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 ...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 1×10^4 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.展开更多
The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three...The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv.oryzae, X. oryzae pv. oryzicola, and Burkholderia glumae. The unique PCR primer sets were designed from portions of a putative glycosyltransferase gene of X. oryzae pv. oryzae, an Avr Rxo gene of X. oryzae pv. oryzicola, and an internal transcribed spacer(ITS) sequence of B. glumae. Using a multiplex PCR assay, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected in one PCR reaction that contained the newly developed primer set mix. Using SYBR Green real-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected at 1, 1, and 10 fg μL-1, respectively. These newly designed molecular assays are sensitive and could be reliable tools for pathogen detection and disease forecasting.展开更多
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 ri...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＇.展开更多
Horizontal gene transfer(HGT)has been proved a major driving force in prokaryotic evolution.However,the molecular functions of these transferred genes in pathogenic bacteria especially plant pathogenic bacteria are st...Horizontal gene transfer(HGT)has been proved a major driving force in prokaryotic evolution.However,the molecular functions of these transferred genes in pathogenic bacteria especially plant pathogenic bacteria are still not fully investigated.In this study,the whole-genome in silico analysis was performed and found a syringopeptin synthetase(syp)homolog in Burkholderia glumae,which can cause bacterial panicle blight in rice,was predicted to be horizontally transferred from Pseudomonas ancestor with solid confidence by phylogenetic analysis.The comprehensive molecular experiments were performed to study the potential role of this gene in B.glumae.Inoculation of rice panicles with the syp mutant resulted in 60%lower disease index compared with the wild type(WT)parent strain,suggesting the requirement of syp for the full virulence of B.glumae.Chromatography analysis of exudates from B.glumae showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants.All these data raise the possibility of HGT phenomenon in shaping the virulence and adaptation of B.glumae over evolutionary time.展开更多
The present study combined the fluoride ion-selective electrode （ISE） method with classical PCR to increase the detection efficiency of Burkholderia glumae. The four antisera of rabbit anti whole cell of B. glumae h...The present study combined the fluoride ion-selective electrode （ISE） method with classical PCR to increase the detection efficiency of Burkholderia glumae. The four antisera of rabbit anti whole cell of B. glumae had been successfully obtained by polyclonal antibody technique, which were ASBgl4, ASBg32, ASBg26 and ASBg04, respectively. The titers of four antisera reached 1：32 in ODD test and 1：5 120 in agglutination reaction. All antisera were identified to be qualified antibodies through combined method. The specificity of ASBgl4 and ASBg32 was very high, but that of ASBg26 and ASBg04 was not very satisfying. The results showed that all tested strains of B. glumae produced 500 bp specific fragments by direct PCR and immunocapture PCR detection, while others showed nega- tive PCR result. Comparing the sensitivity of these two detection methods, direct PCR could detect about 1 x 105 cfu/mL of bacterial suspension while immunocap- ture PCR could detect as few as 1× 103 cfu/mL, 102 times higher than direct PCR in sensitivity. In the detecting experiments of artificially inoculated seeds, at least two seeds were required in direct PCR, while only one seed was enough for immunocapture PCR detection.展开更多
基金supported by National Natural Science Foundation of China(Grant No.30671397 and No.30871655)the Public Beneficial Research Project of Agricultural Ministry,China(Grant No.nyhyzx07-056)
文摘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 1×10^4 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.
基金support of the National 863 Project (2012AA021601)the New Seedling program for graduate students of Zhejiang Province (2012R409012)
文摘The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv.oryzae, X. oryzae pv. oryzicola, and Burkholderia glumae. The unique PCR primer sets were designed from portions of a putative glycosyltransferase gene of X. oryzae pv. oryzae, an Avr Rxo gene of X. oryzae pv. oryzicola, and an internal transcribed spacer(ITS) sequence of B. glumae. Using a multiplex PCR assay, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected in one PCR reaction that contained the newly developed primer set mix. Using SYBR Green real-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected at 1, 1, and 10 fg μL-1, respectively. These newly designed molecular assays are sensitive and could be reliable tools for pathogen detection and disease forecasting.
基金the National Natural Science Foundation of China (Grant No. 30671397)
文摘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＇.
基金the National Key R&D Program of China(2018YFD0201202 and 2017YFD0201108)the Agri-X Interdisciplinary Fund of Shanghai Jiao Tong University,China(Agri-X2017010)+3 种基金the State Key Laboratory for Biology of Plant Diseases and Insect Pests of Shanghai Jiao Tong University(SKLOF201802)the Shanghai Committee of Science and Technology(19390743300)the National Natural Science Foundation of China(31200003 and 31770772)Joint Research Funds for Translational Medicine at Shanghai Jiao Tong University(ZH2018ZDA06).
文摘Horizontal gene transfer(HGT)has been proved a major driving force in prokaryotic evolution.However,the molecular functions of these transferred genes in pathogenic bacteria especially plant pathogenic bacteria are still not fully investigated.In this study,the whole-genome in silico analysis was performed and found a syringopeptin synthetase(syp)homolog in Burkholderia glumae,which can cause bacterial panicle blight in rice,was predicted to be horizontally transferred from Pseudomonas ancestor with solid confidence by phylogenetic analysis.The comprehensive molecular experiments were performed to study the potential role of this gene in B.glumae.Inoculation of rice panicles with the syp mutant resulted in 60%lower disease index compared with the wild type(WT)parent strain,suggesting the requirement of syp for the full virulence of B.glumae.Chromatography analysis of exudates from B.glumae showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants.All these data raise the possibility of HGT phenomenon in shaping the virulence and adaptation of B.glumae over evolutionary time.
文摘The present study combined the fluoride ion-selective electrode （ISE） method with classical PCR to increase the detection efficiency of Burkholderia glumae. The four antisera of rabbit anti whole cell of B. glumae had been successfully obtained by polyclonal antibody technique, which were ASBgl4, ASBg32, ASBg26 and ASBg04, respectively. The titers of four antisera reached 1：32 in ODD test and 1：5 120 in agglutination reaction. All antisera were identified to be qualified antibodies through combined method. The specificity of ASBgl4 and ASBg32 was very high, but that of ASBg26 and ASBg04 was not very satisfying. The results showed that all tested strains of B. glumae produced 500 bp specific fragments by direct PCR and immunocapture PCR detection, while others showed nega- tive PCR result. Comparing the sensitivity of these two detection methods, direct PCR could detect about 1 x 105 cfu/mL of bacterial suspension while immunocap- ture PCR could detect as few as 1× 103 cfu/mL, 102 times higher than direct PCR in sensitivity. In the detecting experiments of artificially inoculated seeds, at least two seeds were required in direct PCR, while only one seed was enough for immunocapture PCR detection.
