n A, a white clover l ec t in . J . Bacteriol . 148:697-711. Hughes, D .Q. , and J .M. Vincent. 1942. Serological studies of the root-nodule bacteria. III. Tests of neighbouring strains of the same species. Proc. Linn. Soc. N.S.W. 67:142-152. Iruthayathas, E . E . , S. Gunasekaran, and K. Vlassak. 1983. Effect of combined inoculation of Azospirillum and Rhizobium on nodulation and N^-fixation of winged bean and soybean. Scientia Hort. 20:231-240. 250 Johnson, V . A . , and P . J . Mattern. 1977. Genetic improvement of productivity and nutrit ional quality of wheat. Report Research Findings U.S. Agency of International Development. 85 pp. Jones, D . G . , and G. Hardarson. 1979. Variation within and between white clover varieties in their preference for strains of Rhizobium t r i f o l i i . Ann. Appl. B i o l . 92:221-228. Josey, D .P . , J . L . Benyon, A.W.B. Johnston, and J . E . Beringer. 1979. Strain identif ication in Rhizobium using intrinsic antibiotic resistance. J . Appl. Bact. 46:343-350. Joy, P . , and A. Laitinen. 1980. Breeding for coadaptation between red clover and timothy. Hankkija's Seed Publ. No. 13. Hankkija Plant Breeding Institute, Finland, pp. 151-157. Kapulnik, Y . , M. Feldman, Y. Okon, and Y. Henis. 1985. Contribution of nitrogen fixed by Azospirillum to the N nutrition of spring wheat in Israel . Soi l B i o l . Biochem. 17:509-515. Kapulnik, Y . , J . Kige l , Y. Okon, I. Nur, and Y. Henis. 1981. Effect of Azospirillum inoculation on some growth parameters and N content of wheat, sorghum, and panicum. Plant and Soi l 61:65-70. Kapulnik, Y . , S. Sarig, I. Nur, and Y. Okon. 1983. Effect of Azospirillum inoculation on yie ld of f ie ld grown wheat. Can. J . Micro. 29:895-899. Katznelson, H. 1965. Nature and importance of the rhizosphere. In: Ecology of Soil-Borne Plant Pathogens. K . F . Baker and W.C. Snyder (Eds.). Univ. of C a l i f . Press, Berkeley. pp. 187-209. Katznelson, H . , and S.E. Cole. 1965. Production of g ibberel l in- l ike substances by bacteria and actinomycetes. Can. J . Micro. 11:733-741. Kavimandan, S.K. 1985. Root nodule bacteria to improve y ie ld of wheat (Tri ticum aestivum L . ) . Plant and Soi l 86: 141-144. Kavimandan, S.K. 1986. Influence of Rhizobial inoculation on y ie ld of wheat (Tri ti cum aestivum L . ) . Plant and Soi l 95:297-300. Keeling, B .L . 1974. Soybean seed rot and the relation of seed exudate to host susceptibi l i ty . Phytopath. 64:1445-1447. Kerr, A. 1980. Biological control of crown gal l through production of Agrocin 84. Plant Dis. 64:24-30. Kingman, A .R . , and J . Moore. 1982. Isolation, purification and quantitation of several growth regulating substances in Ascophyllum nodosum (Phaeophyt a). Botanica Marina 25:149-153. Kingsley, M . T . , and B.B. Bohlool. 1983. Characterization of Rhizobium sp. (Cicer arietinum L.) by immunofluoresence, immunodiffusion, and intr ins ic antibiotic resistance. Can. J . Micro. 29:518-526. Kipe-Nolt, J . A . , U.K. Avalakki, and P . J . Dart. 1985. Root exudation of sorghum and ut i l i zat ion of exudates by nitrogen-fixing bacteria. Soil B i o l . Biochem. 17:859-863. Kishinevsky, B . , and M. Bar-Joseph. 1978. Rhizobium strain identif ication in Arachis hypogaea nodules by enzyme-linked immunosorbent assay (ELISA). Can. J . Micro. 24:1537-1543. Kloepper, J .W. , J . Leong, M. Teintze, and M.N. Schroth. 1980. Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacteria. Nature 286:885-886. Kloepper, J . M . , M.N. Schroth, and T.D. M i l l e r . 1980. Effects of rhizosphere colonization by plant growth-promoting rhizobacteria on potato plant development and y ie ld . Phytopath. 70:1078-1082. Kloepper, J .W. , and M.N. Schroth. 1981. Plant growth-promoting rhizobacteria and plant growth under gnotobiotic conditions. Phytopath. 71:642-644. Klucas, R . V . , and W. Pedersen. 1980. Nitrogen fixation associated with roots of sorghum and wheat. In: Nitrogen Fixation. Vo l . II . W.E. Newton and W.H. Orme-Johnson (Eds.). University Park Press, Baltimore, pp. 243-255. Knowles, R. 1977. The significance of asymbiotic dinitrogen fixation by bacteria. In: A Treatise on Dinitrogen Fixation. Section IV. Agronomy and Ecology. R.W.F. Hardy and A.H. Gibson (Eds.). John Wiley and Sons, New York. pp. 33-83. Kraft, J .M. 1974. The influence of seedling exudates on the resistance of beans to Fusarium and Pythium root rot. Phytopath. 64:190-193. 