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Leguminous tree root nodule nitrogen-fixing bacteria are critical for recuperation of soil C and N cycle processes after disturbance in tropical forests, while other nodule-associated bacteria (NAB) may enhance nodule development and activity, and plant growth. However, little is known of these root nodule microbiomes. Through DNA analysis, we evaluated the bacterial taxa associated with the root nodules of the 1-year-old, 2-year-old, 13-year-old, and old growth Inga punctata trees in a cloud forest. Bradyrhizobium diazoefficiens was the dominant taxon found in all nodules at 63.16% to 85.71% mean percent sequences (MPS) of the total nodule bacterial DNA and was found in the youngest nodules examined (1 year old), suggesting that it is the primary nodular bacteria. There were 26 other NAB genera with collective MPS levels between 7.4% to 12.2%, while 15 of these genera were found in the Bulk Forest soils at collective MPS levels of 4.6%. These bacterial community compositions were different between the NAB and Bulk Forest soils, suggesting the NAB became concentrated within the root nodules, resulting in communities with different compositions from the Bulk Forest soils. Twenty-three of the 26 NAB genera were previously identified with the potential to perform 9 plant growth promoting (PGP) activities, suggesting their importance in root nodule development and plant growth. These NAB communities appeared to successionally develop over time into more complex taxonomic communities, which is consistent with the outcome of advanced microbial communities following succession. The presence of both B. diazoefficiens and the NAB communities in the nodules across all ages of tree roots, and the potential for PGP activities linked with most of the NAB genera, suggest the importance of B. diazoefficiens and the NAB community for nodule development and enhanced development and growth of I. punctata throughout its lifespan, and most critically in the younger plants.

The legume–rhizobia symbiosis is a unique natural phenomenon, which supplies the plant with the necessary mineral nitrogen via fixation of atmospheric dinitrogen. This interaction involves two partners: the legume plant and root nodule bacteria (rhizobia). In the wild, members of the Fabaceae family enter into symbiosis with a polymorphic group of rhizobia specific to them; the mechanism and reasons for the formation of heterogeneity of rhizobia are currently the subject of active research. In the present work, a Rhizobium leguminosarum strain strictly specific to Phaseolus vulgaris L. was used to show that within 30 days upon its introduction into soil, genetic rearrangements occurred in the cells, as was revealed by changes in the pattern of its genetic profile. It was also found that recombination activity of the cells was also affected by the root exudates produced during seed germination, which may indicate involvement of the plant in the formation of polymorphism of its microsymbionts. These findings suggest interpretation of this process not as a spontaneous event, but rather as an event controlled by the plant. [ABSTRACT FROM AUTHOR]

Leguminous tree root nodule nitrogen-fixing bacteria are critical for recuperation of soil C and N cycle processes after disturbance in tropical forests, while other nodule-associated bacteria (NAB) may enhance nodule development and activity, and plant growth. However, little is known of these root nodule microbiomes. Through DNA analysis, we evaluated the bacterial taxa associated with the root nodules of the 1-year-old, 2-year-old, 13-year-old, and old growth Inga punctata trees in a cloud forest. Bradyrhizobium diazoefficiens was the dominant taxon found in all nodules at 63.16% to 85.71% mean percent sequences (MPS) of the total nodule bacterial DNA and was found in the youngest nodules examined (1 year old), suggesting that it is the primary nodular bacteria. There were 26 other NAB genera with collective MPS levels between 7.4% to 12.2%, while 15 of these genera were found in the Bulk Forest soils at collective MPS levels of 4.6%. These bacterial community compositions were different between the NAB and Bulk Forest soils, suggesting the NAB became concentrated within the root nodules, resulting in communities with different compositions from the Bulk Forest soils. Twenty-three of the 26 NAB genera were previously identified with the potential to perform 9 plant growth promoting (PGP) activities, suggesting their importance in root nodule development and plant growth. These NAB communities appeared to successionally develop over time into more complex taxonomic communities, which is consistent with the outcome of advanced microbial communities following succession. The presence of both B. diazoefficiens and the NAB communities in the nodules across all ages of tree roots, and the potential for PGP activities linked with most of the NAB genera, suggest the importance of B. diazoefficiens and the NAB community for nodule development and enhanced development and growth of I. punctata throughout its lifespan, and most critically in the younger plants. [ABSTRACT FROM AUTHOR]

Soybean is one of the most important legumes in the world, and its advantages and disadvantages are well known. As a result of symbiosis with the bacterium Bradyrhizobium japonicum, soybean can assimilate nitrogen from the air and is therefore not fertilized with this element, or if it is, only at small doses. In soybean agriculture practice, an important treatment is the inoculation of seeds with symbiotic bacteria and optimal fertilization with selected nutrients. Therefore, a three-year (2019–2021) field experiment was carried out to investigate the effects of soybean in the field to a seed Rhizobium inoculation or coating and molybdenum foliar fertilization. There were no significant interactions between the tested treatments over the years. It was demonstrated that the best variant was seed inoculation before sowing in combination with foliar molybdenum application. As a result of this treatment, a significant increase in nodulation, soil plant analysis development (SPAD) index, leaf area index (LAI) and seed yield (by 0.61 t·ha−1) was obtained compared to the control. In addition, the content of total protein in the seeds increased, while the content of crude fat decreased, which significantly modified the yield of both components. Sowing coated seeds in the Fix Fertig technology was less effective compared to inoculation, but it was significantly better than that in the control. Coating seeds with B. japonicum, in combination with foliar fertilization with molybdenum, could be recommended for agricultural practice, which was confirmed by economic calculations. Future experiments will assess the soybean's response to seed inoculation or coating and fertilization with other micronutrients. [ABSTRACT FROM AUTHOR]

