Assessing intracellular and extracellular distribution of antibiotic resistance genes in the commercial organic fertilizers.

Compost-based organic fertilizers often contain high levels of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Previous studies focused on quantification of total ARGs and MGEs. For a more accurate risk assessment of the dissemination risk of antibiotic resistance, it is necessary to quantify the intracellular and extracellular distribution of ARGs and MGEs. In the present study, extracellular ARGs and MGEs (eARGs and eMGEs) and intracellular ARGs and MGEs (iARGs and iMGEs) were separately analyzed in 51 commercial composts derived from different raw materials by quantitative polymerase chain reaction (qPCR) and metagenomic sequencing. Results showed that eARGs and eMGEs accounted for 11-56 % and 4-45 % of the total absolute abundance of ARGs and MGEs, respectively. Comparable diversity, host composition and association with MGEs were observed between eARGs and iARGs. Contents of high-risk ARGs were similar between eARGs and iARGs, with high-risk ARGs in the two forms accounting for 6.7 % and 8.2 % of the total abundances, respectively. Twenty-four percent of the overall ARGs were present in plasmids, while 56.7 % of potentially mobile ARGs were found to be associated with plasmids. Variation partitioning analysis, null model and neutral community model indicated that the compositions of both eARGs and iARGs were largely driven by deterministic mechanisms. These results provide important insights into the cellular distribution of ARGs in manure composts that should be paid with specific attention in risk assessment and management.

CFViSA: A comprehensive and free platform for visualization and statistics in omics-data.

INTRODUCTION: The rapid growth of omics technologies has led to the use of bioinformatics as a powerful tool for unravelling scientific puzzles. However, the obstacles of bioinformatics are compounded by the complexity of data processing and the distinct nature of omics data types, particularly in terms of visualization and statistics. OBJECTIVES: We developed a comprehensive and free platform, CFViSA, to facilitate effortless visualization and statistical analysis of omics data by the scientific community. METHODS: CFViSA was constructed using the Scala programming language and utilizes the AKKA toolkit for the web server and MySQL for the database server. The visualization and statistical analysis were performed with the R program. RESULTS: CFViSA integrates two omics data analysis pipelines (microbiome and transcriptome analysis) and an extensive array of 79 analysis tools spanning simple sequence processing, visualization, and statistics available for various omics data, including microbiome and transcriptome data. CFViSA starts from an analysis interface, paralleling a demonstration full course to help users understand operating principles and scientifically set the analysis parameters. Once analysis is conducted, users can enter the task history interface for figure adjustments, and then a complete series of results, including statistics, feature tables and figures. All the graphic layouts were printed with necessary statistics and a traceback function recording the options for analysis and visualization; these statistics were excluded from the five competing methods. CONCLUSION: CFViSA is a user-friendly bioinformatics cloud platform with detailed guidelines for integrating functions in multi-omics analysis with real-time visualization adjustment and complete series of results provision. CFViSA is available at http://www.cloud.biomicroclass.com/en/CFViSA/.

Protist predation selects for the soil resistome.

A key aspect of "One Health" is to comprehend how antibiotic resistomes evolve naturally. In this issue, Nguyen and colleagues pioneered an in situ investigation on the impact of protist predations on the soil microbial community and its antibiotic resistance genes (ARGs). They found that bacterivorous protists consistently increased the abundance of ARGs, such as tetracycline resistant genes. Indeed, antibiotic production is a common strategy for bacteria to evade protist predation. The rise of ARGs can be explained by the balance between antibiotic producers and resisters shaped by predatory selection. This work suggests that ARG enrichment due to biotic interactions may be less worrisome than previously thought. Unless, these ARGs are carried by or disseminated among pathogens. Therefore, it is essential to monitor the occurrence, dissemination and pathogenic hosts of ARGs, enhancing our capacity to combat antibiotic resistance.

Effects of plant tissue permeability on invasion and population bottlenecks of a phytopathogen.

Pathogen genetic diversity varies in response to environmental changes. However, it remains unclear whether plant barriers to invasion could be considered a genetic bottleneck for phytopathogen populations. Here, we implement a barcoding approach to generate a pool of 90 isogenic and individually barcoded Ralstonia solanacearum strains. We used 90 of these strains to inoculate tomato plants with different degrees of physical permeability to invasion (intact roots, wounded roots and xylem inoculation) and quantify the phytopathogen population dynamics during invasion. Our results reveal that the permeability of plant roots impacts the degree of population bottleneck, genetic diversity, and composition of Ralstonia populations. We also find that selection is the main driver structuring pathogen populations when barriers to infection are less permeable, i.e., intact roots, the removal of root physical and immune barriers results in the predominance of stochasticity in population assembly. Taken together, our study suggests that plant root permeability constitutes a bottleneck for phytopathogen invasion and genetic diversity.

