Outer membrane proteins (OMPs) are integral β-barrel proteins of the Gram-negative bacterial cell wall and are crucial to bacterial survival within the macrophages and for eukaryotic cell invasion. Here, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to comprehensively assess the outer membrane proteome of Burkholderia cenocepacia, an opportunistic pathogen causing cystic fibrosis (CF), in conditions mimicking four major ecological niches: water, CF sputum, soil, and plant leaf. Bacterial cells were harvested at late log phase, and OMPs were extracted following the separation of soluble proteins by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE). Protein bands were excised and identified by LC-MS/MS analysis. The proteins identified under various growth conditions were further subjected to in silico analysis of gene ontology (subcellular localization, structural, and functional analyses). Overall, 72 proteins were identified as common to the four culture conditions, while 33, 37, 20, and 10 proteins were exclusively identified in the water, CF sputum, soil, and plant leaf environments, respectively. The functional profiles of the majority of these proteins revealed significant diversity in protein expression between the four environments studied and may indicate that the protein expression profiles are unique for every condition. Comparison of OMPs from one strain in four distinct ecological niches allowed the elucidation of proteins that are essential for survival in each niche, while the commonly expressed OMPs, such as RND efflux system protein, TonB-dependent siderophore receptor, and ABC transporter-like protein, represent promising targets for drug or vaccine development.

Bacillus strains are extensively studied for their beneficial role in plant growth and biological control of tomato bacterial wilt (TBW), however their underlying mechanisms remained unexplored. In this study, four rhizobacterial strains, Bacillus amyloliquefaciens D29, B. amyloliquefaciens Am1, B. subtilis D16 and B. methylotrophicus H8 were investigated for their antibacterial activity against (TBW) pathogen and their ability to stimulate Tomato growth. Results revealed that all four strains were able to form robust biofilm, produce Indole acetic acid (IAA) and siderophores, while only D29, Am1 and H8 have capability to solubilize phosphate. The culture filtrate of each strain significantly suppressed the growth and biofilm of Ralstonia solanacearum, where, the cell wall was severely disrupted, which resulted into cell lysis and subsequent leakage of intracellular cytosolic contents. PCR analysis revealed that all four strains are harboring the antimicrobial associated genes for biosynthesis of Bacyllomicin, Fengycin, Iturin, Surfactin and Bacylisin. Subsequent real-time qPCR analysis revealed that the expression of ituC and srfAA genes in Am1 and D16 was remarkably up-regulated during in vitro interaction with R. solanacearum. This suggest that the potential antibacterial and anti-biofilm related mechanisms are associated to their ability to secret the corresponding lipopeptides in surrounding niche. In greenhouse, a positive correlation (0.777 and 0.686) was noted between the IAA amount produced by Bacillus strains and fresh/dry weight of bacterized tomato plants. This the first report demonstrated the mode of antibacterial effect of Bacillus strains against R. solanacearum, moreover this study will help in understanding the mode of action of Bacillus strains during biological management of TBW and promoting the growth of tomato plants.

Chitosan, a versatile derivative of chitin, is widely used as an antimicrobial agent either alone or mixed with other natural polymers. Burkholderia cenocepacia is a multidrug-resistant bacteria and difficult to eradicate. Our previous studies shown that chitosan had strong antibacterial activity against B. cenocepacia. In the current study, we have investigated the molecular aspects for the susceptibility of B. cenocepacia in response to chitosan antibacterial activity. We have conducted RNA expression analysis of drug efflux system by RT-PCR, membrane protein profiling by SDS–PAGE, and by LC-MS/MS analysis following the validation of selected membrane proteins by real-time PCR analysis. By RT-PCR analysis, it was found that orf3, orf9, and orf13 were expressed at detectable levels, which were similar to control, while rest of the orf did not express. Moreover, shotgun proteomics analysis revealed 21 proteins in chitosan-treated cells and 16 proteins in control. Among them 4 proteins were detected as shared proteins under control and chitosan-treated cells and 17 proteins as uniquely identified proteins under chitosan-treated cells. Among the catalog of uniquely identified proteins, there were proteins involved in electron transport chain and ATP synthase, metabolism of carbohydrates and adaptation to atypical conditions proteins which indicate that utilization and pattern of chitosan is diverse which might be responsible for its antibacterial effects on bacteria. Moreover, our results showed that RND drug efflux system, which display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents, was not determined to play its role in response to chitosan. It might be lipopolysaccharides interaction with chitosan resulted in the destabilization of membrane protein to membrane lyses to cell death. Membrane proteome analysis were also validated by RT-qPCR analysis, which corroborated our results that of membrane proteins.