252 K r a s i l ' n i k o v , N.A. 1958. S o i l Microorganisms and Higher P l a n t s . Acad. S c i . U.S.S.R. Y. H a l p e r i n ( T r a n s l a t o r ) . E n g l i s h e d i t i o n p u b l i s h e d f o r the N a t i o n a l Science Foundation and Department of A g r i c u l t u r e U.S.A. 474 pp. Kundu, B.S., and A.C. Gaur. 1980. Establishment of n i t r o g e n f i x i n g and phosphate s o l u b i l i z i n g b a c t e r i a i n rhizo s p h e r e and t h e i r e f f e c t on y i e l d and n u t r i e n t uptake of wheat crop. P l a n t and S o i l 57:223-230. Kurz, W.G.W., and T.A. La Rue. 1975. Nitrogenase a c t i v i t y i n Rhizobia i n absence of p l a n t host. Nature 256:407-409. Labandera, C. and J.M. V i n c e n t . 1975. Competition between an i n t r o d u c e d s t r a i n and n a t i v e Uruguayan s t r a i n s of Rhizobium trifolii. Plant and S o i l 42:327-347. Law, I . J . , Y. Yamamoto, A.J. Mort, and W.D. Bauer. 1982. Nodulation of soybean by Rhizobium j aponi cum mutants with a l t e r e d capsule s y n t h e s i s . P l a n t a 154:100-109. Le t h b r i d g e , G., and M.S. Davidson. 1983. R o o t - a s s o c i a t e d n i t r o g e n f i x i n g b a c t e r i a and t h e i r r o l e i n the n i t r o g e n n u t r i t i o n of wheat estimated by N iso t o p e d i l u t i o n . S o i l B i o l . Biochem. 15:365-374. L i , D., and D.H. H u b b e l l . 1969. I n f e c t i o n thread formation as a b a s i s of n o d u l a t i o n s p e c i f i c i t y i n Rhi zobi um-strawberry c l o v e r a s s o c i a t i o n s . Can. J . M i c r o . 15:1133-1 136. L i b b e r t , E., and R. M a n t e u f f e l . 1970. I n t e r a c t i o n s between p l a n t s and e p i p h y t i c b a c t e r i a r e g a r d i n g t h e i r auxin metabolism. V I I . The i n f l u e n c e of the e p i p h y t i c b a c t e r i a on the amount of d i f f u s a b l e auxin from corn c o l e o p t i l e s . P h y s i o l . P l a n t . 23:93-98. Lindemann, W.C, E.L. Schmidt, and G.E. Ham. 1 974. Evidence f o r double i n f e c t i o n with soybean nodules. S o i l S c i . 118:274-279. Linderman, R.G. 1986. Managing r h i z o s p h e r e microorganisms i n the p r o d u c t i o n of h o r t i c u l t u r a l c r o p s . H o r t s c i . 21:1299-1302. Lochhead, A.G. 1940. Q u a l i t a t i v e s t u d i e s of s o i l micro-organisms. I I I . I n f l u e n c e of p l a n t growth on the ch a r a c t e r of b a c t e r i a l f l o r a . Can. J . Res. 18:42-53. Lochhead, A.G., and F.E. Chase. 1943. Q u a l i t a t i v e s t u d i e s of s o i l micro-organisms. V. N u t r i t i o n a l requirements of the predominant b a c t e r i a l f l o r a . S o i l S c i . 55:185-195. 253 Loper, J.E., and M.N. S c r o t h . 1986. I n f l u e n c e of b a c t e r i a l sources of i n d o l e - 3 - a c e t i c a c i d on root e l o n g a t i o n of sugar beet. Phytopath. 76:386-389. Mahler, R.L., and D.F. Bezdicek. 1978. D i v e r s i t y of Rhizobium I egumi nosarum i n the Palouse of e a s t e r n Washington. Appl. Env. M i c r o . 36:780-782. Marques-Pinto, C , P.Y. Yao, and J.M. V i n c e n t . 1974. Nod u l a t i n g competitiveness amongst s t r a i n s of Rhizobium meliloti and R. t r i f o l i i . Aust. J . Agr. Res. 25:317-329. Marrack, J.R. 1963. S e n s i t i v i t y of methods d e t e c t i n g a n t i b o d i e s . B r i t . Med. B u l l . 19:178-182. Masterson, C.L., and M.T. Sherwood. 1974. S e l e c t i o n of Rhizobium trifolii s t r a i n s by white and subterranean c l o v e r s . I r . J . A g r i c . Res. 13 : 91-99. Materon, L.A., and C. Hagedorn. 1983. Competitiveness and symbiotic e f f e c t i v e n e s s of f i v e s t r a i n s of Rhizobium trifolii on red c l o v e r . S o i l S c i . Soc. Am. J . 47:491-495. Mayr-Harting, A., A.J. Hedger, and R.C.W. Ber k e l e y . 1972. Methods f o r st u d y i n g b a c t e r i o c i n s . In: Methods i n M i c r o b i o l o g y . V o l . 7A. J.R. N o r r i s and D.W. Ribbons ( E d s . ) . Academic Press, London, pp. 315-422. Means, U.M., H.W. Johnson, and R.A. Date. 1964. Quick s e r o l o g i c a l method of c l a s s i f y i n g s t r a i n s of Rhizobium j aponi cum i n nodules. J . B a c t e r i o l . 87:547-553. Means, U.M., H.W. Johnson, and L.W. Erdman. 1961. Competition between b a c t e r i a l s t r a i n s e f f e c t i n g n o d u l a t i o n i n soybeans. S o i l S c i . Soc. Am. Proc. 25:105-108. M e i j e r , E.G.M. 1982. Development of leguminous root nodules. In: N i t r o g e n F i x a t i o n Volume 2: Rhizobium. W.J. Broughton (Ed.). Clarendon Press, Oxford, pp. 312-331 . M i l l e r , C O . 1965. Evidence f o r the n a t u r a l occurence of z e a t i n and d e r i v a t i v e s . Compounds from maize which promote c e l l d i v i s i o n . Proc. Nat. Acad. S c i . U.S.A. 54:1052-1058. M i l l e t , E. , Y. A v i v i , and M. Feldman. 