Metallothionein1A Regulates Rhizobial Infection and Nodulation in Phaseolus vulgaris.

Metallothioneins (MTs) constitute a heterogeneous family of ubiquitous metal ion-binding proteins. In plants, MTs participate in the regulation of cell growth and proliferation, protection against heavy metal stress, oxidative stress responses, and responses to pathogen attack. Despite their wide variety of functions, the role of MTs in symbiotic associations, specifically nodule-fabacean symbiosis, is poorly understood. Here, we analyzed the role of the PvMT1A gene in Phaseolus vulgaris-Rhizobium tropici symbiosis using bioinformatics and reverse genetics approaches. Using in silico analysis, we identified six genes encoding MTs in P. vulgaris, which were clustered into three of the four classes described in plants. PvMT1A transcript levels were significantly higher in roots inoculated with R. tropici at 7 and 30 days post inoculation (dpi) than in non-inoculated roots. Functional analysis showed that downregulating PvMT1A by RNA interference (RNAi) reduced the number of infection events at 7 and 10 dpi and the number of nodules at 14 and 21 dpi. In addition, nodule development was negatively affected in PvMT1A:RNAi transgenic roots, and these nodules displayed a reduced nitrogen fixation rate at 21 dpi. These results strongly suggest that PvMT1A plays an important role in the infection process and nodule development in P. vulgaris during rhizobial symbiosis.

The Effect of Exogenous Nitrate on LCO Signalling, Cytokinin Accumulation, and Nodule Initiation in Medicago truncatula.

Nitrogen fixation by rhizobia is a highly energy-demanding process. Therefore, nodule initiation in legumes is tightly regulated. Environmental nitrate is a potent inhibitor of nodulation. However, the precise mechanism by which this agent (co)regulates the inhibition of nodulation is not fully understood. Here, we demonstrate that in Medicago truncatula the lipo-chitooligosaccharide-induced accumulation of cytokinins is reduced in response to the application of exogenous nitrate. Under permissive nitrate conditions, perception of rhizobia-secreted signalling molecules leads to an increase in the level of four cytokinins (i.e., iP, iPR, tZ, and tZR). However, under high-nitrate conditions, this increase in cytokinins is reduced. The ethylene-insensitive mutant Mtein2/sickle, as well as wild-type plants grown in the presence of the ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine (AVG), is resistant to the inhibition of nodulation by nitrate. This demonstrates that ethylene biosynthesis and perception are required to inhibit nodule organogenesis under high-nitrate conditions.

Root endophyte-enhanced peanut-rhizobia interaction is associated with regulation of root exudates.

Root exudates play a crucial role in the symbiosis between leguminous plants and rhizobia. Our previous studies have shown that a fungal endophyte Phomopsis liquidambaris promotes peanut-rhizobia nodulation and nitrogen fixation, but the underlying mechanism are largely unknown. Here, we explore the role of peanut root exudates in Ph. liquidambaris-mediated nodulation enhancement. We first collected root exudates from Ph. liquidambaris-inoculated and un-inoculated peanuts and determined their effects on rhizobial growth, biofilm formation, chemotaxis, nodC gene expression, and peanut nodulation. Our results found a positive effect of Ph. liquidambaris-inoculated root exudates on these characteristics of rhizobia. Next, we compared the root exudates profile of Ph. liquidambaris-inoculated and un-inoculated plants and found that Ph. liquidambaris altered the concentrations of phenolic acids, flavonoids, organic acids and amino acids in root exudates. Furthermore, the rhizobial chemotaxis, growth and biofilm formation in response to the changed compounds at different concentrations showed that all of the test compounds induced rhizobial chemotactic behavior, and organic acids (citric acid and oxalic acid) and amino acid (glutamate, glycine and glutamine) at higher concentrations increased rhizobial growth and biofilm formation. Collectively, our results suggest that root exudates alterations contribute to Ph. liquidambaris-mediated peanut-rhizobia nodulation enhancement.

The rhizosphere microbiome plays a role in the resistance to soil-borne pathogens and nutrient uptake of strawberry cultivars under field conditions.