Biological control efficacy of Brevibacillus laterosporus B4 associated with rice rhizosphere was assessed against bacterial brown stripe of rice caused by Acidovorex avenae subsp. avenae. A biochemical bactericide (chitosan) was used as positive control in this experiment. Result of in vitro analysis indicated that B. laterosporus B4 and its culture filtrates (70 %; v/v) exhibited low inhibitory effects than chitosan (5 mg/ml). However, culture suspension of B. laterosporus B4 prepared in 1 % saline solution presented significant ability to control bacterial brown stripe in vivo. Bacterization of rice seeds for 24 h yielded a greater response (71.9 %) for controlling brown stripe in vivo than chitosan (56 %). Studies on mechanisms revealed that B. laterosporus B4 suppressed the biofilm formation and severely disrupted cell membrane integrity of A. avenae subsp. avenae, causing the leakage of intracellular substances. In addition, the expression level of virulence-related genes in pathogen recovered from biocontrol-agent-treated plants showed that the genes responsible for biofilm formation, motility, niche adaptation, membrane functionality and virulence of A. avenae subsp. avenae were down-regulated by B. laterosporus B4 treatment. The biocontrol activity of B. laterosporus B4 was attributed to a substance with protein nature. This protein nature was shown by using ammonium sulfate precipitation and subsequent treatment with protease. The results obtained from this study showed the potential effectiveness of B. laterosporus B4 as biocontrol agent in control of bacterial brown stripe of rice.

The study presents the first report on biocontrol of brown sheath rot disease of rice caused by Pseudomonas fuscovaginae using rhizo-bacterial isolate Bacillus amyloliquefaciens Bk7. Four potential bioactive antagonists were selected from 120 Bacillus isolates. Results obtained from in vitro laboratory assay showed that rhizosphere bacterial strain Bk7 and its metabolites significantly suppressed the growth of Pseudomonas fuscovaginae with 93 % efficacy. In glasshouse experiments, strain Bk7 exhibited biocontrol efficacy of 76.6 % by reducing the disease incidence to 16.9 %, compared to 72.8 % observed in control treatment. In addition, the isolate Bk7 showed the growth promotion efficacy of plant height (GPE, 46.4 %) and fresh weight (GPE, 84.3 %). Characterization of isolate Bk7 revealed its strong capability for biofilm formation, inorganic phosphate solubilization and production of high amounts of Indole-3 acetic acid, siderophores and ammonia in vitro. Results obtained from multiplex PCR assay confirmed the presence of five lipopeptide biosynthetic gene markers (srfAA, fenD, bmyB, bacA and ituC) in the genome of strain Bk7. Moreover, Real-time qPCR of these genes demonstrated that surfactin, iturin and bacylisin coding genes were highly expressed in response to P. fuscovaginae exposure in vitro. Rhizosphere bacterial strain Bk7 was identified as B. amyloliquefaciens strain Bk7 based on the analysis of 16S rDNA internal transcribed spacer sequences and a fatty acid methyl ester analysis. The results obtained from this study showed the potential usefulness of Bk7 as a biocontrol agent in disease control of rice brown sheath rot.

