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Symbiotic nitrogen fixation (SNF) by rhizobium, a Gram-negative soil bacterium, is an essential component in the nitrogen cycle and is a sustainable green way to maintain soil fertility without chemical energy consumption. SNF, which results from the processes of nodulation, rhizobial infection, bacteroid differentiation and nitrogen-fixing reaction, requires the expression of various genes from both symbionts with adaptation to the changing environment. To achieve successful nitrogen fixation, rhizobia and their hosts cooperate closely for precise regulation of symbiotic genes, metabolic processes and internal environment homeostasis. Many researches have progressed to reveal the ample information about regulatory aspects of SNF during recent decades, but the major bottlenecks regarding improvement of nitrogen-fixing efficiency has proven to be complex. In this mini-review, we summarize recent advances that have contributed to understanding the rhizobial regulatory aspects that determine SNF efficiency, focusing on the coordinated regulatory mechanism of symbiotic genes, oxygen, carbon metabolism, amino acid metabolism, combined nitrogen, non-coding RNAs and internal environment homeostasis. Unraveling regulatory determinants of SNF in the nitrogen-fixing protagonist rhizobium is expected to promote an improvement of nitrogen-fixing efficiency in crop production., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microbes regulate soil health by negating ecological disturbances, and improve plant productivity in a sustainable manner. Indiscriminate application of pesticides creates a detrimental impact on the rhizospheric microbiota, thereby affecting soil health. Azadirachtin, earlier believed to be an environment-friendly alternative to chemical pesticides, exhibits a non-target impact on microbial communities. This study aimed to employ potent bacteria to promote the growth of mungbean plant (Vigna radiata), and mitigate the non-target impact of azadirachtin. Bacterial strains were isolated by enrichment from mungbean rhizosphere. A plant growth experiment was performed with mungbean, amended with azadirachtin to assess the impact of bacterial bioinoculants on the rhizospheric microbiota. The impact of azadirachtin on rhizospheric bacterial community was analyzed qualitatively and quantitatively by 16S rRNA PCR-DGGE and qPCR of various markers, respectively. Residual concentration of azadirachtin in the soil was estimated by HPLC. The bacterial inoculants used in combination significantly promoted plant growth and enhanced the diversity and abundance of total bacterial community in the presence of azadirachtin. Further, the abundance of specific bacterial groups (α-Proteobacteria, β-Proteobacteria, Actinobacteria, Acidobacteria, and Firmicutes) were significantly boosted. Compared to the control, the isolates significantly facilitated the reduction in residual concentration of azadirachtin in the mungbean rhizosphere. Bacterial inoculants can serve a tripartite role in reducing the stress imparted by botanical pesticides, together with promoting plant growth and enriching the rhizospheric bacterial community structure., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Legume-cereal intercropping systems, in the context of diversity, ecological function, and better yield have been widely studied. Such systems enhance nutrient phytoavailability by balancing root-rhizosphere interactions. Root exudates (RE) play an important role in the rhizospheric interactions of plant-plant and/or plant-microbiome interaction. However, the influence of the primary metabolites of RE on plant-rhizobia interactions in a legume-cereal intercrop system is not known. To understand the plant communication with rhizobia, Cajanus cajan-Zea mays intercropped plants and the broad host range legume nodulating Ensifer fredii NGR234 as the model plants and rhizobium used respectively. A metabolomics-based approach revealed a clear separation between intercropped and monocropped RE of the two plants. Intercropped C. cajan showed an increase in the myo-inositol, and proline, while intercropped Z. mays showed enhanced galactose, D-glucopyranoside, and arginine in the RE. Physiological assays of NGR234 with the RE of intercropped C. cajan exhibited a significant enhancement in biofilm formation, while intercropped Z. mays RE accelerated the bacterial growth in the late log phase. Further, using label-free proteomics, we identified a total of 2570 proteins of NGR234 covering 50% annotated protein sequences upon exposure to Z. mays RE. Furthermore, intercropped Z. mays RE upregulated bacterioferritin comigratory protein (BCP), putative nitroreductase, IlvD, LeuC, D (branched-chain amino acid proteins), and chaperonin proteins GroEL2. Identification offered new insights into the metabolome of the legume-cereal intercrop and proteome of NGR234-Z. mays interactions that underline the new molecular candidates likely to be involved in the fitness of rhizobium in the intercropping system., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

An endophytic fungus (L3), isolated from the leaf tissues of Saraca asoca was identified as D. longicolla by microscopic and molecular methods. The crude extracts of D. longicolla revealed to harbor seven compounds in GC-MS analysis which was subjected to a thin layer chromatography (TLC) for purification and separation of bioactive ingredients. The partially purified fraction from TLC displayed the presence of 2-tridecene (Z) (RT-14.50), 5-tridecene (E) (RT-16.65) and 2,4-di-tert-butylphenol (RT-13.92) in GC-MS. High-performance liquid chromatography (HPLC) was performed to further purify the constituents which led to the collection of 2,4-di-tert-butyl phenol (RT-2.34) with excellent antioxidant activity and antibacterial activity against methicillin resistance Staphylococcus aureus (MRSA)., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The DNA barcodes were developed from ITS region for the identification of fungal plant pathogens namely, Alternaria alternata and A. tenuissima both causing leaf spots, Ascochyta rabiei causing Ascochyta blight, Fusarium oxysporum f. sp. ciceris causing wilt, Macrophomina phaseolina causing dry root rot, Rhizoctonia solani causing web blight and wet root rot, Sclerotium (Athelia) rolfsii causing collar rot, Sclerotinia sclerotiorum causing stem rot and Cercospora canescens and Pseudocercospora cruenta both causing leaf spots in pulse crops. Barcode compliance for A. alternata (DBTPQ001-18), A. tenuissima (DBTPQ002-18), A. rabiei (DBTPQ003-18), F. oxysporum f. sp. ciceris (DBTPQ004-18), M. phaseolina (DBTPQ005-18), R. solani (DBTPQ006-18), S. rolfsii (DBTPQ007-18), S. sclerotiorum (DBTPQ008-18), C. canescens (DBTPQ009-18) and P. cruenta (DBTPQ029-20) have been generated based on the Barcode of Life Data System (BOLD) system. In addition to ITS, other genomic regions were also explored and on the basis of sequence variation they were ranked as TEF-α > SSU > LSU > β-tubulin. These genes could be considered for secondary barcode and phylogenetic relatedness. ITS-based markers for the detection of A. alternata (BAA2aF and BAA2aR) and R. solani (BRS17cF and BRS17cR) were developed which provided 400 bp and 220 bp amplicons, respectively. While, for F. oxysporum f. sp. ciceris, COX1-based marker (FOCox1F and FOCox3R) was developed which amplified 150 bp. The markers proved highly specific and sensitive with detection limit of 0.0001 ng of template DNA using qPCR and simultaneously detected these three pathogens. The DNA barcodes and diagnostics developed are suitable for quick and reliable detection of these pathogens during quarantine processing and field diagnostics.

