Rhizobial HmuSpSym as a heme-binding factor is required for optimal symbiosis between Mesorhizobium amorphae CCNWGS0123 and Robinia pseudoacacia.

Nitrogen-fixing root nodules are formed by symbiotic association of legume hosts with rhizobia in nitrogen-deprived soils. Successful symbiosis is regulated by signals from both legume hosts and their rhizobial partners. HmuS is a heme degrading factor widely distributed in bacteria, but little is known about the role of rhizobial hmuS in symbiosis with legumes. Here, we found that inactivation of hmuSpSym in the symbiotic plasmid of Mesorhizobium amorphae CCNWGS0123 disrupted rhizobial infection, primordium formation, and nitrogen fixation in symbiosis with Robinia pseudoacacia. Although there was no difference in bacteroids differentiation, infected plant cells were shrunken and bacteroids were disintegrated in nodules of plants infected by the ΔhmuSpSym mutant strain. The balance of defence reaction was also impaired in ΔhmuSpSym strain-infected root nodules. hmuSpSym was strongly expressed in the nitrogen-fixation zone of mature nodules. Furthermore, the HmuSpSym protein could bind to heme but not degrade it. Inactivation of hmuSpSym led to significantly decreased expression levels of oxygen-sensing related genes in nodules. In summary, hmuSpSym of M. amorphae CCNWGS0123 plays an essential role in nodule development and maintenance of bacteroid survival within R. pseudoacacia cells, possibly through heme-binding in symbiosis.

Evolution of specialization in a plant-microbial mutualism is explained by the oscillation theory of speciation.

Specialization in mutualisms is thought to be a major driver of diversification, but few studies have explored how novel specialization evolves, or its relation to the evolution of other niche axes. A fundamental question is whether generalist interactions evolve to become more specialized (i.e., oscillation hypothesis) or if partner switches evolve without any change in niche breadth (i.e., musical chairs hypothesis). We examined alternative models for the evolution of specialization by estimating the mutualistic, climatic, and edaphic niche breadths of sister plant species, combining phylogenetic, environmental, and experimental data on Acmispon strigosus and Acmispon wrangelianus genotypes across their overlapping ranges in California. We found that specialization along all three niche axes was asymmetric across species, such that the species with broader climatic and edaphic niches, Acmispon strigosus, was also able to gain benefit from and invest in associating with a broader set of microbial mutualists. Our data are consistent with the oscillation model of specialization, and a parallel narrowing of the edaphic, climatic, and mutualistic dimensions of the host species niche. Our findings provide novel evidence that the evolution of specialization in mutualism is accompanied by specialization in other niche dimensions.

Chickpea (Cicer arietinum L.) as model legume for decoding the co-existence of Pseudomonas fluorescens and Mesorhizobium sp. as bio-fertilizer under diverse agro-climatic zones.

Microbial co-inoculation strategy utilizes a combination of microbes to stimulate plant growth concomitant with an increased phytopathogen tolerance. In the present study, 15 endophytic bacterial isolates from rhizosphere and roots of wild chickpea accessions (Cicer pinnatifidum, C. judiacum, C. bijugum and C. reticulatum) were characterized for morphological, biochemical and physiological traits. Two promising isolates were identified as Pseudomonas fluorescens strain LRE-2 (KR303708.1) and Pseudomonas argentinensis LPGPR-1 (JX239745.1) based on 16S rRNA gene sequencing. Biocompatibility of selected endophytes with Mesorhizobium sp. CH1233, a standard isolate used as a national check in All India Coordinated Research Project (AICRP) was assessed to develop functional combinations capable of producing Indole acetic acid, gibberellins, siderophores and improving seed vigour (in vitro). In vivo synergistic effect of promising combinations was further evaluated under national AICRP, (Chickpea) at two different agro-climatic zones [North-West plain (Ludhiana and Hisar) and Central zones (Sehore)] for three consecutive Rabi seasons (2015-18) to elucidate their effect on symbiotic, soil quality and yield parameters. On the pooled mean basis across locations over the years, combination of Mrh+LRE-2 significantly enhanced symbiotic, soil quality traits and grain yield over Mrh alone and highly positive correlation was obtained between the nodulation traits and grain yield. Superior B: C ratio (1.12) and additional income of Rs 6,505.18 ha-1 was obtained by application of Mrh+LRE-2 over Mrh alone and un-inoculated control. The results demonstrate that dual combination of Mrh and Pseudomonas sp. from wild Cicer relatives can be exploited as a potential bio-fertilizer for increasing soil fertility and improving chickpea productivity under sustainable agriculture.

