0014-4754/94/100884-0651.50 + 0.20 9 Birkh/iuser Verlag Basel, 1994

Competitiveness and communication for effective inoculation by Rhizobium, Bradyrhizobium and vesieular-arbuscular mycorrhiza fungi
D. Werner, S. Bernard, E. G6rge, A. Jacobi, R. Kape, K. Kosch, P. Miiller, M. Parniske, S. Schenk, P. Schmidt and W. Streit

Fachbereich Biologie der Philipps-Universitiit, Karl-von-Frischstrasse, D-35032 Marburg (Germany)

Abstract. After a short summary on the ecology and rhizosphere biology of symbiotic bacteria and vesicular-arbuscular (VA) mycorrhiza fungi and their application as microbial inocula, results on competitiveness and communication are summarized. Stress factors such as high temperature, low soil pH, aluminium concentrations and phytoalexins produced by the host plants were studied with Rhizobium leguminosarum by. phaseoli and Rhizobium tropici on Phaseolus beans. Quantitative data for competitiveness were obtained by using gus + (glucoronidase) labelled strains, which produce blue-coloured nodules. For Phaseolus-nodulating rhizobia, a group specific D N A probe was also developed, which did not hybridize with more than 20 other common soil and rhizosphere bacteria. Results from several laboratories contributing to knowledge of signal exchange and communication in the Rhizobium/Bradyrhizobium legume system are summarized in a new scheme, including also defense reactions at the early stages of legume nodule initiation. Stimulating effects of flavonoids on germination and growth of VA mycorrhiza fungi were also found. A constitutive antifungal compound in pea roots, /~-isoxazolinonyl-alanine, was characterized. Key words. Bradyrhizobium; communication; competitiveness; Rhizobium; vesicular-arbuscular (VA) mycorrhiza fungi. Introduction
The ecology of symbiotic microorganisms, and their use for inoculation of soils to increase the yield of crop plants by improving their nutrient supply, have recently been reviewed in references 1, 3, 4, 7, 18, 23, 31 and 43. The genetic basis and biochemical characters which control the competitiveness of single strains within one infection group of Rhizobium or Bradyrhizobium are not understood in detail. Often, very efficient nitrogen-fixing and nodulating laboratory strains are not competitive against endogenous strains of the same species under field conditions. On the other hand, there is an increasing understanding of the molecular interactions involved in symbiotic communication 6' ~0. In the following we will summarize and discuss new experimental data from our work to characterize competitiveness and communication in rhizobia, and some results with vesicular arbuscular (VA) mycorrhiza fungi. more sensitive than other strains to lower soil pH, and to tannins and phytoalexins produced by the host plants 45. The conclusion is that it is very difficult to combine the highest resistance against all relevant soil and stress factors into one competitive strain. Sensitivity of the Rhizobium strains towards tannins was greater at acidic pH 46. A large proportion of the arable land in the tropics has acidic soils. Soil pH there is a major factor limiting nodulation and nitrogen fixation in Phaseolus beans 4~. Co-inoculation with different strains at the same time proved not to be a successful strategy to overcome this problem, since the number of nodules formed by specific inoculant strains is affected by the competition of other strains ~1"37.

Quantification of competitiveness: gus technology and gene probes
To improve the methods for quantification of competitiveness a gus + (glucoronidase) strain of R. leguminosarum bv. phaseoIi KIM5s was constructed and used in competition experiments against 17 other strains of R. Ieguminosarum by. phaseoli and three strains of R. tropici. The results of these competition experiments at two pH levels (5.2 and 6.4) are summarized in table 1. The gus technology is very useful for direct identification of a single strain, because the nodules produced by the gus + strain are blue in colour, in contrast to the red or pink nodules produced by the other inoculant strains. For better visibility of the root system, including the nodules,

Competitiveness and stress factors
In Phaseolus beans under field conditions, a response to inoculation with specific strains of Rhizobium Ieguminosarum bv. phaseoli, R. tropici or R. etli, which can all form nodules in these host plants, is more the exception than the rule 41. Studying the resistance of R. leguminosarum by. phaseoli and R. tropici strains towards different stress factors, it was evident that the most competitive strain (KIM5) was rather tolerant against high temperature and aluminium concentrations, but