A novel Gram-strain-negative, beige-pigmented, aerobic, rod-shaped, non-flagellated and non-gliding bacterium, designated strain lm93 T , was isolated from rhizosphere soil of Alhagi sparsifolia obtained from Alar city, located in Xinjiang province, China. Growth optimally occurred at 30 °C, pH 6.5-7.5, and 0-2% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain lm93 T belonged to the genus Chelativorans, with highest sequence similarity to Chelativorans multitrophicus DSM 9103 T (96.9%). Genome sequencing revealed a genome size of 5 689 708 bp and a G + C content of 64.3 mol%. The ANI, POCP and the dDDH between strain lm93 T and C. multitrophicus DSM 9103 T were 76.4%, 54.8% and 0.8%, respectively. The prediction result of secondary metabolites based on genome showed that the strain lm93 T contained one cluster of bacteriocin, one cluster of terpene production, two clusters of ectoine production, one cluster of non-ribosomal peptide synthetase, one cluster of type I polyketide synthases, three clusters of homoserine lactone production, one cluster of N-acetylglutaminylglutamine amide production and one cluster of phosphonate production. The major respiratory quinone was Q-10. The major fatty acids were C 19:0 cyclo ω8c, iso-C 17:0 and summed feature 8 (C 18:1 ω6c and/or C 18:1 ω7c) and its polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, aminoglycolipid, three unknown lipids and diphosphatidylglycerol. On the basis of these data, strain lm93 T is considered to represent a novel species of the genus Chelativorans, for which the name Chelativorans xinjiangense sp. nov. is proposed. The type strain is lm93 T (= KCTC 72857 T  = CCTCC AB2019376 T ).

This study was performed to isolate non-rhizobial endophytic bacteria from the root nodules of Glycine max (soybean), Vigna radiata (mung bean) and Vigna unguiculata (cowpea). The bacteria were characterized for plant growth promoting properties such as indole acetic acid production, phosphate and zinc solubilisation, nitrogen fixation and hydrogen cyanide production. Phylogenetic identification was performed using the Neighbour-Joining method on16S rRNA gene sequences. The impact of salt tolerant isolates on some properties of wheat cv. Chamran was evaluated by a completely randomised factorial design. Nine isolates having some characteristics related to plant growth promotion were identified as Staphylococcus hominis 7E, Streptomyces sp. 11E, Bacillus sp. 13E, Acinetobacter sp. 19E, from mung bean, Bacillus endophyticus 1E from cowpea, Staphylococcus hominis 9E, Bacillus endophyticus 14E, Brevundimonas sp. 16E and Kocuria sp. 26E from soybean nodules. Isolates 7E and 19E caused maximum growth inhibition of Fusarium on PDA plate. All isolates were able to grow at salinity levels of mixtures containing up to 400 mM of NaCl, CaCl 2 and MgCl 2 , but their growth was inhibited by increasing salinity level. Only the growth of isolate 14E increased at three levels of salinity compared with control. Some isolates, i.e. 7E, 14E, 19E and 26E had higher colony diameter at 45 °C after 48 h of incubation compared to the growth at 30 and 40 °C. Inoculation of soil with isolate 1E and isolate 26E caused to ameliorate salinity stress in wheat and increased the weight of 1000-grains as compared with non-inoculated treatments.

The nitrogen-fixing bacterial strain UFLA 01-1174 T was isolated from nodules of Campsiandra laurilifolia Benth. originating from the Amazon region, Brazil. Its taxonomic position was defined using a polyphasic approach. Analysis of the 16S rRNA gene placed the strain in the Bradyrhizobium genus, the closest species being B. guangdongense CCBAU 51649 T and B. guangzhouense CCBAU 51670 T , both with 99.8% similarity. Multilocus sequence analysis (MLSA) of recA, gyrB, glnII, rpoB, atpD, and dnaK indicated that UFLA 01-1174 T  is a new species, most closely related to B. stylosanthis BR 446 T (94.4%) and B. manausense BR 3351 T (93.7%). Average nucleotide identity (ANI) differentiated UFLA 01-1174 T from the closest species with values lower than 90%. The G + C content in the DNA of UFLA 01-1174 T  is 63.6 mol%. Based on this data, we conclude that the strain represents a new species. The name proposed is Bradyrhizobium campsiandrae, with UFLA 01-1174 T (= INPA 394B T  = LMG 10099 T ) as type strain.

In a search for identification of rhizobial strains with superior N 2 -fixation efficiency and improved plant agronomic characteristics upon inoculation, four strains, 4.21, 9.17, 11.2 and 14.1, isolated from root nodules of wild-grown Melilotus indicus have been used to inoculate field-grown common bean, pea, cowpea and fenugreek plants. Uninoculated plants and those inoculated with host-specific commercial inoculants were used as a control. The root length, shoot height, shoot dry weight and root dry weight and the grain yield of the plants were determined after harvest. The content of N, organic C and carbohydrates content of the grain were also recorded. The inoculation with the strains 4.21 and 14.1 increased the grain yield of the fenugreek compared both with the uninoculated plants and those inoculated with the commercial strain ARC-1. The grain yield of the common bean treated with the strains 9.17 and 14.1 was also higher than that of the uninoculated and the commercial strains ARC-301. In contrast, none of the strains increased the grain yield of the pea and cowpea plants compared to the commercial strains ARC-201 and ARC-169, respectively. Significant increases of some agronomical parameters were observed in some plant-bacterium couples, albeit nodulation was not observed. It is possible that the positive effects of rhizobial inoculation on the agronomical parameters of the non-nodule forming legumes could be due to plant growth promotion characteristic of the strains used for inoculation. Analysis of the phylogeny of the almost complete 16S rRNA sequence of the rhizobial inoculants revealed that the strains 4.21 and 9.17 clustered together with R. skierniewicense and R. rosettiformans, respectively, and that the strains 11.2 and 14.1 grouped with E. meliloti. All the four strains produced IAA, and showed biocontrol activity against Rhizotocnia solani, Fusarium oxysporum, Pythium ultimum, Alternaria alternata and Sclerotonia rolsfi, albeit to a different extent.

Part of the native root nodule endophytic microflora referring to members of the genera Proteobacteria and Sphingobacteria were used to test their bioefficacy as seed biopriming. These were quantified for their plant growth promoting (PGP) attributes such as IAA production, P and K-solubilization and ACC deaminase production. Results showed that significantly highest IAA was produced by E. hormaechi RCT10. The highest P-solubilization was observed with S. maltophila RCT31 it was solubilizing all the substrate tri-calcium phosphate, di-calcium phosphate, and zinc phosphate. Significantly highest K-solubilization was observed with S. maltophila RCT31 followed by E. turicensis RCT5. However, the maximum zinc solubilization was reported with S. maltophila RCT31 followed by E. turicensis RCT5. The maximum ACC deaminase was quantified in the bacterium. Results revealed that the E. hormaechi RCT10 utilized seed leechates most effectively while root exudates were maximally utilized by S. maltophila RCT31. The pots experiment proves that S. maltophila RCT31 was the most effective bacterium and it was replication vis-à-vis field experiment. In particular, S. maltophila RCT31 holds strong potential to be possibly used as a bioformulation for the medicinal legume, as an economical and eco-friendly alternative to agrochemicals.

