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Biological control is an effective measure in the green control of rice diseases. To search for biocontrol agents with broad-spectrum and high efficiency against rice diseases, in this study, a strain of antagonistic bacterium BR-01 with strong inhibitory effect against various rice diseases was isolated from Bolbostemmatis Rhizoma by plate confrontation method. The strain was identified as Bacillus velezensis by morphological observation, physiological and biochemical identification, and molecular characterization by 16S rDNA and gyrB gene sequencing analysis. The confrontation test (dual culture) and Oxford cup assays demonstrated that B. velezensis BR-01 had strong antagonistic effects on Magnaporthe oryzae, Ustilaginoidea virens, Fusarium fujikuroi, Xanthomonas oryzae pv. Oryzicola, and Xanthomonas oryzae pv. oryzae, the major rice pathogens. The genes encoding antimicrobial peptides (ituA, ituD, bmyB, bmyC, srfAA, fenB, fenD, bacA, and bacD) were found in B. velezensis BR-01 by PCR amplification with specific primers. B. velezensis BR-01 could produce protease, cellulase, β-1,3-glucanase, chitinase, indoleacetic acid, siderophore, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase, and might produce three lipopeptide antibiotics, surfactin, iturin, and fengycin based on Liquid chromatography–mass spectrometry (LC-MS) results. Furthermore, the plant assays showed that B. velezensis BR-01 had significant control effects on rice bacterial blight and bacterial leaf streak by pot experiments in greenhouse. In conclusion, B. velezensis BR-01 is a broad-spectrum antagonistic bacterium and has the potential as the ideal biocontrol agent in controlling multiple rice diseases with high efficiency.

Xanthomonas oryzae pv. textitoryzae (Xoo) is a causal agent of rice bacterial leaf blight (BLB), the major rice disease, which is seriously constraining rice production in Asia. The interaction between Xoo and rice is in a dynamic process, essentially the co-evolution. Tracking the occurrence of plant diseases and identifying the epidemic pathogens in time are critical to assessing the epidemic disease status and understanding the pathogen evolution. In 2020, the occurrences of rice BLB were spotted in many places of Guangxi, the major rice growing region in China. Two of the 2020-epidemic Xoo strains, namely, GXO20-01 and GXO20-06, were isolated from low land and high mountain paddies in Guangxi, respectively, and were demonstrated to be race R8 of Chinese Xoo strains, but with significantly different virulence on certain susceptible varieties of rice. The HiFi PacBio sequencing revealed that GXO20-01 and GXO20-06 share the highly syntenic genome structures and the major genome contents, but only differ in

In many tunnels in China, the groundwater is acidic and carbonated, causing in challenges in the prevention and control of leakages of the existing tunnels and tunnels under construction. Research on tunnel leakage and the physical and chemical effects of water due to water-rock interactions is a trending topic. In addition, there is a big difference between the waterproof and drainage of multiarch tunnels and separate tunnels. In this study, the mechanism of the interaction between the groundwater and the surrounding rock of a multiarch tunnel were analyzed. The relationship between the leakage of a multiarch tunnel and the interaction between the surrounding rocks and groundwater was determined by analyzing the interaction between the chemical components in the groundwater, rocks, and the concrete lining. A mathematical model was established based on the physical and chemical reactions in the rock surrounding the tunnel, and the RNCDX.FOR program was compiled using the simplex Monte Carlo method. The total free energy in the entire system varied significantly, and the value of objective function (J) reflected the trend of the system's spontaneous reaction. As J decreased, the reaction power of the system increased. The more significant the erosion of the rocks was by the groundwater, the more channels were created by the groundwater in the rock, and the more likely the water leakage was.

