Exploring the therapeutic potential of Thai medicinal plants: in vitro screening and in silico docking of phytoconstituents for novel anti-SARS-CoV-2 agents.

BACKGROUND: The high virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), has triggered global health and economic concerns. The absence of specific antiviral treatments and the side effects of repurposed drugs present persistent challenges. This study explored a promising antiviral herbal extract against SARS-CoV-2 from selected Thai medicinal plants based on in vitro efficacy and evaluated its antiviral lead compounds by molecular docking. METHODS: Twenty-two different ethanolic-aqueous crude extracts (CEs) were rapidly screened for their potential activity against porcine epidemic diarrhea virus (PEDV) as a surrogate using a plaque reduction assay. Extracts achieving ≥ 70% anti-PEDV efficacy proceeded to the anti-SARS-CoV-2 activity test using a 50% tissue culture infectious dose method in Vero E6 cells. Molnupiravir and extract-free media served as positive and negative controls, respectively. Potent CEs underwent water/ethyl acetate fractionation to enhance antiviral efficacy, and the fractions were tested for anti-SARS-CoV-2 performance. The fraction with the highest antiviral potency was identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Molecular docking analyses of these compounds against the main protease (Mpro) of SARS-CoV-2 (6LU7) were performed to identify antiviral lead molecules. The top three hits were further evaluated for their conformational stability in the docked complex using molecular dynamics (MD) simulation. RESULTS: The water fraction of mulberry (Morus alba Linn.) leaf CE (WF-MLCE) exhibited the most potent anti-SARS-CoV-2 efficacy with low cytotoxicity profile (CC50 of ~ 0.7 mg/mL), achieving 99.92% in pre-entry mode and 99.88% in postinfection treatment mode at 0.25 mg/mL. Flavonoids and conjugates were the predominant compounds identified in WF-MLCE. Molecular docking scores of several flavonoids against SARS-CoV-2 Mpro demonstrated their superior antiviral potency compared to molnupiravir. Remarkably, myricetin-3-O-ß-D-galactopyranoside, maragrol B, and quercetin 3-O-robinobioside exhibited binding energies of ~ - 9 kcal/mol. The stability of each ligand-protein complex of these compounds with the Mpro system showed stability during MD simulation. These three molecules were pronounced as antiviral leads of WF-MLCE. Given the low cytotoxicity and high antiviral potency of WF-MLCE, it holds promise as a candidate for future therapeutic development for COVID-19 treatment, especially considering its economic and pharmacological advantages.

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.

Identification of immune-responsive circular RNAs in shrimp (Litopenaeus vannamei) upon yellow head virus infection.

Circular RNAs (circRNAs) are a subclass of non-coding RNAs (ncRNAs) formed through a process known as back-splicing. They play a crucial role in the genetic regulation of various biological processes. Currently, circRNAs have been identified as participants in the antiviral response within mammalian cells. However, circRNAs in shrimp infected with the yellow head virus (YHV) remain largely unexplored. Therefore, this study aims to identify circRNAs in the hemocytes of Litopenaeus vannamei during YHV infection. We discovered 358 differentially expressed circRNAs (DECs), with 177 of them being up-regulated and 181 down-regulated. Subsequently, eight DECs, including circ_alpha-1-inhibitor 3, circ_CDC42 small effector protein 2, circ_hemicentin 2, circ_integrin alpha V, circ_kazal-type proteinase inhibitor, circ_phenoloxidase 3, circ_related protein rab-8B, and circ_protein toll-like, were randomly selected for analysis of their expression patterns during YHV infection using qRT-PCR. Furthermore, the circRNAs' characteristics were confirmed through PCR, RNase R treatment, and Sanger sequencing, all of which were consistent with the features of circRNAs. These findings contribute to a better understanding of circRNAs' involvement in the antiviral response in shrimp.

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.

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.

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.

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.

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.

Transcriptome-based insights into the regulatory role of immune-responsive circular RNAs in Litopanaeus vannamei upon WSSV infection.

Circular RNAs (circRNAs) are non-coding RNAs (ncRNAs) originating from a post-transcriptional modification process called back-splicing. Despite circRNAs being traditionally considered by-products rather than independently functional, circRNAs play many vital roles, such as in host immunity during viral infection. However, in shrimp, these remain largely unexplored. Therefore, this study aims to identify circRNAs in Litopenaeus vannamei in the context of WSSV infection, one of the most eradicative pathogens threatening shrimp populations worldwide. We identified 290 differentially expressed circRNAs (DECs) in L. vannamei upon WSSV infection. Eight DECs were expressed from their parental genes, including alpha-1-inhibitor-3, calpain-B, integrin-V, hemicentin-2, hemocytin, mucin-17, proPO2, and rab11-FIP4. These were examined quantitatively by qRT-PCR, which revealed the relevant expression profiles to those obtained from circRNA-Seq. Furthermore, the structural and chemical validation of the DECs conformed to the characteristics of circRNAs. One of the functional properties of circRNAs as a miRNA sponge was examined via the interaction network between DECs and WSSV-responsive miRNAs, which highlighted the targets of miRNA sponges. Our discovery could provide insight into the participation of these ncRNAs in shrimp antiviral responses.