In addition to rhizobia, other non-symbiotic endophytic bacteria also have been simultaneously isolated from the same root nodules. The existence of non-symbiotic endophytic bacteria in leguminous root nodules is a universal phenomenon. The vast majority of studies have detected endophytic bacteria in other plant tissues. In contrast, little systemic observation has been made on the non-symbiotic endophytic bacteria within leguminous root nodules. The present investigation was carried out to isolate plant growth-promoting endophytic non-symbiotic bacteria from indigenous leguminous Sphaerophysa salsula and their influence on plant growth. A total of 65 endophytic root nodule-associated bacteria were isolated from indigenous legume S. salsula growing in the northwestern arid regions of China. When combining our previous work with the current study, sequence analysis of the nifH gene revealed that the strain belonging to non-nodulating Bacillus pumilus Qtx-10 had genes similar to those of Rhizobium leguminosarum Qtx-10-1. The results indicated that horizontal gene transfer could have occurred between rhizobia and non-symbiotic endophyties. Under pot culture conditions, out of the 20 representative endophytic isolates, 15 with plant growth-promoting traits, such as IAA production, ACC deaminase, phosphate solubilization, chitinase, siderophore, and fungal inhibition activity showed plant growth-promoting activity with respect to various plant parameters such as chlorophyll content, fresh weight of plant, shoot length, nodule number per plant and average nodule weight per plant when co-inoculated with rhizobial bioinoculant Mesorhizobium sp. Zw-19 under N-free culture conditions. Among them, Bacillus pumilus Qtx-10 and Streptomyces bottropensis Gt-10 were excellent plant growth-promoting bacteria, which enhanced the seeding fresh weight by 87.5% and the shoot length by 89.4%, respectively. The number of nodules grew more than 31.89% under field conditions. Our findings indicate the frequent presence of these non-symbiotic endophytic bacteria within root nodules, and that they help to improve nodulation and nitrogen fixation in legume plants through synergistic interactions with rhizobia.

Legume-rhizobia symbiosis has a considerable ecological relevance because it replenishes the soil with fixed-nitrogen (e.g., ammonium) for other plants. Because of this benefit to the environment, the exploitation of the legume-rhizobia symbiosis can contribute to the development of the lower input, sustainable agriculture, thereby, reducing dependency on synthetic fertilizers. To achieve this goal, it is necessary to understand the different levels of regulation of this symbiosis to enhance its nitrogen-fixation efficiency. A different line of evidence attests to the relevance of early molecular events in the establishment of a successful symbiosis between legumes and rhizobia. In this chapter, we will review the early molecular signaling in the legume-rhizobia symbiosis. We will focus on the early molecular responses that are crucial for the recognition of the rhizobia as a potential symbiont.

Nitrogen-fixing root nodule symbiosis can sustain the development of the host plants under nitrogen-limiting conditions. Such symbiosis occurs only in a clade of angiosperms known as the nitrogen-fixing clade (NFC). It has long been proposed that root nodule symbiosis evolved several times (in parallel) in the NFC. Two recent phylogenomic studies compared the genomes of nodulating and related non-nodulating species across the four orders of the NFC and found that genes essential for nodule formation are lost or pseudogenized in the non-nodulating species. As these symbiosis genes are specifically involved in the symbiotic interaction, it means that the presence of pseudogenes and the loss of symbiosis genes strongly suggest that their ancestor, which still had functional genes, most likely had a symbiosis with nitrogen-fixing bacteria. These findings agree with the hypothesis that nodulation evolved once at the common ancestor of the NFC, and challenge the hypothesis of parallel evolution. In this chapter, we will cover the current understandings on actinorhizal-type and legume nodule development, and discuss the evolution of the legume nodule type.

Arbuscular mycorrhizal fungi (AMF) play a central role in rhizosphere functioning as they interact with both plants and soil microbial communities. The conditions in which AMF modify plant physiology and microbial communities in the rhizosphere are still poorly understood. In the present study, four different plant species, (clover, alfalfa, ryegrass, tall fescue) were cultivated in either sterilized (γ ray) or non-sterilized soil and either inoculated with a commercial AMF (Glomus LPA Val 1.) or not. After 20 weeks of cultivation, the mycorrhizal rate and shoot and root biomasses were measured. The abundance and composition of bacteria, archaea, and fungi were analyzed, respectively, by quantitative PCR (qPCR) and fingerprinting techniques. Whilst sterilization did not change the AMF capacity to modify plant biomass, significant changes in microbial communities were observed, depending on the taxon and the associated plant. AMF inoculation decreases both bacterial and archaeal abundance and diversity, with a greatest extent in sterilized samples. These results also show that AMF exert different selections on soil microbial communities according to the plant species they are associated with. This study suggests that the initial abundance and diversity of rhizosphere microbial communities should be considered when introducing AMF to cultures.

Reactive oxygen species are not only harmful for rhizobia but also required for the establishment of symbiotic interactions between rhizobia and their legume hosts. In this work, we first investigated the preliminary role of the bacterioferritin comigratory protein (BCP), a member of the peroxiredoxin family, in the nitrogen-fixing bacterium Azorhizobium caulinodans. Our data revealed that the bcp-deficient strain of A. caulinodans displayed an increased sensitivity to inorganic hydrogen peroxide (H 2 O 2 ) but not to two organic peroxides in a growth-phase-dependent manner. Meanwhile, BCP was found to be involved in catalase activity under relatively low H 2 O 2 conditions. Furthermore, nodulation and N 2 fixation were significantly impaired by mutation of the bcp gene in A. caulinodans. Our work initially documented the importance of BCP in the bacterial defence against H 2 O 2 in the free-living stage of rhizobia and during their symbiotic interactions with legumes. Molecular signalling in vivo is required to decipher the holistic functions of BCP in A. caulinodans as well as in other rhizobia.

This study aimed to identify Ammodendron bifolium endophytic bacteria, and to evaluate promoting mechanism of selected isolates on seed germination and their effects on host osmotic-stress tolerance. Forty-five strains were isolated from A. bifolium and were classified into 13 different genera by 16S rDNA gene sequence analysis. AY3, AY9 and AG18, which were identified as Staphylococcus, Kocuria, Bacillus sp., promoted host seed ethylene release during germination. Ethrel and 1-aminocyclopropane-1-carboxylic acid (ACC) imitated the effect of AY3, AY9 and AG18 on seed germination. The data suggest that ethylene mediates AY3-, AY9-, AG18-induced A. bifolium seed germination. In addition, osmotic stress prevented seed germination and radicle elongation. However, the inhibitory effect of osmotic stress on seed germination and radicle elongation were rescued by AY3, AY9 and AG18. The results show that AY3, AY9 and AG18 increased osmotic-stress tolerance in A. bifolium. AY3, AY9, AG18 induced A. bifolium seed germination through promoting ethylene production during endophytic bacteria-plant interaction, and increase osmotic-stress tolerance in A. bifolium. AY3, AY9 and AG18 are potential candidates for the protection of A. bifolium.

Invasive species often reduce ecosystem services and lead to a serious threat to native biodiversity. Roots of invasive plants are often linked to roots of native plants by common mycorrhizal networks (CMNs) of arbuscular mycorrhizal (AM) fungi, but whether and how CMNs mediate interactions between invasive and native plant species remains largely uninvestigated. We conducted two microcosm experiments, one in which we amended the soil with mineral N and another in which we amended the soil with mineral P. In each experiment, we grew a pair of test plants consisting of Kummerowia striata (native to our research site) and Solidago canadensis (an invasive species). CMNs were established between the plants, and these were either left intact or severed. Intact CMNs increased growth and nutrient acquisition by S. canadensis while they decreased nutrient acquisition by K. striata in comparison with severed CMNs. 15 N and P analyses indicated that compared to severed CMNs, intact CMNs preferentially transferred mineral nutrients to S. canadensis. CMNs produced by different species of AM fungi had slightly different effects on the interaction between these two plant species. These results highlight the role of CMNs in the understanding of interactions between the invasive species S. canadensis and its native neighbor.

