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The family Phyllobacteriaceae is a heterogeneous assemblage of more than 146 species of bacteria assigned to its existing 18 genera. Phylogenetic analyses have shown great phylogenetic diversity and also suggested about incorrect classification of several species that need to be reassessed for their proper phylogenetic classification. However, almost 50% of the family members belong to the genus Mesorhizobium only, of which the majority are symbiotic nitrogen fixers associated with different legumes. Other major genera are Phyllobacterium, Nitratireductor, Aquamicrobium, and Aminobacter. Nitrogen-fixing, legume nodulating members are present in Aminobacter and Phyllobacterium as well. Aquamicrobium spp. can degrade environmental pollutants, like 2,4-dichlorophenol, 4-chloro-2-methylphenol, and 4-chlorophenol. Chelativorans, Pseudaminobacter, Aquibium, and Oricola are the other genera that contain multiple species having diverse metabolic capacities, the rest being single-membered genera isolated from varied environments. In addition, heavy metal and antibiotic resistance, chemolithoautotrophy, poly-β-hydroxybutyrate storage, cellulase production, etc., are the other notable characteristics of some of the family members. In this report, we have comprehensively reviewed each of the species of the family Phyllobacteriaceae in their eco-physiological aspects and found that the family is rich with ecologically and metabolically highly diverse bacteria having great potential for human welfare and environmental clean-up., (© 2023. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Legume plants rely upon multipartite interactions between rhizobia and bacterial endophytes within root nodules to facilitate plant growth. This study aimed to isolate and identify indigenous endophytic bacteria from root nodules of Sulla aculeolata L. in Northeast Morocco. Based on their tri-calcium phosphate (TCP) solubilization capacity, five endophytes were chosen for further evaluation of their plant growth traits. All isolates were hydrogen cyanide (HCN) and siderophore producers, while only BCH24 tested positive for ACC deaminase activity. Indole-3-acetic acid (IAA) synthesis ranged from 1.27 mgL - 1 to 2.89 mgL - 1 , while soluble phosphate concentrations was between 7.99 mg L - 1 and 110.58 mg L - 1 . Additionally, all the endophytes were able to produce more than two lytic enzymes. Based on the analysis of 16 S rRNA gene sequences five isolates were identified as Enterobacter sp (BCH13, BCH2), Pseudomonas sp (BCH16, BCH24), and Serratia sp (BCH10). The strains inhibited the growth of three phytopathogenic fungi, with BCH13 exhibiting the highest rate against Aspergillus ochraceus (45%), followed by BCH24 against Fusarium oxysporum (40%) and Botrytis cinerea (35%), respectively. In vivo inoculation of halotolerant strains Enterobacter hormaechei (BCH13) and Pseudomonas moraviensis (BCH16) under gnotobiotic conditions revealed that co-inoculation with Rhizobium sullae KS6 improved plant development compared to single inoculation, making it a promising eco-friendly bio-inoculant for legume Sulla flexuosa L. production., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Rhizobia are a diazotrophic group of bacteria that are usually isolated form the nodules in roots, stem of leguminous plants and are able to form nodules in the host plant owing to the presence of symbiotic genes. The rhizobial community is highly diverse, and therefore, the taxonomy and genera-wise classification of rhizobia has been constantly changing since the last three decades. This is mainly due to technical advancements, and shifts in definitions, resulting in a changing paradigm of rhizobia taxonomy. Initially, the taxonomic definitions at the species and sub species level were based on phylogenetic analysis of 16S rRNA sequence, followed by polyphasic approach to have phenotypic, biochemical, and genetic analysis including multilocus sequence analysis. Rhizobia mainly belonging to α- and β-proteobacteria, and recently new additions from γ-proteobacteria had been classified. Nowadays rhizobial taxonomy has been replaced by genome-based taxonomy that allows gaining more insights of genomic characteristics. These omics-technologies provide genome specific information that considers nodulation and symbiotic genes, along with molecular markers as taxonomic traits. Taxonomy based on complete genome sequence (genotaxonomy), average nucleotide identity, is now being considered as primary approach, resulting in an ongoing paradigm shift in rhizobial taxonomy. Also, pairwise whole-genome comparisons, phylogenomic analyses offer correlations between DNA and DNA re-association values that have delineated biologically important species. This review elaborates the present classification and taxonomy of rhizobia, vis-a-vis development of technical advancements, parameters and controversies associated with it, and describe the updated information on evolutionary lineages of rhizobia., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Citrus black spot (CBS) and post-bloom fruit drop (PFD), caused by Phyllosticta citricarpa and Colletotrichum abscissum, respectively, are two important citrus diseases worldwide. CBS depreciates the market value and prevents exportation of citrus fruits to Europe. PFD under favorable climatic conditions can cause the abscission of flowers, thereby reducing citrus production by 80%. An ecofriendly alternative to control plant diseases is the use of endophytic microorganisms, or secondary metabolites produced by them. Strain LGMF1631, close related to Diaporthe cf. heveae 1, was isolated from the medicinal plant Stryphnodendron adstringens and showed significant antimicrobial activity, in a previous study. In view of the potential presented by strain LGMF1631, and the absence of chemical data for secondary metabolites produced by D. cf. heveae, we decided to characterize the compounds produced by strain LGMF1631. Based on ITS, TEF1, and TUB phylogenetic analysis, strain LGMF1631 was confirmed to belong to D. cf. heveae 1. Chemical assessment of the fungal strain LGMF1631 revealed one new seco-dihydroisocoumarin [cladosporin B (1)] along with six other related, already known dihydroisocoumarin derivatives and one monoterpene [(-)-(1S,2R,3S,4R)-p-menthane-1,2,3-triol (8)]. Among the isolated metabolites, compound 5 drastically reduced the growth of both phytopathogens in vitro and completely inhibited the development of CBS and PFD in citrus fruits and flowers. In addition, compound 5 did not show toxicity against human cancer cell lines or citrus leaves, at concentrations higher than used for the inhibition of the phytopathogens, suggesting the potential use of (-)-(3R,4R)-cis-4-hydroxy-5-methylmellein (5) to control citrus diseases.

Recent developments in the legume rhizobium symbiotic interaction particularly those related to the emergence of novel strains of bacteria that nodulate and fix nitrogen in legumes is gaining momentum. These novel strains of bacteria were mostly isolated from the root nodules of indigenous and invasive legumes belonging to the sub families Papilionoideae and Mimosoideae in South Africa, South America and South East China. These rhizobia are phylogenetically and taxonomically different from the traditional 'alpha rhizobia' and are termed 'β-rhizobia' as they belong to the β-sub class of Proteobacteria. There are also new reports of novel species of root nodulating bacteria from the α-Proteobacteria, not known for several decades since the discovery of rhizobia. However, in this review focus is given to the emerging β-rhizobia isolated from the indigenous Papilionoid legumes in the Cape Floristic regions in South Africa and the indigenous and invasive Mimosoid legumes in South America and South East Asia respectively. The nodulation of the indigenous South African Papilionoid legumes including that of Aspalathus linearis (rooibos) is discussed in a bit detail. Previous reports indicated that A. linearis is very specific in its rhizobium requirement and was reported to be nodulated by the slow growing Bradyrhizobium spp. This review however summarizes that the bacteria associated with the root nodules of A. linearis belong to members of both the alpha (α) Proteobacteria that include Mesorhizobium, Rhizobium and Bradyrhizobium spp. and the beta (β) Proteobacteria represented by the genus Burkholderia (now reclassified as Paraburkholderia). In addition, the occurrence of Paraburkholderia as the newly emerging root nodule symbionts of various other legumes has been discussed. In doing so, the review highlights that nodulation is no longer restricted to the traditional 'rhizobia' group following the emergence of the new beta rhizobia as potential nodulators of various indigenous legumes. It thus provides some insights on the status of the legume-rhizobium host specificity concept and the loss of this specificity in several symbiotic associations that puts the long held dogma of host specificity of the legume rhizobium symbiosis in a dilemma.

