Non-nodulating Strains Research Articles

Genetic diversity and phylogeny of indigenous rhizobia nodulating faba bean (Vicia faba L.) in Greece

The genetic diversity and phylogeny of fast-growing rhizobia isolated from root nodules of Vicia faba grown in different geographical regions of Greece were assessed. Although Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia spp. in European soils, there is no available information on native rhizobia nodulating faba bean in Greece. Seventy bacterial strains were isolated and grouped into sixteen distinct profiles based on BOX-PCR fingerprinting. The phylogenetic affiliation was further defined by sequence analysis of the rrs and multilocus sequence analysis (MLSA) of three housekeeping genes (recA, atpD and gyrB). Fifty-eight isolates were affiliated with recently described genospecies gsF-2, represented by R. laguerreae FB206T, whereas six isolates were closely related to gsB and two isolates might belong to gsA. Two isolates assigned to R. hidalgonense and another two non-nodulating strains could not be assigned to any validly defined species and possibly belong to a new rhizobial lineage. Interestingly, R. laguerreae strains were commonly found at all sampling sites, suggesting that they could be the main symbionts of faba beans in Greek soils. According to the phylogenies of two symbiosis-related genes (nodC and nifH), all nodulating isolates belonged to symbiovar (sv.) viciae harboring four distinct nodC gene haplotypes and they were grouped into two clades together with strains assigned to R. laguerreae and genospecies of R. leguminosarum isolated from other countries and continents. This is the first report that R. hidalgonense strains belong to sv. viciae. No correlation was observed between the nodC haplotypes, geographic origin and chromosomal background of the isolates in the study.

Phytobeneficial bacteria improve saline stress tolerance in Vicia faba and modulate microbial interaction network

Increased global warming, caused by climate change and human activities, will seriously hinder plant development, such as increasing salt concentrations in soils, which will limit water availability for plants. To ensure optimal plant growth under such changing conditions, microorganisms that improve plant growth and health must be integrated into agricultural practices. In the present work, we examined the fate of Vicia faba microbiota structure and interaction network upon inoculation with plant-nodulating rhizobia (Rhizobium leguminosarum RhOF125) and non-nodulating strains (Paenibacillus mucilaginosus BLA7 and Ensifer meliloti RhOL1) in the presence (or absence) of saline stress. Inoculated strains significantly improved plant tolerance to saline stress, suggesting either a direct or indirect effect on the plant response to such stress. To determine the structure of microbiota associated with V. faba, samples of the root-adhering soil (RAS), and the root tissues (RT) of seedlings inoculated (or not) with equal population size of RhOF125, BLA7 and RhOL1 strains and grown in the presence (or absence) of salt, were used to profile the microbial composition by 16S rRNA gene sequencing. The inoculation did not show a significant impact on the composition of the RT microbiota or RAS microbiota. The saline stress shifted the RAS microbiota composition, which correlated with a decrease in Enterobacteriaceae and an increase in Sphingobacterium, Chryseobacterium, Stenotrophomonas, Agrobacterium and Sinorhizobium. When the microbiota of roots and RAS are considered together, the interaction networks for each treatment are quite different and display different key populations involved in community assembly. These findings indicate that upon seed inoculation, community interaction networks rather than their composition may contribute to helping plants to better tolerate environmental stresses. The way microbial populations interfere with each other can have an impact on their functions and thus on their ability to express the genes required to help plants tolerate stresses.

Microsymbionts of forage peanut under different soil and climate conditions belong to a specific group of Bradyrhizobium strains