Microbe-induced phenotypic variation leads to overyielding in clonal plant populations.

Overyielding, the high productivity of multispecies plant communities, is commonly seen as the result of plant genetic diversity. Here we demonstrate that biodiversity-ecosystem functioning relationships can emerge in clonal plant populations through interaction with microorganisms. Using a model clonal plant species, we found that exposure to volatiles of certain microorganisms led to divergent plant phenotypes. Assembling communities out of plants associated with different microorganisms led to transgressive overyielding in both biomass and seed yield. Our results highlight the importance of belowground microbial diversity in plant biodiversity research and open new avenues for precision ecosystem management.

Positive regulation of the PhcB neighbouring regulator PrhX on expression of the type III secretion system and pathogenesis in Ralstonia solanacearum.

Ralstonia solanacearum PhcB and PhcA control a quorum-sensing (QS) system that globally regulates expression of about one third of all genes, including pathogenesis genes. The PhcB-PhcA QS system positively regulates the production of exopolysaccharide (EPS) and negatively regulates hrp gene expression, which is crucial for the type III secretion system (T3SS). Both EPS and the T3SS are essential for pathogenicity. The gene rsc2734 is located upstream of a phcBSR operon and annotated as a response regulator of a two-component system. Here, we demonstrated that RSc2734, hereafter named PrhX, positively regulated hrp gene expression via a PrhA-PrhIR-PrhJ-HrpG signalling cascade. Moreover, PrhX was crucial for R. solanacearum to invade host roots and grow in planta naturally. prhX expression was independent of the PhcB-PhcA QS system. PrhX did not affect the expression of phcB and phcA and the QS-dependent phenotypes, such as EPS production and biofilm formation. Our results provide novel insights into the complex regulatory network of the T3SS and pathogenesis in R. solanacearum.

RIN enhances plant disease resistance via root exudate-mediated assembly of disease-suppressive rhizosphere microbiota.

The RIPENING-INHIBITOR (RIN) transcriptional factor is a key regulator governing fruit ripening. While RIN also affects other physiological processes, its potential roles in triggering interactions with the rhizosphere microbiome and plant health are unknown. Here we show that RIN affects microbiome-mediated disease resistance via root exudation, leading to recruitment of microbiota that suppress the soil-borne, phytopathogenic Ralstonia solanacearum bacterium. Compared with the wild-type (WT) plant, RIN mutants had different root exudate profiles, which were associated with distinct changes in microbiome composition and diversity. Specifically, the relative abundances of antibiosis-associated genes and pathogen-suppressing Actinobacteria (Streptomyces) were clearly lower in the rhizosphere of rin mutants. The composition, diversity, and suppressiveness of rin plant microbiomes could be restored by the application of 3-hydroxyflavone and riboflavin, which were exuded in much lower concentrations by the rin mutant. Interestingly, RIN-mediated effects on root exudates, Actinobacteria, and disease suppression were evident from the seedling stage, indicating that RIN plays a dual role in the early assembly of disease-suppressive microbiota and late fruit development. Collectively, our work suggests that, while plant disease resistance is a complex trait driven by interactions between the plant, rhizosphere microbiome, and the pathogen, it can be indirectly manipulated using "prebiotic" compounds that promote the recruitment of disease-suppressive microbiota.

DCiPatho: deep cross-fusion networks for genome scale identification of pathogens.

Pathogen detection from biological and environmental samples is important for global disease control. Despite advances in pathogen detection using deep learning, current algorithms have limitations in processing long genomic sequences. Through the deep cross-fusion of cross, residual and deep neural networks, we developed DCiPatho for accurate pathogen detection based on the integrated frequency features of 3-to-7 k-mers. Compared with the existing state-of-the-art algorithms, DCiPatho can be used to accurately identify distinct pathogenic bacteria infecting humans, animals and plants. We evaluated DCiPatho on both learned and unlearned pathogen species using both genomics and metagenomics datasets. DCiPatho is an effective tool for the genomic-scale identification of pathogens by integrating the frequency of k-mers into deep cross-fusion networks. The source code is publicly available at https://github.com/LorMeBioAI/DCiPatho.