1985. E f f e c t s of r h i z o s p h e r i c b a c t e r i a on wheat y i e l d under f i e l d c o n d i t i o n s . P l a n t and S o i l 86:347-355. 254 M i l l e t , E., and M. Feldman. 1984. Y i e l d response of a common s p r i n g wheat c u l t i v a r to i n o c u l a t i o n with Azospirillum brasilense s t r a i n s under temperate c o n d i t i o n s . Can. J . Mic r o . 27:871-877. M i l l s , K.K., and W.D. Bauer. 1985. Rhizobium attachment to c l o v e r r o o t s . J . C e l l . S c i . Suppl. 2:333-345. M i s h u s t i n , E.N. 1963. B a c t e r i a l f e r t i l i z e r s and t h e i r e f f e c t i v e n e s s . M i k r o b i o l o g i y a 32:911-917. M i s h u s t i n , E.N., and A.N. Naumova. 1962. B a c t e r i a l f e r t i l i z e r s , t h e i r e f f e c t i v e n e s s and mode of a c t i o n . M i c r o b i o l o g y 31:442-452. Moore, L.W. 1979. P r a c t i c a l use and success of Agr obact er i um r adi obact er s t r a i n 84 f o r crown g a l l c o n t r o l . In: S o i l - B o r n e P l a n t Pathogens. B. Schippers and W. Gams (Eds.). Academic Press, London/New York/San F r a n s i s c o . pp. 553-568. Moore, L.W., and G. Warren. 1 979. Agr obact er i um radi obact er s t r a i n 84 and c o n t r o l of crown g a l l . Ann. Rev. Phytopath. 17:163-179. Mulder, E.G., and S. Brotonegoro. 1974. F r e e - l i v i n g h e t e r o t r o p h i c n i t r o g e n - f i x i n g b a c t e r i a . In: The B i o l o g y of Nitrogen F i x a t i o n . A. Q u i s p e l (Ed.). North H o l l a n d Pub. Co., Oxford, pp. 37-85. Murakami, Y. 1968. A new r i c e s e e d l i n g t e s t f o r G i b b e r e l l i n s "Microdrop Method". Bot. Mag. (Tokyo) 8:33-34. Murphy, P.M., and C L . Masterton. 1970. Determination of m u l t i p l e forms of e s t e r a s e s i n Rhizobium by paper e l e c t r o p h o r e s i s . J . Gen. M i c r o . 61:121-129. Mytton, L.R. 1975. Plant genotype x Rhizobium s t r a i n i n t e r a c t i o n s i n white c l o v e r . Ann. Appl. B i o l . 80:103-1 07. Mytton, L.R. 1978. Some problems and p o t e n t i a l i t i e s in breeding f o r improved white clover-R h i z o b i u m {Trifolium repens - Rhizobium trifolii) symbiosis. Ann. Appl. B i o l . 88:445-447. Mytton, L.R. 1981. Breeding legumes f o r symbiotic c h a r a c t e r s . In: Current P e r s p e c t i v e s i n Nitr o g e n F i x a t i o n . A.H. Gibson and W.E. Newton ( E d s . ) . A u s t r a l i a n Academy of S c i e n c e s , Canberra, p. 420. 255 Mytton, L.R., J . Br o c k w e l l , and A.H. Gibson. 1984. The p o t e n t i a l f o r breeding an improved lucerne-Rhi zobi um symbiosis. I. Assessment of g e n e t i c v a r i a t i o n . E u p h y t i c a 33:401-410. Mytton, L.R., and J . De F e l i c e . 1977. The e f f e c t of mixtures of Rhizobium s t r a i n s on the dry matter p r o d u c t i o n of white c l o v e r grown i n agar. Ann. Appl. B i o l . 87:83-93. Mytton, L.R., M.H. El-Sherbeeny, and D.A. Lawes. 1977. Symbiotic v a r i a b i l i t y i n Vicia faba. 3. Genetic e f f e c t s of host p l a n t , Rhizobium s t r a i n and of host x s t r a i n i n t e r a c t i o n . Euphytica 26:785-791. Mytton, L.R., and D.G. Jones. 1971. The response to s e l e c t i o n f o r i n c r e a s e d nodule t i s s u e i n white c l o v e r (Trifolium repens L . ) . Plant and S o i l Soec. V o l . pp. 17-25. Mytton, L.R., and D.M. Hughes. 1984. I n o c u l a t i o n of white c l o v e r with d i f f e r e n t s t r a i n s of Rhizobium trifolii on a mi n e r a l h i l l s o i l . J . A g r i c . S c i . 102:455-459. Mytton, L.R., and C.J. L i v e s l y . 1983. S p e c i f i c and general e f f e c t i v e n e s s of Rhizobium trifolii p o p u l a t i o n s from two d i f f e r e n t a g r i c u l t u r a l l o c a t i o n s . P l a n t and S o i l 73:299-305. N a p o l i , C.A., and D.H. H u b b e l l . 1975. U l t r a s t r u c t u r e of Rhi zobi um-induced i n f e c t i o n threads i n c l o v e r root h a i r s . Appl. Env. Micro. 30:1003-1009. Neal, J.R., J r . 1971. A simple method f o r enumeration of a n t i b i o t i c - p r o d u c i n g microorganisms i n the r h i z o s p h e r e . Can. J . Micro. 17:1143-1145. Neal, J.R., J r . , T.G. A t k i n s o n , and R.I. Larson. 1970. Changes i n the r h i z o s p h e r e m i c r o f l o r a of s p r i n g wheat induced by disomic s u b s t i t u t i o n of a chromosome. Can. J . M i c r o . 16:153-158. Neal, J.R., J r . , R.I. Larson, and T.G. A t k i n s o n . 