In this study, two populations of leguminous plants Lathyrus sativus were grown in four soils that were collected from sites differently contaminated by heavy metals. Evaluations included basic soil properties, concentrations of major nutrients and four metals (copper, zinc, lead and cadmium) in these soils. Investigation of Lathyrus sativus response to contamination showed that the increase of heavy metal concentration in soils affected biomass of plant, number of nodules and plant metal uptake. Heavy metal tolerance of 46 isolated bacteria from the root nodules was evaluated and demonstrated that the maximum concentration of Cd, Pb, Cu and Zn tolerated by strains were 0.8, 2.5, 0.2, and 0.5 mM, respectively. Twenty-two isolates were tested for their effects on plant biomass production and nodule formation and showed that only R. leguminosarum nodulated Lathyrus sativus, while some bacteria improved the shoot and root dry biomass. Sequences of their 16S rDNA gene fragments were also obtained and evaluated for tentative identification of the isolates which revealed different bacterial genera represented by Rhizobium sp, Rhizobium leguminosarum, Sinorhizobium meliloti, Pseudomonas sp, Pseudomonas fluorescens, Luteibacter sp, Variovorax sp, Bacillus simplex and Bacillus megaterium. The existence of Pb- and Cd-resistant genes (PbrA and CadA) in these bacteria was determined by PCR, and it showed high homology with PbrA and CadA genes from other bacteria. The tested resistant population was able to accumulate high concentrations of Pb and Cd in all plant parts and, therefore, can be classified as a strong metal accumulator with suitable potential for phytoremediation of Pb and Cd polluted sites. Heavy metal resistant and efficient bacteria isolated from root nodules were chosen with Lathyrus sativus to form symbiotic associations for eventual bioremediation program, which could be tested to remove pollutants from contaminated sites.

Three strains of nitrogen-fixing bacteria isolated from nodules of Inga sp. (INPA54B T ) and Swartzia sp. (INPA86A and INPA01-91A) in soils under native forest in the Brazilian Amazon were previously identified as belonging to the Bradyrhizobium genus. In this study, these strains were characterized using a polyphasic approach to establish their taxonomic position. The three strains shared more than 99.5% sequence similarity of the 16S rRNA gene with the type strains of five Bradyrhizobium species (B. japonicum USDA 6 T , B. liaoningense LMG 18230 T , B. ottawaense OO99 T , B. subterraneum 58 2-1 T and B. yuanmingense LMG 21827 T ). However, multilocus sequence analysis of two (recA and glnII) or three (atpD, gyrB, and recA) housekeeping genes indicated that these three strains represent a new Bradyrhizobium species, which is closely related to B. subterraneum 58 2-1 T and B. yuanmingense LMG 21827 T . DNA-DNA hybridization values between INPA54B T and B. subterraneum 58 2-1 T and B. yuanmingense LMG 21827 T were only 41.5 and 30.9%, respectively. Phenotypic characterization also allowed the differentiation of the novel species from B. subterraneum 58 2-1 T and B. yuanmingense LMG 21827 T . In the phylogenetic analysis of the nodC and nifH genes, the three strains showed similar sequences that were divergent from those of type strains of all Bradyrhizobium species. We concluded that these strains represent a novel species, for which the name Bradyrhizobium forestalis is proposed, with INPA54B T (= LMG 10044 T ) as type strain. The G+C content in the DNA of INPA54B T is 63.7 mol%.

A Gram-stain-negative strain, 23-3-2 T , was isolated from a nodule of Thermopsis lanceolate grown in Northwest China. Phylogenetic analysis of 16S rRNA gene sequence showed that the strain was closely related to Mesorhizobium camelthorni CCNWXJ 40-4 T and M. alhagi CCNWXJ 12-2 T having 98.0 and 97.9% similarities, respectively. Phylogenetic analysis based on the protein-coding genes atpD and glnA showed lower similarity with the same closely related species (94.5 and 89.9%, respectively), which suggest that 23-3-2 T strain represents a distinctly delineated genospecies of the genus Mesorhizobium. The 23-3-2 T strain grew at 20-37 °C temperature (optimum 28 °C) and 5.0-9.0 pH range (optimum pH 7.0). The cells contained Q-10 as the sole respiratory quinone and 18:1ω7c (24.56%) as the major cellular fatty acid. The DNA relatedness between the strain 23-3-2 T and the two reference strains was 39-44%. Based on the phenotypic, chemotaxonomic and phylogenetic properties, strain 23-3-2 T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium zhangyense sp. nov. is proposed. The type strain is 23-3-2 T (= CGMCC 1.15528 T  = NBRC 112337 T ). The respective DPD Taxon Number is TA00147.

The ectomycorrhizal symbiosis was long thought to be restricted to temperate forests. However, as tropical forests have been explored, it has become clear that these habitats host unique ectomycorrhizal (ECM) fungi. We have been exploring tropical dry forests (TDF), which are endangered terrestrial ecosystems and hotspots of endemism. Since Fabaceae is the main plant family in this environment, we hypothesized that trees in this lineage would be the main ECM hosts. We sequenced the ITS rDNA region from fungi and both rbcL and trnL cpDNA from plants to identify both symbiotic partners from root tips. The systematic position of each symbiont was confirmed by Bayesian phylogenetic inference. We identified 20 plant species belonging to 10 families that hosted 19 unique ECM fungal species from 5 lineages. Most ECM fungi were associated with Caryophyllales, not with Fabaceae. Achatocarpus and Guapira, the main hosts, are scattered throughout the forest and are not in monodominant patches. The low ECM fungal diversity can be explained by the low density of host plants and their high specificity. Our results indicate that Caryophyllales is an important order of tropical ECM hosts with at least four independent evolutionary lineages that have evolved the ability to form ectomycorrhizae.

Members of the genus Bradyrhizobium are well-known as nitrogen-fixing microsymbionts of a wide variety of leguminous species, but they have also been found in different environments, notably as endophytes in non-legumes such as sugarcane. This study presents a detailed polyphasic characterization of four Bradyrhizobium strains (type strain BR 10280 T ), previously isolated from roots of sugarcane in Brazil. 16S rRNA sequence analysis, multilocus sequence analysis (MLSA) and analysis of the 16S-23S rRNA internal transcribed spacer showed that these strains form a novel clade close to, but different from B. huanghuaihaiense strain CCBAU 23303 T . Average nucleotide identity (ANI) analyses confirmed that BR 10280 T represents a novel species. Phylogenetic analysis based on nodC gene sequences also placed the strains close to CCBAU 23303 T , but different from this latter strain, the sugarcane strains did not nodulate soybean, although they effectively nodulated Vigna unguiculata, Cajanus cajan and Macroptilium atropurpureum. Physiological traits are in agreement with the placement of the strains in the genus Bradyrhizobium as a novel species for which the name Bradyrhizobium sacchari sp. nov. is proposed.

Coal open pit mining in the South of Santa Catarina state (Brazil) was inappropriately developed, affecting approximately 6.700 ha. Re-vegetation is an alternative for the recovery of these areas. Furthermore, the use of herbaceous legumes inoculated with nitrogen fixing bacteria is motivated due to the difficulty implementing a vegetation cover in these areas, mainly due to low nutrient availability. Therefore, the aim of this work was to evaluate, among 16 autochthonous rhizobia isolated from the coal mining areas, those with the greatest potential to increase growth of the herbaceous legumes Vicia sativa and Calopogonium mucunoides. Tests were conducted in greenhouse containing 17 inoculation treatments (16 autochthonous rhizobia + Brazilian recommended strain for each plant species), plus two treatments without inoculation (with and without mineral nitrogen). After 60 days, nodulation, growth, N uptake, and symbiotic efficiency were evaluated. Isolates characterization was assessed by the production of indole acetic acid, ACC deaminase, siderophores, and inorganic phosphate solubilization. The classification of the isolates was performed by 16 S rDNA gene sequencing. Only isolates UFSC-M4 and UFSC-M8 were able to nodulate C. mucunoides. Among rhizobia capable of nodulating V. sativa, only UFSC-M8 was considered efficient. It was found the presence of more than one growth-promoting attributes in the same organism, and isolate UFSC-M8 presented all of them. Isolates were classified as belonging to Rhizobium, Burkholderia and Curtobacterium. The results suggest the inoculation of Vicia sativa with strain UFSC-M8, classified as Rhizobium sp., as a promising alternative for the revegetation of coal mining degraded areas.