ABSTRACT Members of the genus Mesorhizobium, which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show Mesorhizobium japonicum MAFF 303099 (formerly M. loti) synthesizes and responds to N-[(2E, 4E)-2,4-dodecadienoyl] homoserine lactone (2E, 4E-C12:2-HSL). We show that the 2E, 4E-C12:2-HSL QS circuit involves one of four luxR-luxI-type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among Mesorhizobium species, as R1-I1. We show that two other Mesorhizobium strains also produce 2E, 4E-C12:2-HSL. The 2E, 4E-C12:2-HSL is unique among known AHLs in its arrangement of two trans double bonds. The R1 response to 2E, 4E-C12:2-HSL is extremely selective in comparison with other LuxR homologs, and the trans double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use S-adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the I1 AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a Mesorhizobium japonicum quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits.

The legume-Rhizobium symbiosis is a unique natural phenomenon responsible for providing a legume plant with mineral nitrogen fixed from the atmosphere. In the temperate climatic zone, due to seasonal cycles, plants have to form new interactions with rhizobia at the beginning of each round of vegetation with the formation of specialized structures on the roots, called root nodules, where molecular nitrogen is actually fixed. Nitrogen requirements of the host vary depending on the period of plant growth and development, which may affect its trophic relationships with the microsymbionts. In the present work, using the perennial leguminous plant Lotus corniculatus L. as an example, we assessed the influence of the macrosymbiont vegetation stage on the composition of its root nodule bacteria and carried out the characterization of these microbial communities. The highest microbial genetic diversity was found in the communities developing at the beginning of L. corniculatus vegetation. At the medium stage of plant development, the diversity decreased, while by the end of vegetation it increased again. Analysis of the nitrogen-fixing activity revealed the highest values for microbial strains isolated from the root nodules collected at the early stages of the macrosymbiont development, while it was lower in the strains isolated at the end of vegetation. At different stages of their development legumes may probably use different strategies for selection of microsymbionts. [ABSTRACT FROM AUTHOR]

Soybean is one of the most important legumes in the world, and its advantages and disadvantages are well known. As a result of symbiosis with the bacterium Bradyrhizobium japonicum, soybean can assimilate nitrogen from the air and is therefore not fertilized with this element, or if it is, only at small doses. In soybean agriculture practice, an important treatment is the inoculation of seeds with symbiotic bacteria and optimal fertilization with selected nutrients. Therefore, a three-year (2019–2021) field experiment was carried out to investigate the effects of soybean in the field to a seed Rhizobium inoculation or coating and molybdenum foliar fertilization. There were no significant interactions between the tested treatments over the years. It was demonstrated that the best variant was seed inoculation before sowing in combination with foliar molybdenum application. As a result of this treatment, a significant increase in nodulation, soil plant analysis development (SPAD) index, leaf area index (LAI) and seed yield (by 0.61 t·ha−1) was obtained compared to the control. In addition, the content of total protein in the seeds increased, while the content of crude fat decreased, which significantly modified the yield of both components. Sowing coated seeds in the Fix Fertig technology was less effective compared to inoculation, but it was significantly better than that in the control. Coating seeds with B. japonicum, in combination with foliar fertilization with molybdenum, could be recommended for agricultural practice, which was confirmed by economic calculations. Future experiments will assess the soybean’s response to seed inoculation or coating and fertilization with other micronutrients.

We have investigated the effectiveness of a new soybean fertilization technique, named crack fertilization, which involves the application of nodule bacteria on biochar to soybean roots through cracks formed between planting rows during midterm tillage. In the present study, the factors and timing of crack fertilization were investigated in an upland field converted from a paddy. The variables investigated were: 1) crack formation without the application of any agricultural materials, and 2) the application of biochar or 3) nodule bacteria on biochar into cracks. The treatment periods were: 1) before sowing, 2) during midterm tillage, and 3) during both periods. The combination of crack fertilization and reduced tillage was also tested. The method of crack fertilization that increased yield was the combination of crack formation and the application of biochar, and the most effective period for the treatment was before sowing in the reduced tillage field. Seed yields in conventional and reduced tillage fields were comparable in the upland field converted from the paddy. These results suggest that the application of biochar into cracks after scratching the soil surface to remove weeds before sowing is a practical method of increasing soybean yield in upland fields converted from paddies.