Dysregulation of host-control causes interspecific conflict over host investment into symbiotic organs.

Microbial mutualists provide substantial benefits to hosts that feed back to enhance the fitness of the associated microbes. In many systems, beneficial microbes colonize symbiotic organs, specialized host structures that house symbionts and mediate resources exchanged between parties. Mutualisms are characterized by net benefits exchanged among members of different species, however, inequalities in the magnitude of these exchanges could result in evolutionary conflict, destabilizing the mutualism. We investigated joint fitness effects of root nodule formation, the symbiotic organ of legumes that house nitrogen-fixing rhizobia in planta. We quantified host and symbiont fitness parameters dependent on the number of nodules formed using near-isogenic Lotus japonicus and Mesorhizobium loti mutants, respectively. Empirically estimated fitness functions suggest that legume and rhizobia fitness is aligned as the number of nodules formed increases from zero until the host optimum is reached, a point where aligned fitness interests shift to diverging fitness interests between host and symbiont. However, fitness conflict was only inferred when analyzing wild-type hosts along with their mutants dysregulated for control over nodule formation. These data demonstrate that to avoid conflict, hosts must tightly regulate investment into symbiotic organs maximizing their benefit to cost ratio of associating with microbes.

Synergistic effect of organo-mineral amendments and plant growth-promoting rhizobacteria (PGPR) on the establishment of vegetation cover and amelioration of mine tailings.

Mine tailings pose a huge hazard for environmental and human health, and the establishment of vegetation cover is crucial to reduce pollutant dispersion for the surroundings. However, their hostile physicochemical conditions hamper plant growth, compromising phytoremediation strategies. This study aims to investigate the role of organo-mineral amendments and plant growth-promoting rhizobacteria (PGPR) on the improvement of mine tailings properties and Lolium perenne L. (ryegrass) growth. Plants were grown in mine tailings mixed with an agricultural soil (1:1), 10% compost, and supplied with two different inorganic amendments - rock phosphate (6%) or lime (3%), and inoculated with the rhizobacterial strains Advenellakashmirensis BKM20 (B1) and Mesorhizobium tamadayense BKM04 (B2). The application of organo-mineral amendments ameliorated tailings characteristics, which fostered plant growth and further enhanced soil fertility and microbial activity. These findings were consistent with the increase of total organic carbon levels, with the higher numbers of heterotrophic and phosphate solubilizing bacteria, and higher dehydrogenase and urease activities, found in these substrates after plant establishment. Plant growth was further boosted by PGPR inoculation, most noticeable by co-inoculation of both strains. Moreover, inoculated plants showed increased activities for several antioxidant enzymes (catalase, peroxidase, polyphenoloxidase, and glutathione reductase) which indicate a reinforced antioxidant system. The application of agricultural soil, compost and lime associated with the inoculation of a mixture of PGPR proved to enhance the establishment of vegetation cover, thus promoting the stabilization of Kettara mine tailings. Nonetheless, further studies are needed in order to confirm its effectiveness under field conditions.

Identification of the endosymbionts from Sulla spinosissima growing in a lead mine tailings in Eastern Morocco as Mesorhizobium camelthorni sv. aridi.