Bacterial volatile organic compounds attenuate pathogen virulence via evolutionary trade-offs.

Volatile organic compounds (VOCs) produced by soil bacteria have been shown to exert plant pathogen biocontrol potential owing to their strong antimicrobial activity. While the impact of VOCs on soil microbial ecology is well established, their effect on plant pathogen evolution is yet poorly understood. Here we experimentally investigated how plant-pathogenic Ralstonia solanacearum bacterium adapts to VOC-mixture produced by a biocontrol Bacillus amyloliquefaciens T-5 bacterium and how these adaptations might affect its virulence. We found that VOC selection led to a clear increase in VOC-tolerance, which was accompanied with cross-tolerance to several antibiotics commonly produced by soil bacteria. The increasing VOC-tolerance led to trade-offs with R. solanacearum virulence, resulting in almost complete loss of pathogenicity in planta. At the genetic level, these phenotypic changes were associated with parallel mutations in genes encoding lipopolysaccharide O-antigen (wecA) and type-4 pilus biosynthesis (pilM), which both have been linked with outer membrane permeability to antimicrobials and plant pathogen virulence. Reverse genetic engineering revealed that both mutations were important, with pilM having a relatively larger negative effect on the virulence, while wecA having a relatively larger effect on increased antimicrobial tolerance. Together, our results suggest that microbial VOCs are important drivers of bacterial evolution and could potentially be used in biocontrol to select for less virulent pathogens via evolutionary trade-offs.

Bio-Organic Fertilizer Promotes Pear Yield by Shaping the Rhizosphere Microbiome Composition and Functions.

Bio-organic fertilizers (BOF) containing both organic amendments and beneficial microorganisms have been consistently shown to improve soils fertility and yield. However, the exact mechanisms which link amendments and yields remain disputed, and the complexity of bio-organic fertilizers may work in parallel in several ways. BOF may directly improve yield by replenishing soil nutrients or introducing beneficial microbial genes or indirectly by altering the soil microbiome to enrich native beneficial microorganisms. In this work, we aim to disentangle the relative contributions of direct and indirect effects on pear yield. We treated pear trees with either chemical fertilizer or organic fertilizer with/without the plant-beneficial bacterium Bacillus velezensis SQR9. We then assessed, in detail, soil physicochemical and biological properties (metagenome sequencing) as well as pear yield. We then evaluated the relative importance of direct and indirect effects of soil amendments on pear yield. Both organic treatments increased plant yield by up to 20%, with the addition of bacteria tripling the increase driven by organic fertilizer alone. This increase could be linked to alterations in soil physicochemical properties, bacterial community function, and metabolism. Supplementation of organic fertilizer SQR9 increased rhizosphere microbiome richness and functional diversity. Fertilizer-sensitive microbes and functions responded as whole guilds. Pear yield was most positively associated with the Mitsuaria- and Actinoplanes-dominated ecological clusters and with gene clusters involved in ion transport and secondary metabolite biosynthesis. Together, these results suggested that bio-organic fertilizers mainly act indirectly on plant yield by creating soil chemical properties which promote a plant-beneficial microbiome. IMPORTANCE Bio-organic fertilization is a widely used, eco-friendly, sustainable approach to increasing plant productivity in the agriculture and fruit industries. However, it remains unclear whether the promotion of fruit productivity is related to specific changes in microbial inoculants, the resident microbiome, and/or the physicochemical properties of rhizosphere soils. We found that bio-organic fertilizers alter soil chemical properties, thus manipulating specific microbial taxa and functions within the rhizosphere microbiome of pear plants to promote yield. Our work unveils the ecological mechanisms which underlie the beneficial impacts of bio-organic fertilizers on yield promotion in fruit orchards, which may help in the design of more efficient biofertilizers to promote sustainable fruit production.

WGCNA analysis revealing molecular mechanism that bio-organic fertilizer improves pear fruit quality by increasing sucrose accumulation and reducing citric acid metabolism.