The functional activity of different phylogenetic variants of the transcriptional activator of nitrogen fixation genes in the cells of root nodule bacteria (rhizobia) of different taxa was analyzed. For this purpose, recombinant strains of root nodule bacteria of the genera Mesorhizobium, Ensifer (Sinorhizobium), and Rhizobium were created using genetic engineering constructs based on the plasmid pJN105. This plasmid contains different phylogenetic variants of the nifA gene typical for bacteria of the genera Mesorhizobium, Ensifer, and Rhizobium under the control of the ParaBAD bacterial promoter. It was shown that in free-living recombinant cells of all three bacterial genera, the introduction of an additional active copy of the nifA gene to their genome resulted in emergence of detectable nitrogenase activity. The level of nitrogenase activity was not directly related to the phylogeny of the introduced nifA gene, which is most likely explained by the universality of the product of this gene within the studied group of bacteria. The data obtained indicate the possibility of combinatorial processes in the evolution of nitrogen fixation genes. [ABSTRACT FROM AUTHOR]

The symbiotic system of Legumes and root-nodule bacteria is a significant process for enriching barren and dry soils and supporting plant growth. The aim of this study was to isolate root-nodule bacteria from Medicago falcate and to select high activity strains with resistance to environmental stress. As a result, only MSZ-25 strain grew in the medium containing 0.8M NaCl, with pH4.8-9.8 and at temperatures of 4-45°С. All rhizobial isolates were tolerant to the concentrations of 0.01-0.1% Pb(NO3)2, 0.01% ZnSO4, CdCI2, CuCI2 and NiCI2. The strains were also resisted to penicillin 10μ/ml, erythromycin 10μ/ml, and chlorampenicol 10μ/ml but the growths of all isolates were inhibited by kanamycin 30μ/ml, novobiocin 30μ/ml and tetracycline 10μ/ml.

We have investigated the effectiveness of a new soybean fertilization technique, named crack fertilization, which involves the application of nodule bacteria on biochar to soybean roots through cracks formed between planting rows during midterm tillage. In the present study, the factors and timing of crack fertilization were investigated in an upland field converted from a paddy. The variables investigated were: 1) crack formation without the application of any agricultural materials, and 2) the application of biochar or 3) nodule bacteria on biochar into cracks. The treatment periods were: 1) before sowing, 2) during midterm tillage, and 3) during both periods. The combination of crack fertilization and reduced tillage was also tested. The method of crack fertilization that increased yield was the combination of crack formation and the application of biochar, and the most effective period for the treatment was before sowing in the reduced tillage field. Seed yields in conventional and reduced tillage fields were comparable in the upland field converted from the paddy. These results suggest that the application of biochar into cracks after scratching the soil surface to remove weeds before sowing is a practical method of increasing soybean yield in upland fields converted from paddies. [ABSTRACT FROM AUTHOR]

Abstract Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA.

In order to investigate the effect of the RapA1 protein on growth-stimulating activity, biofilm formation, and root colonization by rhizobia, strains Rhizobium leguminosarum Pvu5, VSy12, Thy2, TPr4 and R. galegae 0702 were transformed with the pJN105TurboRapA1GFP vector, providing for constitutive expression of the rapA1 and gfp genes. Constitutive expression of the rapA1 gene was shown to result in more efficient biofilm formation on inert surfaces and to promote microcolony formation on plant root surfaces. Analysis of auxin production by wild and transformed strains revealed decreased auxin synthesis in all transformed strains, except for R. leguminosarum Thy2. Growth-stimulating activity of the wild and transformed strains did not correlate with the amount of auxins produced in vitro. [ABSTRACT FROM AUTHOR]

Waterlogging is the constraint for soybean growth and yield, because soybean is often cultivated in upland fields converted from paddy in Japan. However, efficient cultivation techniques for alleviating the adverse effects have not been developed. We have proposed the new soybean cultivation technique named crack fertilization which enables yield increase due to enhancing new root growth and N acquisition by increasing nodulation. Waterlogging induces N deficiency due to the suppression of nutrient uptake by the inhibition of root growth and nodule activity. Thus, it is hypothesized that crack fertilization would be effective to alleviate the inhibition of soybean growth and yield. The soybean cultivar of Sachiyutaka was planted in 1/5000 a Wagner pots and root boxes. Two separate waterlogging treatments were imposed to soybean plants at different growth stages, V1 and R4, and crack fertilization was done at V3. After these treatments, soybean plants were sampled at R5 in 2012 and 2013 experiments, respectively. Waterlogging at V1 and R4 inhibited the growth and yield of soybean and nodule growth, and the decreases in physiological parameters of soybean such as photosynthesis, chlorophyll content, and xylem sap exudation rate were observed. The adverse effects of waterlogging at V1 were alleviated by crack fertilization at V3, whereas crack fertilization could not alleviate the adverse effects of waterlogging at R4. Thus, crack fertilization after waterlogging at early vegetative stage would be the cultivation technique that enables to alleviate the adverse effects.

High-value native timber species are being promoted in reforestation in Southeast Asia. However, slow growth during early establishment, coupled with poor soil fertility, poses challenges for promoting tree planting programs. Field trials were undertaken on agroforestry land in Thailand to examine the efficacy of reforestation treatments. The first trial examined the effect of applying microorganisms and fertiliser on directly seeded Acacia mangium Willdenow, Dalbergia cochinchinensis Pierre and Xylia xylocarpa (Roxburgh) Theobald. After 20 months, a mixed inoculum of arbuscular mycorrhizal and ectomycorrhizal fungi improved survival and basal diameter of D. cochinchinensis by 15 and 43%, respectively. The co-inoculation of arbuscular mycorrhizal fungi and N2-fixing bacteria improved survival by 17%. The second trial investigated the effects of fertiliser and a water retention polymer on directly seeded A. mangium, Afzelia xylocarpa (Kurz) Craib, D. cochinchinensis, Eucalyptus camaldulensis Dehnhardt, Sindora cochinchinensis Baillon and X. xylocarpa. Height was improved by 40% at 20 months. Our findings suggest that reforestation is viable, provided that suitable treatments are used. These principles can be applied for reforestation of nutrient-impoverished soils of continental Southeast Asia.