AIMS: To identify the bacteria nodulating Sulla spinosissima growing profusely in a lead and zinc mine tailings in Eastern Morocco. METHODS AND RESULTS: In all, 32 rhizobial cultures, isolated from root nodules of S. spinosissima growing in soils of the mining site, were tolerant to different heavy metals. The ERIC-polymerase chain reaction (PCR) fingerprinting analysis clustered the isolates into seven different groups, and the analysis of the 16S rRNA sequences of four selected representative strains, showed they were related to different species of the genus Mesorhizobium. The atpD, glnII and recA housekeeping genes analysis confirmed the affiliation of the four representative strains to Mesorhizobium camelthorni CCNWXJ40-4T , with similarity percentages varying from 96·30 to 98·30%. The sequences of the nifH gene had 97·33-97·78% similarities with that of M. camelthorni CCNWXJ40-4T ; however, the nodC phylogeny of the four strains diverged from the type and other reference strains of M. camelthorni and formed a separated cluster. The four strains nodulate also Astragalus gombiformis and A. armatus but did not nodulate A. boeticus, Vachellia gummifera, Prosopis chilensis, Cicer arietinum, Lens culinaris, Medicago truncatula, Lupinus luteus or Phaseolus vulgaris. CONCLUSIONS: Based on similarities of the nodC symbiotic gene and differences in the host range, the strains isolated from S. spinosissima growing in soils of the Sidi Boubker mining site may form a different symbiovar within Mesorhizobium for which the name aridi is proposed. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we show that strains of M. camelthorni species nodulating S. spinosissima in the arid area of Eastern Morocco constitute a distinct phylogenetic clade of nodulation genes; we named symbiovar aridi, which encompasses also mesorhizobia from other Mediterranean desert legumes.

Mesorhizobium jarvisii is a dominant and widespread species symbiotically efficient on Astragalus sinicus L. in the Southwest of China.

In order to identify rhizobia of Astragalus sinicus L. and estimate their geographic distribution in the Southwest China, native rhizobia nodulating A. sinicus were isolated and their genetic diversity were studied at 13 sites cultivated in four Chinese provinces. A total of 451 rhizobial isolates were trapped with A. sinicus plants from soils and classified into 8 different genotypes defined by PCR-based restriction fragment length polymorphism (RFLP) of 16S-23S rRNA intergenic spacer (IGS). Twenty-one representative strains were further identified into three defined Mesorhizobium species by phylogenetic analyses of 16S rRNA genes and housekeeping genes (glnII and atpD). M. jarvisii was dominant accounting for 76.3% of the total isolates, 22.8% of the isolates were identified as M. huakuii and five strains belonged to M. qingshengii. All representatives were assigned to the symbiovar astragali by sharing high nodC sequence similarities of more than 99%. Furthermore, the biogeography distribution of these rhizobial genotypes and species was mainly affected by contents of available phosphorus, available potassium, total salts and pH in soils. The most remarkable point was the identification of M. jarvisii as a widespread and predominant species of A. sinicus in southwest of China. These results revealed a novel geographic pattern of rhizobia associated with A. sinicus in China.

Symbiosis islands of Loteae-nodulating Mesorhizobium comprise three radiating lineages with concordant nod gene complements and nodulation host-range groupings.

Mesorhizobium is a genus of soil bacteria, some isolates of which form an endosymbiotic relationship with diverse legumes of the Loteae tribe. The symbiotic genes of these mesorhizobia are generally carried on integrative and conjugative elements termed symbiosis islands (ICESyms). Mesorhizobium strains that nodulate Lotus spp. have been divided into host-range groupings. Group I (GI) strains nodulate L. corniculatus and L. japonicus ecotype Gifu, while group II (GII) strains have a broader host range, which includes L. pedunculatus. To identify the basis of this extended host range, and better understand Mesorhizobium and ICESym genomics, the genomes of eight Mesorhizobium strains were completed using hybrid long- and short-read assembly. Bioinformatic comparison with previously sequenced mesorhizobia genomes indicated host range was not predicted by Mesorhizobium genospecies but rather by the evolutionary relationship between ICESym symbiotic regions. Three radiating lineages of Loteae ICESyms were identified on this basis, which correlate with Lotus spp. host-range grouping and have lineage-specific nod gene complements. Pangenomic analysis of the completed GI and GII ICESyms identified 155 core genes (on average 30.1 % of a given ICESym). Individual GI or GII ICESyms carried diverse accessory genes with an average of 34.6 % of genes unique to a given ICESym. Identification and comparative analysis of NodD symbiotic regulatory motifs - nod boxes - identified 21 branches across the NodD regulons. Four of these branches were associated with seven genes unique to the five GII ICESyms. The nod boxes preceding the host-range gene nodZ in GI and GII ICESyms were disparate, suggesting regulation of nodZ may differ between GI and GII ICESyms. The broad host-range determinant(s) of GII ICESyms that confer nodulation of L. pedunculatus are likely present amongst the 53 GII-unique genes identified.