The use of mixed pastures of grasses with forage peanut (Arachis pintoi) is growing and offers promising results. This leguminous forage plant has a stoloniferous growth habit and tolerates more intense defoliation and shading. However, there are few studies on its symbiosis with nitrogen (N2) fixing bacteria and no studies on genetic identification of its native microsymbionts. The aim of this study was to evaluate phenotypic and genetic diversity and the symbiotic efficiency of bacterial strains isolated from nodules of forage peanut intercropped with grasses in pasture areas. Root nodules were collected from the Belomonte cultivar in Itabela, Bahia, and BRS Mandobi cultivar in Lavras, Minas Gerais, Brazil. The areas of sample collection had contrasting physical and chemical characteristics. Samples of 10 plants per area and 5 nodules per plant were taken. The strains isolated from the nodules were analyzed for culture characteristics (growth period and change in pH of the culture medium), their 16S rRNA, gyrB and nifH genes were partially sequenced, and authentication and analysis of symbiotic efficiency in forage peanut plants were carried out. Both the authentication and symbiotic efficiency experiments were carried out in a greenhouse under axenic conditions, using A. pintoi cv. BRS Mandobi in a completely randomized design with four replications. Authentication was performed in long neck bottles (500 mL) filled with nutrient solution, and symbiotic efficiency was performed in polypropylene seedling plug pots containing a mixture of sand and vermiculite with a nutrient solution. A total of 74 bacterial strains were obtained from isolation of the forage peanut nodules, 40 from Itabela, and 34 from Lavras. Partial sequencing of the 16S rRNA gene was obtained for 48 strains, 30 from Itabela, and 18 from Lavras. The strains isolated from nodules of forage peanut in pasture areas of Itabela, were identified in the following genera: Bradyrhizobium (21), Rhizobium (5), Burkholderia (2), Kocuria (1), and Paenibacillus (1). The strains coming from soils of Lavras, were identified in the following genera: Bradyrhizobium (13), Rhizobium (2), Burkholderia (1), Mucilaginibacter (1), and Enterobacter (1). In the authentication experiment, 17 strains from Itabela, and 13 strains from Lavras, exhibited nodulation. From these authenticated strains, 18 were identified by 16S rRNA sequencing as belonging to the Bradyrhizobium genus. Of these strains, 11 from the two areas exhibited high symbiotic efficiency in A. pintoi, and their results were better than those of the strains currently approved as inoculants for this crop. The non-nodulating strains are probably nodule endophytes. Sequencing of the gyrB housekeeping gene, performed in 13 strains, confirms phylogenetic groups of potential new species among the strains isolated from A. pintoi, which include strains able to nodulate this species, belonging to a specific phylogenetic group of Bradyrhizobium. Sequences of nifH gene of efficient strains belonged to a group different from that formed by non-nodulating strains.

Explaining coexistence of nitrogen fixing and non-fixing rhizobia in legume-rhizobia mutualism using mathematical modeling

In the mutualism established between legumes and soil bacteria known as rhizobia, bacteria from soil infect plants roots and reproduce inside root nodules where they fix atmospheric N2 for plant nutrition, receiving carbohydrates in exchange. Host-plant sanctions against non N2 fixing, cheating bacterial symbionts have been proposed to act in the legume-Rhizobium symbiosis, to preserve the mutualistic relationship. Sanctions include decreased rhizobial survival in nodules occupied by cheating rhizobia. Previously, a simple population model experimentally based showed that the coexistence of fixing and cheating rhizobia strains commonly found in field conditions is possible, and that the inclusion of sanctions leads to the extinction of cheating strains in soil. Here, we extend the previous model to include other factors that could complicate the sanction scenario, like horizontal transmission of symbiotic plasmids, turning non-nodulating strains into nodulating rhizobia, and competition between fixing and cheating strains for nodulation. In agreement with previous results, we show that plant populations persist even in the presence of cheating rhizobia without incorporating any sanction against the cheater populations in the model, under the realistic assumption that plants can at least get some amount of fixed N2 from the effectively mutualistic rhizobia occupying some nodules. Inclusion of plant sanctions leads to the unrealistic extinction of cheater strains in soil. Our results agree with increasing experimental evidence and theoretical work showing that mutualisms can persist in presence of cheating partners.

Nonnodulating Bradyrhizobium spp. Modulate the Benefits of Legume-Rhizobium Mutualism.