Flavonoids excreted by legume roots induce the expression of symbiotically essential nodulation (nod) genes in rhizobia, as well as that of specific protein export systems. In the bean microsymbiont Rhizobium etli CE3, nod genes are induced by the flavonoid naringenin. In this study, we identified 693 proteins in the exoproteome of strain CE3 grown in minimal medium with or without naringenin, with 101 and 100 exoproteins being exclusive to these conditions, respectively. Four hundred ninety-two (71%) of the extracellular proteins were found in both cultures. Of the total exoproteins identified, nearly 35% were also present in the intracellular proteome of R. etli bacteroids, 27% had N-terminal signal sequences and a significant number had previously demonstrated or possible novel roles in symbiosis, including bacterial cell surface modification, adhesins, proteins classified as MAMPs (microbe-associated molecular patterns), such as flagellin and EF-Tu, and several normally cytoplasmic proteins as Ndk and glycolytic enzymes, which are known to have extracellular "moonlighting" roles in bacteria that interact with eukaryotic cells. It is noteworthy that the transmembrane ß (1,2) glucan biosynthesis protein NdvB, an essential symbiotic protein in rhizobia, was found in the R. etli naringenin-induced exoproteome. In addition, potential binding sites for two nod-gene transcriptional regulators (NodD) occurred somewhat more frequently in the promoters of genes encoding naringenin-induced exoproteins in comparison to those ofexoproteins found in the control condition.

Thirteen Gram-negative, aerobic, motile with polar flagella, rod-shaped bacteria were isolated from root nodules of Centrolobium paraense Tul. grown in soils from the Amazon region of Brazil. Growth of strains was observed at temperature range 20-36 °C (optimal 28 °C), pH ranges 5-11 (optimal 6.0-7.0), and 0.1-0.5%NaCl (optimal 0.1-0.3%). Analysis of 16S rRNA gene placed the strains into two groups within Bradyrhizobium. Closest neighbouring species (98.8%) for group I was B. neotropicale while for group II were 12 species with more than 99% of similarity. Multi-locus sequence analysis (MLSA) with dnaK, glnII, recA, and rpoB confirmed B. neotropicale BR 10247 T as the closest type strain for the group I and B. elkanii USDA 76 T and B. pachyrhizi PAC 48 T for group II. Average Nucleotide Identity (ANI) differentiated group I from the B. neotropicale BR 10247 T (79.6%) and group II from B. elkanii USDA 76 T and B. pachyrhizi PAC 48 T (88.1% and 87.9%, respectively). Fatty acid profiles [majority C 16:0 and Summed feature 8 (18:1ω6c/18:1ω7c) for both groups], DNA G + C content, and carbon compound utilization supported the placement of the novel strains in the genus Bradyrhizobium. Gene nodC and nifH of the new strains have in general low similarity with other Bradyrhizobium species. Both groups nodulated plants from the tribes Crotalarieae, Dalbergiae, Genisteae, and Phaseoleae. Based on the presented data, two novel species which the names Bradyrhizobium centrolobii and Bradyrhizobium macuxiense are proposed, with BR 10245 T (=HAMBI 3597 T ) and BR 10303 T (=HAMBI 3602 T ) as the respective-type strains.

The rhizosphere is viewed as a deterministic environment led by the interaction between plants and microorganisms. In the case of semi-arid plants, this interaction is strengthened by the harshness of the environment. We tested the hypothesis that dry season represents a constraint on the bacterial diversity of the rhizosphere from semi-arid plants. To accomplish this, we sampled two leguminous species at five locations during the dry and rainy seasons in the Caatinga biome and characterised bacterial community structures using qPCR and 16S rRNA sequencing. We found that the main differences between seasons were due to reduced phylogenetic diversity caused by dryness. Variation partitioning indicated that environmental characteristics significant impacts in β-diversity. Additionally, distance decay relationship and taxa area relationship indicate a higher spatial turnover at the rainy season. During the dry season, decreased bacterial abundance is likely due to the selection of resistant or resilient microorganisms; with the return of the rain, the sensitive populations start to colonise the rhizosphere by a process that is strongly influenced by environmental characteristics. Thus, we propose that the reduction of PD and strong influence of environmental parameters on the assemblage of these communities make them prone to functional losses caused by climatic disturbances.

To evaluate the interactions among endophytes, plants and heavy metal/arsenic contamination, root endophytic bacteria of Prosopis laevigata (Humb and Bonpl. ex Willd) and Sphaeralcea angustifolia grown in a heavy metal(loid)-contaminated zone in San Luis Potosi, Mexico, were isolated and characterized. Greater abundance and species richness were found in Prosopis than in Sphaeralcea and in the nutrient Pb-Zn-rich hill than in the poor nutrient and As-Cu-rich mine tailing. The 25 species identified among the 60 isolates formed three groups in the correspondence analysis, relating to Prosopis/hill (11 species), Prosopis/mine tailing (4 species) and Sphaeralcea/hill (4 species), with six species ungrouped. Most of the isolates showed high or extremely high resistance to arsenic, such as ≥100 mM for As(V) and ≥20 mM for As(III), in mineral medium. These results demonstrated that the abundance and community composition of root endophytic bacteria were strongly affected by the concentration and type of the heavy metals and metalloids (arsenic), as well as the plant species.

Aiming to investigate the diversity and distribution of rhizobia associated with Ammopiptanthus, an endangered evergreen legume widely distributed in deserts, we characterized a total of 219 nodule isolates from nine sampling sites in Northwest China with different soil characteristics based upon restriction fragment length polymorphism (RFLP) analysis of 16S ribosomal RNA (rRNA) and symbiotic genes (nodC and nifH). Ten isolates representing different 16S rRNA-RFLP types were selected for further sequence analyses of 16S rRNA and four housekeeping genes. As results, nine genospecies belonging to the genera Ensifer, Neorhizobium, Agrobacterium, Pararhizobium, and Rhizobium could be defined among the isolates. The nodC and nifH phylogenies of 14 isolates representing different symbiotic-RFLP types revealed five lineages linked to Ensifer fredii, Ensifer meliloti, Rhizobium leguminosarum, Mesorhizobium amorphae, and Rhizobium gallicum, which demonstrated the various origins and lateral transfers of symbiotic genes between different genera and species. The rhizobial diversities of Ammopiptanthus mongolicus varied among regions, and the community compositions of rhizobia associated with A. mongolicus were significantly different in wild and cultured fields. Constrained correspondence analysis showed that the distribution of A. mongolicus rhizobia could be explained by available potassium content and that the assembly of symbiotic types was mainly affected by available phosphorus content and carbon-nitrogen ratio.

