The Type IV Secretion System (T4SS) Mediates Symbiosis between Bradyrhizobium sp. SUTN9-2 and Legumes.

There has been little study of the type IV secretion system (T4SS) of bradyrhizobia and its role in legume symbiosis. Therefore, broad host range Bradyrhizobium sp. SUTN9-2 was selected for study. The chromosome of Bradyrhizobium sp. SUTN9-2 contains two copies of the T4SS gene, homologous with the tra/trb operons. A phylogenetic tree of the T4SS gene traG was constructed, which exemplified its horizontal transfer among Bradyrhizobium and Mesorhizobium genera. They also showed similar gene arrangements for the tra/trb operons. However, the virD2 gene was not observed in Mesorhizobium, except M. oppotunistum WSM2075. Interestingly, the orientation of copG, traG, and virD2 cluster was unique to the Bradyrhizobium genus. The phylogenetic tree of copG, traG, and virD2 demonstrated that copies 1 and 2 of these genes were grouped in different clades. In addition, the derived mutant and complementation strains of T4SS were investigated in representative legumes Genistoids, Dalbergioids, and Millettiods. When T4SS copy 1 (T4SS1) was deleted, the nodule number and nitrogenase activity decreased. This supports a positive effect of T4SS1 on symbiosis. In addition, delayed nodulation was observed 7 dpi, which was restored by the complementation of T4SS1. Therefore, T4SS plays an important role in the symbiotic interaction between Bradyrhizobium sp. SUTN9-2 and its leguminous hosts. IMPORTANCE SUTN9-2 is a broad host range strain capable of symbiosis with several legumes. Two copies of T4SS clusters belonging to the tra/trb operon are observed on chromosomes with different gene arrangements. We use phylogenetic tree and gene annotation analysis to predict the evolution of the tra/trb operon of rhizobia. Our finding suggests that the gene encoding the T4SS gene among Bradyrhizobium and Mesorhizobium may have coevolution. In addition, Bradyrhizobium has a uniquely arranged copG, traG, and virD2 gene cluster. The results of T4SS1 gene deletion and complementation revealed its positive effect on nodulation. Therefore, T4SS seems to be another determinant for symbiosis. This is the first report on the role of T4SS in Bradyrhizobium symbiosis.

Symbiotic properties of a chimeric Nod-independent photosynthetic Bradyrhizobium strain obtained by conjugative transfer of a symbiotic plasmid.

The lateral transfer of symbiotic genes converting a predisposed soil bacteria into a legume symbiont has occurred repeatedly and independently during the evolution of rhizobia. We experimented the transfer of a symbiotic plasmid between Bradyrhizobium strains. The originality of the DOA9 donor is that it harbours a symbiotic mega-plasmid (pDOA9) containing nod, nif and T3SS genes while the ORS278 recipient has the unique property of inducing nodules on some Aeschynomene species in the absence of Nod factors (NFs). We observed that the chimeric strain ORS278-pDOA9* lost its ability to develop a functional symbiosis with Aeschynomene. indica and Aeschynomene evenia. The mutation of rhcN and nodB led to partial restoration of nodule efficiency, indicating that T3SS effectors and NFs block the establishment of the NF-independent symbiosis. Conversely, ORS278-pDOA9* strain acquired the ability to form nodules on Crotalaria juncea and Macroptillium artropurpureum but not on NF-dependent Aeschynomene (A. afraspera and A. americana), suggesting that the ORS278 strain also harbours incompatible factors that block the interaction with these species. These data indicate that the symbiotic properties of a chimeric rhizobia cannot be anticipated due to new combination of symbiotic and non-symbiotic determinants that may interfere during the interaction with the host plant.

Potential of Rice Stubble as a Reservoir of Bradyrhizobial Inoculum in Rice-Legume Crop Rotation.

Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with leguminous and nonleguminous plants, such as rice. Therefore, it can be expected that rice endophytic bradyrhizobia can be applied in the rice-legume crop rotation system. Some endophytic bradyrhizobial strains were isolated from rice (Oryza sativa L.) tissues. The rice biomass could be enhanced when supplementing bradyrhizobial strain inoculation with KNO3, NH4NO3, or urea, especially in Bradyrhizobium sp. strain SUTN9-2. In contrast, the strains which suppressed rice growth were photosynthetic bradyrhizobia and were found to produce nitric oxide (NO) in the rice root. The expression of genes involved in NO production was conducted using a quantitative reverse transcription-PCR (qRT-PCR) technique. The nirK gene expression level in Bradyrhizobium sp. strain SUT-PR48 with nitrate was higher than that of the norB gene. In contrast, the inoculation of SUTN9-2 resulted in a lower expression of the nirK gene than that of the norB gene. These results suggest that SUT-PR48 may accumulate NO more than SUTN9-2 does. Furthermore, the nifH expression of SUTN9-2 was induced in treatment without nitrogen supplementation in an endophytic association with rice. The indole-3-acetic acid (IAA) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase produced in planta by SUTN9-2 were also detected. Enumeration of rice endophytic bradyrhizobia from rice tissues revealed that SUTN9-2 persisted in rice tissues until rice-harvesting season. The mung bean (Vigna radiata) can be nodulated after rice stubbles were decomposed. Therefore, it is possible that rice stubbles can be used as an inoculum in the rice-legume crop rotation system under both low- and high-organic-matter soil conditions.IMPORTANCE This study shows that some rice endophytic bradyrhizobia could produce IAA and ACC deaminase and have a nitrogen fixation ability during symbiosis inside rice tissues. These characteristics may play an important role in rice growth promotion by endophytic bradyrhizobia. However, the NO-producing strains should be of concern due to a possible deleterious effect of NO on rice growth. In addition, this study reports the application of endophytic bradyrhizobia in rice stubbles, and the rice stubbles were used directly as an inoculum for a leguminous plant (mung bean). The degradation of rice stubbles leads to an increased number of SUTN9-2 in the soil and may result in increased mung bean nodulation. Therefore, the persistence of endophytic bradyrhizobia in rice tissues can be developed to use rice stubbles as an inoculum for mung bean in a rice-legume crop rotation system.

Preferential association of endophytic bradyrhizobia with different rice cultivars and its implications for rice endophyte evolution.

Plant colonization by bradyrhizobia is found not only in leguminous plants but also in nonleguminous species such as rice. To understand the evolution of the endophytic symbiosis of bradyrhizobia, the effect of the ecosystems of rice plantations on their associations was investigated. Samples were collected from various rice (Oryza sativa) tissues and crop rotational systems. The rice endophytic bradyrhizobia were isolated on the basis of oligotrophic properties, selective medium, and nodulation on siratro (Macroptilium atropurpureum). Six bradyrhizobial strains were obtained exclusively from rice grown in a crop rotational system. The isolates were separated into photosynthetic bradyrhizobia (PB) and nonphotosynthetic bradyrhizobia (non-PB). Thai bradyrhizobial strains promoted rice growth of Thai rice cultivars better than the Japanese bradyrhizobial strains. This implies that the rice cultivars possess characteristics that govern rice-bacterium associations. To examine whether leguminous plants in a rice plantation system support the persistence of rice endophytic bradyrhizobia, isolates were tested for legume nodulation. All PB strains formed symbioses with Aeschynomene indica and Aeschynomene evenia. On the other hand, non-PB strains were able to nodulate Aeschynomene americana, Vigna radiata, and M. atropurpureum but unable to nodulate either A. indica or A. evenia. Interestingly, the nodABC genes of all of these bradyrhizobial strains seem to exhibit low levels of similarity to those of Bradyrhizobium diazoefficiens USDA110 and Bradyrhizobium sp. strain ORS285. From these results, we discuss the evolution of the plant-bradyrhizobium association, including nonlegumes, in terms of photosynthetic lifestyle and nod-independent interactions.