Rhizobia are best known for nodulating legume roots and fixing atmospheric nitrogen for the host in exchange for photosynthates. However, the majority of the diverse strains of rhizobia do not form nodules on legumes, often because they lack key loci that are needed to induce nodulation. Nonnodulating rhizobia are robust heterotrophs that can persist in bulk soil, thrive in the rhizosphere, or colonize roots as endophytes, but their role in the legume-rhizobium mutualism remains unclear. Here, we investigated the effects of nonnodulating strains on the native Acmispon-Bradyrhizobium mutualism. To examine the effects on both host performance and symbiont fitness, we performed clonal inoculations of diverse nonnodulating Bradyrhizobium strains on Acmispon strigosus hosts and also coinoculated hosts with mixtures of sympatric nodulating and nonnodulating strains. In isolation, nonnodulating Bradyrhizobium strains did not affect plant performance. In most cases, coinoculation of nodulating and nonnodulating strains reduced host performance compared to that of hosts inoculated with only a symbiotic strain. However, coinoculation increased host performance only under one extreme experimental treatment. Nearly all estimates of nodulating strain fitness were reduced in the presence of nonnodulating strains. We discovered that nonnodulating strains were consistently capable of coinfecting legume nodules in the presence of nodulating strains but that the fitness effects of coinfection for hosts and symbionts were negligible. Our data suggest that nonnodulating strains most often attenuate the Acmispon-Bradyrhizobium mutualism and that this occurs via competitive interactions at the root-soil interface as opposed to in planta Rhizobia are soil bacteria best known for their capacity to form root nodules on legume plants and enhance plant growth through nitrogen fixation. Yet, most rhizobia in soil do not have this capacity, and their effects on this symbiosis are poorly understood. We investigated the effects of diverse nonnodulating rhizobia on a native legume-rhizobium symbiosis. Nonnodulating strains did not affect plant growth in isolation. However, compared to inoculations with symbiotic rhizobia, coinoculations of symbiotic and nonnodulating strains often reduced plant and symbiont fitness. Coinoculation increased host performance only under one extreme treatment. Nonnodulating strains also invaded nodule interiors in the presence of nodulating strains, but this did not affect the fitness of either partner. Our data suggest that nonnodulating strains may be important competitors at the root-soil interface and that their capacity to attenuate this symbiosis should be considered in efforts to use rhizobia as biofertilizers.

Plant–rhizobia interactions alter aphid honeydew composition

Both above- and below-ground interspecific interactions contribute to ecosystem functioning in terrestrial systems, and the integration of below- and above-ground interactions is crucial for deepening our knowledge of nutrient cycling and community dynamics in terrestrial ecosystems. The present study explored the effects of plant–microbe interactions on aphid honeydew quality and quantity and important factors mediating ant–aphid mutualisms and below-ground nutrient dynamics. Soybean aphids (Aphis glycines) were inoculated onto two closely related strains of soybean plants: a nodulating strain that associates with rhizobia and a non-nodulating strain that does not harbor any nitrogen-fixing bacteria. As expected, prior to aphid inoculation, nodulating plants were significantly taller and had more leaves than non-nodulating plants. Aphids feeding on nodulating strains were found to reach slightly larger colony sizes and produce honeydew with significantly different sugar profiles than those feeding on non-nodulating plants. The honeydew collected from aphid colonies feeding on nodulating plants contained 160 % more total sugars than honeydew collected from colonies feeding on non-nodulating plants, but there was no difference in total amino acid-N content in honeydew from colonies feeding on the different plant strains. We discuss the implications of honeydew composition for nutrient cycling and community dynamics and suggest areas of future research to elucidate the consequences of altered aphid honeydew composition on ecosystem properties.

Symbiotic nitrogen-fixing bacterial populations trapped from soils under agroforestry systems in the Western Amazon

Cowpea (Vigna unguiculata) is an important grain-producing legume that can forego nitrogen fertilization by establishing an efficient symbiosis with nitrogen-fixing bacteria. Although inoculating strains have already been selected for this species, little is known about the genotypic and symbiotic diversity of native rhizobia. Recently, Bradyrhizobium has been shown to be the genus most frequently trapped by cowpea in agricultural soils of the Amazon region. We investigated the genetic and symbiotic diversity of 148 bacterial strains with different phenotypic and cultural properties isolated from the nodules of the trap species cowpea, which was inoculated with samples from soils under agroforestry systems from the western Amazon. Sixty non-nodulating strains indicated a high frequency of endophytic strains in the nodules. The 88 authenticated strains had varying symbiotic efficiency. The SPAD (Soil Plant Analysis Development) index (indirect measurement of chlorophyll content) was more efficient at evaluating the contribution of symbiotic N2-fixation than shoot dry matter under axenic conditions. Cowpea-nodulating bacteria exhibited a high level of genetic diversity, with 68 genotypes identified by BOX-PCR. Sequencing of the 16S rRNA gene showed a predominance of the genus Bradyrhizobium, which accounted for 70 % of all strains sequenced. Other genera identified were Rhizobium, Ochrobactrum, Paenibacillus, Bosea, Bacillus, Enterobacter, and Stenotrophomonas. These results support the promiscuity of cowpea and demonstrate the high genetic and symbiotic diversity of rhizobia in soils under agroforestry systems, with some strains exhibiting potential for use as inoculants. The predominance of Bradyrhizobium in land uses with different plant communities and soil characteristics reflects the adaptation of this genus to the Amazon region.