The patterns and drivers of bacterial strain dominance remain poorly understood in natural populations. Here, we cultured 1292 Bradyrhizobium isolates from symbiotic root nodules and the soil root interface of the host plant Acmispon strigosus across a >840-km transect in California. To investigate epidemiology and the potential role of accessory loci as epidemic drivers, isolates were genotyped at two chromosomal loci and were assayed for presence or absence of accessory "symbiosis island" loci that encode capacity to form nodules on hosts. We found that Bradyrhizobium populations were very diverse but dominated by few haplotypes-with a single "epidemic" haplotype constituting nearly 30 % of collected isolates and spreading nearly statewide. In many Bradyrhizobium lineages, we inferred presence and absence of the symbiosis island suggesting recurrent evolutionary gain and or loss of symbiotic capacity. We did not find statistical phylogenetic evidence that the symbiosis island acquisition promotes strain dominance and both symbiotic and non-symbiotic strains exhibited population dominance and spatial spread. Our dataset reveals that a strikingly few Bradyrhizobium genotypes can rapidly spread to dominate a landscape and suggests that these epidemics are not driven by the acquisition of accessory loci as occurs in key human pathogens.

A significant challenge facing the study of arbuscular mycorrhiza is the establishment of suitable non-mycorrhizal treatments that can be compared with mycorrhizal treatments. A number of options are available, including soil disinfection or sterilisation, comparison of constitutively mycorrhizal and non-mycorrhizal plant species, comparison of plants grown in soils with different inoculum potential and the comparison of mycorrhiza-defective mutant genotypes with their mycorrhizal wild-type progenitors. Each option has its inherent advantages and limitations. Here, the potential to use mycorrhiza-defective mutant and wild-type genotype plant pairs as tools to study the functioning of mycorrhiza is reviewed. The emphasis of this review is placed on non-legume plant species, as mycorrhiza-defective plant genotypes in legumes have recently been extensively reviewed. It is concluded that non-legume mycorrhiza-defective mutant and wild-type pairs are useful tools in the study of mycorrhiza. However, the mutant genotypes should be well characterised and, ideally, meet a number of key criteria. The generation of more mycorrhiza-defective mutant genotypes in agronomically important plant species would be of benefit, as would be more research using these genotype pairs, especially under field conditions.

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(T), 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(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) 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.

The efficiency of two mycorrhizal bio-inoculants on the mineral uptake during the growth stages of a Mediterranean forage legume sulla (Hedysarum coronarium L.) was studied in the field on a highly calcareous soil. The first inoculum (Mm) was made up of a mixture of native arbuscular mycorrhizal fungi (AMF) isolated from calcareous soils: Septoglomus constrictum, Funneliformis geosporum, Glomus fuegianum, Rhizophagus irregularis and Glomus sp. The second was a commercial inoculum (Mi) containing one AMF species: R. irregularis. Both mycorrhizal inoculants increased total and arbuscular colonization of sulla roots. Inoculation with Mm showed a positive effect on sulla shoot dry weight (SDW) when compared to Mi and non-inoculated plants (control). Mineral contents (P, Mg, Mn, Fe) were higher in the shoots of sulla plants cultivated on mycorrhiza-inoculated plots compared to non-inoculated ones. This enhancement was observed during the flowering stage for P, Mg and Mn and during the rosette stage for Fe. An increase in P content of 50 % in plants inoculated with Mm compared to non-inoculated ones may be explained by the induction of root alkaline and acid phosphatase activities. Higher efficiency of native AMF species adapted to calcareous soils opens the way towards the development of mycorrhiza bio-fertilizers targeted to improve sustainable fertilization management in such soils.

Amazonian Dark Earths (ADE) or Terra Preta de Índio formed in the past by pre-Columbian populations are highly sustained fertile soils supported by microbial communities that differ from those extant in adjacent soils. These soils are found in the Amazon region and are considered as a model soil when compared to the surrounding and background soils. The aim of this study was to assess the effects of ADE and its surrounding soil on the rhizosphere bacterial communities of two leguminous plant species that frequently occur in the Amazon region in forest sites (Mimosa debilis) and open areas (Senna alata). Bacterial community structure was evaluated using terminal restriction fragment length polymorphism (T-RFLP) and bacterial community composition by V4 16S rRNA gene region pyrosequencing. T-RFLP analysis showed effect of soil types and plant species on rhizosphere bacterial community structure. Differential abundance of bacterial phyla, such as Acidobacteria, Actinobacteria, Verrucomicrobia, and Firmicutes, revealed that soil type contributes to shape the bacterial communities. Furthermore, bacterial phyla such as Firmicutes and Nitrospira were mostly influenced by plant species. Plant roots influenced several soil chemical properties, especially when plants were grown in ADE. These results showed that differences observed in rhizosphere bacterial community structure and composition can be influenced by plant species and soil fertility due to variation in soil attributes.

To analyze macroevolutionary patterns in host use by Bradyrhizobium root-nodule bacteria, 420 strains from 75 legume host genera (sampled in 25 countries) were characterized for portions of six housekeeping genes and the nifD locus in the symbiosis island chromosomal region. Most Bradyrhizobium clades utilized very divergent sets of legume hosts. This suggests that Bradyrhizobium spread across the major legume lineages early in its evolution, with only a few derived clades subsequently developing a narrower pattern of host use. Significant modularity existed in the network structure of recent host jumps (inferred from cases where closely related strain pairs were found on different legume taxa). This implies that recent host switching has occurred most often within particular subgroups of legumes. Nevertheless, the observed link structure would allow a bacterial lineage to reach almost any of the 75 legume host genera in a relatively small number of steps. However, permutation tests also showed that symbionts from certain host plant clades were significantly more similar than would be the case if bacteria were distributed at random on the trees. Related legumes thus harbored related sets of symbionts in some cases, indicating some degree of phylogenetic conservatism in partner selection.

Ant-plant mutualisms are conspicuous and ecologically important components of tropical ecosystems that remain largely unexplored in terms of insect-associated microbial communities. Recent work has revealed that ants in some ant-plant systems cultivate fungi (Chaetothyriales) within their domatia, which are fed to larvae. Using Pseudomyrmex penetrator/Tachigali sp. from French Guiana and Petalomyrmex phylax/Leonardoxa africana and Crematogaster margaritae/Keetia hispida, both from Cameroon, as models, we tested the hypothesis that ant-plant-fungus mutualisms co-occur with culturable Actinobacteria. Using selective media, we isolated 861 putative Actinobacteria from the three systems. All C. margaritae/K. hispida samples had culturable Actinobacteria with a mean of 10.0 colony forming units (CFUs) per sample, while 26 % of P. penetrator/Tachigali samples (mean CFUs 1.3) and 67 % of P. phylax/L. africana samples (mean CFUs 3.6) yielded Actinobacteria. The largest number of CFUs was obtained from P. penetrator workers, P. phylax alates, and C. margaritae pupae. 16S rRNA gene sequencing and phylogenetic analysis revealed the presence of four main clades of Streptomyces and one clade of Nocardioides within these three ant-plant mutualisms. Streptomyces with antifungal properties were isolated from all three systems, suggesting that they could serve as protective symbionts, as found in other insects. In addition, a number of isolates from a clade of Streptomyces associated with P. phylax/L. africana and C. margaritae/K. hispida were capable of degrading cellulose, suggesting that Streptomyces in these systems may serve a nutritional role. Repeated isolation of particular clades of Actinobacteria from two geographically distant locations supports these isolates as residents in ant-plant-fungi niches.

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678(T) which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.

The native legume Adesmia bicolor shows nitrogen fixation efficiency via symbiosis with soil rhizobia. The infection mechanism by means of which rhizobia infect their roots has not been fully elucidated to date. Therefore, the purpose of the present study was to identify the infection mechanism in Adesmia bicolor roots. To this end, inoculated roots were processed following conventional methods as part of our root anatomy study, and the shape and distribution of root nodules were analyzed as well. Neither root hairs nor infection threads were observed in the root system, whereas infection sites-later forming nodules-were observed in the longitudinal sections. Nodules were found to form between the main root and the lateral roots. It can be concluded that in Adesmia bicolor, a bacterial crack entry infection mechanism prevails and that such mechanism could be an adaptive strategy of this species which is typical of arid environments.