Microbial-root associations are important to help plants cope with abiotic and biotic stressors. Managing these interactions offers an opportunity for improving the efficiency and sustainability of agricultural production. By characterizing the bacterial and archaeal community (via 16S rRNA sequencing) associated with bulk and rhizosphere soil of sixteen strawberry cultivars in two controlled field studies, we explored the relationships between the soil microbiome and plant resistance to two soil-borne fungal pathogens (Verticillium dahliae and Macrophomina phaseolina). Overall, the plants had a distinctive and genotype-dependent rhizosphere microbiome with higher abundances of known beneficial bacteria such as Pseudomonads and Rhizobium. The rhizosphere microbiome played a significant role in the resistance to the two soil-borne pathogens as shown by the differences in microbiome between high and low resistance cultivars. Resistant cultivars were characterized by higher abundances of known biocontrol microorganisms including actinobacteria (Arthrobacter, Nocardioides and Gaiella) and unclassified acidobacteria (Gp6, Gp16 and Gp4), in both pathogen trials. Additionally, cultivars that were resistant to V. dahliae had higher rhizosphere abundances of Burkholderia and cultivars resistant to M. phaseolina had higher abundances of Pseudomonas. The mechanisms involved in these beneficial plant-microbial interactions and their plasticity in different environments should be studied further for the design of low-input disease management strategies.

Systematic Analysis of Gibberellin Pathway Components in Medicago truncatula Reveals the Potential Application of Gibberellin in Biomass Improvement.

Gibberellins (GAs), a class of phytohormones, act as an essential natural regulator of plant growth and development. Many studies have shown that GA is related to rhizobial infection and nodule organogenesis in legume species. However, thus far, GA metabolism and signaling components are largely unknown in the model legume Medicago truncatula. In this study, a genome-wide analysis of GA metabolism and signaling genes was carried out. In total 29 components, including 8 MtGA20ox genes, 2 MtGA3ox genes, 13 MtGA2ox genes, 3 MtGID1 genes, and 3 MtDELLA genes were identified in M. truncatula genome. Expression profiles revealed that most members of MtGAox, MtGID1, and MtDELLA showed tissue-specific expression patterns. In addition, the GA biosynthesis and deactivation genes displayed a feedback regulation on GA treatment, respectively. Yeast two-hybrid assays showed that all the three MtGID1s interacted with MtDELLA1 and MtDELLA2, suggesting that the MtGID1s are functional GA receptors. More importantly, M. truncatula exhibited increased plant height and biomass by ectopic expression of the MtGA20ox1, suggesting that enhanced GA response has the potential for forage improvement.

Emergence of ß-rhizobia as new root nodulating bacteria in legumes and current status of the legume-rhizobium host specificity dogma.

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

Effect of common bean seed exudates on growth, lipopolysaccharide production, and lipopolysaccharide transport gene expression of Rhizobium anhuiense.

Lipopolysaccharide (LPS) is essential for successful nodulation during the symbiosis of rhizobia and legumes. However, the detailed mechanism of the LPS in this process has not yet been clearly elucidated. In this study, the effects of common bean seed exudates on the growth, lipopolysaccharide production, and lipopolysaccharide transport genes expression (lpt) of Rhizobium anhuiense were investigated. Rhizobium anhuiense exposed to exudates showed changes in LPS electrophoretic profiles and content, whereby the LPS band was wider and the LPS content was higher in R. anhuiense treated with seed exudates. Exudates enhanced cell growth of R. anhuiense in a concentration-dependent manner; R. anhuiense exposed to higher doses of the exudate showed faster growth. Seven lpt genes of R. anhuiense were amplified and sequenced. Sequences of six lpt genes, except for lptE, were the same as those found in previously analyzed R. anhuiense strains, while lptE shared low sequence similarity with other strains. Exposure to the exudates strongly stimulated the expression of all lpt genes. Approximately 6.7- (lptG) to 301-fold (lptE) increases in the transcriptional levels were observed after only 15 min of exposure to exudates. These results indicate that seed exudates affect the LPS by making the cell wall structure more conducive to symbiotic nodulation.

The role of rhizodeposits in shaping rhizomicrobiome.

Rhizomicrobiome, the communities of microorganisms surrounding the root of the plant, plays a vital role in promoting plant growth and health. The composition of rhizomicrobiome is dynamic both temporally and spatially, and is influenced greatly by the plant host and environmental factors. One of the key influencing factors is rhizodeposits, composed of root-released tissue cells, exudates, lysates, volatile compounds, etc. Rhizodeposits are rich in carbon and nitrogen elements, and able to select and fuel the growth of rhizomicrobiome. In this minireview, we overview the generation, composition and dynamics of rhizodeposits, and discuss recent work describing the general and specific impacts of rhizodeposits on rhizomicrobiome. We focus further on root exudates, the most dynamic component of rhizodeposits, and review recent progresses about the influence of specific root exudates in promoting bacterial root colonization, inducing biofilm development, acting as plant defence and shaping the rhizomicrobiome.

