Two miniature inverted-repeat transposable elements（MITEs）, MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus＇ known to be associated with citrus Huanglongbing（HLB, yellow shoo...Two miniature inverted-repeat transposable elements（MITEs）, MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus＇ known to be associated with citrus Huanglongbing（HLB, yellow shoot disease）. MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus＇ isolates from China, the United States and Brazil. Out of the 12 sequence types, three（T4, T5-2, T6） were reported for the first time. Recombination events were found in the two unique sequence types（T5-2 and T6） which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, S？o Paulo isolates and a few Florida isolates.展开更多
Citrus Huanglongbing （HLB,yellow shoot disease） was first observed in the coastal Chaoshan Plain of Guangdong Province,China,in the late 19th century based on descriptions of yellow shoot symptoms. “Candidatus Libe...Citrus Huanglongbing （HLB,yellow shoot disease） was first observed in the coastal Chaoshan Plain of Guangdong Province,China,in the late 19th century based on descriptions of yellow shoot symptoms. “Candidatus Liberibacter asiaticus”has been considered as a putative pathogen associated with HLB since 1994.Information about the curent prevalence of this bacterium is important for HLB control in Guangdong and also provides useful reference for HLB study elsewhere.In 2007,we collected HLB symptomatic citrus samples from 16 cultivars in 12 prefecture cities,mostly in the north and west regions of Guangdong,where major citrus fruits are currently produced.Among the 359 samples collected,241 （67.1%） were positive for “Ca.L.asiaticus”,distributed in 15 out of the 16 cultivars from all 12 cities,indicating the widespread prevalence of “Ca.L.asiaticus” in Guangdong Province.The detection rates varied from 16.7 to 100% depending on location and cultivar.Lower detection rates were found in newer citrus cultivation cities among the previously less popular but now promoted cultivars.In reviewing the citrus management and pest control practice,we believe that infected nursery stocks play a key role in the current spread of “Ca.L.asiaticus”.展开更多
Candidatus Liberibacter asiaticus （CaLas）, an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing （HLB） in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequ...Candidatus Liberibacter asiaticus （CaLas）, an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing （HLB） in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder （Cuscuta spp.）. To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.展开更多
‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitr...‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘Changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.展开更多
Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The disease is caused by Candidatus Liberibacter spp., which is vectored by the psyllids Diaphorina citri Kuwayama and Trioza erytreae. Secretor...Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The disease is caused by Candidatus Liberibacter spp., which is vectored by the psyllids Diaphorina citri Kuwayama and Trioza erytreae. Secretory proteins are important in bacterial pathogenesis and structure components. Some of them are expressed at a high level. To obtain the highly-expressed secretory protein genes (SPGs) for antiserum preparation, six candidate SPGs were chosen from Candidatus Liberibacter asiaticus by bioinformatic analysis and were further tested by qPCR and RT-qPCR methods, respectively. The result showed that two SPGs, 408 and pap (both are Flp pilus assembly protein genes), have relative high amounts of DNA and RNA transcripts of early HLB-infected green orange leaves. The 408 and pap genes were further constructed into the plant expression vector pCAMBIA1300 (GV1300: GFP) and expressed in tobacco leaf epidermal cells for subcellular localization analysis. The transient expression results indicated that the 408 protein is located in the nuclei and cytoplasm of tobacco leaf cells. However, the pap protein is located in the cytoplasm of tobacco leaf cells, which may help the pathogen invade into plant cells. This research is an important foundation for the preparation of the antiserum against Candidatus Liberibacter asiaticus and the early detection of HLB disease.展开更多