In this study, a Gram-negative, rod-shaped, and non-spore-forming bacterium, which was designated as strain CCBUA 65841 T , was isolated from a root nodule of Calopogonium mucunoides grown in Yunan Province of China. The sequence alignment results of 16S rRNA and four housekeeping genes (including gyrB, recA, dnaK and rpoB) indicated the isolated strain is a member of the genus Microvirga, closely related to Microvirga lotononidis WSM3557 T . In addition, results of genome average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) had revealed the lower values (ANI ≤ 88.72%, dDDH ≤ 39.5%) between strain CCABU 65841 T and other related Microvirga species. The genome of the novel strain exhibits a G + C content of 64.48% and contains 7296 protein-coding genes and 93 RNA genes. The major polar lipids were found to be phosphatidylcholine and phosphatidylethanolamine. The predominant cellar fatty acids were identified to be C16:0, C18:0, C19:0 cyclo ω8c, summed feature 2, summed feature 3 and summed feature 8. Moreover, menaquinone 8 (MK-8) was detected to be the predominant quinone. Based on the phylogenetic and phenotypic dissimilarity, a novel species Microvirga calopogonii sp. nov. is proposed with the type strain CCABU 65841 T (= LMG 25488 T  = HAMBI 3033 T ).

Twelve nodulating Paraburkholderia strains isolated from indigenous South African fynbos legume Hypocalyptus sophoroides were investigated to determine their taxonomic status. Genealogical concordance analysis, based on six loci (16S rRNA, atpD, recA, rpoB, lepA and gltB), revealed that they separate into two consistent and exclusive groups. Average nucleotide identity and DNA-DNA hybridisation comparisons indicated that they were sufficiently divergent from their closest known phylogenetic relatives (Paraburkholderia caledonica and Paraburkholderia terrae, respectively) to be regarded as novel species. This was also supported by the results of fatty acid analysis and metabolic characterisation. For these two isolate groups, we accordingly propose the new species Paraburkholderia strydomiana sp. nov. with WK1.1f T (= LMG 28731 T  = SARCC1213 T ) as its type strain and Paraburkholderia steynii sp. nov. with HC1.1ba T (= LMG 28730 T  = SARCC696 T ) as its type strain. Our data thus showed that H. sophoroides may be considered a promiscuous symbiotic partner due to its ability to associate with multiple species of Paraburkholderia.

Researches involving the use of association between legumes and PGPBs (plant growth-promoting bacteria) in heavy metal phytoremediation process were mainly performed for soils highly contaminated. However, even in agriculture soils, with moderate or low contamination levels, plants can accumulate high rates of heavy metals. So, food chain contamination by these metals presents a real threat to animal and human health. This work aimed to evaluate the use of two legumes/PGPB symbioses; Vicia faba var. minor and Sulla coronaria have been inoculated with specific heavy metal-resistant inocula in a crop rotation system with Lactuca sativa as a following crop, in order to assess their effects on soil fertility, lettuce yield, and heavy metal content. Our results showed that legume inoculation significantly enhanced their biomass production, nitrogen and phosphorus content. The use of our symbioses as green manure before lettuce cultivation, as a rotation cropping system, affected positively soil fertility. In fact, we recorded a higher organic matter content, with rapid decomposition in the soil of inoculated plots. Besides, results demonstrated a greater nitrogen and phosphorus content in this soil, especially in the plot cultivated with inoculated V. faba var. minor. The improvement of soil fertility enhanced lettuce yield and its nitrogen and phosphorus content. Moreover, inoculated legumes extracted and accumulated more heavy metals than non-inoculated legumes. Our symbioses play the role of organic trap for heavy metals, making them unavailable for following crops. These facts were supported by lettuce heavy metal content, showing a significant decrease in metal accumulation, mainly zinc and cadmium, in edible parts. Results showed the usefulness of the studied symbioses, as a main part of a rotation system with lettuce. Our symbioses can be suggested for agriculture soil phytoremediation, aiming to enhance non-legume crop yield and limit heavy metal translocation to food chain.

Phytate is a potent inhibitor of mineral absorption in humans occurring in plant-based food. Application of lactobacilli that produce phytate-degrading enzymes (phytases) to reduce phytate is an interesting yet a not much explored sector of research. Therefore, phytate dephosphorylation by Lactobacillus plantarum MTCC 1325 was evaluated. Cells at stationary phase showed phytase activity which was maximal at 24 h of growth. Glucose concentration and the type of phosphorous source in the media modulated the enzyme activity. Fermentation of cereal and/or legume flours with the strain resulted in phytate reduction with the highest in sorghum (73%) and the lowest in horse gram (34%). Further, the strain showed tolerance to acid, bile, and simulated gastrointestinal fluid. Significant phytase activity in the presence of simulated gastrointestinal fluids along with the ability to produce phytases post-exposure to simulated gastrointestinal fluids is of interest. To the best of our knowledge, this is the first report on the effect of simulated gastrointestinal fluid on cell-associated phytases of lactobacilli. The results of the investigation indicate that L. plantarum MTCC 1325 could be used as a starter in cereal-legume fermentation and as potential probiotics to achieve phytate hydrolysis in food matrices and also in gastrointestinal tract.

Actinomycetes, a Gram positive bacteria, well reported as a source of antibiotics, also possess potential to control various plant pathogens, besides acting as plant growth promoting agent. Chemicals in different forms are extensively being used in vegetable farming, adversely affecting the environment and consumer health. Microbial agent like actinomycetes can substantially replace these harmful chemicals, and have now started finding a place as an important input in to farming practices. Only selected vegetable crops belonging to 11 different families have been explored with use of actinomycetes as biocontrol and plant growth promoting agent till now. It provides ample opportunities to vegetable researchers, to further explore with use of this very important group of microorganisms, in order to achieve even higher production level of safe vegetables. Mycostop and Actinovate are two actinomycetes based formulations globally available for use in vegetable farming as a substitute for chemical formulations. Present review article has summarized the literature available on use of actinomycetes in vegetable farming. Existing wide gap in knowledge, and potential thrust areas for future research have also been projected.