Genetic and Symbiotic Diversity of Nitrogen-Fixing Bacteria Isolated from Agricultural Soils in the Western Amazon by Using Cowpea as the Trap Plant

Cowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. However, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon using cowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodulating strains were predominantly fast growing and acidifying, indicating a high incidence of endophytic strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited various symbiotic efficiencies, with 68% of strains promoting a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA sequence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCR clusters showed a predominance of bacteria from the genus Bradyrhizobium but with high species diversity. Rhizobium, Burkholderia, and Achromobacter species were also identified. These results support observations of cowpea promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.

Effect of N and P nutrition on extracellular secretion of lumichrome, riboflavin and indole acetic acid by N2-fixing bacteria and endophytes isolated from Psoralea nodules

Soils of the Cape Fynbos in South Africa are very low in nutrients, especially N and P, which affect bacterial growth and metabolism. In this study, the effect of supplying nitrate (14.8 and 59.3 mM NO 3 − ), ammonium (28.1 and 112.0 mM NH 4 + ) and phosphorus (1.4 and 5.7 mM P) to five N2-fixing and 11 non-nodulating bacterial strains isolated from root nodules of Psoralea species in the Cape Fynbos was assessed. The data revealed marked variation in the secretion of lumichrome, riboflavin and IAA into culture filtrate. There was generally greater production of lumichrome, riboflavin and IAA by the N2-fixing bacteria than those unable to nodulate P. pinnata and siratro, with much greater concentrations of lumichrome and riboflavin in culture filtrate at high P than low P. At low and high P, symbiotic strain TUT57pp produced 2.2-fold and 3.2-fold more IAA than TUT65prp and TUT33pap respectively, (two non-nodulating strains also with greater IAA production). Although ammonium nutrition has no effect on riboflavin production, it altered lumichrome concentrations in culture filtrate. While ammonium application had no effect, supplying bacterial cells with high nitrate concentration significantly decreased cellular production of lumichrome and riboflavin, two important symbiotic signal molecules. The observed nitrate inhibition of lumichrome and riboflavin biosynthesis and release is in addition to its depressive effect on nodulation and N2 fixation in symbiotic legumes.

Community-wide effects of below-ground rhizobia on above-ground arthropods

1. Plants take nutrients for their growth and reproduction from not only soil but also symbiotic microbes in the rhizosphere, and therefore below-ground microbes may indirectly influence the above-ground arthropod community through changes in the quality and quantity of plants. 2. Rhizobia are root-nodulating bacteria that provide NH4+ to legume plants. We examined bottom-up effects of rhizobia on the community properties of the arthropods on host plants, using a root-nodulating soybean strain (R+) and a non-nodulating strain (R−) in a common garden. 3. R+ plants grew larger and produced a greater number of leaves than R− plants. We observed 28 species of herbivores and three taxonomic groups of predators on R+ and R− plants. The herbivorous species were classified into sap feeders (12 species) and chewers (16 species). 4. The species richness of overall herbivores, sap feeders, and chewers on R+ plants was greater than that on R− plants. Rhizobia positively affected the abundance of chewers. 5. The community composition of herbivores was significantly different between R− and R+ plants, although species diversity and evenness did not differ. 6. Rhizobia-induced bottom-up effects were transmitted to the third trophic level. The abundance, taxonomic richness, and diversity of the predators on R+ plants were greater but evenness was lower than those on R− plants. The community composition of predators was not affected by rhizobia. 7. These results indicate that the below-ground microbes initiated bottom-up effects on above-ground herbivores and predators through trophic levels.