2. tropici strains within a single day. Cooper in Belfast.05. 25. modified from Werner et al. CH-4010 Basel/Switzerland 885 Table 1. nodule occupancy between 4% for the least competitive strain and up to 96% for the most competitive strain could be observed (see table 1). The competitiveness of R. The specificity of this D N A probe is summarized in table 2. The probe hybridizes completely with all t 7 strains tested from the Phaseolus infection group and is negative against 34 other soil bacteria such as Agrobacterium tumefaciens. tropici strains on aromatic compounds was also shown z9.4 pH 5. Growth of R. phaseoli were unable to metabolize ferulate or coumarate.2 ND ND 10 -4-3 67__+20* ND 82 _ 20* 23 • 8 76 __+18" 33 _ 10" 72 __+15 73 _ 22 ND 67 ~ 12 ND 90 __+10 86 ___ 4 95 • 5 93 • 6 86 __+13 96 + 2 CIAT 2510 H2C CIAT 323 CFN 1500 CE3 C-05-II CIAT 7004 CIAT 899 CIAT 895 Tal 182 CIAT 652 Costa 6 CIAT 7014 CFN 227 CIAT 151 CIAT 904 CIAT 163 CIAT 956 CFN 1600 CIAT 611 4+ 2 4• 4 9 _+5 ll • 11 23 • 2 25 • 17 30 __+ 4 41 _+4 54 • 10 68 • 79 _+4 79 _+ 11 82 • 7 85 _+11 89 _+10 91 + 4 91 • 8 93 _+6 94 + 8 96 _+4 the R. Bacillus subtilis. Soils 14 (1992) 140-I44. legurninosarum by. Pseudornonas putida. *p < 0. 28) Figure i. using subtraction hybridization and polymerase chain reaction amplification for the isolation of strainspecific Rhizobium D N A sequences 2.47. whereas. phaseoli and R. Most of the less competitive strains of R. Microsymbiont-legumehost plant communication during the early stages of nodule initiation. phaseoli strains was also affected by pH. For the R. 1). Nodule occupancy in different Rhizobium leguminosarum by. D N A probes were developed. tropicistrains are underlined. The utilization of 43 different carbon sources by these strains was studied. 30) Root hair curling (38) Meristem induction (32) Phytoalexin resistance induction (24) V Phytoalexin degradation (8) C-ring cleavage of flavonoids (29) Utilization of aromatic compounds as C-sources (39) Lectins and ENOD ~ k functions in early nodule development (12) EPS and LPS functions for " ~ infection and competition (14. a soil titer as low as 3 x 104 homologous indigenous rhizobia per g soil could be detected 4~ Means + SD of two replicates of five plants each. To improve the rapid identification of specific Rhizobium species and infection groups. Pseudornonas fluorescens. Biol Fertil.Reviews Experientia 50 (1994). R. . leguminosarum bv. phaseoli and Rhizobium tropici specific D N A probe. legurninosarum bv. ~ t . leguminosarum by. Only one round of subtraction hybridization was necessary for removing all cross-hybridizing sequences.44.4. The patterns offlavonoids present so-called growth pouches made of plastic material are used 39. but a much better competitiveness at pH 5. Birkh/iuser Verlag. References are given in brackets. whereas a 1 2 week test period is needed when plant inoculation is used. tropici studied had a very low nodule occupancy at p H 6. Assays were performed in growth pouches at two pH levels. Xanthomonas campestris and various other Rhizobium and Bradyrhizobium species. With this D N A probe. total DNA preparations from 8 different other Rhizobium species and soil bacteria were pooled and used as a subtracter D N A against total genomic D N A from Microsymbionts Host plants Flavonoids from seed exudation (5) Chemotaxis (t5) j~l~ Flavonoids from root Nod gene induction (9. the highly competitive strains had those catalytic capacities 39. phaseofi strain KIM5s. using the subtraction hybridization method 4~ This method was developed in the laboratory of J. This D N A technology allows the rapid identification of the R.4. With this technology. strain Nodule occupancy/KIM5s (%) pH 6. leguminosarum bv. Requests from more than 30 different countries regarding this D N A probe and probe technique indicate the usefulness of this technology. phaseoli and Rhizobium tropici strains when coinoculated with the Rhizobiurnphaseoli strain KIM5s gus + Rhizobium spp. From: Streit et aL. but no correlation between competitiveness and this capacity was demonstrated. phaseoli and R. The three strains of R. Azotobacter vinelandii. Microsymbiont-legume host plant communication during the early stages of nodule initiation Contributions from a large number of laboratories have made it evident that the signal exchange between microsymbionts and host plants is much more detailed than simply the induction of the nodulation genes (nod) by flavonoids (fig. legurninosarum by. production (36. versus nodule occupancy at pH 6. 26) exudation (27) Nod factor production (6) ~ Phytoalexine induction (35) Stimulation of flavonoid . with a few exceptions.