Plant leaves (phyllosphere) have a great potential for colonization and microbial growth, consisting of a dynamic environment in which several factors can interfere with the microbial population structure. The use of genetically modified (GM) plants has introduced several traits in agriculture, such as the improvement of plant drought tolerance, as observed in the AtAREB1 transcription factor overexpression in soybean (Glycine max L. Merrill). The present study aimed at investigating the taxonomic and functional profile of the leaf microbial community of bacteria found in GM (drought-tolerant event 1Ea2939) and conventional (BR 16) soybean plants. Bacterial DNA was extracted from leaf samples collected from each genotype and used for microbial diversity and richness analysis through the MiSeq Illumina platform. Functional prediction was performed using the PICRUSt tool and the STAMP v 2.1.3 software. The obtainment of the GM event 1Ea2939 showed minimum effects on the microbial community and in the potential for chemical-genetic communication, i.e. in the potential for symbiotic and/or mutualistic interaction between plants and their natural microbiota.

The growing commercial demand for products with medicinal use has caused overexploitation of several plant species worldwide. To prevent the decline of these populations, the collection of these resources should be done in a sustainable way considering the time of its replacement in natural stocks. This study was designed to identify the relationship between different intensities of extraction of bark from the trunk of Stryphnodendron rotundifolium Mart. and its regeneration speed. For this, we selected two areas of Cerrado in the Northeast of Brazil, where a monitoring experiment with duration of 24 months was performed. This experiment consisted in simulating different extractive damage to assess the regeneration of bark. In each area, we selected 20 individuals, among which four treatments with five repetitions were implemented. The data showed that in both study areas, the trees regenerated their shells faster when subjected to higher collection intensities. However, this regeneration was not related to variations in rainfall in the environment.

Key Message: Genetic control of the resistance response against powdery mildew in common bean was studied combining genetic, genomic and transcriptomic analyses. A candidate resistance gene in cultivar Porrillo Sintetico was proposed. The species causing the fungal disease powdery mildew (PM) in the local common bean crop was identified as Erysiphe polygoni through the molecular analysis of the internal transcribed spacer region. A genetic analysis of the resistance in cultivar Porrillo Sintetico was conducted using different F 2:3 populations, and a dominant gene conferring total resistance against a local PM isolate was physically located between 84,188 and 218,664 bp of chromosome Pv04. An in silico analysis of this region, based on the common bean reference sequence, revealed four genes candidate to be involved in the resistance reaction. Relative expression levels of these genes after PM infection showed a significant over-expression of the candidate gene Phvul.004G001500 in the resistant genotype Porrillo Sintetico. This gene was re-sequenced in the parental genotypes X2776 and Porrillo Sintetico to explain their different phenotypic responses against PM. Several substitutions where identified in exon regions, all of them synonymous, so differences in the produced amino acid sequence were not expected. However, a total of 37 mutations were identified in non-coding regions of the gene sequence, suggesting that intron variation could be responsible for the different gene expression levels after PM infection. No evidence of other regulatory mechanisms, such as alternative splicing or methylation, was identified. Candidate resistance gene Phvul.004G001500 codes for an elongation factor that is not a typical gene related to recognition of specific pathogens in plants, suggesting its involvement in the resistance through plant immune system.

A strain 5-1-2 T was isolated from a root nodule of Hedysarum multijugum collected from Zhangye city, Gansu province, north-west China. Phylogenetic analysis based on the 16S rRNA gene sequence and other housekeeping genes (recA and atpD) indicated that the strain represents a novel species in the genus Rhizobium close to the strain Rhizobium subbaraonis JC85 T with similarities of 98.27, 88.92 and 89.62%, respectively. Strain 5-1-2 T contained Q-10 as the predominant ubiquinone. Our results showed that the major fatty acids were feature 8 (C 18:1 ω7c and/or C 18:1 ω6c; 38.90%). In addition, the DNA-DNA hybridizations with the type strains R. subbaraonis JC85 T and Rhizobium halophytocola YC6881 T were 39.2 ± 2.1 and 44.3 ± 1.9, respectively. Therefore, a novel species Rhizobium hedysari sp. nov. is proposed, and 5-1-2 T (=CGMCC1.15677 T  = NBRC112532 T) is designated as the type strain.

To understand the geographic distributions of rhizobia that associated with widely distributed wild legumes, 66 nodules obtained from 41 individuals including three sea-dispersed legumes (Vigna marina, Vigna luteola, and Canavalia rosea) distributed across the tropical and subtropical coastal regions of the world were studied. Partial sequences of 16S rRNA and nodC genes extracted from the nodules showed that only Bradyrhizobium and Sinorhizobium were associated with the pantropical legumes, and some of the symbiont strains were widely distributed over the Pacific. Horizontal gene transfer of nodulation genes were observed within the Bradyrhizobium and Sinorhizobium lineages. BLAST searches in GenBank also identified records of these strains from various legumes across the world, including crop species. However, one of the rhizobial strains was not found in GenBank, which implies the strain may have adapted to the littoral environment. Our results suggested that some rhizobia, which associate with the widespread sea-dispersed legume, distribute across a broad geographic range. By establishing symbiotic relationships with widely distributed rhizobia, the pantropical legumes may also be able to extend their range much further than other legume species.

Isolates from root nodules of Hedysarum flexuosum, sampled from north region of Algeria, were analyzed on the basis of their phenotypic and molecular characteristics. They were tested for their tolerance to NaCl, pH, temperatures, antibiotics and heavy metals resistance. Interestingly, the isolate Hf_04N appeared resistant to ZnCl2 (50 μg/mL) and grew at high saline concentration up to 9 %. The phylogenetic positions of five isolates were studied by comparative sequence analysis of 16S rRNA, recA, nifH and nodD genes. There were grouped close to the Rhizobium sullae type strain in relation to their 16S rRNA, recA and nifH genes-based phylogenies. By contrast, the tree of nodD gene was not congruent with ribosomal, housekeeping and nitrogen fixation genes. We suggest that our strains have a novel nodD gene. The detection of conserved domains of NodD protein and nitrogenase reductase enzyme, confirm their ability to nodulate and fix nitrogen.

The Fusarium solani species complex (FSSC) includes important root pathogens of soybean in the United States, but the evolutionary lineages associated with soybean root rot are unknown. A multilocus phylogeny based on 93 isolates from soybean and pea roots from North Dakota and Minnesota revealed that root rot was associated with three known phylogenetic species, FSSC 3 + 4 (=Fusarium falciforme) (3 % of isolates), FSSC 5 (60 %), FSSC 11 (34 %), and one unknown species, FSSC X (2 %). Of these species FSSC 5 and FSSC 3 + 4 are clinically important while FSSC 11 is a plant pathogen. Isolates from FSSC 11 were pathogenic on soybean, dry bean, pea and lentil, and did not grow at 37 °C. However, isolates from FSSC 5 were weakly to non-pathogenic, but grew at 37 °C. Isolates from both FSSC 5 and FSSC 11 were highly resistant to fludioxonil in vitro. This is the first study revealing the pathogenic robustness of FSSC 11 in causing root rot among Fabaceae crops and also the association of clinically important members of the FSSC with roots of a widely grown field crop in the United States.

