Plant-Growth-Promoting Rhizobacteria Emerging as an Effective Bioinoculant to Improve the Growth, Production, and Stress Tolerance of Vegetable Crops.

Vegetable cultivation is a promising economic activity, and vegetable consumption is important for human health due to the high nutritional content of vegetables. Vegetables are rich in vitamins, minerals, dietary fiber, and several phytochemical compounds. However, the production of vegetables is insufficient to meet the demand of the ever-increasing population. Plant-growth-promoting rhizobacteria (PGPR) facilitate the growth and production of vegetable crops by acquiring nutrients, producing phytohormones, and protecting them from various detrimental effects. In this review, we highlight well-developed and cutting-edge findings focusing on the role of a PGPR-based bioinoculant formulation in enhancing vegetable crop production. We also discuss the role of PGPR in promoting vegetable crop growth and resisting the adverse effects arising from various abiotic (drought, salinity, heat, heavy metals) and biotic (fungi, bacteria, nematodes, and insect pests) stresses.

Genomic characterization and computational phenotyping of nitrogen-fixing bacteria isolated from Colombian sugarcane fields.

Previous studies have shown the sugarcane microbiome harbors diverse plant growth promoting microorganisms, including nitrogen-fixing bacteria (diazotrophs), which can serve as biofertilizers. The genomes of 22 diazotrophs from Colombian sugarcane fields were sequenced to investigate potential biofertilizers. A genome-enabled computational phenotyping approach was developed to prioritize sugarcane associated diazotrophs according to their potential as biofertilizers. This method selects isolates that have potential for nitrogen fixation and other plant growth promoting (PGP) phenotypes while showing low risk for virulence and antibiotic resistance. Intact nitrogenase (nif) genes and operons were found in 18 of the isolates. Isolates also encode phosphate solubilization and siderophore production operons, and other PGP genes. The majority of sugarcane isolates showed uniformly low predicted virulence and antibiotic resistance compared to clinical isolates. Six strains with the highest overall genotype scores were experimentally evaluated for nitrogen fixation, phosphate solubilization, and the production of siderophores, gibberellic acid, and indole acetic acid. Results from the biochemical assays were consistent and validated computational phenotype predictions. A genotypic and phenotypic threshold was observed that separated strains by their potential for PGP versus predicted pathogenicity. Our results indicate that computational phenotyping is a promising tool for the assessment of bacteria detected in agricultural ecosystems.

Symbiosis of soybean with nitrogen fixing bacteria affected by root lesion nematodes in a density-dependent manner.

Early maturing varieties of soybean have a high yield potential in Europe, where the main biotic threat to soybean cultivation are root lesion nematodes (Pratylenchus spp.). Nitrogen fixation in root nodules by highly efficient inoculants of Bradyrhizobium japonicum is an incentive to grow soybean in low-input rotation systems. We investigated density-dependent effects of Pratylenchus penetrans on nitrogen fixation by co-inoculated B. japonicum. Less than 130 inoculated nematodes affected the number and weight of nodules, the density of viable bacteroids in nodules, and nitrogen fixation measured as concentration of ureides in leaves. With more inoculated nematodes, the percentage that invaded the roots increased, and adverse effects on the symbiosis accelerated, leading to non-functional nodules at 4,000 and more nematodes. When P. penetrans invaded roots that had fully established nodules, growth of nodules, density of bacteroids, and nitrogen fixation were affected but not the number of nodules. In contrast, nodulation of already infested roots resulted in a high number of small nodules with decreased densities of bacteroids and nitrogen fixation. P. penetrans invaded and damaged the nodules locally, but they also significantly affected the nodule symbiosis by a plant-mediated mechanism, as shown in an experiment with split-root systems.

Endophytes alleviate the elevated CO2-dependent decrease in photosynthesis in rice, particularly under nitrogen limitation.