1973. Changes i n r h i z o s p h e r e p o p u l a t i o n s of s e l e c t e d p h y s i o l o g i c a l groups of b a c t e r i a r e l a t e d to s u b s t i t u t i o n of s p e c i f i c p a i r s of chromosomes i n s p r i n g wheat. P l a n t and S o i l 39:209-212. Neal, J.R., J r . , and R.I. Larson. 1976. Ac e t y l e n e r e d u c t i o n by b a c t e r i a i s o l a t e d from the rh i z o s p h e r e of wheat. S o i l B i o l . Biochem. 8:151-155. 256 Newman, E.I . , R. Campbell, P. C h r i s t i e , A.J. Heap, and R.A. Lawley. 1979. Root microorganisms i n mixtures and monocultures of g r a s s l a n d p l a n t s . In: The S o i l - R o o t I n t e r f a c e . J.L. Harley and R. Scott Russel ( E d s . ) . Academic Press, London, pp.161-173. Newman, E.I . , and A.D. R o v i r a . 1975. A l l e l o p a t h y among some B r i t i s h g r a s s l a n d s p e c i e s . J . E c o l . 63:722-737. Neyra, C.A., and J . Dobereiner. 1977. Nitrogen f i x a t i o n in gr a s s e s . Adv. Agron. 29:1-38. N i t s c h , J.P., and C. N i t s c h . 1956. Studies on the growth of c o l e o p t i l e and f i r s t internode s e c t i o n s . A new, s e n s i t i v e , s t r a i g h t - g r o w t h t e s t f o r auxins. P l a n t P h y s i o l . 31:94-111. Noel, K.D., and W.J. B r i l l . 1980. D i v e r s i t y and dynamics of indigenous Rhizobium japoni cum p o p u l a t i o n s . Appl. Env. M i c r o. 40:931-938. Norman, A.G. 1955. The e f f e c t of polymyxin on p l a n t r o o t s . Arch. Biochem. Biophys. 58:461-477. Norman, A.G. 1961. M i c r o b i a l products a f f e c t i n g root development. Trans. 7th I n t . Congr. S o i l S c i . 2:531-536. N o r r i s , D.O. 1958. A red s t r a i n of Rhizobium from Lot ononis bainesii Baker. Aust. J . A g r i c . Res. 9:629-632. Nye, P.H. 1966. The e f f e c t of the n u t r i e n t i n t e n s i t y and b u f f e r i n g power of a s o i l and the absorbing power, s i z e , and root h a i r s of a root, on n u t r i e n t a b s o r p t i o n by d i f f u s i o n . P l a n t and S o i l 25:81-105. Obaton, M.M. 1971. U t i l i z a t i o n de mutants spontanes r e s i s t a n t s aux a n t i b i o t i q u e s pour l'etude e c o l o g i q u e des Rhi zobi um. C R . Acad. Sc. P a r i s , t . 272. S e r i e D:2630-2633. Ogan, M.T. 1979. P o t e n t i a l f o r n i t r o g e n f i x a t i o n i n the rhi z o s p h e r e and h a b i t a t i n n a t u r a l stands of the w i l d r i c e Zizania aquatica. Can. J . Bot. 57:1285-1291. O'Hara, G.W., M.R. Davey, and J.A. Lucas. 1981. E f f e c t of i n o c u l a t i o n of Zea mays with Azospirillum brasilense s t r a i n s under temperate c o n d i t i o n s . Can. J . Micro. 27:871-877. Okon, Y., Y. Kapulnik, S. S a r i g , I. Nur, and Y. Henis. 1980. Y i e l d i n c r e a s e s on a commercial s c a l e i n grasses i n o c u l a t e d with Azospirillum i n I s r a e l . In: A b s t r a c t s S o c i e t y f o r A p p l i e d B a c t e r i o l o g y Symposium on B a c t e r i a and P l a n t s . Aberystwyth, Wales, p. 32. 257 Okon, Y., Y. Kapulnik, S. S a r i g , I. Nur, and Y. Henis. 1981. Azospirillum i n c r e a s e s c e r e a l crop y i e l d s i n f i e l d s of I s r a e l . In: Current P e r s p e c t i v e s i n Ni t r o g e n F i x a t i o n . A.H. Gibson and W.E. Newton ( E d s . ) . A u s t r a l i a n Academy of S c i e n c e s , Canberra. p. 492. Old, K.M., and Z.A. P a t r i c k . 1979. Giant s o i l amoebae, p o t e n t i a l b i o c o n t r o l agents. In: S o i l - B o r n e Plant Pathogens. B. Schippers and W. Gams (E d s . ) . Academic Press, London/New York/San F r a n s i s c o . pp. 617-628. Olsen, P.E., and W.A. R i c e . 1983. S t r a i n - s p e c i f i c s e r o l o g i c a l techniques f o r the i d e n t i f i c a t i o n of Rhizobium meliloti i n commercial a l f a l f a i n o c u l a n t s . Can. J . Micro. 29:225-230. Ouchterlony, 0. 1968. Handbook of Immunodiffusion and Immunoelectrophoresis. Ann Arbor Science P u b l i s h e r s Inc., Ann Arbor, Michigan. 215 pp. Parker, C.A., and P.L. Grove. 1970. The r a p i d s e r o l o g i c a l i d e n t i f i c a t i o n of Rhizobia i n small nodules. J . Appl. Bact. 33:248-252. Parker, C.A., M.J. T r i n i c k , and D.L. C h a t e l . 1977. Rhizobia as s o i l and r h i z o s p h e r e i n h a b i t a n t s . In: A T r e a t i s e on D i n i t r o g e n F i x a t i o n . S e c t i o n IV. Agronomy and Ecology. R.W.F. Hardy and A.H. Gibson (Eds. ) . John Wiley and Sons, New York. pp.31 1-352. Parry, J.M., P.C.B. T u r n b u l l , and J.R. Gibson. 1983. A C o l o r A t l a s of Bacillus S p e c i e s . Wolfe Me d i c a l P u b l i c a t i o n s , London. 