Origins of cheating and loss of symbiosis in wildBradyrhizobium

Rhizobial bacteria nodulate legume roots and fix nitrogen in exchange for photosynthates. These symbionts are infectiously acquired from the environment and in such cases selection models predict evolutionary spread of uncooperative mutants. Uncooperative rhizobia - including nonfixing and non-nodulating strains - appear common in agriculture, yet their population biology and origins remain unknown in natural soils. Here, a phylogenetically broad sample of 62 wild-collected rhizobial isolates was experimentally inoculated onto Lotus strigosus to assess their nodulation ability and effects on host growth. A cheater strain was discovered that proliferated in host tissue while offering no benefit; its fitness was superior to that of beneficial strains. Phylogenetic reconstruction of Bradyrhizobium rDNA and transmissible symbiosis-island loci suggest that the cheater evolved via symbiotic gene transfer. Many strains were also identified that failed to nodulate L. strigosus, and it appears that nodulation ability on this host has been recurrently lost in the symbiont population. This is the first study to reveal the adaptive nature of rhizobial cheating and to trace the evolutionary origins of uncooperative rhizobial mutants.

Phylogenetic diversity of non-nodulating Rhizobium  associated with pine ectomycorrhizae

Most Rhizobium species described are symbionts that form nodules on legume roots; however, non-nodulating strains of Rhizobium are also widespread in nature. Unfortunately, knowledge of non-nodulating Rhizobium is quite limited compared with nodulating Rhizobium. Here, we studied the phylogenetic diversity of Rhizobium species that inhabit Japanese red pine roots (Pinus densiflora). Because fine roots of pine trees are usually colonized by ectomycorrhizal fungi in nature, we mainly used ectomycorrhizal root tips for bacterial isolation. Out of 1195 bacteria isolated from 75 independent root samples from the field and greenhouse experiments, 102 isolates were confirmed to be Rhizobium following partial 16S rRNA gene analysis. Rhizobium species were occasionally dominant in culturable bacterial communities, whereas no Rhizobium species were isolated from the soil itself. Molecular phylogenetic analyses using 16S rRNA, atpD, and recA gene sequences revealed that isolated Rhizobium strains were phylogenetically diverse and that several were distantly related to known Rhizobium species. Considering that a single species of pine is associated with unique and phylogenetically diverse Rhizobium populations, we should pay more attention to non-nodulating strains to better understand the diversity, ecology, and evolution of the genus Rhizobium and plant-Rhizobium associations.

Mesorhizobium septentrionale sp. nov. and Mesorhizobium temperatum sp. nov., isolated from Astragalus adsurgens growing in the northern regions of China

Ninety-five rhizobial strains isolated from Astragalus adsurgens growing in the northern regions of China were classified into three main groups, candidate species I, II and III, based on a polyphasic approach. Comparative analysis of full-length 16S rRNA gene sequences of representative strains showed that candidate species I and II were Mesorhizobium, while candidate species III, which consisted of non-nodulating strains, was closely related to Agrobacterium tumefaciens. The phylogenetic relationships of the three candidate species and some related strains were also confirmed by the sequencing of glnA genes, which were used as an alternative chromosomal marker. The DNA-DNA relatedness was between 11.3 and 47.1 % among representative strains of candidate species I and II and the type strains of defined Mesorhizobium species. Candidate III had DNA relatedness of between 4.3 and 25.2 % with type strains of Agrobacterium tumefaciens and Agrobacterium rubi. Two novel species are proposed to accommodate candidate species I and II, Mesorhizobium septentrionale sp. nov. (type strain, SDW014(T)=CCBAU 11014(T)=HAMBI 2582(T)) and Mesorhizobium temperatum sp. nov. (type strain, SDW018(T)=CCBAU 11018(T)=HAMBI 2583(T)), respectively. At least two distinct nodA sequences were identified among the strains. The numerically dominant nodA sequence type was most similar to that from the Mesorhizobium tianshanense type strain and was identified in strains belonging to the two novel species as well as other, as yet, undefined genome types. Host range studies indicate that the different nodA sequences correlate with different host ranges. Further comparative studies with the defined Agrobacterium species are needed to clarify the taxonomic identity of candidate species III.