The Gram-negative bacterium Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS), one of the most destructive diseases of rice (Oryza sativa L.) that is the important staple crop. Xoc can invade host leaves via stomata and wounds and its type three secretion system (T3SS) is pivotal to its pathogenic lifestyle. In this study, using a novel dual RNA-seq approach, we examined transcriptomes of rice and Xoc in samples inoculated with wild type Xoc GX01 and its T3SS defective strain (T3SD), to investigate the global transcriptional changes in both organisms. Compared with T3SD strain, rice inoculated with wild type Xoc GX01 resulted in significant expression changes of a series of plant defence related genes, including ones altered in plant signalling pathway, and downregulated in phenylalanine metabolism, flavonoid and momilactone biosynthesis, suggesting repression of plant defence response and reduction in both callose deposition and phytoalexin accumulation. Also, some known transcription activator-like effector (TALE) targets were induced by Xoc GX01, e.g. OsSultr3;6 which contributes to rice susceptibility. Some cell elongation related genes, including several expansin genes, were induced by GX01 too, suggesting that Xoc may exploit this pathway to weaken cell wall strength, beneficial for bacterial infection. On the other hand, compared with wild type, the T3SD strain transcriptome in planta was characterized by downregulation of ATP, protein and polysaccharide synthesis, and upregulation of antioxidation and detoxification related genes, revealing that T3SD strain faced serious starvation and oxidation stresses in planta without a functional T3SS. In addition, comparative global transcript profiles of Xoc in planta and in medium revealed an upregulation of virulence factor synthesis and secretion in planta in favour of bacterial infection. Collectively, this study provides a comprehensive representation of cross talk between the host and bacterial pathogen, revealing insights into the Xoc-rice pathogenic dynamic and reveals novel strategies exploited by this important pathogen to cause disease.

The black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is a model organism for the study of plant bacterial pathogenesis mechanisms. In bacteria, σ factors serve as important regulatory elements that respond to various environmental signals and cues. Though Xcc encodes 15 putative σ factors little is known about their roles. As an approach to identify the potential role of each σ factor, we constructed mutations in each of the σ-factor genes as well as generating mutants deficient in multiple σ factors to assess these regulators potential additive functions. The work identified two σ70 factors essential for growth. Furthermore, the work discovered a third σ70 factor, RpoE1, important for virulence. Further studies revealed that RpoE1 positively regulates the expression of the hrp gene cluster that encodes the type III secretion system (T3SS) which determines the pathogenicity and hypersensitive response of Xcc on plants. In vivo and in vitro studies demonstrated that RpoE1 could bind to the promoter region and promote transcription of hrpX, a gene encoding a key regulator of the hrp genes. Overall, this systematic analysis reveals important roles in Xcc survival and virulence for previously uncharacterized σ70 factors that may become important targets for disease control.

Two-component regulatory system (TCS), a major type of cellular signal transduction system, is widely used by bacteria to adapt to different conditions and to colonize certain ecological niches in response to environmental stimuli. TCSs are of distinct functional diversity, genetic diversity, and species specificity (pathovar specificity, even strain specificity) across bacterial groups. Although TCSs have been demonstrated to be crucial to the virulence of Xanthomonas, only a few researches have been reported about the studies of TCSs in Xanthomonas oryzae pathovar oryzicola (hereafter Xoc), the pathogen of rice bacterial streak disease. In the genome of Xoc strain GX01, it has been annotated 110 TCSs genes encoding 54 response regulators (RRs), 36 orthodox histidine kinase (HKs) and 20 hybrid histidine kinase (HyHKs). To evaluate the involvement of TCSs in the stress adaptation and virulence of Xoc, we mutated 50 annotated RR genes in Xoc GX01 by homologous vector integration mutagenesis and assessed their phenotypes in given conditions and tested their virulence on host rice. 17 RR genes were identified to be likely involved in virulence of Xoc, of which 10 RR genes are novel virulence genes in Xanthomonas, including three novel virulence genes for bacteria. Of the novel candidate virulence genes, some of which may be involved in the general stress adaptation, exopolysaccharide production, extracellular protease secretion and swarming motility of Xoc. Our results will facilitate further studies on revealing the biological functions of TCS genes in this phytopathogenic bacterium.

The black rot pathogen Xanthomonas campestris utilizes molecules of the diffusible signal factor (DSF) family as signals to regulate diverse processes contributing to virulence. DSF signal synthesis and transduction requires proteins encoded by the rpf gene cluster. RpfF catalyzes DSF synthesis, whereas the RpfCG two-component system links the perception of DSF to alteration in the level of the second messenger cyclic di-GMP. As this nucleotide can exert a regulatory influence at the post-transcriptional and post-translational levels, we have used comparative proteomics to identify Rpf-regulated processes in X. campestris that may not be revealed by transcriptomics. The abundance of a number of proteins was altered in rpfF, rpfC, or rpfG mutants compared with the wild type. These proteins belonged to several functional categories, including biosynthesis and intermediary metabolism, regulation, oxidative stress or antibiotic resistance, and DNA replication. For many of these proteins, the alteration in abundance was not associated with alteration in transcript level. A directed mutational analysis allowed us to describe a number of new virulence factors among these proteins, including elongation factor P and a putative outer membrane protein, which are both widely conserved in bacteria.