The present study focused on the overproducing mutant of a plant growth promoting rhizobacterium (PGPR) Pseudomonas simiae strain AU (MTCC-12057) for significant drought tolerance in mung bean plants. Five mutants namely AU-M1, AU-M2, AU-M3, AU-M4 and AU-M5 were made after treatment of wild type strain with N-methyl-N-nitro-N-nitrosoguanidine. Mutant strain AU-M4 was recorded for enhanced ACC deaminase (ACC-D) activity, indole acetic acid (IAA) production and inorganic phosphate (Pi) solubilization compared to wild strain and other four mutant strains under drought condition. AU-M4 showed higher phosphate solubilization index (8.17) together with higher ACC-D activity (98 nmol/mg/h) and IAA concentration (69.35 µg/ml) compared with the wild type P. simiae strain AU ACC-D activity (79 nmol/mg/h) and IAA concentration (38.98 µg/ml) respectively. In this report, we investigated the effect of both wild and mutant type bacterial strain on mung bean plants under drought stress. Results showed that mutant AU-M4 and wild type strain AU inoculated plants exhibited superior tolerance against drought stress, as shown by their enhanced plant biomass (fresh weight), higher water content, higher proline accumulation and lower osmotic stress injury. Mutant AU-M4 and wild strain AU inoculated plants reduced the ethylene level by 59 and 45% respectively, compared to the control under stress condition. Furthermore, bacterial inoculated plants showed enhanced induced systemic drought tolerance by reducing stomata size and net photosynthesis resulting higher water content in mung bean plants that may help in survival of plants during drought condition. To mitigate the effects of drought stress, use of PGPR will be needed to ensure sufficient production of food from crop plants. Taking current leads available, concerted future research is needed in this area, particularly on field evaluation with application of potential microorganisms.

Lipo-chitooligosaccharides produced by nitrogen-fixing rhizobia are signaling molecules involved in the establishment of an important agronomical and ecological symbiosis with plants. These compounds, known as Nod factors, are biologically active on plant roots at very low concentrations indicating that they are perceived by specific receptors. This article summarizes the main strategies developed for the syntheses of bioactive Nod factors and their derivatives in order to better understand their mode of perception. Different Nod factor receptors and LCO-binding proteins identified by genetic or biochemical approaches are also presented, indicating perception mechanisms that seem to be more complicated than expected, probably involving multi-component receptor complexes.

This study explored the diversity and phylogeny of rhizobia collected from nodules of Sphaerophysa salsula in different geographical regions of Northwest China. The 16S rRNA gene sequences divided the strains into the following distinct groups: Mesorhizobium, Rhizobium and Shinella. The phylogenies of recA and atpD genes showed low correlation with nifH and nodA gene in most species, which indicated that, the gene recombination between species and genera might have been exist. To our knowledge, this is the first study using the multilocus sequencing analysis Sphaerophysa rhizobia in order to understand the relation between genetic diversity and ecology.

Intensive agriculture has resulted in an indiscriminate use of pesticides, which demands in-depth analysis of their impact on indigenous rhizospheric microbial community structure and function. Hence, the objective of the present work was to study the impact of two chemical pesticides (chlorpyrifos and cypermethrin) and one biological pesticide (azadirachtin) at two dosages on the microbial community structure using cultivation-dependent approach and on rhizospheric bacterial communities involved in nitrogen cycle in Vigna radiata rhizosphere through cultivation-independent technique of real-time PCR. Cultivation-dependent study highlighted the adverse effects of both chemical pesticide and biopesticide on rhizospheric bacterial and fungal communities at different plant growth stages. Also, an adverse effect on number of genes and transcripts of nifH (nitrogen fixation); amoA (nitrification); and narG, nirK, and nirS (denitrification) was observed. The results from the present study highlighted two points, firstly that nontarget effects of pesticides are significantly detrimental to soil microflora, and despite being of biological origin, azadirachtin exerted negative impact on rhizospheric microbial community of V. radiata behaving similar to chemical pesticides. Hence, such nontarget effects of chemical pesticide and biopesticide in plants' rhizosphere, which bring out the larger picture in terms of their ecotoxicological effect, demand a proper risk assessment before application of pesticides as agricultural amendments.

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18(T) 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(T) 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(T) and Vaf-43 had notable amounts of C16:0 (4.8-6.0 %), C16:0 3-OH (6.4-6.6 %), C16:1 ω5c (8.8-9.0 %), C17:0 cyclo (13.5-13.9 %), C18:1 ω7c (43.4-45.4 %), C19:0 cyclo ω8c (10.5-12.6 %) and Summed Feature (SF) 3 (6.4-8.0 %). The DNA-DNA relatedness between the strains Vaf-18(T) and B. lathyri LMG 26379(T) was 24.0 %. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129(T) = LMG 28367(T) = Vaf-18(T)) is proposed.

Anthyllis vulneraria was highlighted here as a Zn-hyperaccumulator for the development of a pilot phytoextraction process in the mine site of Les Avinières in the district of Saint-Laurent-Le-Minier. A. vulneraria appeared to hyperaccumulate the highest concentration of Zn in shoots with a better metal selectivity relative to Cd and Pb than the reference Zn-hyperaccumulator Noccea caerulescens. A bigger biomass production associated to a higher Zn concentration conducted A. vulneraria to the highest total zinc gain per hectare per year. As a legume, A. vulneraria was infected by rhizobia symbionts. Inoculation of A. vulneraria seeds showed a positive impact on Zn hyperaccumulation. A large-scale culture process of symbiotic rhizobia of A. vulneraria was investigated and optimized to allow large-scale inoculation process. Contaminated shoots of A. vulneraria were not considered as wastes and were recovered as Eco-Zn catalyst in particular, examples of organic synthesis, electrophilic aromatic substitution. Eco-Zn catalyst was much more efficient than conventional catalysts and allowed greener chemical processes.

The aim of this work was using the legume plant Anthyllis vulneraria L. (ecotype metallicolous) as a trap plant, in order to isolate metal-tolerant rhizobial strains from metal-contaminated soils from Morocco, with pollution indexes spanning three orders of magnitude. As bioindicator, soil bacterial density was inversely correlated to the pollution index. Forty-three bulk soil bacteria and sixty two bacteria from nodules were isolated. The resistance of bacteria from nodules to heavy metals was four to ten times higher than that of bulk soil bacteria, reaching high maximum tolerable concentrations for Cd (2 mM), Cu (2 mM), Pb (7 mM), and Zn (3 mM). Besides, some strains show multiple metal-tolerant abilities and great metal biosorption onto the bacterial surface. Amplification and restriction analysis of ribosomal 16S rDNA (ARDRA) and 16S ribosomal DNA (rDNA) sequencing were used to assess biodiversity and phylogenetic position among bacteria present in nodules. Our results suggest that a great diversity of non-rhizobial bacteria (alpha- and gamma-proteobacteria) colonize nodules of Anthyllis plants in contaminated soils. Taking together, our results evidence that, in polluted soils, rhizobia can be displaced by non-rhizobial (and hence, non-fixing) strains from nodules. Thus, the selection of metal-resistant rhizobia is a key step for using A. vulneraria symbioses for in situ phytoremediation.