The positive effects of high atmospheric CO2 concentrations [CO2] decrease over time in most C3 plants because of down-regulation of photosynthesis. A notable exception to this trend is plants hosting N-fixing bacteria. The decrease in photosynthetic capacity associated with an extended exposure to high [CO2] was therefore studied in non-nodulating rice that can establish endophytic interactions. Rice plants were inoculated with diazotrophic endophytes isolated from the Salicaceae and CO2 response curves of photosynthesis were determined in the absence or presence of endophytes at the panicle initiation stage. Non-inoculated plants grown under elevated [CO2] showed a down-regulation of photosynthesis compared to those grown under ambient [CO2]. In contrast, the endophyte-inoculated plants did not show a decrease in photosynthesis associated with high [CO2], and they exhibited higher photosynthetic electron transport and mesophyll conductance rates than non-inoculated plants under high [CO2]. The endophyte-dependent alleviation of decreases in photosynthesis under high [CO2] led to an increase in water-use efficiency. These effects were most pronounced when the N supply was limited. The results suggest that inoculation with N-fixing endophytes could be an effective means of improving plant growth under high [CO2] by alleviating N limitations.

Are we there yet? The long walk towards the development of efficient symbiotic associations between nitrogen-fixing bacteria and non-leguminous crops.

Nitrogen is an essential element of life, and nitrogen availability often limits crop yields. Since the Green Revolution, massive amounts of synthetic nitrogen fertilizers have been produced from atmospheric nitrogen and natural gas, threatening the sustainability of global food production and degrading the environment. There is a need for alternative means of bringing nitrogen to crops, and taking greater advantage of biological nitrogen fixation seems a logical option. Legumes are used in most cropping systems around the world because of the nitrogen-fixing symbiosis with rhizobia. However, the world's three major cereal crops-rice, wheat, and maize-do not associate with rhizobia. In this review, we will survey how genetic approaches in rhizobia and their legume hosts allowed tremendous progress in understanding the molecular mechanisms controlling root nodule symbioses, and how this knowledge paves the way for engineering such associations in non-legume crops. We will also discuss challenges in bringing these systems into the field and how they can be surmounted by interdisciplinary collaborations between synthetic biologists, microbiologists, plant biologists, breeders, agronomists, and policymakers.

Interactive Effects of Nitrogen-Fixing Bacteria Inoculation and Nitrogen Fertilization on Soybean Yield in Unfavorable Edaphoclimatic Environments.

The objective of this work was to evaluate the effect of nitrogen (N) fertilization and the biological nitrogen fixation efficiency in soybean crops grown in unfavorable environments (high temperature, low fertility, and areas with sandy soil). Six field experiments were conducted between 2015 and 2018. Two experiments were performed per year. They were conducted in two separate areas. One was previously covered with degraded pasture (post-pasture area). The other was previously used to plant soybean (post-soybean crop area). The treatments consisted of inoculations with N-fixing bacteria (NFB) (0, 4, 8, and 12 doses ha-1) and N fertilization with rates of 0, 25, 50, and 100 kg ha-1. N fertilization and inoculation with NFB increased soil N, though the levels were still low. Among the tested groups, those with the application of eight doses of inoculant recorded the highest grain yields in post-soybean areas. They showed 10% (237 kg ha-1) and 15% (336 kg ha-1) higher grain yields when compared to crops treated without inoculant and crops with four doses of inoculant, respectively. N fertilization with 25 and 100 kg ha-1 decreased the root nodules of soybean plants grown in the post-soybean and post-pasture areas, respectively. Soybean crops grown on degraded pasture areas also showed good response to N fertilization (50 kg ha-1) when combined with NFB inoculation (12 doses ha-1). These showed grain yields 22% (439 kg ha-1) higher than those of plants treated with just 12 doses of inoculant and no N fertilization.

Nitrogen-fixing bacteria and Oxalis - evidence for a vertically inherited bacterial symbiosis.