The present study was undertaken to evaluate the effects of phosphorus (P) application and arbuscular mycorrhizal (AM) fungi (Funneliformis mosseae) on growth, foliar nitrogen mobilization, and phosphorus partitioning in cowpea (Vigna unguiculata cv. Vita-5) plants. The experiment was conducted in a greenhouse in pots containing a mixture of vermiculite and sterilized quartz sand. Mycorrhizal and non-mycorrhizal cowpea plants were supplied with three levels of soluble P (0.1 (low P), 0.5 (medium P), or 1.0 mM (high P)).Cowpea plants supplied with low P fertilization showed significantly (p < 0.05) higher root colonization than those with medium and high P fertilization at both the vegetative and pod-filling stages. P uptake and growth parameters of cowpea plants were positively influenced by mycorrhizal inoculation only in the medium P fertilization treatment at the vegetative stage. Lack of these effects in the other treatments may be linked to either a very low P supply (in the low P treatment at the vegetative stage) or the availability of optimal levels of freely diffusible P in the substrate towards the pod-filling stage due to accumulation with time. The N concentration in leaves of all cowpea plants were lower at the pod-filling stage than at the vegetative stage, presumably as a result of N mobilization from vegetative organs to the developing pods. This was however not influenced by AM fungal inoculation and may be a consequence of the lack of an improved plant P acquisition by the fungus at the pod-filling stage.

Arbuscular mycorrhizal (AM) symbiosis in vascular plant roots is an ancient mutualistic interaction that evolved with land plants. More recently evolved root mutualisms have recruited components of the AM signalling pathway as identified with molecular approaches in model legume research. Earlier we reported that the reduced mycorrhizal colonisation (rmc) mutation of tomato mapped to chromosome 8. Here we report additional functional characterisation of the rmc mutation using genotype grafts and proteomic and transcriptomic analyses. Our results led to identification of the precise genome location of the Rmc locus from which we identified the mutation by sequencing. The rmc phenotype results from a deletion that disrupts five predicted gene sequences, one of which has close sequence match to the CYCLOPS/IPD3 gene identified in legumes as an essential intracellular regulator of both AM and rhizobial symbioses. Identification of two other genes not located at the rmc locus but with altered expression in the rmc genotype is also described. Possible roles of the other four disrupted genes in the deleted region are discussed. Our results support the identification of CYCLOPS/IPD3 in legumes and rice as a key gene required for AM symbiosis. The extensive characterisation of rmc in comparison with its 'parent' 76R, which has a normal mycorrhizal phenotype, has validated these lines as an important comparative model for glasshouse and field studies of AM and non-mycorrhizal plants with respect to plant competition and microbial interactions with vascular plant roots.

The objective of the present study was to isolate and characterize nodulating bacteria associated with wild legumes. For this purpose, we recovered twenty isolates from root nodules of five wild legume species: Melilotus alles, Melilotus officinalis, Trifolium pratense, Trifolium repens and Medicago sp. Most of the isolates were morphologically analogous with only few exceptions in colony shape, appearance and incubation time. All isolates were Gram negative except T.P2-4. Random amplification of polymorphic DNA showed genetic variation among isolates. The 16S rRNA sequence analysis revealed these isolates as Rhizobium, Sinorhizobium and Paenibacillus. Each of these was also screened for nod D and nod F genes with marked variation at these loci; however, the nucleotide sequence analysis confirmed the presence of nod genes. The assignment of strains to their hosts revealed a unique symbiotic association of Paenibacillus sp. nodulating T .pratense which is being reported here for the first time.

In many wild legumes, attempts to cultivate nodule bacteria fail. We hypothesized that the limited culturability could be related to injury from oxidative stress caused by disruption of plant tissues during isolation. To test that, we isolated bacteria from nodules of Hedysarum spinosissimum and Tetragonolobus purpureus using buffers supplemented with scavenging systems to prevent damage from reactive oxygen species (ROS). Treatments included the following: antioxidants (glutathione, ascorbate, EDTA) or enzymes (catalase, peroxidase, superoxide dismutase), tested either as modified squashing buffers or added in plates. Some combinations yielded dramatic increases of culturability. Different endophytes were found, including additional Rhizobiaceae that were not the primary symbiont and were unable to nodulate. Their H2O2 tolerance in broth culture showed differences consistent with the unequal culturability observed. In wild legumes species, ROS generation during extraction appears to be a major factor limiting microbiota isolation, and protocols presented here significantly improve the recovery of culturable bacterial endophytes from plants.

Labeled nitrogen ((15)N) was applied to a soil-based substrate in order to study the uptake of N by Glomus intraradices extraradical mycelium (ERM) from different mineral N (NO(3)(-) vs. NH(4)(+)) sources and the subsequent transfer to cowpea plants. Fungal compartments (FCs) were placed within the plant growth substrate to simulate soil patches containing root-inaccessible, but mycorrhiza-accessible, N. The fungus was able to take up both N-forms, NO(3)(-) and NH(4)(+). However, the amount of N transferred from the FC to the plant was higher when NO(3)(-) was applied to the FC. In contrast, analysis of ERM harvested from the FC showed a higher (15)N enrichment when the FC was supplied with (15)NH(4)(+) compared with (15)NO(3)(-). The (15)N shoot/root ratio of plants supplied with (15)NO(3)(-) was much higher than that of plants supplied with (15)NH(4)(+), indicative of a faster transfer of (15)NO(3)(-) from the root to the shoot and a higher accumulation of (15)NH (4)(+) in the root and/or intraradical mycelium. It is concluded that hyphae of the arbuscular mycorrhizal fungus may absorb NH(4)(+) preferentially over NO(3)(-) but that export of N from the hyphae to the root and shoot may be greater following NO(3)(-) uptake. The need for NH(4)(+) to be assimilated into organically bound N prior to transport into the plant is discussed.

Soil nutrient availability and colonization by arbuscular mycorrhizal fungi are important and potentially interacting factors shaping vegetation composition and succession. We investigated the effect of carbon (C) addition, aimed at reducing soil nutrient availability, on arbuscular mycorrhizal colonization. Seedlings of 27 plant species with different sets of life-history traits (functional group affiliation, life history strategy and nitrophilic status) were grown in pots filled with soil from a nutrient-rich set-aside field and amended with different amounts of C. Mycorrhizal colonization was progressively reduced along the gradient of increasing C addition in 17 out of 27 species, but not in the remaining species. Grasses had lower colonization levels than forbs and legumes and the decline in AM fungal colonization was more pronounced in legumes than in other forbs and grasses. Mycorrhizal colonization did not differ between annual and perennial species, but decreased more rapidly along the gradient of increasing C addition in plants with high Ellenberg N values than in plants with low Ellenberg N values. Soil C addition not only limits plant growth through a reduction in available nutrients, but also reduces mycorrhizal colonization of plant roots. The effect of C addition on mycorrhizal colonization varies among plant functional groups, with legumes experiencing an overproportional reduction in AM fungal colonization along the gradient of increasing C addition. We therefore propose that for a better understanding of vegetation succession on set-aside fields one may consider the interrelationship between plant growth, soil nutrient availability and mycorrhizal colonization of plant roots.