It's been long known that the application of organic fertilizer (OF) and bio-organic fertilizer (BF) which containing beneficial microorganisms to pear trees can both significantly improve fruit quality and yield. In order to reveal the mechanism of BF and OF regulating fruit growth and quality in pear, the effects of BF and OF on the photosynthetic characteristics and the accumulation of major sugars and organic acids of the pear fruit were quantified compared with chemical fertilizer (CF). Additionally, the molecular mechanisms regulating pear fruit development and quality were studied through transcriptome analysis. The three treatments were conducted based on the same amounts of nitrogen supply. The results showed that compared with CF, BF and OF treatments increased the fruit yield, and also significantly improved the photosynthesis efficiency in pear. BF and OF both significantly increased the sucrose content but significantly decreased the fructose and glucose content within the pear fruit. The amount of malic acid was significantly higher in OF treatment. Compared with CF and OF, BF significantly increased the sugar-acid ratio and thus improved the fruit quality. Transcriptome analysis and weighted correlation network analysis (WGCNA) revealed that the sugar metabolism of fruits applied with the BF was enhanced compared with those applied with CF or OF. More specifically, the expression of SDH (Sorbitol dehydrogenase) was higher in BF, which converts sorbitol into fructose. For both of the OF and BF, the transcript abundance of sugar transporter genes was significantly increased, such as SOT (Sorbitol transporter), SUT14 (Sugar transport 14), UDP-GLUT4 (UDP-glucose transporter 4), UDP-SUT (UDP-sugar transporter), SUC4 (Sucrose transport 4), SUT7 (Sugar transporter 7), SWEET10 and SWEET15 (Bidirectional sugar transporter), which ensures sugar transportation. The genes involved in organic acid metabolism showed decreased transcripts abundance in both BF and OF treatments, such as VAP (Vesicle-associated protein) and cyACO (Cytosolic aconitase), which reduce the conversion from succinate to citric acid, and decrease the conversion from citric acid to malic acid in the TCA cycle (Tricarboxylic Acid cycle) through Pept6 (Oligopeptide transporter). In conclusion, the application of BF and OF improved fruit quality by regulating the expression of sugar and organic acid metabolism-related genes and thus altering the sugar acid metabolism. Both BF and OF promote sucrose accumulation and citric acid degradation in fruits, which may be an important reason for improving pear fruit quality. The possible mechanism of bio-organic fertilizer to improve fruit quality was discussed.

Exploring rhizo-microbiome transplants as a tool for protective plant-microbiome manipulation.

The development of strategies for effectively manipulating and engineering beneficial plant-associated microbiomes is a major challenge in microbial ecology. In this sense, the efficacy and potential implications of rhizosphere microbiome transplant (RMT) in plant disease management have only scarcely been explored in the literature. Here, we initially investigated potential differences in rhizosphere microbiomes of 12 Solanaceae eggplant varieties and accessed their level of resistance promoted against bacterial wilt disease caused by the pathogen Ralstonia solanacearum, in a 3-year field trial. We elected 6 resistant microbiomes and further tested the broad feasibility of using RMT from these donor varieties to a susceptible model Solanaceae tomato variety MicroTom. Overall, we found the rhizosphere microbiome of resistant varieties to enrich for distinct and specific bacterial taxa, of which some displayed significant associations with the disease suppression. Quantification of the RMT efficacy using source tracking analysis revealed more than 60% of the donor microbial communities to successfully colonize and establish in the rhizosphere of recipient plants. RTM from distinct resistant donors resulted in different levels of wilt disease suppression, reaching up to 47% of reduction in disease incidence. Last, we provide a culture-dependent validation of potential bacterial taxa associated with antagonistic interactions with the pathogen, thus contributing to a better understanding of the potential mechanism associated with the disease suppression. Our study shows RMT from appropriate resistant donors to be a promising tool to effectively modulate protective microbiomes and promote plant health. Together we advocate for future studies aiming at understanding the ecological processes and mechanisms mediating rates of coalescence between donor and recipient microbiomes in the plant rhizosphere.

Indigenous and commercial isolates of arbuscular mycorrhizal fungi display differential effects in Pyrus betulaefolia roots and elicit divergent transcriptomic and metabolomic responses.