The GntR family is one of the most abundant and widely distributed groups of helix-turn-helix transcriptional regulators in bacteria. Six open reading frames in the genome of the plant pathogen Xanthomonas campestris pv. campestris were predicted to encode GntR regulators. All six of the predicted GntR-encoding genes were individually mutagenized and mutants from five of them were successfully obtained. Plant disease response assays revealed that one, whose product belongs to the YtrA subfamily and has been named HpaR1, is involved in the hypersensitive response (HR) and virulence. Electrophoretic mobility shift assays and in vitro transcription assays revealed that HpaR1 could repress its own transcription level through binding to its promoter sequence, indicating an autoregulatory feedback inhibition mechanism for HpaR1 expression. Promoter-gusA reporter and reverse-transcription polymerase chain reaction analyses revealed that HpaR1 positively and negatively affects the expression of HR and pathogenicity (hrp) genes in host plant and standard media, respectively. Constitutive expression of the key hrp regulator, hrpG, in the hpaR1 mutant could bypass the requirement of HpaR1 for the induction of wild-type HR, suggesting that HpaR1 regulates the expression of hrp genes that encode the type III secretion system via hrpG.

Xanthomonas campestris pv. campestris produces a membrane-bound yellow pigment called xanthomonadin. A diffusible factor (DF) has been reported to regulate xanthomonadin biosynthesis. In this study, DF was purified from bacterial culture supernatants using a combination of solvent extraction, flash chromatography, and high-performance liquid chromatography. Mass spectrometry and nuclear magnetic resonance analyses resolved the DF chemical structure as 3-hydroxybenzoic acid (3-HBA), which was further confirmed by synthetic 3-HBA. Significantly, bioassay and in silico analysis suggest that DF production is widely conserved in a range of bacterial species. Analysis of DF derivatives established the hydroxyl group and its position as the key structural features for the role of DF in xanthomonadin biosynthesis. In addition, we showed that DF is also associated with bacterial survival, H2O2 resistance, and systemic invasion. Furthermore, evidence was also presented that DF and diffusible signaling factor have overlapping functions in modulation of bacterial survival, H2O2 resistance, and virulence. Utilization of different mechanisms to modulate similar virulence traits may provide X. campestris pv. campestris with plasticity in response to various environmental cues.

Xanthomonas campestris pv. campestris is the pathogen of black rot of cruciferous plants. The pathogenicity of the pathogen depends on the type III secretion system (T3SS) that translocates directly effector proteins into plant cells, where they play important roles in the molecular interaction between the pathogen and its hosts. The T3SS of Xanthomonas spp. is encoded by a cluster of hypersensitive response and pathogenicity (hrp) genes. It has been demonstrated that the expression of hrp genes and some type III secreted (T3S)-effector genes is coactivated by the key hrp regulatory protein HrpX. The regulation by HrpX can be mediated by the binding of HrpX protein to a cis-regulatory element named the plant-inducible promoter (PIP) box present in the promoter region of HrpX-regulated genes. A genome screen revealed that X. campestris pv. campestris 8004 possesses 56 predicted genes with the PIP box. Nine of these genes have been shown to encode T3S effectors, Hrp, and Hrp-associated proteins. In this study, we employed an established T3S effector translocation assay with the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter to characterize the remaining 47 genes with the PIP box and showed that 6 of them, designated as XopXccE1, XopXccP, XopXccQ, XopXccR1, XopXccLR, and AvrXccB, harbor a functional translocation signal in their N-terminal regions, indicating that they are T3S effectors of X. campestris pv. campestris. We provided evidence to demonstrate that all these effectors are expressed in an HrpX-dependent manner and their translocation into plant cells relies on the translocon protein HrpF and the chaperone HpaB. Mutational analyses demonstrated that all these effectors, except AvrXccB, are individually required for full virulence and growth of X. campestris pv. campestris in the host plant Chinese radish.

In bacteria, Zur is a key regulator for zinc homeostasis. Our previous work has shown that, in the phytopathogen Xanthomonas campestris pv. campestris, in addition to regulating zinc homeostasis, Zur is essential for full virulence. Here, we demonstrate that the X. campestris pv. campestris Zur is involved in hypersensitive response (HR) and positively regulates the transcription of hrpA to hrpF operons and hrpX but not hrpG. Constitutively expressing hrpX but not hrpG in the zur mutant could bypass the requirement of Zur for the expression of hrpA to hrpF operons and the induction of wild-type HR, indicating that Zur controls the expression of hrp cluster via hrpX. Promoter-gusA reporter and semiquantitative reverse-transcription polymerase chain reaction analyses revealed that HrpG controls the expression of hrpX and HrpX regulates the expression of all the six hrp operons (hrpA to hrpF) in X. campestris pv. campestris.