M. Cuernavaca (Mexico). 4. NigTal. Lotz. Dr. legummosarum biovar phaseoli KIM5s (3) R. BirkbSuser Vertag. Dr. (lupinus) USDA 3055 (8) Azorhizobium caulinodans ORS 57I Bradyrhizobium japonicum USDA 123 (8) Bradyrhizobium sp. Handelsman. Bohlool. 3. M. Wisconsin (USA). Springer-Verlag. Dr. activation of cell cycle m a c h i n e r y a n d meristem i n d u c t i o n 32. US Dept. Berlin. legummosarum biovar phaseoli CIAT 904 (2) R. B. 5. The microsymb i o n t s themselves induce phytoalexin p r o d u c t i o n in the host plants. Bacteria were obtained from: 1. Ecol. Marburg (Germany). tropici C-O5-I1 (2) Agrobacterium rhizogenes DSM 30148 (6) Agrobacterium tumefaciens DSM 30150 (6) Agrobacterium tumefaciens C6-6 (5) Alcaligenes eutrophus DSM 517 (6) Ar~hrobacter globiformis (5) Azotobacter vinelandii DSM 43l (6) Bacillus subtilis DSM i970 (6) Citrobacter freundii DSM 30039T (6) E. this is perhaps a protection . refs 9. (Centrosema plumieri) CIAT 3101 (2) B~adyrhizobium sp. (Centrosema macrocarpum) CIAT 3011 (2) crosshybridization. legummosarum biovar phaseoli CIAT 652 (2) R.no crosshybridization. meliloti. Centro Internacional de Agriculture Tropical. legummosarum biovar phaseoli COSTA 6 (2) R tropici CFN 1500 (1) R_ tropiei CIAT 899 (2) R. a n d also to a stimulation of flavonoid p r o d u c t i o n 36. legummosarurn biovar phaseoli CIAT 163 (2) R tegurnmosarum biovar phaseoli CIAT 141 (2) R. I n d u c tion of nod a n d nol genes is the centre of interest in a large n u m b e r of publications (e. Braunschweig (Germany). DNA from R. 7. legummosarum biovar phaseoli CIAT 613 (2) R. Prof. legummosarum biovar phaseoli CIAT 2510 (2) R. R6hm. of Agriculture.5 (6) Pseudomonasfluorescens DSM 1694 (6) Pseudomonas facilis DSM 620 (6) Pseudomonas putida DSM 3226 (6) Paraeoccus denitrificans DSM 1404 (6) Proteus mirabilis DSM 4479T (6) Shevanella putrefaciens NCIMB 10471 (6) Xanthomonas campestris DSM 50859 (6) Rhizobium leguminosarum biovar trifolii K9 (5) Rhizobium Ieguminosarum biovar viciae F28 (4) Rhizobium leguminosarum biovar viciae 248 (4) Rhizobiumfredii HH 103 (7) Rhizobium meliloti 2011 (7) Rhizobium sp.886 Experientia 50 (1994). legummosarum biovar phaseoli CIAT 323 (2) R. 2. Cali (Colombia). Cross hybridization of the developed DNA probe in dot Not hybridizations with total genomie DNA from different bacteria Bacteria Hybridizing with + + + + + + + + + + + + + + + + + R. 8. can have chemotactic activity is' 16. Dr. 13 (1993) 59-68. legummosarum biovar phaseoli CIAT 7033 (2) R. or the precursor c i n n a m i c acid. F l a v o n o i d s . F l a v o n o i d s can be enriched in the outer m e m b r a n e o f R. (Pueraria phaseolide) CIAT 3918 (2) Bradyrhizobium sp. . (Robinia pseudoacacia) R I I I (5) Bradyrhizobium sp. legummosarum biovar phaseoli CIAT 611 (2) R. From: Streit et al. Deutsche Sammlung von Mikroorganismen. (Robinia pseudoaeaeia) R I (5) Rhizobium sp. Nod factor p r o d u c t i o n 6 leads to root hair curling 3s. subtracter strains are underlined. (Centrosema maerocarpum) CIAT 3111 (2) Bradyrhizobium sp. leguminosarum biovar phaseoli KIM5s was used as probe-DNA. Centro de Investigacion sobre Fijacion de Nitrogeno. FEMS Microbiol. Hawaii (USA). b u t only d u r i n g the first 6 to 12 hours of i n o c u l a t i o n ~5. coli K12 DSM 498 (6) Enterobacter agglomerans (5) Klebsiella pneumoniae K11 (new isolate) Pseudomonas stutzeri ATCC 14405 (6) Pseudomonas carboxydovorans O. 6. in seeds 5 a n d in roots are very different in terms of quality a n d q u a n t i t y 27. Erlangen (Germany). Madison. legummosarum biovar phaseoli CFN 1600 (1) R [egummosarum biovar phaseoli TAL 182(2) R. legummosarum biovar phaseoli CIAT 895 (2) R. Beltsville (USA). 26). CH-4010 Basel/Switzerland Reviews Table 2. J.g.