Many legumes have been selected as model plants to degrade organic contaminants with their special associated rhizosphere microbes in soil. However, the function of root nodules during microbe-assisted phytoremediation is not clear. A pot study was conducted to examine phenanthrene (PHE) utilizing bacteria associated with root nodules and the effects of cowpea root nodules on phytoremediation in two different types of soils (freshly contaminated soil and aged contaminated soil). Cowpea nodules in freshly-contaminated soil showed less damage in comparison to the aged-contaminated soil, both morphologically and ultra-structurally by scanning electron microscopy. The study of polycyclic aromatic hydrocarbon (PAH) attenuation conducted by high performance liquid chromatography revealed that more PAH was eliminated from liquid culture around nodulated roots than nodule-free roots. PAH sublimation and denaturation gradient gel electrophoresis were applied to analyze the capability and diversity of PAH degrading bacteria from the following four parts of rhizo-microzone: bulk soil, root surface, nodule surface and nodule inside. The results indicated that the surface and inside of cowpea root nodules were colonized with bacterial consortia that utilized PHE. Our results demonstrated that root nodules not only fixed nitrogen, but also enriched PAH-utilizing microorganisms both inside and outside of the nodules. Legume nodules may have biotechnological values for PAH degradation.

Arbuscular mycorrhizal (AM) fungi have traditionally been considered generalist symbionts. However, an increasing number of studies are pointing out the selectivity potential of plant hosts. Plant life form, determined by plant life history traits, seems to drive the AM fungal community composition. The AM fungi also exhibit a wide diversity of functional traits known to be responsible for their distribution in natural ecosystems. However, little is known about the role of plant and fungal traits driving the resultant symbiotic assemblages. With the aim of testing the feedback relationship between plant and fungal traits on the resulting AM fungal community, we inoculated three different plant life forms, i.e. annual herbs, perennial herbs and perennial semi-woody plants, with AM fungal communities sampled in different seasons. We hypothesized that the annual climate variation will induce changes in the mean traits of the AM fungal communities present in the soil throughout the year. Furthermore, the association of plants with different life forms with AM fungi with contrasting life history traits will show certain preferences according to reciprocal traits of the plants and fungi. We found changes in the AM fungal community throughout the year, which were differentially disrupted by disturbance and altered by plant growth form and plant biomass. Both plant and fungal traits clearly contributed to the resultant AM fungal communities. The revealed process can have implications for the functioning of ecosystems since changes in dominant plant life forms or climatic variables could influence the traits of AM fungal communities in soil and hence ecosystem processes.

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4-6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di- and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA-RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP-16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.

Four hundred seventy Rhizoctonia solani isolates from different leguminous hosts originating from 16 agro-ecological regions of India covering 21 states and 72 districts were collected. The disease incidence caused by R. solani varied from 6.8 to 22.2 % in the areas surveyed. Deccan plateau and central highlands, hot sub-humid ecoregion followed by northern plain and central highlands and hot semi-arid ecoregion showed the highest disease incidence. R. solani isolates were highly variable in growth diameter, number, size and pattern of sclerotia formation as well as hyphal width. The isolates obtained from aerial part of the infected plants showing web blight symptoms produced sclerotia of 1-2 mm in size whereas, the isolates obtained from infected root of the plants showing wet root rot symptoms produced microsclerotia (<1 mm). Majority of R. solani isolates showed <8 μm hyphal diameter. Based on morphological characters the isolates were categorized into 49 groups. Seven anastomosis groups (AGs) were identified among the populations of R. solani associated with the pulse crops. The frequency (25.6 %) of AG3 was the highest followed by AG2-3 (20.9 %) and AG5 (17.4 %). The cropping sequence of rice/sorghum/wheat-chickpea/mungbean/urdbean/cowpea/ricebean influenced the dominance of AG1 (16.3 %). Phylogenetic analysis utilizing ITS-5.8S rDNA gene sequences indicated high level of genetic similarity among isolates representing different AGs, crops and regions. ITS groups did not correspond to the morphological characters. The sequence data from this article has been deposited with NCBI data libraries with JF701707 to JF701795 accession numbers.

Introduced plants may out-compete natives by belowground allelopathic effects on soil communities including the symbionts of native plants. We tested for an allelopathic effect of an introduced crucifer, Raphanus sativus, on a common neighboring legume, Lupinus nanus, on the legume's rhizobium affiliates, and on the broader soil community. In both field observations and a greenhouse experiment, we found that R. sativus decreased the density of nodules on L. nanus roots. However, in the greenhouse experiment, R. sativus soils only decreased the density of small, likely non-beneficial rhizobium nodules. In the same experiment, R. sativus soils decreased fungivorous nematode abundance, though there was no effect of R. sativus introduction on fungal density. In the greenhouse experiment, R. sativus soils had a net positive effect on L. nanus biomass. One explanation of this effect is that R. sativus introduction might alter the mutualistic/parasitic relationship between L. nanus and its rhizobial associates with a net benefit to L. nanus. Our results suggest that introduced brassicas can quickly alter belowground communities, but that the net effect of this on neighboring plants is not necessarily negative.

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. viciae 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.

The taxonomic status of a bacterium, strain NCCP-246(T), isolated from rhizosphere of Vigna mungo, was determined using a polyphasic taxonomic approach. The strain NCCP-246(T) can grow at 16-37 °C (optimum 32 °C), at pH ranges of 6-8 (optimum growth occurs at pH 7) and in 0-4 % (w/v) NaCl. Phylogenetic analysis based upon on 16S rRNA gene sequence comparison revealed that strain NCCP-246(T) belonged to genus Sphingobacterium. Strain NCCP-246(T) showed highest similarity to the type strain of Sphingobacterium canadense CR11(T) (97.67 %) and less than 97 % with other species of the genus. The DNA-DNA relatedness value of strain NCCP-246(T) with S. canadense CR11(T) and Sphingobacterium thalpophilum JCM 21153(T) was 55 and 44.4 %, respectively. The chemotaxonomic data revealed the major menaquinone as MK-7 and dominant cellular fatty acids were summed feature 3 [C16:1 ω7c/C16:1 ω6c] (37.07 %), iso-C15:0 (28.03 %), C16:0 (11.85 %), C17:0 cyclo (8.84 %) and C14:0 (2.42 %). The G+C content of the strain was 39.2 mol%. On the basis of DNA-DNA hybridization, phylogenetic analyses, physiological and, biochemical data, strain NCCP-246(T) can be differentiated from the validly named members of genus Sphingobacterium and thus represents as a new species, for which the name, Sphingobacterium pakistanensis sp. nov. is proposed with the type strain NCCP-246(T) (= JCM18974 (T) = KCTC 23914(T)).