Enhancing Resistance to Cercospora Leaf Spot in Mung Bean (Vigna radiata L.) through Bradyrhizobium sp. DOA9 Priming: Molecular Insights and Bio-Priming Potential.

Mung bean (Vigna radiata L.), a vital legume in Asia with significant nutritional benefits, is highly susceptible to Cercospora leaf spot (CLS) caused by Cercospora canescens, leading to significant yield losses. As an alternative to chemical fungicides, bio-priming with rhizobacteria can enhance plant resistance. This study explores the potential of Bradyrhizobium sp. strain DOA9 to augment resistance in mung bean against CLS via root priming. The results reveal that short (3 days) and double (17 and 3 days) priming with DOA9 before fungal infection considerably reduces lesion size on infected leaves by activating defense-related genes, including Pti1, Pti6, EDS1, NDR1, PR-1, PR-2, Prx, and CHS, or by suppressing the inhibition of PR-5 and enhancing peroxidase (POD) activity in leaves. Interestingly, the Type 3 secretion system (T3SS) of DOA9 may play a role in establishing resistance in V. radiata CN72. These findings suggest that DOA9 primes V. radiata CN72's defense mechanisms, offering an effective bio-priming strategy to alleviate CLS. Hence, our insights propose the potential use of DOA9 as a bio-priming agent to manage CLS in V. radiata CN72, providing a sustainable alternative to chemical fungicide applications.

CopG1, a Novel Transcriptional Regulator Affecting Symbiosis in Bradyrhizobium sp. SUTN9-2.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of T4SS1 were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild-type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.

Exploring the cellular surface polysaccharide and root nodule symbiosis characteristics of the rpoN mutants of Bradyrhizobium sp. DOA9 using synchrotron-based Fourier transform infrared microspectroscopy in conjunction with X-ray absorption spectroscopy.

The functional significance of rpoN genes that encode two sigma factors in the Bradyrhizobium sp. strain DOA9 has been reported to affect colony formation, root nodulation characteristics, and symbiotic interactions with Aeschynomene americana. rpoN mutant strains are defective in cellular surface polysaccharide (CSP) production compared with the wild-type (WT) strain, and they accordingly exhibit smaller colonies and diminished symbiotic effectiveness. To gain deeper insights into the changes in CSP composition and the nodules of rpoN mutants, we employed synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy. FTIR analysis of the CSP revealed the absence of specific components in the rpoN mutants, including lipids, carboxylic groups, polysaccharide-pyranose rings, and ß-galactopyranosyl residues. Nodules formed by DOA9WT exhibited a uniform distribution of lipids, proteins, and carbohydrates; mutant strains, particularly DOA9∆rpoNp:ΩrpoNc, exhibited decreased distribution uniformity and a lower concentration of C=O groups. Furthermore, Fe K-edge X-ray absorption near-edge structure and extended X-ray absorption fine structure analyses revealed deficiencies in the nitrogenase enzyme in the nodules of DOA9∆rpoNc and DOA9∆rpoNp:ΩrpoNc mutants; nodules from DOA9WT and DOA9∆rpoNp exhibited both leghemoglobin and the nitrogenase enzyme. IMPORTANCE This work provides valuable insights into how two rpoN genes affect the composition of cellular surface polysaccharides (CSPs) in Bradyrhizobium sp., which subsequently dictates root nodule chemical characteristics and nitrogenase production. We used advanced synchrotron methods, including synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy (XAS), for the first time in this field to analyze CSP components and reveal the biochemical changes occurring within nodules. These cutting-edge techniques confer significant advantages by providing detailed molecular information, enabling the identification of specific functional groups, chemical bonds, and biomolecule changes. This research not only contributes to our understanding of plant-microbe interactions but also establishes a foundation for future investigations and potential applications in this field. The combined use of the synchrotron-based FTIR and XAS techniques represents a significant advancement in facilitating a comprehensive exploration of bacterial CSPs and their implications in plant-microbe interactions.

Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth.

RpoN is an alternative sigma factor (sigma 54) that recruits the core RNA polymerase to promoters of genes. In bacteria, RpoN has diverse physiological functions. In rhizobia, RpoN plays a key role in the transcription of nitrogen fixation (nif) genes. The Bradyrhizobium sp. DOA9 strain contains a chromosomal (c) and plasmid (p) encoded RpoN protein. We used single and double rpoN mutants and reporter strains to investigate the role of the two RpoN proteins under free-living and symbiotic conditions. We observed that the inactivation of rpoNc or rpoNp severely impacts the physiology of the bacteria under free-living conditions, such as the bacterial motility, carbon and nitrogen utilization profiles, exopolysaccharide (EPS) production, and biofilm formation. However, free-living nitrogen fixation appears to be under the primary control of RpoNc. Interestingly, drastic effects of rpoNc and rpoNp mutations were also observed during symbiosis with Aeschynomene americana. Indeed, inoculation with rpoNp, rpoNc, and double rpoN mutant strains resulted in decreases of 39, 64, and 82% in the number of nodules, respectively, as well as a reduction in nitrogen fixation efficiency and a loss of the bacterium's ability to survive intracellularly. Taken together, the results show that the chromosomal and plasmid encoded RpoN proteins in the DOA9 strain both play a pleiotropic role during free-living and symbiotic states.

Bacillus velezensis S141, a soybean growth-promoting bacterium, hydrolyzes isoflavone glycosides into aglycones.

Bacillus velezensis S141, a plant growth-promoting rhizobacteria (PGPR), was isolated from a soybean field in Thailand. Previous studies demonstrated that S141 enhanced soybean growth, stimulating nodulation for symbiotic nitrogen fixation with soybean root nodule bacteria, including Bradyrhizobium diazoefficience USDA110. Isoflavone glycosides are produced in soybean roots and hydrolyzed into their aglycones, triggering nodulation. This study revealed that S141 efficiently hydrolyzed two isoflavone glycosides in soybean roots (daidzin and genistin) to their aglycones (daidzein and genistein, respectively). However, S141, Bacillus subtilis 168, NCIB3610, and B. velezensis FZB42 hydrolyzed isoflavone glucosides into aglycones. A BLASTp search suggested that S141 and the other three strains shared four genes encoding ß-glucosidases corresponding to bglA, bglC, bglH, and gmuD in B. subtilis 168. The gene inactivation analysis of B. subtilis 168 revealed that bglC encoded the major ß-glucosidase, contributing about half of the total activity to hydrolyze isoflavone glycosides and that bglA, bglH, and gmuD, all barely committed to the hydrolysis of isoflavone glycosides. Thus, an unknown ß-glucosidase exists, and our genetic knowledge of ß-glucosidases was insufficient to evaluate the ability to hydrolyze isoflavone glycosides. Nevertheless, S141 could predominate in the soybean rhizosphere, releasing isoflavone aglycones to enhance soybean nodulation.

Insights into Antifungal Mechanisms of Bacillus velezensis S141 against Cercospora Leaf Spot in Mungbean (V. radiata).