as indicated by the effect of various flavonoids on spore germination and hyphal growth of Glomus species (Kape et a1. +. However. in alfalfa. Structure of ]?-isoxazolinonyl-alanine (BIA) and susceptibility of fungi against this heterocyclic non-protein amino acid. moderate (inhibition zone 5-10 mm). which can make up between 5 and 20% of the dry weight of cells of Bradyrhizobium japonicum 2~ may play an important role in osmotic adaptation and thereby also in competition in the soil under varying water conditions. leguminosarum by. legurninosarum with a phytoalexin degradation of the phytoalexin wyerone s. again the strategies in Bradyrhizobium and Rhizobium appear to be different ]4"2s'28. however. C-ring cleavage of flavonoids 29 is a strategy of the microsymbionts to change the concentration of signalling compounds. Birkh/iuser Verlag. with the latter triggering phytoalexin concentrations that are lower by two orders of magnitude. 22 with other host plants.and 6-fold increase of the phytoalexin concentration in roots of Vicia faba infected with mycorrhiza ( G. Also. aggregatum. betaines such as trigonellin and stachydrin also have a gene-inducing activity. a major result of these data is that there is a large difference between a phytopathogenic interaction and a response to VA mycorrhiza.. Strong (inhibition zone 20 30 ram).17). G. compared to uninfected plants 17. phaseoli have also been shown to utilize different aromatic compounds as carbon sources to a larger extent than less competitive strains 39. . There are two strategies against phytoalexins in the host rhizosphere. Cyclic glucans.Reviews Experientia 50 (1994). Bradyrhizobiurn and legume host plants. in principle we can assume that there are some similar mechanisms. CH-4010 Basel/Switzerland 887 mechanism for the inner membrane 13. Significant increase of both growth parameters was observed by application of myricetin and quercetin. Flavonoids and VA mycorrhiza development Compared to our understanding of the molecular mechanisms of communication between Rhizobium. not significant (inhibition zone < 1 mm) Figure 2. More competitive strains of R. For example. Not much is known about how lectins and early nodulines produced by the host plants 12 are affected by microsymbiont strains with different competitiveness. a 2. However. Exopolysaccharides (EPS) and lipopolsaccharides (LPS) also have important functions for infection and competition. knowledge of the VA mycorrhizal symbiosis is far behind. Constitutive antifungal components in roots A major role in the competitiveness of symbiotic microorganisms and their communication with the host CH2 I ' CH NH2 C02H I Species Strain DSM 70449 (typestrain) MUCL 27835 MUCL 27835 MUCL 30058 MUCL 29853 MUCL30238 MUCL 30004 New isolate New isolate MUCL 30249 DSM 4636 DSM 70398 DSM 70825 Inhibition a + + + + + + + + + + + + + Saccharomyces cerevisiae Saecharomyces uvarum Saccharomyces exiguus Candida utilis Candida lipolytica Kluyveromyees polysporus Pichia membranaefaeiens Botrytis cinerea Pythium ultimum Rhodosporidium toruloides Leucosporidium scottii Rhodotorula glutinis Rhodotorula rubra ++ ++ _ _ _ __ "+ +. This confirms previous results by Morandi 21 and Morandi et al. Bradyrhizobium can react by an induced resistance 24 and R. though at a much higher concentration than most flavonoids 27. . There are many variations from a generalized scheme such as that in figure 1. macrocarpum and G. mosseae) was found in different host cultivars.

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