While the occurrence of Betaproteobacteria occupying the nodules of tropical legumes has been shown, little is known about subtropical areas. Araucaria Forest is a subtropical endangered ecosystem, and a better understanding of the legume-rhizobial symbionts may allow their use in land reclamation. The 16S rRNA gene of bacteria isolated from nine leguminous species was sequenced and their nodulation tested in Mimosa scabrella and Phaseolus vulgaris. 196 isolates were identified as eight genotypes: Pantoea, Pseudomonas, Bradyrhizobium sp1-2, Rhizobium, and Burkholderia sp1-3. The majority of the isolates from native plants (87 %) were taxonomically related to β-rhizobia, namely Burkholderia, however the legumes Galactia crassifolia and Collea speciosa were nodulated by both α and β-rhizobia, and Acacia dealbata, an exotic plant, only by α-rhizobia. The nifH genes of some isolates were sequenced and N-fixing potential shown by the acetylene reduction test. Most of the isolates nodulated the test plants, some were effective in M. scabrella, but all presented low efficiency in the exotic promiscuous legume P. vulgaris. Pantoea and Pseudomonas did not nodulate and probably are endophytic bacteria. The presented data shows diversity of α, β and γ-Proteobacteria in nodules of subtropical legumes, and suggests host specificity with β-rhizobia. Potential isolates were found for M. scabrella, indicating that a high N-fixing strain may be further inoculated in plants for use in reforestation.

Leucaena leucocephala was introduced into Panxi, Sichuan, China, in the 1980s and 1990s for afforestation and preventing water loss and soil erosion in this area. The co-introduction of rhizobial symbionts of introduced plants has drawn attention since they may influence local soil communities. We studied the phylogenetic position of the L. leucocephala isolates and assessed if the rhizobia were introduced together with the host to Panxi, Sichuan, China. The glnII and atpD genes of fifteen representative isolates were sequenced and analyzed, and applied multilocus sequence analyses in which the housekeeping genes recA, glnII and atpD were included. Furthermore, we estimated the within species diversity directly with 23S rDNA and IGS RFLP and indirectly through phenotypic analysis of forty L. leucocephala isolates. The isolates represented seven species and 38 diversified strains in the genera Ensifer, Mesorhizobium, Bradyrhizobium and Rhizobium. The within species diversity of the Ensifer isolates was large, proposing a potential to occupy novel niches. There was not conclusive evidence to show that any of the strains would have been co-introduced with L. leucocephala. On the contrary, we came to a conclusion that the possible introduction should not be inferred from sequence data alone.

Increasing concern regarding mining area environmental contamination with heavy metals has resulted in an emphasis of current research on phytoremediation. The aim of the present study was to assess the efficiency of symbiotic Cupriavidus necator strains on different leguminous plants in soil contaminated with heavy metals following the application of inorganic materials. The application of limestone and calcium silicate induced a significant increase in soil pH, with reductions in zinc and cadmium availability of 99 and 94 %, respectively. In addition, improved nodulation of Mimosa caesalpiniaefolia, Leucaena leucocephala and Mimosa pudica in soil with different levels of contamination was observed. Significant increases in the nitrogen content of the aerial parts of the plant were observed upon nodulation of the root system of Leucaena leucocephala and Mimosa pudica by strain UFLA01-659 (36 and 40 g kg(-1)) and by strain UFLA02-71 in Mimosa caesalpiniaefolia (39 g kg(-1)). The alleviating effect of calcium silicate resulted in higher production of dry matter from the aerial part of the plant, an increase in nodule number and an increase in the nitrogen fixation rate. The results of the present study demonstrate that the combination of rhizobia, leguminous plants and calcium silicate may represent a key factor in the remediation of areas contaminated by heavy metals.

Arid environments are regular and well distributed over all continents and display drought characteristics whether full-time or seasonal. This study aims to characterize how the microbial communities of the rhizosphere of two leguminous trees from the Brazilian semi-arid biome the Caatinga are geographically and seasonally shaped, as well as the factors driving this variation. With that purpose, the soil rhizosphere from two leguminous trees (Mimosa tenuiflora and Piptadenia stipulacea (Benth.) Ducke) were sampled in two different seasons: rainy and drought at five different sites. Assessment of bacterial and archaeal communities occurred by T-RFLP analysis of 16S rRNA and archaeal amoA genes. By these means, it was observed that the seasons (wet and dry periods) are the factors that most influence the composition of the microbial community from both analyzed plants, except for the results obtained from the CCA applied to Archaeas. Furthermore, soil physical-chemical factors also had a significant influence on the community and indicated a geographical pattern of the bacterial community. It was not possible to observe significant modifications in the composition in relation to the plant species. We have seen that soil characteristics and rainfall were the factors that most influenced the microbial composition. Also, the bacterial community had a significant correlation with soil characteristics that indicates that these rhizosphere communities might be selected by environmental characteristics. Furthermore, the data suggest that climate plays a key role in structuring the microbial community of this biome.

Four strains representing three novel anamorphic yeast species were isolated from the external surface of sugarcane leaves (DMKU-RK254(T)), corn leaves (DMKU-RK548(T)), bean leaves (K129) in Thailand and hengchun pencilwood leaves (TrB1-1(T)) in Taiwan. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene, the internal transcribed spacer (ITS) region, the actin gene (ACT1) and the elongation factor 2 gene (EF2), the four strains were determined to represent novel Yamadazyma species although formation of ascospores was not observed. Strain DMKU-RK254(T) was determined to be related to Candida diddensiae, Candida naeodendra and Candida kanchanaburiensis but with 1.8, 1.8 and 2.0 % nucleotide substitutions in the D1/D2 region of the LSU rRNA gene, respectively. It was assigned to Yamadazyma siamensis sp. nov. (type strain DMKU-RK254(T) = BCC 50730(T) = NBRC 108901(T) = CBS 12573(T)). The sequences of the D1/D2 region of the LSU rRNA gene, the ITS region, ACT1 gene and EF2 gene of two strains (DMKU-RK548(T) and K129) were identical but differed from that of strain TrB1-1(T) by 0.6, 1.0, 3.3 and 5.9 % nucleotide substitutions, respectively. Therefore, the two strains (DMKU-RK548(T) and K129) and strain TrB1-1(T) were assigned to be two separate species. The closest species in terms of pairwise sequences similarity of the D1/D2 region to the two novel species was Yamadazyma philogaea but with 1.1-1.7 % nucleotide substitutions. The two strains (DMKU-RK548(T) and K129) were assigned to Yamadazyma phyllophila sp. nov. (type strain DMKU-RK548(T) = BCC 50736(T) = NBRC 108906(T) = CBS 12572(T)) and the strain TrB1-1(T) was named Yamadazyma paraphyllophila sp. nov. (type strain TrB1-1(T) = BCRC 23030(T) = CCTCC AY 204005(T) = CBS 9928(T)).

A phosphate solubilizing fungus, Aspergillus awamori S29 was isolated from rhizoshpere of mungbean. The phosphate solubilizing activity of A. awamori S29 in liquid was 1,110 mg/L for tricalcium phosphate (TCP). The organism was able to solubilize various inorganic forms of phosphate at a wide range of temperatures. Among various insoluble phosphate sources tested, di-calcium phosphate was solubilized the most, followed by TCP. A. awamori S29 had significant effect (p < 0.05) on mungbean growth, total P and plant biomass under pot conditions, although no obvious difference in available P in soil and number of leaves was found compared to the control.