Cercospora leaf spot (CLS) is caused by Cercospora canescens and is one of the most important diseases of mungbean (Vigna radiata). Cercospora leaf spot may result in economic loss in production areas. The present study investigated the potential of Bacillus velezensis S141 as a biocontrol agent for C. canescens PAK1 growth on culture plates. Cell-free secretions from a dual culture of S141+PAK1 inhibited fungal growth more than those from a single culture of S141. The biocontrol efficiency of S141 against Cercospora leaf spot on mungbean was then evaluated by spraying. The disease severity of Cercospora leaf spot was significantly reduced in plants treated with S141, with a control efficiency of 83% after 2 days of infection. Comparative transcriptomics and qRT-PCR ana-lyses of S141 during C. canescens inhibition were performed to elucidate the antifungal mechanisms underlying its antifungal activity against Cercospora leaf spot. According to the differentially expressed genes, most up-regulated genes involved in the biosynthetic genes encoding enzymatic hydrolases, including protease, ß-glucanase, and N-acyl glucosaminase, were detected in strain S141 following its interaction. Moreover, genes related to secondary metabolites (surfactin, bacilysin, and bacillomycin D) were up-regulated. Collectively, these results suggest that S141 exhibited strong antifungal activity against C. canescens due to multiple enzymatic hydrolases and secondary metabolites. Therefore, the present study provides insights into the biological network responsible for the antifungal activity of B. velezensis S141 against C. canescens.

Genetic diversity, symbiotic evolution, and proposed infection process of Bradyrhizobium strains isolated from root nodules of Aeschynomene americana L. in Thailand.

The diversity of bacteria nodulating Aeschynomene americana L. in Thailand was determined from phenotypic characteristics and multilocus sequence analysis of the 16S rRNA gene and 3 housekeeping genes (dnaK, recA, and glnB). The isolated strains were nonphotosynthetic bacteria and were assigned to the genus Bradyrhizobium, in which B. yuanmingense was the dominant species. Some of the other species, including B. japonicum, B. liaoningense, and B. canariense, were minor species. These isolated strains were divided into 2 groups-nod-containing and divergent nod-containing strains-based on Southern blot hybridization and PCR amplification of nodABC genes. The divergent nod genes could not be PCR amplified and failed to hybridize nod gene probes designed from B. japonicum USDA110, but hybridized to probes from other bradyrhizobial strains under low-stringency conditions. The grouping based on sequence similarity of nod genes was well correlated with the grouping based on that of nifH gene, in which the nod-containing and divergent nod-containing strains were obviously distinguished. The divergent nod-containing strains and photosynthetic bradyrhizobia shared close nifH sequence similarity and an ability to fix nitrogen in the free-living state. Surprisingly, the strains isolated from A. americana could nodulate Aeschynomene plants that belong to different cross-inoculation (CI) groups, including A. afraspera and A. indica. This is the first discovery of bradyrhizobia (nonphotosynthetic and nod-containing strain) originating from CI group 1 nodulating roots of A. indica (CI group 3). An infection process used to establish symbiosis on Aeschynomene different from the classical one is proposed.

Symbiosis Contribution of Non-nodulating Bradyrhizobium cosmicum S23321 after Transferal of the Symbiotic Plasmid pDOA9.

The symbiotic properties of rhizobial bacteria are driven by the horizontal gene transfer of symbiotic genes, which are located in symbiosis islands or on plasmids. The symbiotic megaplasmid pDOA9 of Bradyrhizobium sp. DOA9, carrying the nod, nif, fix, and type three secretion system (T3SS) genes, has been conjugatively transferred to different Bradyrhizobium strains. In the present study, non-nodulating B. cosmicum S23321, which shows a close phylogenetic relationship with Bradyrhizobium sp. DOA9, but lacks symbiotic properties, was used to carry pDOA9 (annotated as chimeric S2:pDOA9). The results obtained showed that pDOA9 conferred symbiotic properties on S23321; however, nodulation phenotypes varied among the DOA9, chimeric ORS278:pDOA9, and S2:pDOA9 strains even though they all carried symbiotic pDOA9 plasmid. S23321 appeared to gain symbiotic nodulation from pDOA9 by processing nodulation genes and broadening the host range. The present results also showed the successful formation of active nodules in Arachis hypogaea (Dalbergoid) and Vigna radiata (Millitoid) by chimeric S2:pDOA9, while Crotalaria juncea (Genistoid) and Macroptilium atropurpureum (Millitoid) formed nodule-like structures. The formation of nodules and nodule-like structures occurred in a nod factor-dependent manner because the nod factor-lacking strain (S2:pDOA9ΩnodB) completely abolished nodulation in all legumes tested. Moreover, T3SS carried by S2:pDOA9 exerted negative effects on symbiosis with Crotalaria juncea, which was consistent with the results obtained on DOA9. T3SS exhibited symbiotic compatibility with V. radiata when nodulated by S23321. These outcomes implied that pDOA9 underwent changes during legume evolution that broadened host specificity and the compatibility of nodulation in a manner that was dependent on the chromosomal background of the recipient as well as legume host restrictions.