Genomic diversity of chickpea-nodulating rhizobia in Ningxia (north central China) and gene flow within symbiotic Mesorhizobium muleiense populations.

Diversity and taxonomic affiliation of chickpea rhizobia were investigated from Ningxia in north central China and their genomic relationships were compared with those from northwestern adjacent regions (Gansu and Xinjiang). Rhizobia were isolated from root-nodules after trapping by chickpea grown in soils from a single site of Ningxia and typed by IGS PCR-RFLP. Representative strains were phylogenetically analyzed on the basis of the 16S rRNA, housekeeping (atpD, recA and glnII) and symbiosis (nodC and nifH) genes. Genetic differentiation and gene flow were estimated among the chickpea microsymbionts from Ningxia, Gansu and Xinjiang. Fifty chickpea rhizobial isolates were obtained and identified as Mesorhizobium muleiense. Their symbiosis genes nodC and nifH were highly similar (98.4 to 100%) to those of other chickpea microsymbionts, except for one representative strain (NG24) that showed low nifH similarities with all the defined Mesorhizobium species. The rhizobial population from Ningxia was genetically similar to that from Gansu, but different from that in Xinjiang as shown by high chromosomal gene flow/low differentiation with the Gansu population but the reverse with the Xinjiang population. This reveals a biogeographic pattern with two main populations in M. muleiense, the Xinjiang population being chromosomally differentiated from Ningxia-Gansu one. M. muleiense was found as the sole main chickpea-nodulating rhizobial symbiont of Ningxia and it was also found in Gansu sharing alkaline-saline soils with Ningxia. Introduction of chickpea in recently cultivated areas in China seems to select from alkaline-saline soils of M. muleiense that acquired symbiotic genes from symbiovar ciceri.

A Methionine Sulfoxide Reductase B Is Required for the Establishment of Astragalus sinicus-Mesorhizobium Symbiosis.

Methionine sulfoxide reductase B (MsrB) is involved in oxidative stress or defense responses in plants. However, little is known about its role in legume-rhizobium symbiosis. In this study, an MsrB gene was identified from Astragalus sinicus and its function in symbiosis was characterized. AsMsrB was induced under phosphorus starvation and displayed different expression patterns under symbiotic and nonsymbiotic conditions. Hydrogen peroxide or methyl viologen treatment enhanced the transcript level of AsMsrB in roots and nodules. Subcellular localization showed that AsMsrB was localized in the cytoplasm of onion epidermal cells and co-localized with rhizobia in nodules. Plants with AsMsrB-RNAi hairy roots exhibited significant decreases in nodule number, nodule nitrogenase activity and fresh weight of the aerial part, as well as an abnormal nodule and symbiosome development. Statistical analysis of infection events showed that plants with AsMsrB-RNAi hairy roots had significant decreases in the number of root hair curling events, infection threads and nodule primordia compared with the control. The content of hydrogen peroxide increased in AsMsrB-RNAi roots but decreased in AsMsrB overexpression roots at the early stage of infection. The transcriptome analysis showed synergistic modulations of the expression of genes involved in reactive oxygen species generation and scavenging, defense and pathogenesis and early nodulation. In addition, a candidate protein interacting with AsMsrB was identified and confirmed by bimolecular fluorescence complementation. Taken together, our results indicate that AsMsrB plays an essential role in nodule development and symbiotic nitrogen fixation by affecting the redox homeostasis in roots and nodules.

