Polymerase chain reaction (PCR) approach based on newly designed primers, JYF5/JYR5, wasapplied for specific detection of Xanthomonas axonopodis pv.citri(Xac). The efficiencyand reliability of PCR method were compared...Polymerase chain reaction (PCR) approach based on newly designed primers, JYF5/JYR5, wasapplied for specific detection of Xanthomonas axonopodis pv.citri(Xac). The efficiencyand reliability of PCR method were compared with dot immunobinding assay (DIA) andclassical pathogenicity test techniques for detecting suspensions of pure cells of Xacand soaking sap of citrus tissues. Detection sensitivity of PCR was about 4.5 cells or1.56 pg target DNA per reaction which was higher than that of DIA (ca. 450 cells per dot).These three techniques (PCR assay, DIA and Pathogenecity test) could always detect Xacfrom symptomatic citrus samples. Different performances were obtained from citrusmaterials without symptoms, and the positive detection frequency was PCR, DIA andpathogenicity test.展开更多
The local auxin distribution characteristics in the roots,stems,and leaves of stably transformed plantlets of trifoliate orange(Poncirus trifoliata)with auxin reporter system DR5::GUS-YFP were elucidated in this resea...The local auxin distribution characteristics in the roots,stems,and leaves of stably transformed plantlets of trifoliate orange(Poncirus trifoliata)with auxin reporter system DR5::GUS-YFP were elucidated in this research.The auxin response maxima could be observed in the apex of the root tip,primary phloem of the tender stem,and the margin of the young leaves according to the activity of theβ-glucuronidase(GUS)reporter gene triggered by the auxin responsive DR5 promoter.Auxin responses in the apex of the root tips increased when treated with synthetic auxin 1-naphthylacetic acid(NAA),but decreased when treated with the auxin polar transportation inhibitor 2,3,5-triiodobenzoic acid(TIBA).These results indicated that the DR5 reporter system worked in P.trifoliata for auxin distribution and response observation.Trifoliate orange is highly susceptible to citrus canker disease.Auxin accumulation was observed visually in the invasion sites of the detached leaves inoculated with Xanthomonas axonopodis pv.citri(Xac)by GUS staining;the upregulated expression of the YFP,GH3.1,GH3.9,and SAUR genes assessed by quantitative real-time PCR(qRT-PCR)also identified auxin accumulation in the inoculated tissues following Xac infection.Overall,these findings indicated that the plantlets of P.trifoliata engineered with the auxin reporter gene provided a promising system for studying auxin responses during Xac infection.展开更多
Xanthomonas citri pv.citri(Xcc),a gram-negative bacterium,is the causal agent of citrus canker,one of the most devastating diseases threatening the citrus industry worldwide.Understanding the diversity and population ...Xanthomonas citri pv.citri(Xcc),a gram-negative bacterium,is the causal agent of citrus canker,one of the most devastating diseases threatening the citrus industry worldwide.Understanding the diversity and population structure of Xcc is a prerequisite for disease epidemiological monitoring and effective disease management.Recent characterization of the clustered regularly interspaced short palindromic repeats(CRISPR)/cas(CRISPR-associated proteins genes)system with a highly variable repeat number among species provides a new molecular typing method for bacterial genetic analysis.In this study,we performed systematic in silico analyses of 28 Xcc genomes and identified a credible CRISPR/cas in Xcc strains.Further analysis of CRISPR polymorphisms(repeat number and spacer types)in 129 Xcc A strains collected from six provinces in China identified 15 types of CRISPR arrays with 25 spacers.Phylogenetic analysis of Xcc strains based on the CRISPR locus produced a more reliable and accurate typing result compared to the commonly used loci.In addition,seven associated cas genes—cas1,cas2,cas3,cas4,cas5,cas7(csd2),and cas8(csd1)—were found located adjacent to the CRISPR array.BLAST results showed>99%similarity of seven cas genes among Xcc strains.Homology analysis of spacer sequences showed that six spacers had possible phage/prophage origin.The characterization of the CRISPR/cas system among Xcc strains provided an updated strain typing method for Xcc diversity analysis and yielded a panoramic view of CRISPR evolution for further studies of Xcc-phage interactions.展开更多