Root colonization by heavy metal resistant Enterobacter and its influence on metal induced oxidative stress on Cajanus cajan.

BACKGROUND: Heavy metal resistant bacterium Enterobacter sp. C1D was evaluated for cadmium (Cd) mediated exopolysaccharide production, biofilm formation and legume root colonization ability under Cd stress to alleviate metal induced stress. RESULTS: The plant was sensitive to Cd (IC50 3-4 µg mL-1 ), whereas the bacterium showed high Cd tolerance (MIC99 120 µg mL-1 ). Confocal laser scanning microscopy of the Cajanus cajan roots showed heavy loads of green fluorescence protein labelled Enterobacter sp. C1D on the surface of plant root, specifically at the point of root hair/lateral root formation along with cortex, even under metal stress. The root colonizing ability of Enterobacter sp. C1D was not affected by the presence of Rhizobium and the bacteria could be observed after 30 days of incubation in soil. Various plant growth parameters, antioxidant metabolites and oxidative stress indicator were significantly influenced by bacterial treatment, which, overall, reduced the adverse effect of Cd. CONCLUSION: Heavy metal tolerant bacteria may be a good choice for the development of biofertilizers and may work well with the native soil microbes such as Rhizobium under the metal polluted soil. © 2019 Society of Chemical Industry.

The role of volatiles in Rhizobium tolerance to cadmium: Effects of aldehydes and alcohols on growth and biochemical endpoints.

Rhizobia have a significant agronomic and environmental role and are eminent contributors to soil fertility. However, this group of microorganisms are affected by various environmental stresses, such as Cd contamination. High Cd concentrations change bacterial metabolism. During this metabolic shift, bacteria alter their volatilome (the set of volatile metabolites synthesized by an organism). In the presence of Cd, peak areas of saturated aldehydes and alcohols were previously reported to increase, and the consequences of this increase to cells are poorly known. In this study, Rhizobium sp. strain E20-8 cells were exposed to Cd and aldehydes or their conjugated alcohols. Exposure to Cd (100 µM) inhibited cell growth and induced several biomarkers of oxidative stress. The present study also evidenced the higher toxicity of most aldehydes relatively to the corresponding alcohol in the presence of Cd, suggesting that reduction of aldehydes into alcohols may be an effective mechanism to restrain aldehydes toxicity in Rhizobium cells under Cd toxicity. Nonetheless, the protective effect was dependent on the pair aldehyde-respective alcohol considered and it differed between Cd stressed and non-stressed cells. Differences in the ability to convert aldehydes to alcohols may emerge as a new feature helping explain the oxidative tolerance variability among bacteria.

Rhizobia population was favoured during in situ phytoremediation of vanadium-titanium magnetite mine tailings dam using Pongamia pinnata.

Mine tailings contain toxic metals and can lead to serious pollution of soil environment. Phytoremediation using legumes has been regarded as an eco-friendly way for the rehabilitation of tailings-laden lands but little is known about the changes of microbial structure during the process. In the present study, we monitored the dynamic change of microbiota in the rhizosphere of Pongamia pinnata during a 2-year on-site remediation of vanadium-titanium magnetite tailings. After remediation, overall soil health conditions were significantly improved as increased available N and P contents and enzyme activities were discovered. There was also an increase of microbial carbon and nitrogen contents. The Illumina sequencing technique revealed that the abundance of taxa under Proteobacteria was increased and rhizobia-related OTUs were preferentially enriched. A significant difference was discovered for sample groups before and after remediation. Rhizobium and Nordella were identified as the keystone taxa at genus rank. The functional prediction indicated that nitrogen fixation was enhanced, corresponding well with qPCR results which showed a significant increase of nifH gene copy numbers by the 2nd year. Our findings for the first time elucidated that legume phytoremediation can effectively cause microbial communities to shift in favour of rhizobia in heavy metal contaminated soil.

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation.

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.

Rhizobium spp exopolysaccharides production and xanthan lyase use on its structural modification.