Mesorhizobium terrae sp. nov., a novel species isolated from soil in Jangsu, Korea.

A gram-negative, white-pigmented, aerobic, rod-shaped bacterium, designated as strain NIBRBAC000500504T, was isolated from soil in Jangsu, Korea. Optimal growth of this strain was observed at 25 °C, pH 7.0, and in the presence of 0% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain NIBRBAC000500504T belonged to the genus Mesorhizobium and was closely related to Mesorhizobium shangrilense LMG 24762T (98.3% sequence similarity), Mesorhizobium australicum LMG 24608T (98.2%), Mesorhizobium qingshengii LMG 26793T (98.1%), Mesorhizobium ciceri ATCC 51585T (98.0%), Mesorhizobium loti DSM 2626T (98.0%), Mesorhizobium sophorae LMG 28223T (97.9%), Mesorhizobium waitakense LMG 28227T (97.8%), and Mesorhizobium cantuariense LMG 28225T (97.8%). Next-generation sequencing analysis indicated that the genome of strain NIBRBAC000500504T comprised a circular chromosome (5,731,152 bp, G+C content: 63.26%) and a plasmid (293,638 bp, G+C content: 61.39%) with 5672 coding sequences, 50 tRNAs, and 6 rRNAs. The major respiratory isoprenoid quinone was Q10; the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine; the major fatty acids were summed feature 8 (comprising C18:1 ω7c/C18:1 ω6c), C19:0 cyclo ω8c, C16:0, and C18:1 ω7c 11-methyl; and the G+C content of the genomic DNA was 62.9 mol%. The DNA-DNA relatedness values between NIBRBAC000500504T and its closest type strains were low. On the basis of these polyphasic taxonomic data, it is proposed that strain NIBRBAC000500504T represents a novel species of the genus Mesorhizobium, with the type strain being NIBRBAC000500504T (= KCTC 72278T = JCM 33432T).

Symbiotic nitrogen fixation and endophytic bacterial community structure in Bt-transgenic chickpea (Cicer arietinum L).

Symbiotic nitrogen fixation (SNF) of transgenic grain legumes might be influenced either by the site of transgene integration into the host genome or due to constitutive expression of transgenes and antibiotic-resistant marker genes. The present investigation confirmed proper nodulation of five tested Bt-chickpea events (IPCa2, IPCa4, IPCT3, IPCT10, and IPCT13) by native Mesorhizobium under field environment. Quantitative variations for nodulation traits among Bt-chickpea were determined and IPCT3 was found superior for nodule number and nodule biomass. Diversity, as well as richness indices, confirmed the changes in bacterial community structure of root and root-nodules from Bt-chickpea events IPCa2 and IPCT10. Especially, Gram-positive bacteria belonging to Firmicutes and Actinobacteria were selectively eliminated from root colonization of IPCa2. Richness indices (CHAO1 and ACE) of the root-associated bacterial community of IPCa2 was 13-14 times lesser than that of parent cv DCP92-3. Root nodule associated bacterial community of IPCT10 was unique with high diversity and richness, similar to the roots of non-Bt and Bt-chickpea. It indicated that the root nodules of IPCT10 might have lost their peculiar characteristics and recorded poor colonization of Mesorhizobium with a low relative abundance of 0.27. The impact of Bt-transgene on bacterial community structure and nodulation traits should be analyzed across the years and locations to understand and stabilize symbiotic efficiency for ecosystem sustainability.

Mesorhizobium alexandrii sp. nov., isolated from phycosphere microbiota of PSTs-producing marine dinoflagellate Alexandrium minutum amtk4.

