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.

Ethylene is synthesized at accelerated rate in response to stress conditions, and regarded as a stress hormone that inhibits overall plant growth. Apparently, the amount of ethylene can be reduced by using silver thiosulfate (STS) or bacteria containing 1-amino-cyclopropane-1-carboxylate (ACC) deaminase. In this study, we examined whether different concentrations of STS differently prevent legume growth under various environment stress conditions, and whether co-inoculation of mungbean (Vigna radiata (L.) R. Wilczek) by Bradyrhizobium and rhizobacteria containing ACC deaminase also alleviates legume stress. Mungbean is one of the most responsive plants among tested legume species, and different varieties of mungbean also respond differently to STS. Stress conditions and inappropriate concentrations of STS affect plant growth and symbiosis, while a suitable concentration of STS supports plant growth under stress conditions. Three isolates of bacteria containing ACC deaminase, Enterobacter sp. ACC1, Enterobacter sp. ACC2, and Chryseobacterium sp. ACC3, were selected according to their ACC deaminase activity and resistance abilities to high temperature, drought, and salt stress conditions. Interestingly, the gene encoding ACC deaminase, acdS, of selected ACC deaminase bacteria was highly induced when the cell culture was exposed to stress conditions. It coincided with co-inoculation of plants by Bradyrhizobium and rhizobacteria containing ACC deaminase. This co-inoculation obviously alleviated the stress of plants growing under stress conditions. These results revealed the role of bacteria containing ACC deaminase that can adjust its expression of ACC deaminase to different levels of stress conditions, which will be useful in agriculture under global warming.

The Bradyrhizobium sp. DOA9 strain displays the unusual properties to have a symbiotic plasmid and to fix nitrogen during both free-living and symbiotic growth. Sequence genome analysis shows that this strain contains the structural genes of dinitrogenase (nifDK) and the nifA regulatory gene on both the plasmid and chromosome. It was previously shown that both nifDK clusters are differentially expressed depending on growth conditions, suggesting different mechanisms of regulation. In this study, we examined the functional regulatory role of the two nifA genes found on the plasmid (nifAp) and chromosome (nifAc) that encode proteins with a moderate level of identity (55%) and different structural architectures. Using gusA (β-glucuronidase) reporter strains, we showed that both nifA genes were expressed during both the free-living and symbiotic growth stages. During symbiosis with Aeschynomene americana, mutants in only one nifA gene were not altered in their symbiotic properties, while a double nifA mutant was drastically impaired in nitrogen fixation, indicating that the two NifA proteins are functionally redundant during this culture condition. In contrast, under in vitro conditions, the nifAc mutant was unable to fix nitrogen, and no effect of the nifAp mutation was detected, indicating that NifAc is essential to activate nif genes during free-living growth. In accordance, the nitrogenase fixation deficiency of this mutant could be restored by the introduction of nifAc but not by nifAp or by two chimeric nifA genes encoding hybrid proteins with the N-terminus part of NifAc and the C-terminus of NifAp. Furthermore, transcriptional analysis by RT-qPCR of the WT and two nifA mutant backgrounds showed that NifAc and NifAp activated the expression of both chromosome and plasmid structural nifDK genes during symbiosis, while only NifAc activated the expression of nifDKc during free-living conditions. In summary, this study provides a better overview of the complex mechanisms of regulation of the nitrogenase genes in the DOA9 strain that involve two distinct NifA proteins, which are exchangeable during symbiosis for the activation of nif genes but not during free-living growth where NifAc is essential for the activation of nifDKc.

Cannabis sativa L. has a variety of uses, including fiber production, food, oil, and medicine. In response to environmental concerns regarding chemical fertilizers, Bacillus velezensis S141 was examined as a plant-growth-promoting bacterium (PGPB) for cannabis. This study evaluated the effects of S141 on cannabis growth and utilized transcriptomic analysis to identify the responsive pathways. Inoculation with S141 significantly increased growth in laboratory and field environments, with most of the bacteria residing in the leaves, followed by the stems and roots, as determined by quantitative polymerase chain reaction (qPCR). Transcriptomic analysis revealed 976 differentially expressed genes. Upregulated genes were associated with metabolism, cellular processes, and catalytic activities, especially in the biosynthesis of phenylpropanoid, plant–pathogen interactions, and hormone signaling pathways. S141 mutants deficient in the production of auxin and cytokinin displayed reduced growth enhancement, which affirmed the roles of these hormones in cannabis development. These findings emphasize the potential of S141 as a sustainable growth promoter for cannabis and provide insights into the underlying pathways it influences.

Bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase is a key factor for alleviating the plant ethylene biosynthesis, which is induced by stress. The ACC deaminase-improved strains of the rice endophytic Bradyrhizobium sp. SUTN9-2, SUTN9-2 (ACCDadap) and SUTN9-2:pMG103::acdRS, exhibit 1.4- and 8.9-fold higher ACC deaminase activity than the wild type, respectively (Sarapat S, Songwattana P, Longtonglang A, Umnajkitikorn K, Girdthai T, Tittabutr P, Boonkerd N, Teaumroong N. 2020. Effects of Increased 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Activity in Bradyrhizobium sp. SUTN9-2 on Mung Bean Symbiosis under Water Deficit Conditions. Microbes Environ. 35). The effects of these on rice growth under water deficit conditions were evaluated. The bacterial inoculations reduced ethylene synthesis, leading to a reduction in membrane destruction and the chlorophyll content of rice. Furthermore, the bacterial inoculations improved the leaf relative water content, survival, recovery rates, and improved the crop yield in field conditions. Therefore, the water deficit tolerance of rice was improved by controlling ethylene biosynthesis by improving ACC deaminase activity with endophytic SUTN9-2. Moreover, the SUTN9-2 (ACCDadap) strain can be used as a bio-inoculant under field conditions to enhance rice growth, grain yield, and enhance drought tolerance.

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.