Pisonia grandis (Nyctaginaceae), a widespread tree of Pacific coral atolls and islands, displays one of the more restrictive ranges of ectomycorrhizal (ECM) fungus associates among autotrophic plants. Only five ECM fungi are currently known associates; our study adds one. In many habitats, P. grandis is restricted to large seabird colonies where nitrogen and phosphorus inputs in the form of guano are substantial. It has been suggested that the ECM specificity displayed by P. grandis is the result of the unusual nutrient-rich habitat in which P. grandis grows. On Rota, Commonwealth of the Northern Mariana Islands, P. grandis grows in habitats heavily influenced by guano additions and also in upland forests where seabirds do not roost or nest. To test the hypothesis that the ECM specificity displayed by P. grandis is the result of nutrient-related or toxicity-related factors associated with guano inputs, we sampled P. grandis growing in both guano-rich and guano-poor habitats on Rota, Commonwealth of the Northern Mariana Islands. We identified ECM symbionts of P. grandis from both habitats as well as two symbionts of Intsia bijuga (Fabaceae) from nutrient-rich habitats. We identified three ECM symbionts of P. grandis from Rota; all three were found in both guano-rich and guano-poor habitats. No differences in community diversity were detected between guano-rich and guano-poor habitats. We also detected two ECM fungal species associating with I. bijuga but not associating with P. grandis inside guano-rich habitats. From these results, we infer that edaphic factors are not responsible for limiting the ECM community associating with P. grandis to its observed level of specificity.

Studying rhizobia in the root nodules of Sphaerophysa salsula (Pall.) DC in the northwest of China, we obtained five strains classified as genus Rhizobium on the basis of their 16S rRNA gene sequences. The sequence similarity of strain CCNWQTX14(T) with the most related species was 99.0%. Further phylogenetic analysis of housekeeping genes (recA and atpD) suggested the five strains comprised a novel lineage within Rhizobium. The nifH and nodD gene sequences of CCNWQTX14(T) were phylogenetically closely related with those of Sinorhizobium kummerowiae and R. sphaerophysae, respectively. The five strains isolated from different places were also distinct from related Rhizobium species using ERIC fingerprint profiles. The DNA-DNA hybridization value was 41.8% between CCNWQTX14(T) and Rhizobium sphaerophysae CCNWGS0238(T). Our novel strains were only able to form effective nodules on its original host Sphaerophysa salsula. Our data showed that the five Rhizobium strains formed a unique genomic species, for which a novel species Rhizobium helanshanense sp. nov. is proposed. The type strain is CCNWQTX14(T) (=ACCC 16237(T) =HAMBI 3083(T)).

The diversity, ecology and function of ectomycorrhizal (EM) fungi and ectomycorrhizas (ECMs) on tropical African tree species are reviewed here. While ECMs are the most frequent mycorrhizal type in temperate and boreal forests, they concern an economically and ecologically important minority of plants in African tropical forests. In these African tropical forests, ECMs are found mainly on caesalpionioid legumes, Sarcolaenaceae, Dipterocarpaceae, Asterpeiaceae, Phyllantaceae, Sapotaceae, Papilionoideae, Gnetaceae and Proteaceae, and distributed in open, gallery and rainforests of the Guineo-Congolian basin, Zambezian Miombo woodlands of East and South-Central Africa and Sudanian savannah woodlands of the sub-sahara. Overall, EM status was confirmed in 93 (26%) among 354 tree species belonging to EM genera. In addition, 195 fungal taxa were identified using morphological descriptions and sequencing of the ML5/ML6 fragment of sporocarps and ECMs from West Africa. Analyses of the belowground EM fungal communities mostly based on fungal internal transcribed spacer sequences of ECMs from Continental Africa, Madagascar and the Seychelles also revealed more than 350 putative species of EM fungi belonging mainly to 18 phylogenetic lineages. As in temperate forests, the /russula-lactarius and /tomentella-thelephora lineages dominated EM fungal flora in tropical Africa. A low level of host preference and dominance of multi-host fungal taxa on different African adult tree species and their seedlings were revealed, suggesting a potential for the formation of common ectomycorrhizal networks. Moreover, the EM inoculum potential in terms of types and density of propagules (spores, sclerotia, EM root fragments and fragments of mycelia strands) in the soil allowed opportunistic root colonisation as well as long-term survival in the soil during the dry season. These are important characteristics when choosing an EM fungus for field application. In this respect, Thelephoroid fungal sp. XM002, an efficient and competitive broad host range EM fungus, possessed these characteristics and appeared to be a good candidate for artificial inoculation of Caesalps and Phyllanthaceae seedlings in nurseries. However, further efforts should be made to assess the genetic and functional diversity of African EM fungi as well as the EM status of unstudied plant species and to strengthen the use of efficient and competitive EM fungi to improve production of ecologically and economically important African multipurpose trees in plantations.

Eighteen isolates of rhizobia isolated from root nodules of Colutea arborescens (Bladder senna) grown in different soils of the eastern area of Morocco were characterized by phenotypic and genomic analyses. All the isolates characterized were fast growers. This is may be due to the isolation procedures used. The phenotypic, symbiotic and cultural characteristics analyzed allowed the description of a wide physiological diversity among tested isolates. The results obtained suggest that the phenotype of these rhizobia might have convergent evolved to adapt the local conditions. The genetic characterization consisted in an analysis of the rep-PCR fingerprints and the PCR-based RFLP of the 16S rDNA patterns. The 16S rDNA of six isolates representing the main ribotypes obtained by the PCR-based RFLP was sequenced. A large diversity was observed among these rhizobia, and they were classified as different species of the genera Rhizobium, Sinorhizobium and Mesorhizobium. The nodC gene was also sequenced, and the results confirmed the three lineages corresponding to the three genera. The results of the sequencing of nodC and 16S rDNA genes suggest that the nodulation genes and chromosome might have co-evolved among these bacteria.

Three arbuscular mycorrhizal (AM) fungi (Glomus mosseae, Glomus claroideum, and Glomus intraradices) were compared for their root colonizing ability and activity in the root of Astragalus sinicus L. under salt-stressed soil conditions. Mycorrhizal formation, activity of fungal succinate dehydrogenase, and alkaline phosphatase, as well as plant biomass, were evaluated after 7 weeks of plant growth. Increasing the concentration of NaCl in soil generally decreased the dry weight of shoots and roots. Inoculation with AM fungi significantly alleviated inhibitory effect of salt stress. G. intraradices was the most efficient AM fungus compared with the other two fungi in terms of root colonization and enzyme activity. Nested PCR revealed that in root system of plants inoculated with a mix of the three AM fungi and grown under salt stress, the majority of mycorrhizal root fragments were colonized by one or two AM fungi, and some roots were colonized by all the three. Compared to inoculation alone, the frequency of G. mosseae in roots increased in the presence of the other two fungal species and highest level of NaCl, suggesting a synergistic interaction between these fungi under salt stress.

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.

The associations among rhizobia chromosomal background, nodulation genes, legume plants, and geographical regions are very attractive but still unclear. To address this question, we analyzed the interactions among rhizobia rDNA genotypes, nodC genotypes, legume genera, as well as geographical regions in the present study. Complex relationships were observed among them, which may be the genuine nature of their associations. The statistical analyses indicate that legume plant is the key factor shaping both rhizobia genetic and symbiotic diversity. In the most cases of our results, the nodC lineages are clearly associated with rhizobial genomic species, demonstrating that nodulation genes have co-evolved with chromosomal background, though the lateral transfer of nodulation genes occurred in some cases in a minority. Our results also support the hypothesis that the endemic rhizobial populations to a certain geographical area prefer to have a wide spectrum of hosts, which might be an important event for the success of both legumes and rhizobia in an isolated region.