An aerobic, Gram-stain-negative, motile and rod-shaped bacterial strain, designated as Z1-4T, was isolated from the phycosphere microbiota of marine dinoflagellate Alexandrium minutum that produces paralytic shellfish poisoning toxins. Phylogenetic analysis based on 16S rRNA gene sequences showed that the new isolate belongs to the genus Mesorhizobium, and it was closely related to Mesorhizobium waimense LMG 28228T and Mesorhizobium amorphae LMG 18977T with both 16S rRNA gene sequence similarities of 97.3%. The values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) relatedness between strain Z1-4T and its relatives are both well below the thresholds used for the delineation of a new species. A genome-based phylogenetic tree constructed by up-to-date bacterial core gene set (UBCG) indicates that strain Z1-4T forms an independent branch within the genus Mesorhizobium. The respiratory quinone of strain Z1-4T was Q-10. The major fatty acids were similar to other members of the genus Mesorhizobium containing the summed feature 8, C16:0, C19:0cycloω8c, C17:0 and summed feature 3. The polar lipids are phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, five glycolipids and seven unknown polar lipids. The DNA G + C content was determined to be 62.1 mol % based on its genomic sequence. Combined evidences based on the genotypic, chemotaxonomic and phenotypic characteristics clearly indicates that strain Z1-4T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium alexandrii sp. nov. is proposed. The type strain is Z1-4T (= KCTC 72512T = CCTCC AB 2019101T).

Novel putative Mesorhizobium and Ensifer genomospecies together with a novel symbiovar psoraleae nodulate legumes of agronomic interest grown in Tunisia.

Forty rhizobial strains were isolated from Lotus creticus, L. pusillus and Bituminaria bituminosa endemic to Tunisia, and they belonged to the Mesorhizobium and Ensifer genera based on 16S rDNA sequence phylogeny. According to the concatenated recA and glnII sequence-based phylogeny, four Bituminaria isolates Pb5, Pb12, Pb8 and Pb17 formed a monophyletic group with Mesorhizobium chacoense ICMP14587T, whereas four other strains Pb1, Pb6, Pb13 and Pb15 formed two separate lineages within the Ensifer genus. Among the L. pusillus strains, Lpus9 and Lpus10 showed a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas six other strains could belong to previously undescribed Mesorhizobium and Ensifer species. For L. creticus strains, Lcus37, Lcus39 and Lcus44 showed 98% sequence identity with Ensifer aridi JNVU TP6, and Lcus42 shared a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas another four strains were divergent from all the described Ensifer and Mesorhizobium species. The analysis of the nodC gene-based phylogeny identified four symbiovar groups; Mesorhizobium sp. sv. anthyllidis (Lpus3 and Lpus11 from L. pusillus, Lcus43 from L. creticus), Ensifer medicae sv. meliloti (four strains from L. creticus and two strains from L. pusillus), E. meliloti sv. meliloti (four from L. creticus, four from L. pusillus and four from B. bituminosa). In addition, four B. bituminosa strains (Pb5, Pb8, Pb12, and Pb17) displayed a distinctive nodC sequence distant from those of other symbiovars described to date. According to their symbiotic gene sequences and host range, the B. bituminosa symbionts (Pb5, Pb8, Pb12 and Pb17) would represent a new symbiovar of M. chacoense for which sv. psoraleae is proposed.

Mesorhizobium intechi sp. nov. isolated from nodules of Lotus tenuis in soils of the Flooding Pampa, Argentina.

Three symbiotic nitrogen-fixing bacteria (BD68T, BD66 and BD73) isolated from root nodules of Lotus tenuis in lowland soils of the Flooding Pampa (Argentina), previously classified as members of the Mesorhizobium genus, were characterized in this study. Phylogenetic analysis of their 16S rRNA gene sequences showed a close relationship to M. japonicum MAFF 303099T, M. erdmanii USDA 3471T, M. carmichaelinearum ICMP 18942T, M. opportunistum WSM 2975T and M. jarvisii ATCC 33699T, with sequence identities of 99.72%-100%. Multilocus sequence analysis of other housekeeping genes revealed that the three isolates belonged to a phylogenetically distinct clade within the genus Mesorhizobium. Strain BD68T was designated as the group representative and its genome was fully sequenced. The average nucleotide identity and in silico DNA-DNA hybridization comparisons between BD68T and the most related type strains showed values below the accepted threshold for species discrimination. Phenotypic and chemotaxonomic features were also studied. Based on these results, BD68T, BD66 and BD73 could be considered to represent a novel species of the genus Mesorhizobium, for which the name Mesorhizobium intechi sp. nov. is hereby proposed. The type strain of this species is BD68T (=CECT 9304T=LMG 30179T).