Enhancing the Efficiency of Soybean Inoculant for Nodulation under Multi-Environmental Stress Conditions.

The development of rhizobial inoculants with increased resistance to abiotic stress is critical to mitigating the challenges related to climate change. This study aims at developing a soybean stress-tolerant Bradyrhizobium inoculant to be used under the mixed stress conditions of acidity, high temperature, and drought. Six isolates of Bradyrhizobium with high symbiotic performance on soybean were tested to determine their growth or survival abilities under in vitro conditions. The representative stress-tolerant Bradyrhizobium isolates 184, 188, and 194 were selected to test their ability to promote soybean growth under stress conditions compared to the type strain Bradyrhizobium diazoefficiens USDA110. The plant experiment indicated that isolate 194 performed better in symbiosis with soybean than other Bradyrhizobium strains under stress conditions. Based on the stress tolerance index, soybeans inoculated with isolate 194 showed a high growth performance and significantly better nodulation competition ability than USDA110 under several stress conditions. Interestingly, supplementation of sucrose in the culture medium significantly enhances the survival of the isolate and leads to improved plant biomass under various stress conditions. Analysis of the intra-cellular sugars of isolate 194 supplemented with sucrose showed the accumulation of compatible solutes, such as trehalose and glycerol, that may act as osmoprotectants. This study indicates that inoculation of stress-tolerant Bradyrhizobium together with sucrose supplementation in a medium could enhance bacterial survival and symbiosis efficiency under stress conditions. Although it can be applied for inoculant production, this strategy requires validation of its performance in field conditions before adopting this technology.

The Bradyrhizobium diazoefficiens type III effector NopE modulates the regulation of plant hormones towards nodulation in Vigna radiata.

Host-specific legume-rhizobium symbiosis is strictly controlled by rhizobial type III effectors (T3Es) in some cases. Here, we demonstrated that the symbiosis of Vigna radiata (mung bean) with Bradyrhizobium diazoefficiens USDA110 is determined by NopE, and this symbiosis is highly dependent on host genotype. NopE specifically triggered incompatibility with V. radiata cv. KPS2, but it promoted nodulation in other varieties of V. radiata, including KPS1. Interestingly, NopE1 and its paralogue NopE2, which exhibits calcium-dependent autocleavage, yield similar results in modulating KPS1 nodulation. Furthermore, NopE is required for early infection and nodule organogenesis in compatible plants. Evolutionary analysis revealed that NopE is highly conserved among bradyrhizobia and plant-associated endophytic and pathogenic bacteria. Our findings suggest that V. radiata and B. diazoefficiens USDA110 may use NopE to optimize their symbiotic interactions by reducing phytohormone-mediated ETI-type (PmETI) responses via salicylic acid (SA) biosynthesis suppression.

Arthrobacter sp. strain KU001 isolated from a Thai soil degrades atrazine in the presence of inorganic nitrogen sources.