Infections by Enterobacter species are common and are multidrug resistant. The use of bactericidal surface materials such as copper has lately gained attention as an effective antimicrobial agent due to its deadly effects on bacteria, yeast, and viruses. The aim of the current study was to assess the antibacterial activity of copper surfaces against Enterobacter species. The antibacterial activity of copper surfaces was tested by overlying 5×106 CFU/ml suspensions of representative Enterobacter strains and comparing bacterial survival counts on copper surfaces at room temperature. Iron, stainless steel, and polyvinylchloride (PVC) were used as controls. The mechanisms responsible for bacterial killing on copper surfaces were investigated by a mutagenicity assay of the D-cycloserin (cyclA gene), single cell gel electrophoresis, a staining technique, and inductively coupled plasma mass spectroscopy. Copper yielded a significant decrease in the viable bacterial counts at 2 h exposure and a highly significant decrease at 4 h. Loss of cell integrity and a significantly higher influx of copper into bacterial cells exposed to copper surfaces, as compared to those exposed to the controls, were documented. There was no increase in mutation rate and DNA damage indicating that copper contributes to bacterial killing by adversely affecting cellular structure without directly targeting the genomic DNA. These findings suggest that copper’s antibacterial activity against Enterobacter species could be utilized in health care facilities and in food processing plants to reduce the bioburden, which would increase protection for susceptible members of the community.

The antifungal properties and mechanism of three types of chitosan against the rice sheath blight pathogen, Rhizoctonia solani, were evaluated. Each chitosan had strong antifungal activity against R. solani and protected rice seedlings from sheath blight, in particular, two types of acid-soluble chitosan caused a 60–91 % inhibition in mycelial growth, 31–84 % inhibition of disease incidence, and 66–91 % inhibition in lesion length. The mechanism of chitosan in protection of rice from R. solani pathogen was attributed to direct destruction of the mycelium, evidenced by scanning and transmission electron microscopic observations and pathogenicity testing; indirect induced resistance was evidenced by the changes in the activities of the defense-related phenylalanine ammonia lyase, peroxidase and polyphenol oxidase in rice seedling. To our knowledge, this is the first report on the antifungal activity of chitosan against rice R. solani.

This study was conducted to evaluate the potential of biofumigation in three Brassica crops including Brassica napus, Brassica juncea and Brassica campestris against potato stem rot caused by Sclerotinia sclerotiorum in field tests. Results from field trials carried out in three naturally infected potato fields during three cropping seasons of 2008–2010 showed that the Brassica crops used as green manure cover crops were able to significantly reduce disease incidence and mean percentage of dead plants (as a proportion of infected plants). Although results varied somewhat by field site and year, B. juncea generally provided the highest level of control, averaging greater than 55.6 % reduction in disease incidence over all fields and years, compared to average disease reductions of 31.6 and 45.8 % for the B. napus and B. campestris crop treatments, respectively. Furthermore reduction of dead plants averaged 61.6, 39.2 and 32.1 % for B. juncea, B. napus, and B. campestris, respectively. In this study, Brassica crops showed various significant inhibitory effects in different fields and years indicating that disease development is affected by other factors including environmental conditions.

The Gram-negative bacterium Acidovorax avenae subsp. avenae is the causal agent of bacterial brown stripe (BBS), which can cause severe diseases in many plants, including rice, with huge economic importance. Type IV pili (TFP) are hair-like appendages involved in several bacterial activities such as bacterial surface motility, surface adherence, colonization, biofilm formation, and virulence. The aim of our study is to characterize the association of A. avenae subsp. avenae TFP with BBS in rice. We generated a transposon (Tn5) mutant library. Then, an insertional mutagenesis on the background of this bacterium was identified as reduced pathogenicity. The confirmed inserted genetic region was into gene pilP, which encodes a TFP assembly protein. The pilP-deficient mutant strain seriously affected the motility twitching ability, biofilm formation and virulence. Collectively, our results clearly indicated that the pilP gene and TFP in A. avenae subsp. avenae play a key role in plant pathogenicity, twitching motility, and biofilm formation.

