Selection and evaluation of Bradyrhizobium inoculum for peanut, Arachis hypogea production in the Lao People’s Democratic Republic

The interaction between leguminous plants and Bradyrhizobium is limited, known as host specificity. Therefore, the selection of an appropriate Bradyrhizobia for use as biofertilizer inoculum for legumes is necessary. The Arachis hypogea L. is the most popular legume produced in the Lao People's Democratic Republic (PDR). Therefore, this research aimed to obtain the appropriate Bradyrhizobia that provides high efficiency in A. hypogea production in the Lao PDR. The 14 isolates were obtained from root nodules of A. hypogea L. trapped with Lao PDR soil samples. Three were the top isolates PMVTL-01, SMVTL-02, and BLXBL-03 showing high efficiency for peanut growth promotion. Strains PMVTL-01 and SMVTL-02 were closely related to the Bradyrhizobium geno sp. SA-3 Rp7b and B. zhanjiangense, respectively, whilst strain BLXBL-03 was closely related to Bradyrhizobium sp. CCBAU51745 and B. manausense BR3351. The competitiveness of these strains with Bradyrhizobium sp. SUTN9-2::GFP was analyzed, and only Bradyrhizobium sp. SMVTL-02 performed a number of occupied nodules higher than SUTN9-2::GFP. In addition, the competitiveness of the selected strain Bradyrhizobium sp. SMVTL-02 in a soil sample from the Lao PDR in the pot level was employed by tagging the SMVTL-02 with the DsRed gene. The results demonstrated that the DsRed-expressing tagged strain showed higher nodule occupancy than indigenous strains. Moreover, the results of the acetylene reduction assay (ARA), nodule number, nodule dry weight, and total plant dry weight from the pot experiment that inoculated with the SMVTL-02::DsRed were presented as having high potential to promote peanut growth as compared to non-inoculation. Thus, Bradyrhizobium sp. SMVTL-02 could be considered a potential biofertilizer inoculum for A. hypogea production in the Lao PDR

Type 3 Secretion System (T3SS) of Bradyrhizobium sp. DOA9 and Its Roles in Legume Symbiosis and Rice Endophytic Association.

The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod, nif, and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod-box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (ΩrhcN) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four (Arachis hypogea, Vigna radiata, Crotalaria juncea, and Macroptilium atropurpureum) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one (Stylosanthes hamata) responded negatively (fewer nodules and less nitrogen fixation) and four species (Aeschynomene americana, Aeschynomene afraspera, Indigofera tinctoria, and Desmodium tortuosum) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice

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 freeliving and symbiotic states

Application of Light-Emitting Diodes with Plant Growth-Promoting Rhizobacteria and Arbuscular Mycorrhiza Fungi for Tomato Seedling Production

Various technologies, such as light-emitting diodes (LEDs) and beneficial plant micro-organisms, have been applied to enhance plant growth and development. We aimed to develop appropriate technology by incorporating the benefits of LED light, plant growth promoting rhizobacteria (PGPR), and arbuscular mycorrhiza fungi (AMF) into sweet girl cherry tomato (Solanum lycopersicum L.) seedling production. Our results demonstrated that incorporating red (R) and blue (B) LED lights, PGPR, and AMF positively affected tomato seedling growth. The optimal lighting conditions for tomato seedling growth were LEDs at 200 μmol/m2/s with a ratio of R60:B40 and 20 h/d exposure. The optimum LED-illuminated tomato seedlings significantly upregulated photosynthesis-related genes, including psbA, psbB, fdx, atpB, and rbcL. Plants inoculated with PGPR Bradyrhizobium sp. SUTN9-2, Bacillus velezensis SD10 and B. megaterium A20 had a high health index after inoculation. Furthermore, the optimized LED-illuminated tomato seedlings inoculated with SD10 had the highest health index. In addition, the optimum LED-illuminated tomato seedlings inoculated with SD10 and AMF had the highest biomass. Our experiment demonstrated that tomato seedlings produced under optimized LED lights inoculated with SD10 and AMF increased yield by about 16% under field conditions. Therefore, these results provided the optimum conditions for a high-quality tomato seedling production system

Mutualistic co-evolution of T3SSs during the establishment of symbiotic relationships between <em>Vigna radiata</em> and Bradyrhizobia

International audienceThis study supports the idea that the evolution of type III secretion system (T3SS) is one of the factors that controls Vigna radiata-bradyrhizobia symbiosis. Based on phylogenetic tree data and gene arrangements, it seems that the T3SSs of the Thai bradyrhizobial strains SUTN9-2, DOA1, and DOA9 and the Senegalese strain ORS3257 may share the same origin. Therefore, strains SUTN9-2, DOA1, DOA9, and ORS3257 may have evolved their T3SSs independently from other bradyrhizobia, depending on biological and/or geological events. For functional analyses, the rhcJ genes of ORS3257, SUTN9-2, DOA9, and USDA110 were disrupted. These mutations had cultivar-specific effects on nodulation properties. The T3SSs of ORS3257 and DOA9 showed negative effects on V. radiata nodulation, while the T3SS of SUTN9-2 showed no effect on V. radiata symbiosis. In the roots of V. radiata CN72, the expression levels of the PR1 gene after inoculation with ORS3257 and DOA9 were significantly higher than those after inoculation with ORS3257 omega T3SS, DOA9 omega T3SS, and SUTN9-2. The T3Es from ORS3257 and DOA9 could trigger PR1 expression, which ultimately leads to abort nodulation. In contrast, the T3E from SUTN9-2 reduced PR1 expression. It seems that the mutualistic relationship between SUTN9-2 and V. radiata may have led to the selection of the most well-adapted combination of T3SS and symbiotic bradyrhizobial genotype

Type 3 secretion system (T3SS) of <em>Bradyrhizobium</em> sp. DOA9 and its roles in legume symbiosis and rice endophytic association

International audienceThe Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod, nif, and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod-box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (Omega rhcN) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four (Arachis hypogea, Vigna radiata, Crotalaria juncea, and Macroptilium atropurpureum) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one (Stylosanthes hamata) responded negatively (fewer nodules and less nitrogen fixation) and four species (Aeschynomene americana, Aeschynomene afraspera, Indigofera tinctoria, and Desmodium tortuosum) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice

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

International audienceThe 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 β-galactopyrano syl 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. Further more, 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 nod ules 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