Enzymes can be very useful on exopolysaccharides (EPS) research, can be used at elucidation and also to modify the polysaccharides' structure in order to alter their physical properties. Thus, the reduction of the molecular mass could increase applications of these biopolymers. Therefore, the EPS production of different rhizobia and the action of xanthan lyase on its structures were evaluated. The strains produced significant amounts of EPS, and it was noticed that are heteropolysaccharides, composed galactose and glucose. Both EPS and xanthan were modified on ß-glycosidic bonds, the mannose was removed of xanthan had but the EPS was affected in the CO stretching vibration, where the glucuronic acid removed from of your structure. The ester/carboxylic acid portions affected functional groups of the acetate/succinate, methyl carbons of the O-acetyl and pyruvate methyl groups in addition to affect the carbons the main pyranoid. The Resistance to temperature increase of the EPS was observed, made possible by the activity of the lyase. EPS has the ability to form stable gels at higher temperatures and anionic feature can be used on solubilization and controlled release of substances. Modified EPS knowledge will presently facilitate future investigations relating the structure of the rhizobia polysaccharide against rheological properties.

Rhizobacteria Mediate the Phytotoxicity of a Range of Biorefinery-Relevant Compounds.

Advances in engineering biology have expanded the list of renewable compounds that can be produced at scale via biological routes from plant biomass. In most cases, these chemical products have not been evaluated for effects on biological systems, defined in the present study as bioactivity, that may be relevant to their manufacture. For sustainable chemical and fuel production, the industry needs to transition from fossil to renewable carbon sources, resulting in unprecedented expansion in the production and environmental distribution of chemicals used in biomanufacturing. Further, although some chemicals have been assessed for mammalian toxicity, environmental and agricultural hazards are largely unknown. We assessed 6 compounds that are representative of the emerging biofuel and bioproduct manufacturing process for their effect on model plants (Arabidopsis thaliana, Sorghum bicolor) and show that several alter plant seedling physiology at submillimolar concentrations. However, these responses change in the presence of individual bacterial species from the A. thaliana root microbiome. We identified 2 individual microbes that change the effect of chemical treatment on root architecture and a pooled microbial community with different effects relative to its constituents individually. The present study indicates that screening industrial chemicals for bioactivity on model organisms in the presence of their microbiomes is important for biologically and ecologically relevant risk analyses. Environ Toxicol Chem 2019;38:1911-1922. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Effect of Vicia faba L. var. minor and Sulla coronaria (L.) Medik associated with plant growth-promoting bacteria on lettuce cropping system and heavy metal phytoremediation under field conditions.

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

LACK OF SYMBIONT ACCOMMODATION controls intracellular symbiont accommodation in root nodule and arbuscular mycorrhizal symbiosis in Lotus japonicus.

Nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi (AMF) form symbioses with plant roots and these are established by precise regulation of symbiont accommodation within host plant cells. In model legumes such as Lotus japonicus and Medicago truncatula, rhizobia enter into roots through an intracellular invasion system that depends on the formation of a root-hair infection thread (IT). While IT-mediated intracellular rhizobia invasion is thought to be the most evolutionarily derived invasion system, some studies have indicated that a basal intercellular invasion system can replace it when some nodulation-related factors are genetically modified. In addition, intracellular rhizobia accommodation is suggested to have a similar mechanism as AMF accommodation. Nevertheless, our understanding of the underlying genetic mechanisms is incomplete. Here we identify a L. japonicus nodulation-deficient mutant, with a mutation in the LACK OF SYMBIONT ACCOMMODATION (LAN) gene, in which root-hair IT formation is strongly reduced, but intercellular rhizobial invasion eventually results in functional nodule formation. LjLAN encodes a protein that is homologous to Arabidopsis MEDIATOR 2/29/32 possibly acting as a subunit of a Mediator complex, a multiprotein complex required for gene transcription. We also show that LjLAN acts in parallel with a signaling pathway including LjCYCLOPS. In addition, the lan mutation drastically reduces the colonization levels of AMF. Taken together, our data provide a new factor that has a common role in symbiont accommodation process during root nodule and AM symbiosis.

Heavy metal accumulation in Lathyrus sativus growing in contaminated soils and identification of symbiotic resistant bacteria.

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.

Aqueous peat extract exposes rhizobia to sub-lethal stress which may prime cells for improved desiccation tolerance.