Background: Arbuscular mycorrhizal fungi (AMF) are beneficial soil fungi which can effectively help plants with acquisition of mineral nutrients and water and promote their growth and development. The effects of indigenous and commercial isolates of arbuscular mycorrhizal fungi on pear (Pyrus betulaefolia) trees, however, remains unclear. Methods: Trifolium repens was used to propagate indigenous AMF to simulate spore propagation in natural soils in three ways: 1. the collected soil was mixed with fine roots (R), 2. fine roots were removed from the collected soil (S), and 3. the collected soil was sterilized with 50 kGy 60Co γ-radiation (CK). To study the effects of indigenous AMF on root growth and metabolism of pear trees, CK (sterilized soil from CK in T. repens mixed with sterilized standard soil), indigenous AMF (R, soil from R in T. repens mixed with sterilized standard soil; S, soil from S in T. repens mixed with sterilized standard soil), and two commercial AMF isolates (Rhizophagus intraradices(Ri) and Funneliformis mosseae (Fm)) inoculated in the media with pear roots. Effects on plant growth, root morphology, mineral nutrient accumulation, metabolite composition and abundance, and gene expression were analyzed. Results: AMF treatment significantly increased growth performance, and altered root morphology and mineral nutrient accumulation in this study, with the S treatment displaying overall better performance. In addition, indigenous AMF and commercial AMF isolates displayed common and divergent responses on metabolite and gene expression in pear roots. Compared with CK, most types of flavones, isoflavones, and carbohydrates decreased in the AMF treatment, whereas most types of fatty acids, amino acids, glycerolipids, and glycerophospholipids increased in response to the AMF treatments. Further, the relative abundance of amino acids, flavonoids and carbohydrates displayed different trends between indigenous and commercial AMF isolates. The Fm and S treatments altered gene expression in relation to root metabolism resulting in enriched fructose and mannose metabolism (ko00051), fatty acid biosynthesis (ko00061) and flavonoid biosynthesis (ko00941). Conclusions: This study demonstrates that indigenous AMF and commercial AMF isolates elicited different effects in pear plants through divergent responses from gene transcription to metabolite accumulation.

Compositional and functional succession of bacterial and fungal communities is associated with changes in abiotic properties during pig manure composting.

While both bacteria and fungi are important for the degradation and humification of organic matter during composting, it is unclear to what extent their roles are associated with abiotic compost properties. This study evaluated changes in abiotic compost properties and the succession of bacterial and fungal communities during pig manure composting for 90 days. The compost rapidly reached thermophilic phase (>58 ℃), which lasted for 15 days. Both bacterial and fungal community compositions changed drastically during composting and while bacterial diversity increased, the fungal diversity decreased during the thermophilic phase of composting. Two taxa dominated both bacterial (Bacillales and Clostridiales) and fungal (Eurotiales and Glomerellales) communities and these showed alternating abundance fluctuations following different phases of composting. The abundance fluctuations of most dominant bacterial and fungal taxa could be further associated with decreases in the concentrations of fulvic acid, cellulose, hemicellulose and overall biodegradation potential in the compost. Moreover, bacterial predicted metabolic gene abundances dominated the first three phases of composting, while predicted fungal saprotrophic functional genes increased consistently, reaching highest abundances towards the end of composting. Finally, redundancy analysis (RDA) showed that changes in abiotic compost properties correlated with the bacterial community diversity and carbohydrate metabolism and fungal wood saprotrophic function. Together these results suggests that bacterial and fungal community succession was associated with temporal changes in abiotic compost properties, potentially explaining alternating taxa abundance patterns during pig manure composting.

Isolation of a novel psychrotrophic fungus for efficient low-temperature composting.

This study aimed to isolate psychrotrophic cellulose-degrading fungi and to investigate their application potential for composting in cold climate regions in China. One out of five psychrotrophic cellulose-degrading fungal isolates was identified as a novel fungal species, Aureobasidium paleasum sp. nov., with a strong straw degradation potential. Enzyme activity assays and FITR spectroscopy revealed high cellulolytic activities of this psychrotrophic fungus at lower temperatures, with high thermal adaptability from 5 °C to 50 °C (optimum at 10 °C). A. paleasum efficiently decomposed rice straws and cellulose at 10 °C compared to the common cellulose-degrading fungus Penicillium oxalicum. In comparison to P. oxalicum, A. paleasum shortened the thermophilic stage, enhanced compost maturity and improved compost quality. Our work suggests that the psychrotrophic fungus A. paleasum is efficient for rice straw degradation and composting at low temperatures, highlighting its application potential for composting in colder regions.

Glutaredoxins Play an Important Role in the Redox Homeostasis and Symbiotic Capacity of Azorhizobium caulinodans ORS571.