An endophytic Streptomyces sp. GMKU 3100 isolated from roots of a Thai jasmine rice plant (Oryza sativa L. cv. KDML105) showed the highest siderophore production on CAS agar while phosphate solubilization and IAA production were not detected. A mutant of Streptomyces sp. GMKU 3100 deficient in just one of the plant growth promoting traits, siderophore production, was generated by inactivation of a desD-like gene encoding a key enzyme controlling the final step of siderophore biosynthesis. Pot culture experiments revealed that rice and mungbean plants inoculated with the wild type gave the best enhancement of plant growth and significantly increased root and shoot biomass and lengths compared with untreated controls and siderophore-deficient mutant treatments. Application of the wild type in the presence or absence of ferric citrate significantly promoted plant growth of both plants. The siderophore-deficient mutant clearly showed the effect of this important trait involved in plant-microbe interaction in enhancement of growth in rice and mungbean plants supplied with sequestered iron. Our results highlight the value of a substantial understanding of the relationship of the plant growth promoting properties of endophytic actinomycetes to the plants. Endophytic actinomycetes, therefore, can be applied as potentially safe and environmentally friendly biofertilizers in agriculture.

The degree of specialization in the legume-rhizobium mutualism and the variation in the response to different potential symbionts are crucial factors for understanding the process of invasion by exotic legumes and the consequences for the native resident plants and bacteria. The enhanced novel mutualism hypothesis predicts that exotic invasive legumes would take advantage of native rhizobia present in the invaded soils. However, recent studies have shown that exotic legumes might become invasive by using exotic introduced microsymbionts, and that they could be a source of exotic bacteria for native legumes. To unravel the role of novel and old symbioses in the progress of invasion, nodulation and symbiotic effectiveness were analyzed for exotic invasive plants and native co-occurring legumes in a Mediterranean coastal dune ecosystem. Although most of the studied species nodulated with bacteria from distant origins these novel mutualisms were less effective in terms of nodulation, nitrogenase activity and plant growth than the interactions of plants and bacteria from the same origin. The relative effect of exotic bradyrhizobia was strongly positive for exotic invasive legumes and detrimental for native shrubs. We conclude that (1) the studied invasive legumes do not rely on novel mutualisms but rather need the co-introduction of compatible symbionts, and (2) since exotic rhizobia colonize native legumes in invaded areas, the lack of effectiveness of these novel symbiosis demonstrated here suggests that invasion can disrupt native belowground mutualisms and reduce native legumes fitness.

Plant growth promoting rhizobacteria (PGPR) are an attractive eco-friendly alternative to chemicals in agriculture. While the rhizospheres of crop plants have been well studied with the objective of screening PGPR, weeds, which play an important role in maintaining ecological balance, have largely been ignored. The rhizosphere of a luxuriantly growing, medicinal weed, Cassia occidentalis was analysed by enumerating PGPR on N free media from the most diverse stage of plant (determined by profiles obtained on denaturing gradient gel electrophoresis). Each isolate was tested for other plant growth promotion assays including production of cellulase, indole acetic acid (IAA), ammonia, HCN, siderophore and chitinase to select for ones possessing multi-trait plant growth promoting (PGP) properties. Selected isolates were used for bacterization of Vigna radiata and Vigna mungo to evaluate their efficacy in promoting plant's growth in seedling germination and axenic pot conditions. Thirty five isolates were analysed further for the array of PGP properties they exhibit. A total of 6 isolates were shortlisted on the basis of maximum traits positive, amount of phosphate solubilized and IAA produced. V. radiata responded well to seed bacterization during seedling germination. A maximum increase of approximately 36 and 60 % was observed for shoot and root length, respectively in V. radiata in axenic pot culture over control plants. Extensive branching of roots was also observed with isolate NL, which produced the maximum amount of IAA. Present study investigated the plant growth promoting isolates obtained on N free media in the rhizosphere of C. occidentalis, which have the potential to be used as inoculants for other crops. This provides a new dimension to the significance of weeds in agricultural ecosystems. The study opens up possibilities for utilization of this property of weeds in plant growth promotion, and subsequent enhancement of yield for agricultural crops.

Three strains (LM008(T), LM068 and LM078(T)), representing two novel yeast species were isolated from the phylloplane of three plant species by an enrichment technique. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analysis of the D1/D2 domain of the large subunit rRNA gene and the internal spacer region, the three strains were assigned as two novel Candida species. Strain LM008(T) was assigned to be Candida sirachaensis sp. nov. (type strain LM008(T) = BCC 47628(T) = NBRC 108605(T) CBS 12094(T)) in the Starmerella clade. Two strains (LM068 and LM078(T)) represent a single species in the Lodderomyces-Spathaspora clade for which the name Candida sakaeoensis sp. nov. is proposed with the type strain LM078(T) = BCC 47632(T) = NBRC 108895(T) = CBS 12318(T).

Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099(T)), Pseudomonas aureofaciens (NCIM2026(T)), and Pseudomonas aeruginosa (MTCC2582(T)). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH(4))(2)SO(4) precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn(2+), Cu(2+), and Ni(2+) ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.

In the present investigation, the effect of three beneficial organisms (root endophytic fungus Piriformospora indica (Pi) and pseudomonads strains R62 and R81) and their four different consortia (Pi+R62, Pi+R81, R62+R81, Pi+R62+R81) was investigated on the plant Vigna mungo through their inorganic carrier-based (talcum powder and vermiculite) formulations. All the treatments resulted in significant increase in growth parameters under glasshouse as well as field conditions and showed a consistency in their performance on moving from glasshouse to field conditions. In glasshouse conditions, a maximum increase of 4.5-fold in dry root weight and 3.9-fold in dry shoot weight compared to control was obtained with vermiculite-based consortium formulation of Pi+R81. In field studies using vermiculite as carrier, a maximum enhancement of 3.2-fold in dry root weight, 3.0-fold in dry shoot weight, 8.4-fold in number of nodules and 4.0-fold in number of pods in comparison to control was obtained with the bio-inoculant formulation containing consortium of Pi+R81. The same treatment also caused the highest improvement of 1.9-fold in nitrogen content and 1.7-fold in phosphorus content, while the highest increase of 1.4-fold in potassium content was obtained with Pi alone.