Inoculation of legume seed with rhizobia is an efficient and cost-effective means of distributing elite rhizobial strains to broad-acre crops and pastures. However, necessary drying steps after coating seed expose rhizobia to desiccation stress reducing survival and limiting potential nitrogen fixation by legumes. Rhizobial tolerance to desiccation varies with strain and with growth conditions prior to drying. Cells grown in peat generally survive desiccation better than cells grown in liquid broth. We aimed to identify peat-induced proteomic changes in rhizobia that may be linked to desiccation tolerance. Proteins expressed differentially after growth in peat extract when compared with a minimal defined medium were measured in four rhizobial strains. Proteins showing the greatest increase in abundance were those involved in amino acid and carbohydrate transport and metabolism. Proteins involved in posttranslational modification and cell defence mechanisms were also upregulated. Many of the proteins identified in this study have been previously linked to stress responses. In addition, analysis using nucleic acid stains SYTO9 and propidium iodide indicated that membranes had been compromised after growth in peat extract. We targeted the membrane repair protein PspA (ΔRL3579) which was upregulated in Rhizobium leguminosarum bv. viceae 3841 after growth in peat extract to validate whether the inability to repair membrane damage after growth in peat extract reduced desiccation tolerance. The ΔRL3579 mutant grown in peat extract had significantly lower survival under desiccation stress, whereas no difference in survival between wild-type and mutant strains was observed after growth in tryptone yeast (TY) or minimal medium (JMM) media. Staining mutant and wild-type strains with SYTO9 and propidium iodide indicated that membranes of the mutant were compromised after growth in peat extract and to a lesser extent in TY. This study shows that growth in peat extract causes damage to cell membranes and exposes rhizobia to sub-lethal stress resulting in differential expression of several stress-induced proteins. The induction of these proteins may prime and protect the cells when subjected to subsequent stress such as desiccation. Identifying the key proteins involved in desiccation tolerance and properties of peat that stimulate this response will be important to inform development of new inoculant technology that maximises survival of rhizobia during delivery to legume crops and pastures.

Alleviation of drought stress in pulse crops with ACC deaminase producing rhizobacteria isolated from acidic soil of Northeast India.

The agricultural crops are often affected by the scarcity of fresh water. Seasonal drought is a major constraint on Northeast Indian agriculture. Almost 80% of the agricultural land in this region is acidic and facing severe drought during the winter period. Apart from classical breeding and transgenic approaches, the application of plant-growth-promoting bacteria (PGPB) is an alternative strategy for improving plant fitness under stressful conditions. The 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing PGPB offer drought stress tolerance by regulating plant ethylene levels. The aim of the present study was to evaluate the consortium effect of three ACC-deaminase producing rhizobacteria - Ochrobactrum pseudogrignonenseRJ12, Pseudomonas sp.RJ15 and Bacillus subtilisRJ46 on drought stress alleviation in Vigna mungo L. and Pisum sativum L. Consortium treatment significantly increase seed germination percentage, root length, shoot length, and dry weight of treated plants. An elevated production of reactive oxygen species scavenging enzymes and cellular osmolytes; higher leaf chlorophyll content; increase in relative water content and root recovery intension were observed after consortium treatment in comparison with the uninoculated plants under drought conditions. The consortium treatment decreased the ACC accumulation and down-regulated ACC-oxidase gene expression. This consortium could be an effective bio-formulator for crop health improvement in drought-affected acidic agricultural fields.

Old meets new: most probable number validation of metagenomic and metatranscriptomic datasets in soil.

Metagenomics and metatranscriptomics provide insights into biological processes in complex substrates such as soil, but linking the presence and expression of genes with functions can be difficult. Here, we obtain traditional most probable number estimates (MPN) of Rhizobium abundance in soil as a form of sample validation. Our work shows that in the Highfield experiment at Rothamsted, which has three contrasting conditions (>50 years continual bare fallow, wheat and grassland), MPN based on host plant nodulation assays corroborate metagenomic and metatranscriptomic estimates for Rhizobium leguminosarum sv. trifolii abundance. This validation is important to legitimize soil metagenomics and metatranscriptomics for the study of complex relationships between gene function and phylogeny. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated for the first time a functional assay validation of metagenomic and metatranscriptomic datasets by utilizing the clover and Rhizobium leguminosarum sv. trifolii mutualism. The results show that the Most Probable Number results corroborate the results of the 'omics approaches and gives confidence to the study of other biological systems where such a cross-check is not available.