Glutaredoxin (GRX) plays an essential role in the control of the cellular redox state and related pathways in many organisms. There is limited information on GRXs from the model nitrogen (N2)-fixing bacterium Azorhizobium caulinodans. In the present work, we identified and performed functional analyses of monothiol and dithiol GRXs in A. caulinodans in the free-living state and during symbiosis with Sesbania rostrata. Our data show that monothiol GRXs may be very important for bacterial growth under normal conditions and in response to oxidative stress due to imbalance of the redox state in grx mutants of A. caulinodans. Functional redundancies were also observed within monothiol and dithiol GRXs in terms of different physiological functions. The changes in catalase activity and iron content in grx mutants were assumed to favor the maintenance of bacterial resistance against oxidants, nodulation, and N2 fixation efficiency in this bacterium. Furthermore, the monothiol GRX12 and dithiol GRX34 play a collective role in symbiotic associations between A. caulinodans and Sesbania rostrata. Our study provided systematic evidence that further investigations are required to understand the importance of glutaredoxins in A. caulinodans and other rhizobia.

Ohr and OhrR Are Critical for Organic Peroxide Resistance and Symbiosis in Azorhizobium caulinodans ORS571.

Azorhizobium caulinodans is a symbiotic nitrogen-fixing bacterium that forms both root and stem nodules on Sesbania rostrata. During nodule formation, bacteria have to withstand organic peroxides that are produced by plant. Previous studies have elaborated on resistance to these oxygen radicals in several bacteria; however, to the best of our knowledge, none have investigated this process in A. caulinodans. In this study, we identified and characterised the organic hydroperoxide resistance gene ohr (AZC_2977) and its regulator ohrR (AZC_3555) in A. caulinodans ORS571. Hypersensitivity to organic hydroperoxide was observed in an ohr mutant. While using a lacZ-based reporter system, we revealed that OhrR repressed the expression of ohr. Moreover, electrophoretic mobility shift assays demonstrated that OhrR regulated ohr by direct binding to its promoter region. We showed that this binding was prevented by OhrR oxidation under aerobic conditions, which promoted OhrR dimerization and the activation of ohr. Furthermore, we showed that one of the two conserved cysteine residues in OhrR, Cys11, was critical for the sensitivity to organic hydroperoxides. Plant assays revealed that the inactivation of Ohr decreased the number of stem nodules and nitrogenase activity. Our data demonstrated that Ohr and OhrR are required for protecting A. caulinodans from organic hydroperoxide stress and play an important role in the interaction of the bacterium with plants. The results that were obtained in our study suggested that a thiol-based switch in A. caulinodans might sense host organic peroxide signals and enhance symbiosis.

Medicago-Sinorhizobium-Ralstonia Co-infection Reveals Legume Nodules as Pathogen Confined Infection Sites Developing Weak Defenses.

Legumes have the capacity to develop root nodules hosting nitrogen-fixing bacteria, called rhizobia. For the plant, the benefit of the symbiosis is important in nitrogen-deprived conditions, but it requires hosting and feeding massive numbers of rhizobia. Recent studies suggest that innate immunity is reduced or suppressed within nodules [1-10]; this likely maintains viable rhizobial populations. To evaluate the potential consequences and risks associated with an altered immuni`ty in the symbiotic organ, we developed a tripartite system with the model legume Medicago truncatula [11, 12], its nodulating symbiont of the genus Sinorhizobium (syn. Ensifer) [13, 14], and the pathogenic soil-borne bacterium Ralstonia solanacearum [15-18]. We show that nodules are frequent infection sites where pathogen multiplication is comparable to that in the root tips and independent of nodule ability to fix nitrogen. Transcriptomic analyses indicate that, despite the presence of the hosted rhizobia, nodules are able to develop weak defense reactions against pathogenic R. solanacearum. Nodule defense response displays specificity compared to that activated in roots. In agreement with nodule innate immunity, optimal R. solanacearum growth requires pathogen virulence factors. Finally, our data indicate that the high susceptibility of nodules is counterbalanced by the existence of a diffusion barrier preventing pathogen spreading from nodules to the rest of the plant.

Bacterioferritin comigratory protein is important in hydrogen peroxide resistance, nodulation, and nitrogen fixation in Azorhizobium caulinodans.

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 (H2O2) 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 H2O2 conditions. Furthermore, nodulation and N2 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 H2O2 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.