Highlights • First study to show that there is a large diversity of Bradyrhizobium species associated with legumes on Christmas Island. • Naturalised rhizobia are present for the crop legumes cowpea, mungbean, peanut and lablab. • No nodules were detected on common bean, chickpea, and soybean, and hence inoculation is required for these crops. • Mimosa species are nodulated by C. alkaliphilus -like strains and C. taiwanensis. • Vertical transmission of symbiosis genes in the alpha-rhizobia but horizontal transmission within the beta-rhizobia. Abstract Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer , Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH , the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island. [ABSTRACT FROM AUTHOR]

Abstract—The importance of root nodule bacteria in biotechnology is determined by their distinctive feature: symbiotic nitrogen fixation resulting in the production of organic nitrogen-containing compounds. While interacting with host legume plants, the cells of these bacteria undergo global changes at all levels of expression of genetic information leading to the formation in root nodules of so-called bacteroids functioning as nitrogen fixation factories. The molecular mechanisms underlying plant-microbial symbiosis are actively investigated, and one of the most interesting and poorly studied aspects of this problem is the species-specificity of interaction between root nodule bacteria and host plants. In this work we have performed the proteomic analysis of the Sinorhizobium meliloti bacteroids isolated from two legume species: alfalfa (Medicago sativa L.) and yellow sweet clover (Melilotus officinalis L.). It has been shown that the S. meliloti bacteroids produce a lot of proteins (many of them associated with symbiosis) in a host-specific manner, i.e., only in certain host plant species. It has been demonstrated for the first time that the levels of expression in bacteroids of the genes encoding the ExoZ and MscL proteins responsible for the synthesis of surface lipopolysaccharides and formation of a large conductance mechanosensitive channel, respectively, depend on a host plant species that confirms the results of proteomic analysis. Overall, our data show that the regulation of bacteroid development by the host plant has species-specific features. [ABSTRACT FROM AUTHOR]

In silico analysis of open reading frames of genomic islands of Sinorhizobium meliloti Rm1021 was performed. This strain is a typical representative of soil bacteria forming nitrogen-fixing symbiosis with legume host plants from the alfalfa cross-inoculation group. It was demonstrated that genomic islands had mosaic structure, in which blocks of functional genes, IS-elements and noncoding RNA alternated. Genomic islands contained as well the components of T4SS and T4CP systems, and lacked systems of conjugative mobilization of islands. It was concluded that two of the three islands could be the variants of reduced integrative conjugative elements, and the third island represented a reduced integrated conjugative transposon. Site-specific integration of islands occurred into a 15-31 bp sequence (depending on the island) localized at the 3′-end of the tRNA gene, which is then shifted rightward, while the remaining part of the tRNA gene is completed by a similar sequence that exists in the island. A suggestion on the existence of “speciesspecific insertion hotspots” for genomic islands of root nodule bacteria was put forward. [ABSTRACT FROM AUTHOR]

Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA. [ABSTRACT FROM AUTHOR]

Many relict and endemic legumes are often rare and endangered species, located in remote and almost impenetrable areas. This makes it difficult to study their symbiosis with nodule bacteria. The aim of this work was to obtain root nodules under laboratory conditions ofHedysarum zundukii, Oxytropis popoviana, Oxytropis triphylla, Oxytropis tragacanthoides, Astragalus chorinensisandGlycyrrhiza uralensisbelonging to the Miocene-Pliocene relicts, usually growing in restricted habitats of the Baikal Lake region. A pot experiment was conducted with soils and seeds collected in distribution areas of these legumes. The collected soils had low content of N and P, suggesting importance of nitrogen-fixing symbiosis for successful maintenance of plant populations. The symbiotic nodules were observed on roots of all plant species tested and 52 bacterial strains assumed as root nodule bacteria (rhizobia) were isolated from nodules. The proposed method avoids collection of native plants and prevents decrease in plant populations. The isolated bacteria will be used to search for ancestral genes responsible for the specificity and efficiency of rhizobia-legume symbiosis and can contribute to the conservation of rare and endangered species of relict legumes in the Baikal region. [ABSTRACT FROM AUTHOR]

Members of the genus Mesorhizobium, which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show Mesorhizobium japonicum MAFF 303099 (formerly M. loti ) synthesizes and responds to N -[(2 E , 4 E )-2,4-dodecadienoyl] homoserine lactone (2 E, 4 E- C 12:2 -HSL). We show that the 2 E, 4 E- C 12:2 -HSL QS circuit involves one of four luxR-luxI -type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among Mesorhizobium species, as R1-I1. We show that two other Mesorhizobium strains also produce 2 E, 4 E- C 12:2 -HSL. The 2 E, 4 E- C 12:2 -HSL is unique among known AHLs in its arrangement of two trans double bonds. The R1 response to 2 E, 4 E- C 12:2 -HSL is extremely selective in comparison with other LuxR homologs, and the trans double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use S -adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the I1 AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a Mesorhizobium japonicum quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits., Competing Interests: The authors declare no conflict of interest.