The in vitro antibacterial activity and mechanism of action of two kinds of acid-soluble chitosan and one water-soluble chitosan against apricot fruit rot pathogen Burkholderia seminalis was examined in this study. Results showed that water-soluble chitosan displayed limited antibacterial activity at four tested concentrations. However, two kinds of acid-soluble chitosan solution at 2.0 mg/mL had strong antibacterial activity against B. seminalis although weak antibacterial activity was observed at a concentration lower than 1 mg/mL. The antibacterial activity of acid-soluble chitosan may be due to membrane disruption, cell lysis, abnormal osmotic pressure, and additional chitosan coating around the bacteria based on integrity of cell membranes test, out membrane permeability assays and transmission electron microscopy observation. In addition, biofilm biomass were markedly reduced after treating with two kinds of acid-soluble chitosan at concentrations of 2.0 and 1.0 mg/mL for 3 and 12 h, indicating the importance of biofilm formation in the antibacterial mechanism of chitosan. Overall, the results clearly indicated that two kinds of acid-soluble chitosan had a potential to control the contamination of apricot fruits caused by B. seminalis.

The effect of rhizobacterium Burkholderia sp. strain R456 on the inhibition of Rhizoctonia solani, sheath blight of rice was examined. Results from this study indicated that strain R456 not only suppressed the in vitro mycelial growth of R. solani, but also reduced the incidence and severity of rice sheath blight under greenhouse conditions. However, similar to plant pathogenic strain LMG 1222T of Burkholderia cepacia, the type species of the genus, infiltration of tobacco leaves with cell suspension of strain R456 resulted in typical hypersensitivity reactions while the two bacterial strains were unable to cause disease symptoms on rice seedlings. The fatty acid methyl ester profile, sole carbon source utilization, and biochemical tests confirmed that the antagonistic rhizobacterium R456 is a member of the genus Burkholderia. Furthermore, strain R456 was differentiated from B. cepacia LMG 1222T and was identified as Burkholderia seminalis based on recA gene sequence analysis and multilocus sequence typing. In addition, this rhizobacterium had a lower proteolytic activity compared with that of the pathogenic B. cepacia LMG 1222T while no cblA and esmR marker genes were detected for the two bacterial strains. Overall, this is the first characterization of rhizobacterium B. seminalis that protected rice seedlings from infection by R. solani.

In 2010, the outbreak of a disease with symptoms similar to bacterial brown stripe was observed in rice seedlings planted in a perlite culture system in China. The causal bacterium was identified as Acidovorax avenae subsp. avenae based on its biochemical and physiological characteristics, cellular fatty acid composition, Biolog data, specific PCR detection and 16S rRNA gene sequence analysis. The bacterial isolates caused similar symptoms after inoculation of rice seedlings. This report is the first of bacterial brown stripe of rice in a soil-less culture system caused by A. avenae subsp. avenae in China.

Thirty-six pathogenetic bacterial strains were isolated from wilted mulberry plants in Hangzhou, Zhejiang province of China. The six representative strains were confirmed to be involved in more than one Enterobacter species by common bacteriological test, electron microscope observation, hypersensitive reaction, Koch’s postulates, physiological and biochemical test, biolog, fatty acid methyl esters analysis (FAMEs), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), 16s rRNA sequences analysis, and comparative analysis with 7 type strains and 3 reference strains. This is the first report on mulberry disease caused by Enterobacter spp. in the world providing new evidence on induction of the plant disease in this genus. The results are not only important in the mulberry disease management but also have significant scientific value for further studies of opportunistic human pathogens and environmental strains in Enterobacter.

A survey of Burkholderia cepacia complex (Bcc) species was conducted in sputum from cystic fibrosis (CF) patients in China. One hundred and four bacterial isolates were recovered on B. cepacia selective agar and 42 of them were assigned to Bcc by PCR assays. The species composition of the Bcc isolates from CF sputum was analyzed by a combination of recA-restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequencing. The results revealed that the 42 Bcc isolates belong to B. cepacia, B. cenocepacia and B. contaminans while predominant Bcc species was B. cenocepacia. This is the first report of B. contaminans from CF sputum in China. In addition, results from this study showed that chitosan solution at 10, 25, 50 and 100 μg/ml markedly inhibited the growth of the 16 representative isolates from the three different Bcc species, which indicated that chitosan was a potential bactericide against Bcc bacteria.