272 pp. Pegg, G.F. 1985. Pathogenic and non-pathogenic microorganisms and i n s e c t s . In: E n c y c l o p e d i a of P l a n t P h y s i o l o g y . New S e r i e s V o l . I I . Hormonal Re g u l a t i o n of Developement I I I . Role of Environmental F a c t o r s . R.P. P h a r i s and D.M. Reid ( E d s . ) . S p r i n g e r - V e r l a g , New York, pp. 599-624. Person, C. 1959. Gene-for-gene r e l a t i o n s h i p s i n h o s t : p a r a s i t e systems. Can. J . Bot. 37:1101-1130. Peters e n , E.A., J.C. S i r o i s , W.B. Berndt, and R.W. M i l l e r . 1983. E v a l u a t i o n of c o m p e t i t i v e a b i l i t y of Rhizobium meliloti s t r a i n s f o r n o d u l a t i o n i n a l f a l f a . Can. J . M i c r o . 29:541-545. P h i l l i p s , D.A., and J.G. T o r r e y . 1972. S t u d i e s on c y t o k i n i n p r o d u c t i o n by Rhizobium. P l a n t P h y s i o l . 49:11-15. Postgate, J.R. 1982. The Fundamentals of N i t r o g e n F i x a t i o n . Cambridge U n i v e r s i t y Press, Cambridge. 252 pp. 258 Primrose, S.B. 1976. Ethylene-forming b a c t e r i a from s o i l and water. J . Gen. M i c r o . 97:343-346. Purchase, H.F., and J.M. V i n c e n t . 1949. A d e t a i l e d study of the f i e l d d i s t r i b u t i o n of s t r a i n s of c l o v e r nodule b a c t e r i a . Proc. L i n n . Soc. N.S.W. 74:227-236. Rennie, R.J. 1980. D i n i t r o g e n - f i x i n g b a c t e r i a : computer-a s s i s t e d i d e n t i f i c a t i o n of s o i l i s o l a t e s . Can. J . Micro. 26:1275-1283. Rennie, R.J. 1981a. A s i n g l e medium f o r the i s o l a t i o n of a c e t y l e n e - r e d u c i n g ( d i n i t r o g e n - f i x i n g ) b a c t e r i a from s o i l s . Can. J . M i c r o . 27:8-14. Rennie, R.J. 1981b. P o t e n t i a l use of induced mutations to improve symbiosis of crop p l a n t s with N f i x i n g b a c t e r i a . In: Induced Mutations - A T o o l i n Plant Breeding. I n t . Atomic Agency, Vienna. pp. 293-321. Rennie, R.J., and R.I. Larson. 1979. Ni t r o g e n f i x a t i o n a s s o c i a t e d with disomic chromosome s u b s t i t u t i o n l i n e s of s p r i n g wheat. Can. J . Bot. 57:2771-2775. Rennie, R.J., and R.I. Larson. 1981. D i n i t r o g e n f i x a t i o n a s s o c i a t e d with disomic chromosome s u b s t i t u t i o n l i n e of s p r i n g wheat i n the phytotron and i n the f i e l d . In: A s s o c i a t i v e N 2 - F i x a t i o n . V o l . 1. P.B. Vose and A.P. Ruschel (Eds.7. CRC Press, Boca Raton, F l o r d i a . pp. 145-154. Rennje, R.J., D.A. Rennie, and M. F r i e d . 1977. Concepts of N usage i n d i n i t r o g e n f i x a t i o n s t u d i e s . In: Isotopes i n B i o l o g i c a l D i n i t r o g e n F i x a t i o n ( P r o c . ) . I n t . Atomic Energy Agency, Vienna, pp. 107-133. Rennie, R.J., and D.A. Rennie. 1983. Techniques f o r q u a n t i f y i n g N 2 f i x a t i o n i n a s s o c i a t i o n with nonlegumes under f i e l d and greenhouse c o n d i t i o n s . Can. J . Micro. 29:1022-1035. Reynders, L., and K. V l a s s a k . 1982. Use of Azospirillum brasilense as b i o f e r t i l i z e r i n i n t e n s i v e wheat cropping. P l a n t and S o i l 66:217-223. Roberts, G.P., W.T. Leps, L.E. S i l v e r , and W.J. B r i l l . 1980. Use of two-dimensional p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s to i d e n t i f y and c l a s s i f y Rhizobium s t r a i n s . Appl. Env. Micro. 39:414-422. Robinson, A.C. 1967. The i n f l u e n c e of host on s o i l and r h i z o s p h e r e p o p u l a t i o n s of c l o v e r and lu c e r n e nodule b a c t e r i a in the f i e l d . J . Aust. I n s t . A g r i c . S c i . 33:207-209. Robinson, A.C. 1969. Host s e l e c t i o n f o r e f f e c t i v e Rhizobium trifolii by red c l o v e r and subterranean c l o v e r i n the f i e l d . Aust. J . Agr. Res. 20:1053-1060. Rouatt, J.W., and H. K a t z n e l s o n . 1961. A study of b a c t e r i a on the root s u r f a c e and i n the r h i z o s p h e r e s o i l of crop p l a n t s . J . Appl. Bact. 24:164-171. R o v i r a , A.D. 1959. Root e x c r e t i o n s i n r e l a t i o n to the r h i z o s p h e r e e f f e c t . IV. I n f l u e n c e of p l a n t s p e c i e s , age of p l a n t , l i g h t , temperature, and c a l c i u m n u t r i t i o n on exudation. Plant and S o i l 11:53-64. R o v i r a , A.D. 1961. Rhi zobi um numbers in the r h i z o s p h e r e of red c l o v e r and Pas pal um in r e l a t i o n to s o i l treatment and the numbers of b a c t e r i a and f u n g i . Aust. J . Agr. Res. 12:77-83. Ro v i r a , A.D. 1963. M i c r o b i a l i n o c u l a t i o n of p l a n t s . I. Establishment of f r e e - l i v i n