BACKGROUND: Plant-endophyte symbioses often revolve around nitrogen metabolism, and involve varying degrees of intimacy. Although evidence for vertical inheritance of nitrogen-fixing endophytic bacteria is increasing, it is confined mostly to crop plants, and to date no such system has been reported for geophytes. METHODS: Bacterial endophytes associated with Oxalis, the most species-rich geophytic genus form the Cape Flora in southern Africa was studied. Culturable endophytes were isolated from surface-sterilized vegetative and reproductive plant organs for six host species at three locations. Colonies of microbes on various artificial media were morphotyped, enumerated and identified using sequence data. Filter exclusion experiments were conducted to determine if endophytes were vertically transmitted to seeds, determine if mucilage plays a role to actively attract microbes from the soil and to assess microbial richness isolated from the mucilage of Oxalis seedlings. Fluorescent microscopy was implemented in order to visualize endophytic bacteria in cryo-sectioned seeds. RESULTS: Evidence for a novel, vertically transmitted symbiosis was reported. Communities of nitrogen-fixing and plant growth-promoting Bacillus endophytes were found to associate with selected Oxalis hosts from nitrogen-deficient environments of the Cape. Bacillus endophytes were ubiquitous and diverse across species and plant bodies, and were prominent in seeds. Three common nitrogen-fixing Bacillus have known oxalotrophic properties and appear to be housed inside specialised cavities (containing oxalates) within the plant body and seeds. CONCLUSIONS: The discovery of vertical transmission and potential benefits to both host and endophyte suggest a particularly tight mutualism in the Oxalis-endophyte system. This discovery suggests unexpected ways in which geophytes might avoid nitrogen deficiency, and suggest that such symbioses are more common than previously expected.

Urban traffic changes the biodiversity, abundance, and activity of phyllospheric nitrogen-fixing bacteria.

The phyllosphere provides appropriate conditions for colonization by microorganisms, including diazotrophic bacteria. However, a poor understanding of the effects of the atmospheric environment on the phyllospheric diazotrophic communities persists. We detected the biodiversity, abundance, and activity of nitrogen-fixing bacteria in the phyllospheres of two evergreen shrubs, Nerium indicum Mill. and Osmanthus sp., sampled from urban areas with heavy traffic, a college campus, and a forest. Quantitative PCR analysis indicated that the copy numbers of nifH sequences were highest in the phyllospheres of both plants in heavy-traffic urban areas and correlated with the recorded nitrogenase activities of the phyllospheres. Similarly, the phyllosphere from heavy-traffic urban areas also possessed the highest biodiversity indices of diazotrophic communities from both the two plants. Pyrosequencing analysis revealed a diversity of nifH sequences in phyllosphere that were mostly uniquely found in the phyllosphere, and many of these were proteobacteria-like and cyanobacteria-like. Members of the Proteobacteria, mostly of which were not closely related to unknown organisms, were detected exclusively in the phyllosphere and represented substantial fractions of their associated diazotrophic communities. Our study provides initial insight into the shifts in the biodiversity and community structure of N2-fixing microorganisms in the phyllospheres of different atmospheric environments.

Comprehensive analysis of phenotype, microstructure and global transcriptional profiling to unravel the effect of excess copper on the symbiosis between nitrogen-fixing bacteria and Medicago lupulina.

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. However, knowledge of the molecular mechanisms, especially the microstructure and global transcriptional profiling, of the symbiosis process under heavy metal contamination is limited. In this study, a heavy metal-tolerant legume, Medicago lupulina, was treated with different concentrations of copper (Cu). The results showed that the early infection process was inhibited and the nodule ultrastructure was changed under 200 mg kg-1 Cu stress. Most infection threads (ITs) were prevented from entering the nodule cells, and few rhizobia were released into the host cells, in which thickening of the plant cell wall and IT wall was observed, demonstrating that rhizobial invasion was inhibited under Cu stress. RNA-seq analysis indicated that a strong shift in gene expression occurred (3257 differentially expressed genes, DEGs). The most pronounced effect was the upregulation of a set of 71 of 73 DEGs for nodule-specific cysteine-rich peptides, which have been shown to control the terminal differentiation of rhizobia in the nodules and to have antimicrobial activity. Various genes for metal transport, chelation binding and antioxidant defence were regulated. In particular, the DEGs for Cu trafficking and detoxification were induced during nodule formation. The DEGs for ethylene (ET) biosynthesis and signalling were also differentially expressed during nodulation, suggesting that the inhibition of nodulation by Cu occurred partially through ET signalling. Furthermore, the genes related to the cell wall were mostly upregulated and most likely involved in cell wall thickening. These findings provide an integrated understanding of the effects of Cu on legume nodule symbiosis at the molecular and phenotypic levels.