Mulberry wilt disease (MWD) was recently identified in Hangzhou, Zhejiang province, China. Typical symptoms of the disease are browning of vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the symptoms of bacterial wilt disease caused by Ralstonia solanacearum, symptoms of MWD generally started from the bottom of the plants and moved upward. In inoculation experiments, four selected MWD strains caused mulberry shoot leaf wilt, discoloration, and defoliation. They also induced whole plant leaf wilt, defoliation and dark brown discoloration of vascular tissue. Based on Biolog metabolic profiles, fatty acid methyl ester analysis (FAME) and sequence analysis of the partial 16S rDNA and rpoB genes four MWD strains were identified as members of the genus Enterobacter. The 16S rDNA and rpoB gene sequences revealed a close relationship among two isolates, R2-2 and R6-2, and the E. asburiae type strain JCM6051. The isolates showed >98% similarity to E. asburiae JCM6051 in their rpoB gene. These results indicated that isolates R2-2 and R6-2 belonged to E. asburiae. No similarity in 16S rDNA sequences above 97% was found between either of the remaining isolates, R11-2 or R18-2, and any recognized Enterobacter species, suggesting that the two isolates may represent novel Enterobacter species. rpoB gene similarity values between the isolates and Enterobacter spp. type strains were <98%, providing further evidence that the two isolates may represent a novel species within the Enterobacter. The causal agent for MWD was previously reported to be E. cloacae, however, this study found that other Enterobacter spp. (E. asburiae and Enterobacter sp.) also cause MWD.

The antibacterial activity of chitosan solution against Xanthomonas pathogenic bacteria isolated from Euphorbia pulcherrima was investigated in this study. Results showed that chitosan solution at 0.10 mg/mL markedly inhibited the growth of Xanthomonas pathogenic bacteria from different geographical origins. The antibacterial activity of chitosan solution against the selected strain R22580 of Xanthomonas axonopodis pv. poinsettiicola increased with the increase of chitosan concentration up to 0.10 mg/mL. The antibacterial activity of chitosan solution was also enhanced by the addition of NaCl, but was unaffected by the type of nutrient and sterilization. In addition, chitosan solution exhibited strong antibacterial activity against strain R22580 over the pH range of 5.5–7.0. Overall, the results indicated that chitosan was a potential bactericide against bacterial pathogens of E. pulcherrima under different environmental conditions.

Burkholderia cepacia is regarded as a genetically distinct but phenotypically similar bacteria group referring to Burkholderia cepacia complex (Bcc), which is found not only in clinic but also in rice growing environment. It is very important in microbial safety of rice for us to understand the genomovar status of Bcc. Genomovar analysis was performed among 87 Bcc isolates by means of Hae ?-recA RFLP assays and species-specific PCR tests. Three genomovars were found from the rice rhizosphere including?, ?B and ?, and genomovar ? was predominant. Genomovars?, ?A and ?B existed in the clinical samples, and genomovar ?A was the most popular. It showed that genomovar composition was different between the Bcc strains from the rice rhizosphere and clinical environment. Simultaneously, the results revealed the genetic diversity of Bcc strains from the rice rhizosphere, and genomovar ? referred as virulent species in clinic also existed in the rice rhizosphere.

•Chitosans and acetyl salicylic acid are able to induce systemic resistance against carrot rot.•The fungicidal effect of chitosans increased when the degree of N-deacetylation decreased.•Chitosans and acetyl salicylic acid increased SAR enzyme activities in inoculated carrots.This study was conducted to evaluate antifungal activity of three chemically different chitosans named as A, B and C at different concentrations against three isolates of Sclerotinia sclerotiorum, the causal agent of storage carrot rot, by in vitro and in vivo tests. In addition, potential of SAR (systemic acquired resistance) induced by the chitosans and acetyl salicylic acid (ASA) was assessed against the disease. The degree of N-deacetylation (DN) and the molecular weight of chitosans A, B and C were 85%–1129 kDa, 95%–521 kDa and 75%–607 kDa, respectively. The concentrations 2 g/L and 5 g/L of all chitosans were significantly able to decrease mycelial growth, sclerotia formation and carpogenic germination of the pathogen. Four hours and three days before inoculation with the pathogen, application of chitosans and ASA reduced severity of carrot rot in all isolates. However, mycelial growth and sclerotia formation of isolate 2 was more reduced than other isolates. In accordance with in vitro tests, chitosan B showed the highest inhibitory efficacy against the disease. The fungicidal effect of chitosan increased when the DN decreased and there was a negative correlation between the mycelial growth inhibition and the molecular weight of chitosans. The enzyme analysis showed that the activity of phenylalanine ammonia layse, polyphenoloxidase and peroxidase increase in the inoculated carrots after application of different chitosans and ASA.