Growth promoting effect of Azospirillum brasilense on Casuarina cunninghamiana Miq. seedlings

The growth of Casuarina cunninghamiana seedlings was stimulated when inoculated with Azospirillum brasilense. This resulted in a higher biomass production than in uninoculated controls in the presence or absence of a non-nodulating strain of Frankia.

Competitive interaction between non-nodulating and nodulating strains for nodulation of cowpea (Vigna unguiculata)

Competitive interaction between non-nodulating and nodulating strains for nodulation of cowpea (Vigna unguiculata)

Competitive interaction between non-nodulating and nodulating strains for nodulation of cowpea (Vigna unguiculata)

Abstract We have examined the effect of a non-nodulating mutant (JRW3-Sm D ) on the nodulation ability of cowpea rhizobia ( Bradyrhizobium sp.) strains JRW3 and IRC256. Nodulation of cowpea ( Vigna unguiculata ) by a nodulating Rhizobium strain is suppressed by the presence of a non-nodulating mutant. The msgnitude of suppression for nodule formation by nodulating strains varied between 40% and 80% depending on the strain and the time of inoculation.

Split-Root Assays Using Trifolium subterraneum Show that Rhizobium Infection Induces a Systemic Response That Can Inhibit Nodulation of Another Invasive Rhizobium Strain

Subterranean clover plants possessing two equally infectible and robust lateral root systems ("split roots") were used in conjunction with several specific mutant strains (derived from Rhizobium trifolii ANU843) to investigate a systemic plant response induced by infective Rhizobium strains. This plant response controls and inhibits subsequent nodulation on the plant. When strain ANU843 was inoculated onto both root systems simultaneously or 24, 48, 72, or 96 h apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum but only when the delay period between inocula was greater than 24 h. Equal numbers of nodules were generated on both roots when ANU843 was inoculated simultaneously or 24 h apart. The ability to infect subterranean clover plants was required to initiate the plant inhibitory response since preexposure of one root system to non-nodulating strains did not retard the ability of the wild-type strain to nodulate the opposing root system (even when the delay period was 96 h). Moreover, the use of specific Tn5-induced mutants subtly impaired in their ability to nodulate demonstrated that the plant could effectively and rapidly discriminate between infections initiated by either the parent or the mutant strains. When inoculated alone onto clover plants, these mutant strains were able to infect the most susceptible plant cells at the time of inoculation and induce nitrogen-fixing nodules. However, the separate but simultaneous inoculation on opposing root systems of the parent and the mutant strains resulted in the almost complete inhibition of the nodulation ability of the mutant strains. We concluded that the mutants were affected in their competitive ability, and this finding was reflected by poor nodule occupancy when the mutants were coinoculated with the parent strain onto a single root system. Thus the split-root system may form the basis of a simple screening method for the ranking of competitiveness of various rhizobia on small seeded legumes.

Degradation of glucuronic acid-containing exopoly-saccharides from rhizobium by the hakomori methylation procedure

The uronic acid-containing polysaccharides secreted by some fast-growing, nodulating and non-nodulating strains of Rhizobium underwent marked degradation when methylated by the Hakomori procedure. The polysaccharide produced by Rh. meliloti, which lacks uronic acid, was not degraded.

The structure of the acidic, exocellular polysaccharide produced by Rhizobium trifolii bart A

The repeating unit of the acidic polysaccharide secreted by Rhizobium trifolii Bart A, a non-nodulating strain, is an octasaccharide containing O-acetyl and pyruvic acetal groups. The polysaccharide contains d-glucose, d-galactose, d-glucuronic acid, pyruvic acid, and acetic acid in the molar ratios 5:1:2:2:3. The glycosyl residues are all pyranoid and only the linkages between the repeating units are α. The repeating unit is different from that found in another non-nodulating strain but the same as that found in a nodulating strain of the same bacterial species, and the following structure is proposed.

Growth regulators, Rhizobium and nodulation in peas

Indole-3-acetic acid (IAA) has been identified in the culture medium of nodulating and non-nodulating strains of Rhizobium lebuminosarum by gas chromatography-mass spectrometry. The levels of IAA produced by the different strains have been quantified using multiple ion monitoring and a deuterated internal standard. Indole-3-acetic acid is produced in the absence of exogenous tryptophan in all strains but its level is greatly stimulated by applied tryptophan. No correlation has been established between the ability to nodulate peas and the ability to produce IAA.