g n i t r o g e n - f i x i n g b a c t e r i a i n the rhizosphere and t h e i r e f f e c t s on maize, tomato, and wheat. Pl a n t and S o i l 19:304-314. R o v i r a , A.D. 1969. P l a n t root exudates. Bot. Rev. 35:35-57. R o v i r a , A.D. 1971. P l a n t root exudates. In: Biochemical I n t e r a c t i o n s among P l a n t s . Environmental P h y s i o l o g y Subcommittee. U.S. N a t i o n a l Committee I.B.P. Work Conference, 1968. Nat. Acad. S c i . , Wash. D.C. pp. 19-24. R o v i r a , A.D. 1978. M i c r o b i o l o g y of pasture s o i l s and some e f f e c t s of microorganisms on pasture p l a n t s . In: P l a n t R e l a t i o n s i n P a s t u r e s . J.R. Wilson (Ed.). Commonwealth S c i e n t i f i c and I n d u s t r i a l Research O r g a n i s a t i o n , East Melbourne. pp. 95-110. R o v i r a , A.D., and C B . Davey. 1974. B i o l o g y of the r h i z o s p h e r e . In: The P l a n t Root and i t s Environment. E.W. Carson (Ed.). Univ. Press of V i r g i n i a , C h a r l o t t e s v i l l e . pp. 153-204. R o v i r a , A.D., and W.R. S t e r n . 1961. Rhizosphere b a c t e r i a i n g r a s s - c l o v e r a s s o c i a t i o n s . Aust. J . Agr. Res. 12:1108-1118. R u s s e l l , E.W. 1973. S o i l C o n d i t i o n s and P l a n t Growth, 10th e d i t i o n . Longmans, London. 849 pp. R u s s e l l , P.E., and D.G. Jones. 1975. V a r i a t i o n i n the s e l e c t i o n of Rhi zobi um trifolii by v a r i e t i e s of red and white c l o v e r . S o i l B i o l . Biochem. 7:15-18. Schmidt, E.L. 1978. Ecology of the legume root nodule b a c t e r i a . In: I n t e r a c t i o n s Between Non-Pathogenic S o i l Microorganisms and P l a n t s . Y.R. Dommergues and S.V. Krupa ( E d s . ) . E l s e v i e r S c i e n t i f i c , Amsterdam/Oxford/New York. pp. 269-303. Schroth, M.N., and J.G. Hancock. 1981. S e l e c t e d t o p i c s i n b i o l o g i c a l c o n t r o l . Ann. Rev. Micro. 35:453-476. Schroth, M.N., and A.R. Weinhold. 1986. R o o t - c o l o n i z i n g b a c t e r i a and p l a n t h e a l t h . H o r t s c i . 21:1295-1298. Schwinghamer, E.A. 1967. E f f e c t i v e n e s s of Rhizobium as mo d i f i e d by mutation f o r r e s i s t a n c e to a n t i b i o t i c s . Antonie van Leeuwenhoek 33:121-136. Schwinghamer, E.A. 1977. Genetic a s p e c t s of n o d u l a t i o n and d i n i t r o g e n f i x a t i o n by legumes: The microsymbiont. In: A T r e a t i s e on D i n i t r o g e n F i x a t i o n . S e c t i o n I I I . B i o l o g y . R.W.F. Hardy (General E d i t o r ) and W.S. S i l v e r ( S e c t i o n E d i t o r ) . John Wiley and Sons, New York. pp. 577-622. Schwinghamer, E.A., and W.F. Dudman. 1980. Methods f o r i d e n t i f y i n g s t r a i n s of d i a z o t r o p h s . In: Methods f o r E v a l u a t i n g B i o l o g i c a l N i t r o g e n F i x a t i o n . F . J . Bergersen (Ed.). John Wiley and Sons, New York. pp. 337-365. S i n c l a i r , M.J., and A.R.J. Eaglesham. 1984. I n t r i n s i c a n t i b i o t i c r e s i s t a n c e i n r e l a t i o n to colony morphology in three p o p u l a t i o n s of west A f r i c a n cowpea Rhi zobi a. S o i l B i o l . Biochem. 16:247-251. Singh, C.S., and N.S. Subba Rao. 1979. A s s o c i a t i v e e f f e c t of Azospirillum brasilense and Rhizobium j aponi cum on n o d u l a t i o n and y i e l d of soybean {Glycine max). P l a n t and S o i l 53:387-392. S k r e d l e t a , V., and J . Karimova. 1969. Competition between two somatic serotypes of Rhi zobi um japonicum used as d o u b l e - s t r a i n i n o c u l a i n v a r y i n g p r o p o r t i o n s . Arch. M i k r o b i o l . 66:25-28. Smith, J.H., F.E. A l l i s o n , and D.A. S o u l i d e s . 1961. E v a l u a t i o n of phosphobacteria as a s o i l i n o c u l a n t . S o i l S c i . Soc. Am. Proc. 25:109-111. Smith, G.R., W.E. Knight, H.H. Petersen, and C. Hagedorn. 1982. The e f f e c t of Rhi zobi um trifolii s t r a i n s and crimson c l o v e r genotypes on N f i x a t i o n . Crop S c i . 22:970-972. Smith, M.S., and CW. R i c e . 1986. The r o l e microorganisms i n the s o i l n i t r o g e n c y c l e . In: M i c r o f l o r a l and Faunal I n t e r a c t i o n s i n N a t u r a l and Agro-Ecosystems. M.J. M i t c h e l l and J.P. Nakas ( E d s . ) . Martinus N i j h o f f / D r W. Junk P u b l i s h e r s , Dordrecht/Boston/Lancaster. pp. 245-284. Sperber, J . I . , and A.D. R o v i r a . 