Waterlogging is the constraint for soybean growth and yield, because soybean is often cultivated in upland fields converted from paddy in Japan. However, efficient cultivation techniques for alleviating the adverse effects have not been developed. We have proposed the new soybean cultivation technique named crack fertilization which enables yield increase due to enhancing new root growth and N acquisition by increasing nodulation. Waterlogging induces N deficiency due to the suppression of nutrient uptake by the inhibition of root growth and nodule activity. Thus, it is hypothesized that crack fertilization would be effective to alleviate the inhibition of soybean growth and yield. The soybean cultivar of Sachiyutaka was planted in 1/5000 a Wagner pots and root boxes. Two separate waterlogging treatments were imposed to soybean plants at different growth stages, V1 and R4, and crack fertilization was done at V3. After these treatments, soybean plants were sampled at R5 in 2012 and 2013 experiments, respectively. Waterlogging at V1 and R4 inhibited the growth and yield of soybean and nodule growth, and the decreases in physiological parameters of soybean such as photosynthesis, chlorophyll content, and xylem sap exudation rate were observed. The adverse effects of waterlogging at V1 were alleviated by crack fertilization at V3, whereas crack fertilization could not alleviate the adverse effects of waterlogging at R4. Thus, crack fertilization after waterlogging at early vegetative stage would be the cultivation technique that enables to alleviate the adverse effects. [ABSTRACT FROM AUTHOR]

The root nodule bacteria were isolated from three different leguminous plants belonging to the Fabaceae family such as Cajanus cajan, Lablab purpureus and Vigna unguiculata using YEMA medium and identified. The isolates were submitted to 16S rRNA and PCR- RFLP typing. A representative sample was further submitted to sequence analysis of 16S rRNA. Isolates were assigned to two genera and were related to Rhizobium spp. (75%) and Bacillus spp. (25%) respectively. This study opens the doors to researchers to do specific studies on this root nodule bacteria which are symbiotic with these legume plants in this area for their antibacterial activity, medicine and food application, and sustainable agriculture. Rhizobium and other beneficial microbes may be used as biofertilizers as a substitute for chemical fertilizers. [ABSTRACT FROM AUTHOR]

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467, while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467. However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467 was 69.6 % minimum. Two symbiosis-related genes ( nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation. [ABSTRACT FROM AUTHOR]

Phylogenomic analyses showed two major superclades within the family Rhizobiaceae that corresponded to the Rhizobium / Agrobacterium and Shinella / Ensifer groups. Within the Rhizobium / Agrobacterium group, four highly supported clades were evident that could correspond to distinct genera. The Shinella / Ensifer group encompassed not only the genera Shinella and Ensifer but also a separate clade containing the type strain of Rhizobium giardinii . Ensifer adhaerens (Casida A T ) was an outlier within its group, separated from the rest of the Ensifer strains. The phylogenomic analysis presented provided support for the revival of Allorhizobium as a bona fide genus within the Rhizobiaceae , the distinctiveness of Agrobacterium and the recently proposed Neorhizobium genus, and suggested that R. giardinii may be transferred to a novel genus. Genomics has provided data for defining bacterial-species limits from estimates of average nucleotide identity (ANI) and in silico DNA–DNA hybridization (DDH). ANI reference values are becoming the gold standard in rhizobial taxonomy and are being used to recognize novel rhizobial lineages and species that seem to be biologically coherent, as shown in this study. [ABSTRACT FROM AUTHOR]

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18 and Vaf-43 were isolated from the nodules of Vavilovia formosa plants growing in the hard-to-reach mountainous region of the North Ossetian State Natural Reserve (north Caucasus, Russian Federation). The sequencing of 16S rDNA ( rrs), ITS region and five housekeeping genes ( atpD, dnaK, recA, gyrB and rpoB) showed that the isolated strains were most closely related to the species Bosea lathyri (class Alphaproteobacteria, family Bradyrhizobiaceae) which was described for isolates from root nodules of Lathyrus latifolius. However the sequence similarity between the isolated strains and the type strain B. lathyri LMG 26379 for the ITS region was 90 % and for the housekeeping genes it was ranged from 92 to 95 %. All phylogenetic trees, except for the rrs-dendrogram showed that the isolates from V. formosa formed well-separated clusters within the Bosea group. Differences in phenotypic properties of the B. lathyri type strain and the isolates from V. formosa were studied using the microassay system GENIII MicroPlate BioLog. Whole-cell fatty acid analysis showed that the strains Vaf-17, Vaf-18 and Vaf-43 had notable amounts of C (4.8-6.0 %), C 3-OH (6.4-6.6 %), C ω5c (8.8-9.0 %), C cyclo (13.5-13.9 %), C ω7c (43.4-45.4 %), C cyclo ω8c (10.5-12.6 %) and Summed Feature (SF) 3 (6.4-8.0 %). The DNA-DNA relatedness between the strains Vaf-18 and B. lathyri LMG 26379 was 24.0 %. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129 = LMG 28367 = Vaf-18) is proposed. [ABSTRACT FROM AUTHOR]

Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. v iciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas ( Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species. [ABSTRACT FROM AUTHOR]

The ability of wild indigenous legumes to form root nodules capable of biological nitrogen (N) fixation has rarely been demonstrated for species in natural ecosystems in large parts of Europe . In order to understand and manage these ecosystems, it is important to demonstrate nodulation across a diverse range of environments, sites and climates. This study surveyed nodulation at a number of sites in Scotland and Sweden. Presence of nodules was noted and nodule structure and indicators of nitrogen fixation capacity were assessed using light and transmission electron microscopy. Soils from several sites were also sampled for carbon and nitrogen analysis. The collections comprised 24 species in Scotland, and 30 taxa in Sweden; 17 of these in common for both countries. Highest species numbers occurred in meadows, farmland margins, hedgerows, roadsides and wasteland. Coastal sites and sites in the mountainous region above the Arctic Circle hosted several rare species. All sampled species had features of N-fixing nodules such as pink colour (leghaemoglobin) when dissected and bacteroids. Nodule structure for a number of species is here reported for the first time and presence of the N-fixing enzyme nitrogenase is demonstrated in three previously not studied Swedish legume species. North European legumes may make significant contributions to the N-budgets of their ecosystems. Such species (and their symbionts) represent unique germplasm that may be adopted to empower advances in agriculture and conservation aimed at mitigation and adaptation to the effects of climate change. [ABSTRACT FROM AUTHOR]

Summary Inoculation with beneficial soil microorganisms has the potential to enhance success of restoration, particularly in harsh Mediterranean-type ecosystems (MTEs). We investigated the effects of microorganisms (mycorrhizal fungi and root nodule bacteria) and planting material (seed and nursery-raised seedlings) on early establishment and growth of two key postdisturbance colonizing species with different life histories, life forms and functional types ( Eucalyptus gomphocephala and Acacia saligna) under field conditions. Establishment and growth were monitored at 13 months, following the first MTE drought period. For E. gomphocephala, establishment was higher for seedlings (81%) than for seeding (7.5%). Inoculation with ectomycorrhizal fungal spores was not beneficial. For A. saligna, establishment was also higher for seedlings (84%) than for seeding (42.5%). Mycorrhizal fungal inoculum had no effect on establishment or growth. This study has shown that in harsh MTE conditions, the use of seedlings is more effective than seeding in degraded woodlands even when attempting to reintroduce key colonizing species. The microorganism treatments tested did not result in significant improvement in establishment or growth. [ABSTRACT FROM AUTHOR]

Inactivation of the genes involved in dicarboxylic acid transport in the alfalfa rhizobia Sinorhizobium meliloti-the structural gene of succinate permease dctA and its transcriptional regulators dctBD, nifA, and ntrA-leads to complete or partial loss of N-fixing activity, while amplification of these genes leads to an increase in this activity. This considerably elevates the accumulation of nitrogen and carbon in alfalfa; however, the plant weight increases to a lesser degree, which is associated with an incomplete translocation of nitrogen to the aboveground organs. Factor analysis has demonstrated that the amplification of dctABD genes significantly influences these characteristics of symbiotic efficiency in all the performed experiments, while the effects of dctA, nifA, and ntrA depend on the plant cultivar and vegetation conditions. [ABSTRACT FROM AUTHOR]

The genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine Lathyrus vernus (L.) Bernh. ( Fabaceae) growing in various regions of the Republic of Bashkortostan were studied. RAPD analysis revealed a high degree of polymorphism of the DNA of the isolated strains giving evidence of the heterogeneity of the microorganisms in question. The study of the phylogeny of microsymbionts based on comparative analysis of the nucleotide sequences of 16S rRNA genes showed that the bacteria isolated from the plant nodules of L. vernus growing on the territory of Ufa and Beloretsk raions belonged to the species Rhizobium leguminosarum, whereas the microsymbionts of L. vernus growing on the territory of Tatyshly raion belonged to the species Rhizobium tropici,@ except for several strains of Rhizobium leguminosarum [ABSTRACT FROM AUTHOR]

Abstract: The vktA catalase gene, which had been cloned from Vibrio rumoiensis S-1T having extraordinarily high catalase activity, was introduced into the root nodule bacterium, Rhizobium leguminosarum bv. phaseoli USDA 2676. The catalase activity of the vktA-transformed R. leguminosarum cells (free-living) was three orders in magnitude higher than that of the parent cells and this transformant could grow in a higher concentration of exogenous hydrogen peroxide (H2O2). The vktA-transformant was inoculated to the host plant (Phaseolus vulgaris L.) and the nodulation efficiency was evaluated. The results showed that the nitrogen-fixing activity of nodules was increased 1.7 to 2.3 times as compared to the parent. The levels of H2O2 in nodules formed by the vktA-transformant were decreased by around 73%, while those of leghemoglobins (Lba and Lbb) were increased by 1.2 (Lba) and 2.1 (Lbb) times compared with the parent. These results indicated that the increase of catalase activity in rhizobia could be useful to improve the nitrogen-fixing efficiency of nodules by the reduction of H2O2 content concomitantly with the enhancement of leghemoglobins contents. [ABSTRACT FROM AUTHOR]