An atrazine degrading bacterium, strain KU001, was obtained from a sugarcane field at the Cane and Sugar Research and Development Center at the Kasetsart University, Kamphaeng Saen campus, Thailand. Strain KU001 had a rod-to-coccus morphological cycle during growth. Biolog carbon source analysis indicated that the isolated bacterium was Arthrobacter histidinolovorans. Sequence analysis of PCR product indicated the 16S rRNA gene in strain KU001 was 99% identical to the same region in Arthrobacter sp.. The atrazine degradation pathway in strain KU001 consists of the catabolic genes, trzN, atzB and, atzC. Strain KU001 was able to use atrazine as a sole nitrogen source for growth, and surprisingly, atrazine degradation was not inhibited in cells grown on ammonium, nitrate or urea, as compared to cells cultivated on growth-limiting nitrogen sources. During the atrazine degradation process, the supplementation of nitrate completely inhibits atrazine degradation activity in strain KU001, whereas ammonium and urea had no effect on atrazine degradation activity. The addition of strain KU001 to sterile or nonsterile soils resulted in the disappearance of atrazine at a rate that was 4 to 5-fold more than that achieved by the indigenous microbial community. The addition of citrate to soils resulted in enhanced atrazine degradation, 80% of atrazine disappeared within one day following nutrient supplementation.

Type III Secretion System of Bradyrhizobium sp. SUTN9-2 Obstructs Symbiosis with Lotus spp.

The rhizobial type III secretion system secretes effector proteins into host plant cells, which may either promote or inhibit symbiosis with legumes. We herein demonstrated that the type III secretion system of Bradyrhizobium sp. SUTN9-2 obstructed symbiosis with Lotus japonicus Miyakojima, L. japonicus Gifu, and Lotus burttii. A mutant of SUTN9-2 that is unable to secrete effector proteins showed better nodulation and plant growth promotion than wild-type SUTN9-2 when paired with these Lotus spp. We propose that SUTN9-2 is a useful strain for understanding the mechanisms by which effector proteins obstruct symbiosis between Bradyrhizobium and Lotus spp.

Effects of Increased 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity in Bradyrhizobium sp. SUTN9-2 on Mung Bean Symbiosis under Water Deficit Conditions.

Bacteria exhibiting 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, which inhibits the biosynthesis of ethylene in higher plants, promote plant growth through the degradation of ethylene precursors, such as ACC. ACC deaminase activity in Bradyrhizobium sp. SUTN9-2 was enhanced by genetic engineering and adaptive laboratory evolution (ALE)-based methods. The transferal of a plasmid containing the acdR and acdS genes into SUTN9-2 was genetic engineering improved, while the ALE method was performed based on the accumulation of an adaptive bacterial population that continuously grew under specified growth conditions for a long time. ACC deaminase enzyme activity was 8.9-fold higher in SUTN9-2:pMG103::acdRS and 1.4-fold higher in SUTN9-2 (ACCDadap) than in the wild-type strain. The effects of increased activity were examined in the host plant (Vigna radiata (L.) R.Wilczek SUT1). The improved strains enhanced nodulation in early stage of plant growth. SUTN9-2:pMG103::acdRS also maintained nitrogen fixation under water deficit conditions and increased the plant biomass after rehydration. Changes in nucleotides and amino acids in the AcdS protein of SUTN9-2 (ACCDadap) were then investigated. Some nucleotides predicted to be located in the ACC-binding site were mutated. These mutations may have increased ACC deaminase activity, which enhanced both symbiotic interactions and drought tolerance and promoted recovery after rehydration more than lower ACC deaminase activity. Adaptive evolution represents a promising strategy for further applications in the field.

Co-Inoculation of Bacillus velezensis Strain S141 and Bradyrhizobium Strains Promotes Nodule Growth and Nitrogen Fixation.