Induction of Systemic Resistance in Chickpea (Cicer arietinum L.) Against Fusarium oxysporum f. sp. ciceris by Antagonistic Rhizobacteria in Assistance with Native Mesorhizobium.

In the present study five potent rhizobacterial antagonists of Fusarium oxysporum f. sp. ciceris alone and in combination with Mesorhizobium (M) were evaluated for their potential to elicit the defence response reactions to reduce the total loss of plants and enhance the growth of two chickpea cultivars i.e. resistant GPF-2 and susceptible JG-41. Observations revealed that maximum phenolic, peroxidase (PO) and polyphenol oxidase (PPO) activity was induced after 30th day of germination. Maximum phenol concentration of 745.8 and 724.1 µg/gfw root tissues was recorded by Ps45 when co-inoculated with Mesorhizobium in both the varieties i.e. GPF-2 and JG-41 respectively. Isolates Ps45, Ps47 and Ps44 were found most promising to induce PO and PPO activity, in combination with Mesorhizobium and recorded superior over the fungicide with respect to negative control. Similar results were recorded for the phenylalanine ammonia lyase (PAL), maximally induced on 20th day after germination, where dual inoculation of Ps44+M and Ps45+M induced 57.0 and 54.2 nmol of cinnamic acid min-1 gfw-1 in GPF-2. However in case of JG-41, Ps45 and Ba1a exhibited highest PAL activity of 54.2 and 41.4 nmol of cinnamic acid min-1 gfw-1. Malonic aldehyde concentration in stem tissues at 30th day revealed that lipid peroxidation was effectively reduced in rhizobacterial treated plants compared to fungicide and negative control, signifying the role of antagonistic plant growth promoting rhizobacteria in reducing the stress and enhancing the plant's defence response to reduce the disease incidence and thus improving the plant growth and yield. Moreover the dual inoculations were observed superior over the fungicide treatment as well as single inoculations in terms of growth (root/shoot length and weight), signifying the synergistic effect of screened antagonists and native Mesorhizobium in suppressing the pathogen and thereby enhancing the plant growth.

A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus.

Legumes develop root nodules in symbiosis with nitrogen-fixing rhizobial bacteria. Rhizobia evoke cell division of differentiated cortical cells into root nodule primordia for accommodating bacterial symbionts. In this study, we show that NODULE INCEPTION (NIN), a transcription factor in Lotus japonicus that is essential for initiating cortical cell divisions during nodulation, regulates the gene ASYMMETRIC LEAVES 2-LIKE 18/LATERAL ORGAN BOUNDARIES DOMAIN 16a (ASL18/LBD16a). Orthologs of ASL18/LBD16a in nonlegume plants are required for lateral root development. Coexpression of ASL18a and the CCAAT box-binding protein Nuclear Factor-Y (NF-Y) subunits, which are also directly targeted by NIN, partially suppressed the nodulation-defective phenotype of L. japonicus daphne mutants, in which cortical expression of NIN was attenuated. Our results demonstrate that ASL18a and NF-Y together regulate nodule organogenesis. Thus, a lateral root developmental pathway is incorporated downstream of NIN to drive nodule symbiosis.

Shedding light on the presymbiontic phase of C. arietinum.