The South African legumes Lotononis bainesii, L. listii and L. solitudinis are specifically nodulated by highly effective, pink-pigmented bacteria that are most closely related to Methylobacterium nodulans on the basis of 16S rRNA gene homology. Methylobacterium spp. are characterized by their ability to utilize methanol and other C1 compounds, but 11 Lotononis isolates neither grew on methanol as a sole carbon source nor were able to metabolize it. No product was obtained for PCR amplification of mxaF, the gene encoding the large subunit of methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to utilize methanol may be due to the absence of the mxa operon. While methylotrophy appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, our results indicate that the ability to utilize methanol is not a factor in the Lotononis/ Methylobacterium symbiosis. [ABSTRACT FROM AUTHOR]

Abstract: The mathematical model for evolution of the plant–microbe facultative mutualistic interactions based on the partners’ symbiotic feedbacks is constructed. Using the example of rhizobia–legume symbiosis, we addressed these feedbacks in terms of the metabolic exchange resulting in the parallel improvements of the partners’ fitness (positive feedbacks). These improvements are correlated to the symbiotic efficiency dependent on the ratio of N2-fixing bacterial strains (“genuine mutualists”) to the non- N2-fixing strains (“symbiotic cheaters”) in the root nodules. The computer experiments demonstrated that an interplay between the frequency-dependent selection (FDS) and the Darwinian (frequency-independent) selection pressures implemented in the partners’ populations ensures an anchoring or even domination for the newly generated host-specific mutualists (which form N2-fixing nodules only with one of two available plant genotypes) more successfully than for the non-host-specific mutualists (which form N2-fixing nodules with both plant genotypes). The created model allows us to consider the mutualistic symbiosis as a finely balanced polymorphic system wherein the equilibrium in bacterial population may be shifted in favor of “genuine mutualists” due to the partner-stipulated selection for an improved symbiotic efficiency implemented in the plant population. [Copyright &y& Elsevier]

Different species of Rhizobium were successfully introduced into the extracellular slime of Nostoc paludosum (Kütz) Elenk, strain 18; cyanobacteria did not eliminate them and exhibited no specificity to the introduced species. Both partners were shown to exist in a self-sufficient manner in an artificial consortium, the stability of which is determined by the technology of growing the cultures in collections. Cyanobacteria act as carriers of introduced satellites, providing contact with the inoculated material through the slime, and increase the nitrogen-fixing ability of legume plants due to the increase of the number and activity of nodules. The fact of penetration of cyanobacterial hormogonia into the nodules has been noted. The treatment of seeds by the consortium resulted in an increase of the harvest as compared with the standard methods of nitragin treatment of legumes. [ABSTRACT FROM AUTHOR]

Abstract: The effects of Glomus intraradices, Rhizobium sp. and Pseudomonas straita on the root-rot disease complex of chickpea caused by Meloidogyne incognita and Macrophomina phaseolina were observed. Inoculation of G. intraradices, P. straita and Rhizobium caused a significant increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents of pathogen-inoculated plants. Inoculation of Rhizobium caused a greater increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents of pathogen-inoculated plants than caused by P. straita or G. intraradices. Combined inoculation of G. intraradices with P. straita plus Rhizobium to pathogen-inoculated plants caused greater increase in plant growth, number of pods, chlorophyll, nitrogen, phosphorus and potassium contents than by inoculation of G. intraradices plus Rhizobium or G. intraradices plus P. straita. The numbers of nodules per root system were significantly higher in plants inoculated with Rhizobium compared with uninoculated ones. Inoculation of Rhizobium with P. straita/G. intraradices further increases nodulation per root system over plants inoculated with Rhizobium alone. Root colonization by G. intraradices was high in plants inoculated alone. In the presence of P. straita and Rhizobium, root colonization by G. intraradices was increased, while inoculation of pathogens reduced colonization by G. intraradices. Inoculation of Rhizobium caused higher reduction in galling and nematode multiplication, followed by P. straita and G. intraradices. Maximum reduction in galling and nematode multiplication was observed when G. intraradices was inoculated with both bacteria. Biocontrol of root-rot disease complex of chickpea may be achieved by the combined use of Rhizobium, G. intraradices and P. straita or use of Rhizobium plus P. straita. [Copyright &y& Elsevier]