Signalling in actinorhizal root nodule symbioses.

Plants able to establish a nitrogen-fixing root nodule symbiosis with the actinobacterium Frankia are called actinorhizal. These interactions lead to the formation of new root organs, called actinorhizal nodules, where the bacteria are hosted intracellularly and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. Like other symbiotic interactions, actinorhizal nodulation involves elaborate signalling between both partners of the symbiosis, leading to specific recognition between the plant and its compatible microbial partner, its accommodation inside plant cells and the development of functional root nodules. Actinorhizal nodulation shares many features with rhizobial nodulation but our knowledge on the molecular mechanisms involved in actinorhizal nodulation remains very scarce. However recent technical achievements for several actinorhizal species are allowing major discoveries in this field. In this review, we provide an outline on signalling molecules involved at different stages of actinorhizal nodule formation and the corresponding signalling pathways and gene networks.

Bioelectrochemical Nitrogen fixation (e-BNF): Electro-stimulation of enriched biofilm communities drives autotrophic nitrogen and carbon fixation.

A new approach to microbial electrosynthesis is proposed, aimed at producing whole biomass from N2 and inorganic carbon, by electrostimulation of complex microbial communities. On a carbon-based conductor under constant polarization (-0.7 V vs SHE), an electroactive biofilm was enriched with autotrophic nitrogen fixing microorganims and led to biomass synthesis at higher amounts (up to 18 fold), as compared to controls kept at open circuit (OC). After 110 days, the electron transfer had increased by 30-fold, as compared to abiotic conditions. Metagenomics evidenced Nif genes associated with autotrophs (both Archaea and Bacteria) only in polarized biofilms, but not in OC. With this first proof of concept experiment, we propose to call this promising field 'bioelectrochemical nitrogen fixation' (e-BNF): a possible way to 'power' biological nitrogen fixation, organic carbon storage and soil fertility against desertification, and possibly a new tool to study the development of early prokaryotic life in extreme environments.

Nitrogen-Fixing Bacteria Associated with Peltigera Cyanolichens and Cladonia Chlorolichens.

Lichens have been extensively studied and described; however, recent evidence suggests that members of the bacterial community associated with them could contribute new functions to the symbiotic interaction. In this work, we compare the nitrogen-fixing guild associated with bipartite terricolous lichens with different types of photobiont: Peltigera cyanolichens and Cladonia chlorolichens. Since cyanobacteria contribute nitrogen to the symbiosis, we propose that chlorolichens have more diverse bacteria with the ability to fix nitrogen compared to cyanolichens. In addition, since part of these bacteria could be recruited from the substrate where lichens grow, we propose that thalli and substrates share some bacteria in common. The structure of the nitrogen-fixing guild in the lichen and substrate bacterial communities of both lichens was determined by terminal restriction fragment length polymorphism (TRFLP) of the nifH gene. Multivariate analyses showed that the nitrogen-fixing bacteria associated with both types of lichen were distinguishable from those present in their substrates. Likewise, the structure of the nitrogen-fixing bacteria present in the cyanolichens was different from that of chlorolichens. Finally, the diversity of this bacterial guild calculated using the Shannon index confirms the hypothesis that chlorolichens have a higher diversity of nitrogen-fixing bacteria than cyanolichens.