Bacterial panicle blight caused by Burkholderia glumae is one of the most severe seed-borne bacterial diseases of rice in the world. Currently, this disease has affected many countries of Asia, Africa, South and North America. It is a typical example of the shifting from minor plant disease to major disease due to the changes of environmental conditions. Some virulent factors of B. glumae have been identified, including toxoflavins and lipases, whose productions are dependent on the TofI/TofR quorum-sensing system, and type III effectors. In spite of its economic significance, neither effective control measure for this disease nor resistant rice variety is currently available. In recent years, genomics, transcriptomics and other molecular methods have provided useful information for better understanding the molecular mechanisms underlying B. glumae virulence and the rice defence mechanisms against pathogens. For the prevention of this pathogen, our laboratory has developed a rapid and sensitive multiplex PCR assay for detecting and distinguishing B. glumae from other Burkholderia species. This improved understanding of B. glumae will shed new light on bacterial panicle blight disease management.

Burkholderia glumae causing seedling rot and grain rot of rice was listed as a plant quarantine disease of China in 2007. It's quite necessary to set up effective detection methods for the pathogen to manage further dispersal of this disease. The present study combined the real-time PCR method with classical PCR to increase the detecting efficiency, and to develop an accurate, rapid and sensitive method to detect the pathogen in the seed quarantine for effective management of the disease. The results showed that all the tested strains of B. glumae produced about 139 bp specific fragments by the real-time PCR and the general PCR methods, while others showed negative PCR result. The bacteria could be detected at the concentrations of 1×104 CFU/mL by general PCR method and at the concentrations below 100 CFU/mL by real-time fluorescence PCR method. B. glumae could be detected when the inoculated and healthy seeds were mixed with a proportion of 1:100.

A survey on isolation and detection of the casual organism of bacterial grain rot of rice was conducted during 1997–2006. In 2006, six pathogenic bacterial strains were isolated from two symptomless seed samples of rice (Oryza sativa L.) originally produced in Hainan Province and then planted in Zhejiang Province, China. They were identified as Burkholderia glumae which is the causal organism of bacterial grain rot of rice by physiological characteristics, colony morphology, pathogenicity test, Biolog, fatty acid methyl ester (FAME) analysis and RAPD-PCR compared with the four standard reference strains. It is confirmed that there is the infection of B. glumae in so-called ‘health looking seeds’.

The Burkholderia cepacia complex (Bcc) consists of 17 closely related multidrug resistant bacterial species that are difficult to eradicate. Copper has recently gained attention as an antimicrobial agent because of its inhibitory effects on bacteria, yeast, and viruses. The objective of this study was to evaluate the antibacterial activity of copper surfaces and copper powder against members of the B. cepacia complex. The antibacterial activity of different copper surfaces was evaluated by incubating them with Bcc strain suspensions (5 × 107 cfu/ml). The bacterial survival counts were calculated and the data for various copper surfaces were compared to the data for stainless steel and polyvinylchloride, which were used as control surfaces. The antibacterial activity of copper powder was determined with the diffusimetrical technique and the zone of inhibition was evaluated with paper disks. A single cell gel electrophoresis assay, staining assays, and inductively coupled plasma mass spectroscopy were performed to determine the mechanism responsible for the bactericidal activity. The results showed a significant decrease in the viable bacterial count after exposure to copper surfaces. Moreover, the copper powder produced a large zone of inhibition and there was a significantly higher influx of copper ions into the bacterial cells that were exposed to copper surfaces compared to the controls. The present study demonstrates that metallic copper has an antibacterial effect against Bcc bacteria and that copper adversely affects the bacterial cellular structure, thus resulting in cell death. These findings suggest that copper could be utilized in health care facilities to reduce the bioburden of Bcc species, which may protect susceptible members of the community from bacterial infection.