Abstract: Siderophores are ferric specific ligands that are involved in receptor specific iron transport into bacteria. Partially purified siderophores from 37 different rhizospheric bacterial isolates, and 17 Cajanus cajan root nodule isolates were tested for their utilization by the 17 nodule isolates as test organisms. Significant variation in the siderophore cross-utilization pattern was observed. Catecholate siderophores were produced predominantly by nodule isolates whereas rhizospheric isolates produced catecholates, as well as hydroxamates. The quantity of hydroxamate produced was higher than that of catecholates. Considerable variation was observed with respect to siderophore cross-utilization amongst nodule isolates obtained from a particular location. Isolates collected from different nodules of same plant also showed considerable variation with respect to siderophore cross-utilization. Isolates, which showed, higher siderophores cross-utilizing ability, when tested for homologous (from other nodule bacteria) siderophores also gave good cross-utilization of heterologous (from non-nodule rhizospheric bacteria) siderophores. Isolate GHU(iii), the highest cross-utilizer of homologous and heterologous siderophores showed a significant increase in growth in the presence of exogenously added siderophores. Similar results were observed during the co-inoculation studies with rhizospheric isolate under iron-limited condition. Both isolates GAU(1)1 and AP(6)4, low cross-utilizers of siderophores, failed to show growth increase in response to co-inoculation and addition of siderophores. The iron regulated outer membrane protein (IROMP) profiles of strains showing maximum or least cross-utilization did not differ. The significance of these findings in context to survivability under natural conditions and the possible reasons accounting for high siderophore cross-utilizing status of isolate GHU(iii) is discussed. [Copyright &y& Elsevier]

Abstract: Coffee (Coffea arabica) production provides a source of income for small-scale farmers in Chiapas, Mexico. Organic production regulations prohibit the use of synthetic nitrogen (N) fertilizers, therefore farmers are dependent upon N-sources accepted by organic certification agencies. In the state of Chiapas, little is known about effectiveness of rhizobia and nodule development, location and structure of the common coffee shade tree genus Inga. The objectives of this study were to (1) evaluate the symbiotic effectiveness of selected rhizobia isolates as inoculants for Inga oerstediana under greenhouse and field conditions, (2) describe the morphological and histochemical characteristics of I. oerstediana root nodules, and (3) apply the 15N natural abundance technique to investigate nitrogen fixation in two stands of I. oerstediana of different ages intercropped with C. arabica. To meet objectives one and two, we assessed shoot biomass, nodule number, nodule mass and total shoot N of inoculated I. oerstediana seedlings at 90 and 150 days after planting (DAP) in the greenhouse and field. Light microscopy, and in situ hybridization of nodule sections for leghemoglobin and Nif H cDNA determination were used to describe nodule morphology and histology. Results indicated that tested isolates appear not to be fixing N2 150 DAP and inoculation with isolated bacteria, and that inoculated treatment nodules lacked leghemoglobin and Nif H mRNA transcript, however contained infected bacteroids. An unidentified brown-pigmented granular substance was present in all nodules examined. I. oerstediana appears slow to nodulate, with negligible nodulation at 90 DAP, and limited nodulation at 150 DAP. Using the natural abundance method to meet objective three, recycling of fixed N in older 5–7 year plots is thought to have caused great 15N value variation in both reference and leguminous trees and data could not be used to estimate % N derived from Biological Nitrogen Fixation (BNF). I. oerstediana found in younger 1–3 year old plots was found to derive 20% of its N from BNF. As fixation appears to be low in young Inga, recommendations for organic C. arabica shade tree management include supplementation of N during early growth of Inga-C. arabica intercrop, and longer-term nodulation studies combined with additional N2-fixation assessment using 15N natural abundance methods. [Copyright &y& Elsevier]

Abstract: The leguminous tree genus Inga is thought to be critical for providing N to certified organic coffee shrubs that commonly grow beneath its shade in the state of Chiapas, Mexico. Organic certification prohibits use of synthetic N fertilizers, necessitating understanding of the Inga–rhizobia symbiosis. The objectives of this study were to: (1) establish a collection of isolates from Inga sp. nodules found on organic coffee farms in Chiapas, Mexico; (2) use phenotypic and genotypic approaches to characterize these isolates, and (3) examine cross-inoculation patterns of strains isolated from Inga sp. with other Inga species and N2-fixing trees. Eighty-three strains were isolated from root nodules of Inga oerstediana, Inga pavoniana and an unknown species of Inga. Aside from isolates of I. pavoniana, Inga isolates demonstrate characteristics consistent with Bradyrhizobium. Dendrograms generated using phylogenetic and phenotypic traits showed strains to exhibit significant diversity, even among strains originating from the same farm. Strains isolated from I. pavoniana were 95% similar, and displayed strikingly different characteristics from strains isolated from other species. Cross-inoculation experiments used three Inga strains (cowpea miscellany CB756, and isolates from I. oerstediana and I. sp.) as inoculants with I. edulis, I. fuellii, Acacia mangium, Acacia koa, Vigna unguculata and Gliricidia sepium. All three strains formed nodules containing leghaemoglobin with V. unguculata, while isolates from I. oerstediana, Inga sp. and cowpea miscellany formed nodules with I. feulli and I. edulis, but not with A. mangium and G. sepium. A. koa formed nodules containing leghaemoglobin when inoculated by cowpea miscellany strain CB756 and the strain from Inga sp., but nodules formed with the strain isolated from I. oerstediana did not contain leghaemoglobin. One and two-way ANOVA results showed that no mean biomass of inoculated seedlings was significantly greater than the −N control for each of A. koa, G. sepium, A. mangium (p <0.001), supporting our hypothesis that inoculation with non-Inga strains would have no effect on biomass production. We suggest that great spatial variation exists among Inga isolates from organic coffee farms in the Chiapas highland region, both between and within farms, and that more research is needed to identify reasons for the genetic distribution and function of Inga symbionts. [Copyright &y& Elsevier]