Multiple mutualist effects on genomewide expression in the tripartite association between Medicago truncatula, nitrogen-fixing bacteria and mycorrhizal fungi.

While all species interact with multiple mutualists, the fitness consequences and molecular mechanisms underlying these interactions remain largely unknown. We combined factorial ecological experiments with genomewide expression analyses to examine the phenotypic and transcriptomic responses of model legume Medicago truncatula to rhizobia and mycorrhizal fungi. We found synergistic effects of these mutualists on plant performance and examined unique features of plant gene expression responses to multiple mutualists. There were genomewide signatures of mutualists and multiple mutualists on expression, with partners often affecting unique sets of genes. Mycorrhizal fungi had stronger effects on plant expression than rhizobia, with 70% of differentially expressed genes affected by fungi. Fungal and bacterial mutualists had joint effects on 10% of differentially expressed genes, including unexpected, nonadditive effects on some genes with important functions such as nutrient metabolism. For a subset of genes, interacting with multiple mutualists even led to reversals in the direction of expression (shifts from up to downregulation) compared to interacting with single mutualists. Rhizobia also affected the expression of several mycorrhizal genes, including those involved in nutrient transfer to host plants, indicating that partner species can also impact each other's molecular phenotypes. Collectively, these data illustrate the diverse molecular mechanisms and transcriptional responses associated with the synergistic benefits of multiple mutualists.

Effect of vegetation types on soil arbuscular mycorrhizal fungi and nitrogen-fixing bacterial communities in a karst region.

Arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria play important roles in plant growth and recovery in degraded ecosystems. The desertification in karst regions has become more severe in recent decades. Evaluation of the fungal and bacterial diversity of such regions during vegetation restoration is required for effective protection and restoration in these regions. Therefore, we analyzed relationships among AM fungi and nitrogen-fixing bacteria abundances, plant species diversity, and soil properties in four typical ecosystems of vegetation restoration (tussock (TK), shrub (SB), secondary forest (SF), and primary forest (PF)) in a karst region of southwest China. Abundance of AM fungi and nitrogen-fixing bacteria, plant species diversity, and soil nutrient levels increased from the tussock to the primary forest. The AM fungus, nitrogen-fixing bacterium, and plant community composition differed significantly between vegetation types (p < 0.05). Plant richness and pH were linked to the community composition of fungi and nitrogen-fixing bacteria, respectively. Available phosphorus, total nitrogen, and soil organic carbon levels and plant richness were positively correlated with the abundance of AM fungi and nitrogen-fixing bacteria (p < 0.05). The results suggested that abundance of AM fungi and nitrogen-fixing bacteria increased from the tussock to the primary forest and highlight the essentiality of these communities for vegetation restoration.

Impact of a Number of Factors on Physiological and Biochemical Activity of Strains ­ Components of Azogran, a Complex Bacterial Preparation.

The use of microbial preparations in plant-growing can be due to the correction of biological processes in agroecosystems and stimulates growth and development of plants. The efficiency of this process is dependent on biotic and abiotic factors, however their influence on introduction microorganisms in phytosphere is insufficiently studied. The article summarizes some results of recent studies, related to the impact of a number of environmental factors on physiological and biochemical activity of nitrogen-fixing bacteria Azotobacter vinelandii IMV B-7076 and phosphate-mobilizing strain Bacillus subtilis IMV B-7023 ­ components of Azogran, a complex bacterial preparation for plant growing. The dependence of the physiological and biochemical activity of these bacteria, including their antioxidant potential, on biotic and abiotic environmental agents was determined. The impact of a number of factors on chemotaxis, energy metabolism of these bacteria, their synthesis of substances of phenol nature, and other biologically active substances, which may influence the efficiency of using this preparation in plant growing, was studied. Azogran inhibits the spread of phytopathogens and some kinds of phytophages in agroecosystems, is capable of protecting plants from the oxidative stress and enhancing on 16­37 % their crop productivity.