The genotypic diversity of rhizospheric bacteria of 3 legumes including Vigna radiata, Arachis hypogaea and Acacia mangium was compared by using cultivation-dependent and cultivation-independent methods. For cultivation-dependent method, Random amplified polymorphic DNA (RAPD) profiles revealed that the bacterial genetic diversity of V. radiata and A. mangium rhizospheres was higher than that of A. hypogaea rhizosphere. For cultivation-independent method, Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes revealed the difference in bacterial community and diversity of rhizospheres collected from 3 legumes. The ribotype richness which indicates species diversity, was highest in V. radiata rhizosphere, followed by A. hypogaea and A. mangium rhizospheres, respectively. Three kinds of media were used to cultivate different target groups of bacteria. The result indicates that the communities of cultivable bacteria in 3 rhizospheres recovered from nutrient agar (NA) medium were mostly different from each other, while Bradyrhizobium selective medium (BJSM) and nitrogen-free medium shaped the communities of cultivable bacteria. Nine isolates grown on BJSM were identified by 16S rRNA gene sequence analysis. These isolates were very closely related (with 96% to 99% identities) to either one of the three groups including Cupriavidus-Ralstonia group, Bacillus group and Bradyrhizobium-Bosea-Afipia group. The rhizospheres were also examined for their enzymatic patterns. Of 19 enzymes tested, 3 rhizospheres were distinguishable by the presence or the absence of leucine acrylamidase and acid phosphatase. The selected cultivable bacteria recovered from NA varied in their abilities to produce indole-acetic acid and ammnonia. The resistance to 10 antibiotics was indistinguishable among bacteria isolated from different rhizospheres.

The availability and quality of mutualists beyond a species' range edge may limit range expansion. With the legume Chamaecrista fasciculata, we asked to what extent the availability and quality of rhizobia beyond the range edge limits host range expansion. We tested the effect of rhizobia availability on plant growth by transplanting seed from three locations into five sites spanning C. fasciculata's range (interior, at the northern and western range edges, and beyond the range edges), and inoculating half the seeds with rhizobia. We recorded growth of all surviving plants, and, for the uninoculated plants, whether they had formed nodules or not. We isolated rhizobia from nodules collected on the uninoculated plants, and cross-inoculated seed from four populations (both range edge and interior populations) in the greenhouse to determine whether the quality of rhizobia differed between regions. We found that seeds transplanted beyond the range edge were less likely to be nodulated when they were not experimentally inoculated, and there was benefit to inoculation at all sites. In the greenhouse, the three inocula that formed nodules on plants, from the range interior, northern edge and beyond the northern edge, did not detectably differ in their effect on plant growth. These results suggest that low densities of suitable rhizobia beyond the range edge may limit range expansion of legume species.

Four gram-negative, aerobic, motile, non-spore, forming rods with a wide pH and temperature range for growth (pH 7.0-11.0, optimum pH 8.0; 20-45°C, optimum 28°C) strains were isolated from root nodules of Sphaerophysa salsula and characterized by means of a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the four strains formed a new lineage related to the genus Rhizobium and the sequence similarities between the isolate and the most related type strain Rhizobium giardinii was 96.5%. These strains also formed a distinctive group from the reference strains for defined Rhizobium species based on housekeeping gene sequences (atpD and recA), BOX-PCR fingerprinting, phenotypic features and symbiotic properties. The representative strain CCNWGS0238(T) has DNA-DNA relatedness of less than 33.4% with the most closely related species R. giardinii. It is therefore proposed as a new species, Rhizobium sphaerophysae sp. nov., with isolate CCNWGS0238(T) (=ACCC17498(T) = HAMBI3074(T)) as the type strain.

Bacteria not proficient in nitrogen fixing symbiosis were proven able to invade root nodules of three wild legumes of the genus Hedysarum in Algeria and to be multiplying in these in place of the natural rhizobium symbionts. The involved species featured taxa known as human pathogens including: Enterobacter cloacae, Enterobacter kobei, Escherichia vulneris, Pantoea agglomerans and Leclercia adecarboxylata. A direct screening of the phenotypic determinants of virulence using human cultured cells tested positive for the traits of cytotoxicity, vital stain exclusion and adhesion to epithelia. Antibiogram analyses revealed also a complex pattern of multiple antibiotic resistances. The data suggest that legume root nodules can be a site of survival and of active multiplication for populations of mammalian pathogens, which could thus alternate between the target animal and a number of neutral plant hosts. The worldwide distribution of as yet uninvestigated legumes raises the concern that these represent a general niche that could enhance the hazards posed by microorganisms of clinical nature.

The establishment of nitrogen-fixing symbiosis between a legume plant and its rhizobial symbiont requires that the bacterium adapt to changing conditions that occur with the host plant that both promotes and allows infection of the host root nodule cell, regulates and resists the host defense response, permits the exchange of metabolites, and contributes to the overall health of the host. This adaptive process involves changes to the bacterial cell surface and, therefore, structural modifications to the lipopolysaccharide (LPS). In this chapter, we describe the structures of the LPSs from symbiont members of the Rhizobiales, the genetics and mechanism of their biosynthesis, the modifications that occur during symbiosis, and their possible functions.

A multilocus phylogenetic approach was applied to elucidate the phylogeny of Astragalus cicer rhizobia derived from Poland, Ukraine, and Canada. The strains selected for the studies represented three main geographically different phenons of these bacteria. Phylogenetic analyses were performed with three chromosomal housekeeping loci (16S rRNA, atpD, glnII) and three symbiotic genes located on a plasmid (nodA, nodC, nifH). The "core" and "auxiliary" gene trees revealed that A. cicer nodule isolates were intermingled with the strains of Mesorhizobium species, which implies that they are descendents of the same ancestor as mesorhizobia and fall into the Mesorhizobium genus. The noted congruence of the housekeeping and symbiotic gene phylogenies of A. cicer microsymbionts indicates that sym loci are transferred to these bacteria through vertical transmission without a significant participation of intergeneric horizontal gene spread. All the three sym gene sequences of the Polish and Ukrainian A. cicer nodule isolates were more closely related to one another than to the corresponding sequences of the Canadian isolates. The phylogeographic patterns of the sym genes of intercontinental strains point to their relatively long, separate, evolutionary history.

Resistance to nine races of the pathogenic fungus Colletotrichum lindemuthianum, causal agent of anthracnose, was evaluated in F(3) families derived from the cross between the anthracnose differential bean cultivars TU (resistant to races, 3, 6, 7, 31, 38, 39, 102, and 449) and MDRK (resistant to races, 449, and 1545). Molecular marker analyses were carried out in the F(2) individuals in order to map and characterize the anthracnose resistance genes or gene clusters present in these two differential cultivars. The results of the combined segregation indicate that at least three independent loci conferring resistance to anthracnose are present in TU. One of them, corresponding to the previously described anthracnose resistance locus Co-5, is located in linkage group B7, and is formed by a cluster of different genes conferring specific resistance to races, 3, 6, 7, 31, 38, 39, 102, and 449. Evidence of intra-cluster recombination between these specific resistance genes was found. The second locus present in TU confers specific resistance to races 31 and 102, and the third locus confers specific resistance to race 102, the location of these two loci remains unknown. The resistance to race 1545 present in MDRK is due to two independent dominant genes. The results of the combined segregation of two F(4) families showing monogenic segregation for resistance to race 1545 indicates that one of these two genes is linked to marker OF10(530), located in linkage group B1, and corresponds to the previously described anthracnose resistance locus Co-1. The second gene conferring resistance to race 1545 in MDRK is linked to marker Pv-ctt001, located in linkage group B4, and corresponds to the Co-3/Co-9 cluster. The resistance to race 449 present in MDRK is conferred by a single gene, located in linkage group B4, probably included in the same Co-3/Co-9 cluster.