A complex network of symbiotic events between plants and bacteria allows the biosphere to exploit the atmospheric reservoir of molecular nitrogen. In seeds, a series of presymbiotic steps are already identified during imbibition, while interactions between the host and its symbiont begin in the early stages of germination. In the present study, a detailed analysis of the substances' complex delivered by Cicer arietinum seeds during imbibition showed a relevant presence of proteins and amino acids, which, except for cysteine, occurred with the whole proteinogenic pool. The imbibing solution was found to provide essential probiotic properties able to sustain the growth of the specific chickpea symbiont Mesorhizobium ciceri. Moreover, the imbibing solution, behaving as a complete medium, was found to be critically important for the symbiont's attraction, a fact this that is strictly related to the presence of the amino acids glycine, serine, and threonine. Here, the presence of these amino acids is constantly supported by the presence of the enzymes serine hydroxymethyltransferase and formyltetrahydrofolate deformylase, which are both involved in their biosynthesis. The reported findings are discussed in the light of the pivotal role played by the imbibing solution in attracting and sustaining symbiosis between the host and its symbiont.

Mesorhizobium composti sp. nov., isolated from compost.

A polyphasic taxonomic approach was used to characterize a presumptively novel diazotrophic bacterium, designated strain CC-YTH430T, isolated from a compost sample in Taiwan. Cells of strain CC-YTH430T were found to be Gram-stain negative, facultative anaerobic rods that formed yellow-colored colonies on nutrient agar. Cell growth occurred at 15-40 °C, pH 5.0-9.0 and in the presence of 0-2% NaCl. Strain CC-YTH430T resembled Mesorhizobium species while sharing high pair-wise 16S rRNA gene sequence similarities with Mesorhizobium silamurunense, Mesorhizobium thiogangeticum, Mesorhizobium plurifarium, Mesorhizobium tamadayense, Mesorhizobium amorphae (96.9% each), Mesorhizobium sediminum (96.8%), and Mesorhizobium soli (96.5%) and < 96.5% similarity to other species. Strain CC-YTH430T showed 78.8-79.7% average nucleotide identity compared to the type strains of M. amorphae, M. plurifarium, M. soli, M. tamadayense and M. wenxiniae. The N2-fixing activity of strain CC-YTH430T was 0.2 nmol ethylene h-1 at 30 °C. The respiratory system was ubiquinone 10 (Q-10) and the DNA G+C content was 62.0 ± 0.2 mol%. The major fatty acids (> 5%) were C16:0, C17:0 cyclo, C19:0 cyclo ω8c, C14:0 3OH/C16:1 iso I and C18:1ω7c/C18:1ω6c. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine and an unidentified aminolipid in major amounts. In addition, phosphatidylethanolamine, an unidentified lipid and several unidentified polar lipids were also found in moderate-to-trace amounts. Based on the phylogenetic, phenotypic and chemotaxonomic features, strain CC-YTH430T is proposed to represent a novel Mesorhizobium species, for which the name Mesorhizobium composti sp. nov. (type strain CC-YTH430T = BCRC 81024T = JCM 31762T) is proposed.

Biodiversity of rhizobia present in plant nodules of Biserrula pelecinus across Southwest Spain.

Biodiversity studies of native Mesorhizobium spp. strains able to nodulate the annual herbaceous legume Biserrula pelecinus L. in soils from Southwest Spain have been carried out. One or two isolates per plant, 30 in total, were randomly selected for further characterization. There was no association between the presence of mesorhizobia nodulating-B. pelecinus and the chemical or textural properties of the soils. The isolates were tested for their symbiotic effectiveness on this forage legume under greenhouse conditions and characterized on the basis of physiological parameters: carbon source utilisation (API 50CH), 16S rRNA sequencing and ERIC-PCR, lipopolysaccharide, protein and plasmid profiles. Our results show that in spite of the great diversity found among the native isolates, most of them belong to the genus Mesorhizobium, the exception being strain B24 which sequence matches 97.52% with Neorhizobium huautlense; this is the first description of a Neorhizobium strain effectively nodulating-biserrula plants. Results of a field trial indicated that some of these isolates could be recommended as inoculants for this legume. B24=DSM 28743=CECT 8815; ENA (HF955513) 16S rRNA sequences of isolates B13, B18, B26, B30 and B1 are deposited at ENA under numbers LS999402 to LS999406, respectively.