A mutant library of the bacterium Xanthomonas citri subsp. citri strain 306 pathotype A (Xac), the causative agent of most aggressive Asiatic type A citrus canker, was screened regarding altered canker symptoms after ...A mutant library of the bacterium Xanthomonas citri subsp. citri strain 306 pathotype A (Xac), the causative agent of most aggressive Asiatic type A citrus canker, was screened regarding altered canker symptoms after inoculations into Citrus sinensis and Citrus limonia host leaves. Twenty-six mutants have shown phenotypic virulence changes and have respectively knocked out gene identified by sequencing. In vivo growth curves were obtained for nine mutants to quantify how the mutations could affect pathogen’s adaptability to growth inside and attack host plant infected tissue. Among identified genes in mutated strains, we could find those that until now had not been reported as being involved in Xac adaptation and/or virulence, such as predicted to encode for xylose repressor-like protein (XACΔxylR), Fe-S oxidoredutase (XACΔaslB), helicase IV (XACΔhelD), ubiquinol cytochrome c oxidoreductase iron-sulfur subunit (XACΔpetA), chromosome partitioning protein (XACΔparB) and cell division protein FtsB (XACΔftsB), in addition to genes predicted to encode for hypothetical proteins. The new genes found in this study as being relevant to adaptation and virulence, improve the understanding of Xac fitness during citrus plant attack and canker symptoms development.展开更多
Mango bacterial canker is caused by Xanthomonas campestris pv. mangiferaeindicae. During 2009 and 2013,leaves,twigs and fruits of mango were collected from commercial and experimental mango fields with typical canker ...Mango bacterial canker is caused by Xanthomonas campestris pv. mangiferaeindicae. During 2009 and 2013,leaves,twigs and fruits of mango were collected from commercial and experimental mango fields with typical canker symptoms in Hainan,Guangxi,Guangdong and Szechwan Provinces of China. The causal agent was identified as X. campestris pv. mangiferaeindicae through KC semi-selective medium isolation,pathogenicity tests,and sequencing of the gyrB gene.展开更多
文摘Polymerase chain reaction (PCR) approach based on newly designed primers, JYF5/JYR5, wasapplied for specific detection of Xanthomonas axonopodis pv.citri(Xac). The efficiencyand reliability of PCR method were compared with dot immunobinding assay (DIA) andclassical pathogenicity test techniques for detecting suspensions of pure cells of Xacand soaking sap of citrus tissues. Detection sensitivity of PCR was about 4.5 cells or1.56 pg target DNA per reaction which was higher than that of DIA (ca. 450 cells per dot).These three techniques (PCR assay, DIA and Pathogenecity test) could always detect Xacfrom symptomatic citrus samples. Different performances were obtained from citrusmaterials without symptoms, and the positive detection frequency was PCR, DIA andpathogenicity test.
基金the National Natural Science Foundation of China(Grant No.31660564)the science and technology project of Jiangxi province(Grant Nos.20161BBF60063,151008).
文摘The local auxin distribution characteristics in the roots,stems,and leaves of stably transformed plantlets of trifoliate orange(Poncirus trifoliata)with auxin reporter system DR5::GUS-YFP were elucidated in this research.The auxin response maxima could be observed in the apex of the root tip,primary phloem of the tender stem,and the margin of the young leaves according to the activity of theβ-glucuronidase(GUS)reporter gene triggered by the auxin responsive DR5 promoter.Auxin responses in the apex of the root tips increased when treated with synthetic auxin 1-naphthylacetic acid(NAA),but decreased when treated with the auxin polar transportation inhibitor 2,3,5-triiodobenzoic acid(TIBA).These results indicated that the DR5 reporter system worked in P.trifoliata for auxin distribution and response observation.Trifoliate orange is highly susceptible to citrus canker disease.Auxin accumulation was observed visually in the invasion sites of the detached leaves inoculated with Xanthomonas axonopodis pv.citri(Xac)by GUS staining;the upregulated expression of the YFP,GH3.1,GH3.9,and SAUR genes assessed by quantitative real-time PCR(qRT-PCR)also identified auxin accumulation in the inoculated tissues following Xac infection.Overall,these findings indicated that the plantlets of P.trifoliata engineered with the auxin reporter gene provided a promising system for studying auxin responses during Xac infection.