The objective of this research was to evaluate the PGPR effect on nodulation and nitrogen-fixing efficiency of soybean (Glycine max (L.) Merr.) by co-inoculation with Bradyrhizobium diazoefficiens USDA110. Co-inoculation of Bacillus velezensis S141 with USDA110 into soybean resulted in enhanced nodulation and N2-fixing efficiency by producing larger nodules. To understand the role of S141 on soybean and USDA110 symbiosis, putative genes related to IAA biosynthesis were disrupted, suggesting that co-inoculation of USDA110 with S141ΔyhcX reduces the number of large size nodules. It was revealed that yhcX may play a major role in IAA biosynthesis in S141 as well as provide a major impact on soybean growth promotion. The disruption of genes related to cytokinin biosynthesis and co-inoculation of USDA110 with S141ΔIPI reduced the number of very large size nodules, and it appears that IPI might play an important role in nodule size of soybean-Bradyrhizobium symbiosis. However, it was possible that not only IAA and cytokinin but also some other substances secreted from S141 facilitate Bradyrhizobium to trigger bigger nodule formation, resulting in enhanced N2-fixation. Therefore, the ability of S141 with Bradyrhizobium co-inoculation to enhance soybean N2-fixation strategy could be further developed for supreme soybean inoculants.

Mechanisms of Rice Endophytic Bradyrhizobial Cell Differentiation and Its Role in Nitrogen Fixation.

Bradyrhizobium sp. strain SUTN9-2 is a symbiotic and endophytic diazotrophic bacterium found in legume and rice plants and has the potential to promote growth. The present results revealed that SUTN9-2 underwent cell enlargement, increased its DNA content, and efficiently performed nitrogen fixation in response to rice extract. Some factors in rice extract induced the expression of cell cycle and nitrogen fixation genes. According to differentially expressed genes (DEGs) from the transcriptomic analysis, SUTN9-2 was affected by rice extract and the deletion of the bclA gene. The up-regulated DEGs encoding a class of oxidoreductases, which act with oxygen atoms and may have a role in controlling oxygen at an appropriate level for nitrogenase activity, followed by GroESL chaperonins are required for the function of nitrogenase. These results indicate that following its exposure to rice extract, nitrogen fixation by SUTN9-2 is induced by the collective effects of GroESL and oxidoreductases. The expression of the sensitivity to antimicrobial peptides transporter (sapDF) was also up-regulated, resulting in cell differentiation, even when bclA (sapDF) was mutated. This result implies similarities in the production of defensin-like antimicrobial peptides (DEFs) by rice and nodule-specific cysteine-rich (NCR) peptides in legume plants, which affect bacterial cell differentiation.

Homocitrate Synthase Genes of Two Wide-Host-Range Bradyrhizobium Strains are Differently Required for Symbiosis Depending on Host Plants.

The nifV gene encodes homocitrate synthase, the enzyme that catalyzes the formation of homocitrate, which is essential for arranging the FeMo-cofactor in the catalytic center of nitrogenase. Some host plants, such as Lotus japonicus, supply homocitrate to their symbionts, in this case, Mesorhizobium loti, which lacks nifV. In contrast, Bradyrhizobium ORS285, a symbiont of Aeschynomene cross-inoculation (CI) groups 2 and 3, requires nifV for symbiosis with Aeschynomene species that belong to CI group 3, and some species belonging to CI group 2. However, it currently remains unclear whether rhizobial nifV is required for symbiosis with Aeschynomene species belonging to CI group 1 or with other legumes. We generated nifV-disruption (ΔnifV) mutants of two wide-host-range rhizobia, Bradyrhizobium SUTN9-2 and DOA9, to investigate whether they require nifV for symbiosis. Both ΔnifV mutant strains showed significantly less nitrogenase activity in a free-living state than the respective wild-type strains. The symbiotic phenotypes of SUTN9-2, DOA9, and their ΔnifV mutants were examined with four legumes, Aeschynomene americana, Stylosanthes hamata, Indigofera tinctoria, and Desmodium tortuosum. nifV was required for the efficient symbiosis of SUTN9-2 with A. americana (CI group 1), but not for that of DOA9. SUTN9-2 established symbiosis with all three other legumes; nifV was required for symbiosis with I. tinctoria and D. tortuosum. These results suggest that, in addition to Aeschynomene CI groups 2 and 3, CI group 1 and several other legumes require the rhizobial nifV for symbiosis.