1959. A study of the b a c t e r i a a s s o c i a t e d with the roo t s of subterranean c l o v e r and Wimmera r y e - g r a s s . J . Ap p l . Bact. 22:85-95. S t e i n , M. , E.S.P. B r o m f i e l d , and M. Dye. 1982. An assessment of a method based on i n t r i n s i c a n t i b i o t i c r e s i s t a n c e f o r i d e n t i f y i n g Rhizobium s t r a i n s . Ann. Appl. B i o l . 101 :261-267. Subba Rao, N.S. 1980. Crop response t o m i c r o b i a l i n o c u l a t i o n . In: Recent Advances i n B i o l o g i c a l N i t r o g e n F i x a t i o n . N.S. Subba Rao (Ed.). A r n o l d , London. pp. 406-420. Subba Rao, N.S. 1982. Phosphate s o l u b i l i z a t i o n by s o i l microorganisms. In: Advances i n A g r i c u l t u r a l M i c r o b i o l o g y . N.S. Subba Rao (Ed.). Buttersworth S c i e n t i f i c , Toronto. pp. 295-303. Subba Rao, N.S., K.V.B.R. T i l a k , C S . Singh, and M. Lakshmi-Kumari. 1979. Response to a few economic s p e c i e s of graminaceous p l a n t s to i n o c u l a t i o n with Azospirillum brasilense. Current Science 48:133-134. Subba Rao, N.S., K.V.B.R. T i l a k , and C S . Singh. 1985. F i e l d response of crops to i n o c u l a t i o n with Azospirillum brasilense i n I n d i a . Z b l . M i k r o b i o l . 140:97-102. Subba Rao, N.S., K.V.B.R. T i l a k , and C S . Singh. 1986. Dual i n o c u l a t i o n with Rhizobium sp. and Glomus fasciculatum enhances n o d u l a t i o n , y i e l d , and n i t r o g e n f i x a t i o n i n chickpea (Cicer arietinum L i n n . ) . P l a n t and S o i l 95:351-359. Sundara-Rao, W.V.B., P.D. B a j p a i , J.P. Sharma, and B.V. Subbiah. 1963. S o l u b i l i z a t i o n of phosphates by phosphorus s o l u b i l i z i n g organisms u s i n g P as t r a c e r and the i n f l u e n c e of seed b a c t e r i z a t i o n on the uptake by the crop. Ind. Soc. S o i l S c i . 11:209-219. Suslow, T.V. 1982. Role of r o o t - c o l o n i z i n g b a c t e r i a i n p l a n t growth. In: Phytopathogenic P r o k a r y o t e s . V o l . 1. M.S. Mount and G.H. Lacy ( E d s . ) . Academic Press, New York. pp. 187-223. Suslow, T.V., and M.N. Schroth. 1982. Role of d e l e t e r i o u s r h i z o b a c t e r i a as minor pathogens i n reducing crop growth. Phytopath. 72:111-115. S z y b a l s k i , W . , and V. Bryson. 1952. Genetic s t u d i e s on the m i c r o b i a l c r o s s r e s i s t a n c e to t o x i c agents. I. Cross r e s i s t a n c e of Escherichia coli to f i f t e e n a n t i b i o t i c s . J . Bact. 64:489-499. Tarrand, J . J . , N.R. K r i e g , and J . Doberiener. 1978. A taxonomic study of the Spirillum lipoferum group, with d e s c r i p t i o n s of a new genera, Azospirillum gen. nov. and two s p e c i e s , Azospirillum lipoferum ( B e i j e r i n c k ) comb. nov. and Azospirillum brasilense sp. nov. Can. J . M i c ro. 24:967-980. T i e n , T.M., M.H. Gaskins, and D.H. Hu b b e l l . 1979. Pla n t growth substances produced by Azospirillum brasilense and t h e i r e f f e c t on the growth of p e a r l m i l l e t (Pennisetum americanum L . ) . Appl. Env. Mi c r o . 37:1016-1024. T i n k e r , P.B. 1984. The r o l e of microorganisms i n mediating and f a c i l i t a t i n g the uptake of p l a n t n u t r i e n t s from s o i l . P l a n t and S o i l 76:77 - 9 1 . T r i n i c k , M.J. 1969. I d e n t i f i c a t i o n of legume nodule b a c t e r o i d s by the f l u o r e s c e n t antibody r e a c t i o n . J . Appl. Bact. 32:181-186. T r i n i c k , M.J. 1982. B i o l o g y . In: Nitrogen F i x a t i o n Volume 2: Rhizobium. W.J. Broughton (Ed.). Clarendon Press, Oxford, pp 76-146. Turkington, R. 1985. V a r i a t i o n and d i f f e r e n t i a t i o n i n po p u l a t i o n s of Trifolium repens i n permanent p a s t u r e s . In: S t u d i e s on Plant Demography: A F e s t s c h r i f t f o r John L. Harper. J . White (Ed.). Academic Press, London. pp. 69-82. Turkington, R., and J.L. Harper. 1979. The growth, d i s t r i b u t i o n , and neighbour r e l a t i o n s h i p s of Trifolium repens i n a permanent p a s t u r e . IV. F i n e - s c a l e b i o t i c d i f f e r e n t i a t i o n . J . E c o l . 67:245-254. Turner, S.M., and E . I . Newman. 1984. Growth of b a c t e r i a on r o o t s of grasse s : i n f l u e n c e of min e r a l n u t r i e n t supply and i n t e r a c t i o n s between s p e c i e s . J . Gen. Micro. 130:505-512. Tuzimura, K., and I. Watanabe. 1961. The growth of Rhi zobi um i n the rh i z o s p h e r e of the host p l a n t . E c o l o g i c a l s t u d i e s of root nodule b a c t e r i a (Part 2). S o i l S c i . P l a n t Nutr. (Tokyo) 8:19-24. Tuzimura, K., and I. Watanabe. 