基金supported by Chinese Modern Agricultural Technology Systems (Grant No.CARS-26)。
文摘Xanthomonas citri pv.citri(Xcc),a gram-negative bacterium,is the causal agent of citrus canker,one of the most devastating diseases threatening the citrus industry worldwide.Understanding the diversity and population structure of Xcc is a prerequisite for disease epidemiological monitoring and effective disease management.Recent characterization of the clustered regularly interspaced short palindromic repeats(CRISPR)/cas(CRISPR-associated proteins genes)system with a highly variable repeat number among species provides a new molecular typing method for bacterial genetic analysis.In this study,we performed systematic in silico analyses of 28 Xcc genomes and identified a credible CRISPR/cas in Xcc strains.Further analysis of CRISPR polymorphisms(repeat number and spacer types)in 129 Xcc A strains collected from six provinces in China identified 15 types of CRISPR arrays with 25 spacers.Phylogenetic analysis of Xcc strains based on the CRISPR locus produced a more reliable and accurate typing result compared to the commonly used loci.In addition,seven associated cas genes—cas1,cas2,cas3,cas4,cas5,cas7(csd2),and cas8(csd1)—were found located adjacent to the CRISPR array.BLAST results showed>99%similarity of seven cas genes among Xcc strains.Homology analysis of spacer sequences showed that six spacers had possible phage/prophage origin.The characterization of the CRISPR/cas system among Xcc strains provided an updated strain typing method for Xcc diversity analysis and yielded a panoramic view of CRISPR evolution for further studies of Xcc-phage interactions.
文摘A mutant library of the bacterium Xanthomonas citri subsp. citri strain 306 pathotype A (Xac), the causative agent of most aggressive Asiatic type A citrus canker, was screened regarding altered canker symptoms after inoculations into Citrus sinensis and Citrus limonia host leaves. Twenty-six mutants have shown phenotypic virulence changes and have respectively knocked out gene identified by sequencing. In vivo growth curves were obtained for nine mutants to quantify how the mutations could affect pathogen’s adaptability to growth inside and attack host plant infected tissue. Among identified genes in mutated strains, we could find those that until now had not been reported as being involved in Xac adaptation and/or virulence, such as predicted to encode for xylose repressor-like protein (XACΔxylR), Fe-S oxidoredutase (XACΔaslB), helicase IV (XACΔhelD), ubiquinol cytochrome c oxidoreductase iron-sulfur subunit (XACΔpetA), chromosome partitioning protein (XACΔparB) and cell division protein FtsB (XACΔftsB), in addition to genes predicted to encode for hypothetical proteins. The new genes found in this study as being relevant to adaptation and virulence, improve the understanding of Xac fitness during citrus plant attack and canker symptoms development.
基金Supported by the Ministry of Science and Technology and the Ministry of Agriculture of China(2014hzs1J007-2)
文摘Mango bacterial canker is caused by Xanthomonas campestris pv. mangiferaeindicae. During 2009 and 2013,leaves,twigs and fruits of mango were collected from commercial and experimental mango fields with typical canker symptoms in Hainan,Guangxi,Guangdong and Szechwan Provinces of China. The causal agent was identified as X. campestris pv. mangiferaeindicae through KC semi-selective medium isolation,pathogenicity tests,and sequencing of the gyrB gene.