The DsbA/DsbB oxidation pathway is one of the two pathways that catalyze disulfide bond formation of proteins in the periplasm of gram-negative bacteria. It has been demonstrated that DsbA is essential for multiple virulence factors of several animal bacterial pathogens. In this article, we present genetic evidence to show that the open reading frame XC_3314 encodes a DsbB protein that is involved in disulfide bond formation in periplasm of Xanthomonas campestris pv. campestris, the causative agent of crucifer black rot disease. The dsbB mutant of X. campestris pv. campestris exhibited attenuation in virulence, hypersensitive response, cell motility, and bacterial growth in planta. Furthermore, mutation in the dsbB gene resulted in ineffective type II and type III secretion systems as well as flagellar assembly. These findings reveal that DsbB is required for the pathogenesis process of X. campestris pv. campestris.

RsmA is an RNA-binding protein functioning as a global post-transcriptional regulator of various cellular processes in bacteria and has been demonstrated to be an important virulence regulator in many animal bacterial pathogens. However, its function in other phytopathogenic bacteria is unclear, except for the Erwinia carotovora RsmA, which acts as a negative virulence regulator. In this work, we investigated the function of the rsmA-like gene, named rsmAXcc, of the phytopathogen Xanthomonas campestris pv. campestris. Deletion of rsmAXcc resulted in complete loss of virulence on the host plant Chinese radish, hypersensitive response on the nonhost plant pepper ECW-10R, and motility on the surface of an agar plate. The rsmAXcc mutant displayed a significant reduction in the production of extracellular amylase, endoglucanase, and polysaccharide, but a significant increase in intracellular glycogen accumulation and an enhanced bacterial aggregation and cell adhesion. Microarray hybridization and semiquantitative reverse-transcription polymerase chain reaction analysis showed that deletion of rsmAXcc led to significantly reduced expression of genes encoding the type III secretion system (T3SS), T3SS-effectors, and the bacterial aggregate dispersing enzyme endo-β-1,4-mannanase. These results suggest that rsmAXcc is involved in the control of various cellular processes, including pathogenesis of X. campestris pv. campestris.

Macrophage infectivity potentiators (Mips) are FKBP domain-containing proteins reported as virulence factors in several human pathogens, such as members of genera Legionella, Salmonella and Chlamydia. The putative peptidyl-prolyl cis-trans isomerase (PPIase) encoded by XC2699 of the plant bacterial pathogen Xanthomonas campestris pv. campestris 8004 exhibits a 49% similarity at the aminoacid level to the Mip protein of Legionella pneumophila. This mip-like gene, XC2699, was overexpressed in Es-cherichia coli and the purified (His)6-tagged Mip-like protein encoded by XC2699 exhibited a PPIase activity specifically inhibited by FK-506. A mutation in the mip-like gene XC2699 led to significant reductions in virulence and replication capacity in the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.). Furthermore, the production of exopolysaccharide and the activity of extracellular proteases, virulence factors X. campestris pv. campestris, were significantly decreased in the mip-like mutant. These results reveal that the mip-like gene is involved in the pathogenesis of X. campestris pv. campestris through an effect on the production of these virulence factors.

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 μM and in nonrich medium supplemented with Zn2+ at a concentration of 110 μM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 μM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.

In order to improve the Internet coverage ratio and provide connectivity guarantee, based on sensor opportunistic coverage mechanism and cooperative cloud service, we proposed the coverage connectivity guarantee protocol for mobile Internet. In this scheme, based on the opportunistic covering rules, the network coverage algorithm of high reliability and real-time security was achieved by using the opportunity of sensor nodes and the Internet mobile node. Then, the cloud service business support platform is created based on the Internet application service management capabilities and wireless sensor network communication service capabilities, which is the architecture of the cloud support layer. The cooperative cloud service aware model was proposed. Finally, we proposed the mobile Internet coverage connectivity guarantee protocol. The results of experiments demonstrate that the proposed algorithm has excellent performance, in terms of the security of the Internet and the stability, as well as coverage connectivity ability.