The interferon-like proteins, Vagos, in Fenneropenaeus merguiensis elicit antimicrobial responses against WSSV and VPAHPND infection.

The Vago interferon-like protein participates in the interplay between interferon regulatory factors and the expression of immune-responsive genes. Vago was initially perceived to participate only in the antiviral activation through JAK/STAT pathway. However, certain isoforms of Vago can stimulate antimicrobial responses. Here we identify Vago isoforms in Fenneropenaeus merguiensis (FmVagos) and how they function in antiviral and antibacterial responses against highly invasive pathogens, including white spot syndrome virus (WSSV) and Vibrio parahaemolyticus (VPAHPND). Three isoforms of FmVagos were identified: FmVago4, FmVago5a, and FmVago5b, and expressed throughout tissues of the shrimp. During infection, FmVago4, FmVago5a, and FmVago5b, were up-regulated after WSSV and VPAHPND challenges at certain time points. Pre-injection of purified recombinant FmVago4 (rVago4), FmVago5a (rVago5a), and FmVago5b (rVago5b) proteins could significantly reduce the mortality of shrimp upon WSSV infection, while the increase of survival rate of VPAHPND-infected shrimp was observed only in rVago4 treatment. The immunity routes that FmVagos might instigate in response to the pathogens were examined by qRT-PCR, revealing that the JAK/STAT pathway was activated after introducing rVago4, rVago5a, and rVago5b, while the Toll/IMD pathway and proPO system, combined with PO activity, were provoked only in the rVago4-treated shrimp. Our finding suggests cross-talk between Vago's antiviral and antimicrobial responses in shrimp immunity. These findings complement previous studies in which Vago and its specific isoform could promote viral and bacterial clearance in shrimp.

The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes.

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade.

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.

Co-culture of microalga Chlorella sorokiniana with syntrophic Streptomyces thermocarboxydus in cassava wastewater for wastewater treatment and biodiesel production.

This study aimed to conduct a co-culture of a microalga and syntrophic actinomycetes to treat cassava a biogas effluent wastewater and enhance biodiesel production. Streptomyces thermocarboxydus strain BMI 10 produced the greatest effect on biomass production by Chlorella sorokiniana strain P21. Maximal algal biomass production and total lipid yield were increased when strain BMI 10 was co-cultured (by 21 and 25 %, respectively). Furthermore, the nutrient removal efficiency of P21 was not significantly different under sterilized and unsterilized conditions. Harvestability of the strain was also increased under both conditions. Analysis of the amount and composition of fatty acids from this co-culture biomass revealed that it was quite satisfactory for biodiesel production (54.11-61.52% saturated fatty acids with a 0.59-0.82 degree of unsaturation). Overall, the results showed the co-culture of the alga and bacterium is a holistic enhancement that couples wastewater treatment with biodiesel production.

Enhancing Teak (Tectona grandis) Seedling Growth by Rhizosphere Microbes: A Sustainable Way to Optimize Agroforestry.

With its premium wood quality and resistance to pests, teak is a valuable tree species remarkably required for timber trading and agroforestry. The nursery stage of teak plantation needs critical care to warrant its long-term productivity. This study aimed to search for beneficial teak rhizosphere microbes and assess their teak-growth-promoting potentials during nursery stock preparation. Three teak rhizosphere/root-associated microbes, including two teak rhizobacteria (a nitrogen-fixing teak root endophyte-Agrobacterium sp. CGC-5 and a teak rhizosphere actinobacterium-Kitasatospora sp. TCM1-050) and an arbuscular mycorrhizal fungus (Claroideoglomus sp. PBT03), were isolated and used in this study. Both teak rhizobacteria could produce in vitro phytohormones (auxins) and catalase. With the pot-scale assessments, applying these rhizosphere microbes in the form of consortia offered better teak-growth-promoting activities than the individual applications, supported by significantly increased teak seedling biomass. Moreover, teak-growth-promoting roles of the arbuscular mycorrhizal fungus were highly dependent upon the support by other teak rhizobacteria. Based on our findings, establishing the synergistic interactions between beneficial rhizosphere microbes and teak roots was a promising sustainable strategy to enhance teak growth and development at the nursery stage and reduce chemical inputs in agroforestry.