1962. The e f f e c t of rhi z o s p h e r e of v a r i o u s p l a n t s on the growth of Rhizobium. E c o l o g i c a l s t u d i e s of root nodule b a c t e r i a (Part 3 ) . S o i l S c i . P l a n t Nutr. (Tokyo) 8:13-17. van Egeraat, A.W.S.M. 1975. The p o s s i b l e r o l e of homoserine i n the development of Rhi zobi um Iegumi nosarum i n the rh i z o s p h e r e of pea s e e d l i n g s . Plant and S o i l 42:381-386. Venkateswarlu, B., and A.V. Rao. 1983. Response of p e a r l m i l l e t to i n o c u l a t i o n with d i f f e r e n t s t r a i n s of Azospirillum brasilense. P l a n t and S o i l 74:379-386. Vesper, S.J., and W.D. Bauer. 1986. Role of p i l i ( f i m b r i a e ) i n attachment of Bradyrhi zobi um j aponi cum to soybean r o o t s . Appl. Env. Micro. 52:134-141. Vidaver, A.K. 1976. Prospects f o r c o n t r o l of phytopathogenic b a c t e r i a by bacteriophages and b a c t e r i o c i n s . Ann. Rev. Phytopath. 14:451-465. Vi n c e n t , J.M. 1941. S e r o l o g i c a l s t u d i e s of the r o o t -nodule b a c t e r i a . Proc. L i n n . Soc. N.S.W. 66:145-154. Vi n c e n t , J.M. 1970. A Manual f o r the P r a c t i c a l Study of Root-Nbdule B a c t e r i a . I BP Handbook No. 15. B l a c k w e l l S c i e n t i f i c , Oxford. 164 pp. Vinc e n t , J.M. 1982. Serology. In: Nitrogen F i x a t i o n Volume 2: Rhi zobi um. W.J. Broughton (Ed.). Clarendon Press, Oxford. pp. 235-273. Vi n c e n t , J.M., and L.M. Waters. 1953. The i n f l u e n c e of the host on competition amongst c l o v e r root-nodule b a c t e r i a . J . Gen. Micro. 9:357-370. Vlassak, K., and L. Reynders. 1981. Azospirillum r h i z o c o e n o s i s i n a g r i c u l t u r a l p r a c t i s e . In: Current P e r s p e c t i v e s i n Nitrogen F i x a t i o n . A.H. Gibson and W.E. Newton ( E d s . ) . A u s t r a l i a n Academy of Sc i e n c e s , Canberra. p. 494. V o l l e r , A., A. B a r t l e t t , D.E. B i d w e l i , M.F. C l a r k , and A.N. Adams. 1976. The d e t e c t i o n of v i r u s e s by enzyme-linked immunosorbent assay (ELISA). J . Gen. V i r . 33:165-167. Vose, P.B., and A.P. Ruschel ( E d i t o r s ) . 1981. A s s o c i a t i v e N 9 - F i x a t i o n . CRC Press, Boca Raton, F l o r i d a . VolT I, 215 pp.; V o l . I I , 269 pp. Walton, P.D. 1983. Produ c t i o n and management of c u l t i v a t e d f o r a g e s . Reston P u b l i s h i n g Co., Reston, V i r g i n i a . 336 pp. 264 Warembourg, F.R., and R.A.A. M o r r a l l . 1878. Energy flow i n the plant-microorganism system. In: I n t e r a c t i o n s between Non-Pathogenic S o i l Microorganisms and P l a n t s . Y.R. Domergues and S.V. Krupa (Eds.). E l s e v i e r S c i e n t i f i c , Amsterdam/Oxford/New York. pp. 205-268. Witty, J.F. 1979. Overestimate of N»-fixation i n the rhi z o s p h e r e by the a c e t y l e n e r e d u c t i o n method. In: The S o i l - R o o t I n t e r f a c e . J.L. Hardy and R. S c o t t Russel (Eds.). Academic Press, London/New York. pp. 137-144. Witty , J ' F ^ c 1983. E s t i m a t i n g N 2 f i x a t i o n i n the f i e l d u s i n g N - l a b e l l e d f e r t i l i z e r : Some problems and s o l u t i o n s . S o i l B i o l . Biochem. 15:631-639. Witty , J.F., and J.M. Day. 1977. Use of 1 5 N „ in e v a l u a t i n g asymbiotic N 2 f i x a t i o n . In: Isotopes i n B i o l o g i c a l D i n i t r o g e n F i x a t i o n ( P r o c . ) . I n t . Atomic Energy Agency, Vienna, pp. 135-150. Woods, F.W_, and Brock. 1964. I n t e r s p e c i f i c t r a n s f e r of Ca and P by root systems. Ecology 63:886-889. Wright, S.F., J.G. F o s t e r , and O.L. Bennett. 1986. Produc t i o n and use of monoclonal a n t i b o d i e s f o r i d e n t i f i c a t i o n of s t r a i n s of Rhizobium trifolii. A p p l . Env. Mi c r o . 52:119-123. W u l l s t e i n , L.H., M.L. Bruening, and W.B. B o l l e n . 1979. Nit r o g e n f i x a t i o n a s s o c i a t e d with sand g r a i n root sheaths ( r h i z o s h e a t h s ) of c e r t a i n x e r i c g r a s s e s . P h y s i o l . P l a n t . 46:1-4. Young, N.R., and L.R. Mytton. 1983. The response of white c l o v e r to d i f f e r e n t s t r a i n s of Rhi zobi um trifolii i n h i l l l a n d r e s e e d i n g . Grass and Forage S c i . 38:13-19. Yuen, G.Y., and M.N. Schroth. 1986. I n t e r a c t i o n s of Pseudomonas fluorescens s t r a i n E6 with ornamental p l a n t s and i t s e f f e c t on the composition of r o o t -c o l o n i z i n g m i c r o f l o r a . Phytopath. 76:176-180. Zelazna-Kowalska, I. 1971. C o r r e l a t i o n between streptomycin r e s i s t a n c e and i n f e c t i v e n e s s i n Rhizobium trifolii. P l a n t and S o i l Spec. V o l . pp. 67-71. 

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