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.

Soil bacterial communities and their associated functions for forest restoration on a limestone mine in northern Thailand.

Opencast mining removes topsoil and associated bacterial communities that play crucial roles in soil ecosystem functioning. Understanding the community composition and functioning of these organisms may lead to improve mine-rehabilitation practices. We used a culture-dependent method, combined with Illumina sequencing, to compare the taxonomic richness and composition of living bacterial communities in opencast mine substrates and young mine-rehabilitation plots, with those of soil in adjacent remnant forest at a limestone mine in northern Thailand. We further investigated the effects of soil physico-chemical factors and ground-flora cover on the same. Although, loosened subsoil, brought in to initiate rehabilitation, improved water retention and facilitated plant re-establishment, it did not increase the population density of living microbes substantially within 9 months. Planted trees and sparse ground flora in young rehabilitation plots had not ameliorated the micro-habitat enough to change the taxonomic composition of the soil bacteria compared with non-rehabilitated mine sites. Viable microbes were significantly more abundant in forest soil than in mine substrates. The living bacterial community composition differed significantly, between the forest plots and both the mine and rehabilitation plots. Proteobacteria dominated in forest soil, whereas Firmicutes dominated in samples from both mine and rehabilitation plots. Although, several bacterial taxa could survive in the mine substrate, soil ecosystem functions were greatly reduced. Bacteria, capable of chitinolysis, aromatic compound degradation, ammonification and nitrate reduction were all absent or rare in the mine substrate. Functional redundancy of the bacterial communities in both mine substrate and young mine-rehabilitation soil was substantially reduced, compared with that of forest soil. Promoting the recovery of microbial biomass and functional diversity, early during mine rehabilitation, is recommended, to accelerate soil ecosystem restoration and support vegetation recovery. Moreover, if inoculation is included in mine rehabilitation programs, the genera: Bacillus, Streptomyces and Arthrobacter are likely to be of particular interest, since these genera can be cultivated easily and this study showed that they can survive under the extreme conditions that prevail on opencast mines.

Elevated Nitrogen Priming Induced Oxinitro-Responses and Water Deficit Tolerance in Rice.

Under water deficit conditions, the essential macronutrient nitrogen becomes limited as a result of reduced dissolved nitrogen and root nitrogen uptake. An elevated nitrogen level might be able to mitigate these effects, integrated with the idea of using nitric oxide as abiotic stress tolerant inducers. In this study, we evaluated the potential of using elevated nitrogen priming prior to water shortage to mitigate plant stress through nitric oxide accumulation. We grew rice plants in 300 mg L-1 nitrogen for 10 weeks, then we primed plants with four different nitrogen concentrations: 100, 300 (control), 500 and 1000 mg L-1 nitrogen prior to inducing water deficit conditions. Plants primed with 500 mg L-1 nitrogen possessed a higher photosynthetic rate, relative water content, electrolyte leakage and lipid peroxidation under water deficit conditions, compared to control plants. The induction of water deficit tolerance was supported with the activation of antioxidant defense system, induced by the accumulation of nitric oxide in leaves and roots of rice plants. We originally demonstrated the accumulation of nitric oxide in leaves of rice plants. The elevated nitrogen priming can be used to enhance water deficit tolerance in irrigated paddy fields, instead of nitric oxide donors.

Identification of type III effectors modulating the symbiotic properties of Bradyrhizobium vignae strain ORS3257 with various Vigna species.

The Bradyrhizobium vignae strain ORS3257 is an elite strain recommended for cowpea inoculation in Senegal. This strain was recently shown to establish symbioses on some Aeschynomene species using a cocktail of Type III effectors (T3Es) secreted by the T3SS machinery. In this study, using a collection of mutants in different T3Es genes, we sought to identify the effectors that modulate the symbiotic properties of ORS3257 in three Vigna species (V. unguiculata, V. radiata and V. mungo). While the T3SS had a positive impact on the symbiotic efficiency of the strain in V. unguiculata and V. mungo, it blocked symbiosis with V. radiata. The combination of effectors promoting nodulation in V. unguiculata and V. mungo differed, in both cases, NopT and NopAB were involved, suggesting they are key determinants for nodulation, and to a lesser extent, NopM1 and NopP1, which are additionally required for optimal symbiosis with V. mungo. In contrast, only one effector, NopP2, was identified as the cause of the incompatibility between ORS3257 and V. radiata. The identification of key effectors which promote symbiotic efficiency or render the interaction incompatible is important for the development of inoculation strategies to improve the growth of Vigna species cultivated in Africa and Asia.