An active miniature inverted-repeat transposable element(MITE),MCL as-A,was previously identified from'Candidatus Liberibacter asiaticus'known to be associated with citrus Huanglongbing(HL B,yellow shoot disea...An active miniature inverted-repeat transposable element(MITE),MCL as-A,was previously identified from'Candidatus Liberibacter asiaticus'known to be associated with citrus Huanglongbing(HL B,yellow shoot disease).To explore the recent transposition status of MCLas-A,389'Ca.L.asiaticus'strains collected from nine regions in China were amplifed using a specific primer set and three representative'Ca.L.asiaticus'strains were analyzed by next-generation sequencing(NGS)approach.PCR and genomic analysis showed that the entire MCLas A was only present in 1.80%(7/389)and the jumping-out type of the MITE was predominant(8123%)in samples tested,suggesting high frequency transposition occurred in'Ca.L.asiaticus'strains recently collected from China.Biological roles of transposition of the active MITE remain to be determined.展开更多
The psyllid Bactericera cockerelli was first reported in New Zealand in 2006 and spread quickly throughout all potato growing regions.In 2009,B.cockerelli was associated with the plant pathogenic bacterium Candidatus ...The psyllid Bactericera cockerelli was first reported in New Zealand in 2006 and spread quickly throughout all potato growing regions.In 2009,B.cockerelli was associated with the plant pathogenic bacterium Candidatus Liberibacter solanacearum,the putative causal agent of zebra chip disease in potato.Both the psyllid and the bacterium have non-crop host plants which can serve as reservoirs when the crop is not available.Growers apply different management strategies that fit integrated pest management programmes to manage B.cockerelli and subsequently lower incidence of zebra chip disease in potato crops.Despite best management efforts,complete control of B.cockerelli and zero incidence of zebra chip disease are not achievable at the current time.展开更多
Huanglongbing(HLB)is the most devastating disease for citrus worldwide.Candidatus Liberibacter asiaticus(C Las),vectored by Asian citrus psyllid(ACP,Diaphorina citri Kuwayama),is the most common pathogen causing the d...Huanglongbing(HLB)is the most devastating disease for citrus worldwide.Candidatus Liberibacter asiaticus(C Las),vectored by Asian citrus psyllid(ACP,Diaphorina citri Kuwayama),is the most common pathogen causing the disease.Commercial citrus varieties are highly susceptible to HLB,whereas trifoliate orange(Poncirus trifoliata)is considered highly tolerant to HLB.An F1 segregating population and their parent trifoliate orange and sweet orange,which had been exposed to intense HLB pressure for three years,was evaluated for disease symptoms,ACP colonization,C Las titer and tree vigor repeatedly for two to three years.Trifoliate orange and sweet orange showed significant differences for most of the phenotypic traits,and the F1 population exhibited a large variation.A high-density SNP-based genetic map with 1402 markers was constructed for trifoliate orange,which exhibited high synteny and high coverage of its reference genome.A total of 26 quantitative trait locus(QTLs)were identified in four linkage groups LG-t6,LG-t7,LG-t8 and LG-t9,of which four QTL clusters exhibit a clear co-localization of QTLs associated with different traits.Through genome-wide analysis of gene expression in response to C Las infection in‘Flying Dragon’and‘Larger-Flower DPI-50-7’trifoliate orange,85 differentially expressed genes were found located within the QTL clusters.Among them,seven genes were classified as defense or immunity protein which exhibited the highest transcriptional change after C Las infection.Our results indicate a quantitative genetic nature of HLB tolerance and identified candidate genes that should be valuable for searching for genetic solutions to HLB through breeding or genetic engineering.展开更多
基金provided by the Special Fund for Agro-Scientific Research in the Public Interest, China (201003067-02)the Natural Science Foundation Project of CQ CSTC (cstc2012jj A80025)the Fundamental Research Funds for the Central Universities, China (XDJK2014A001, XDJK2014D004)
文摘Two miniature inverted-repeat transposable elements（MITEs）, MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus＇ known to be associated with citrus Huanglongbing（HLB, yellow shoot disease）. MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus＇ isolates from China, the United States and Brazil. Out of the 12 sequence types, three（T4, T5-2, T6） were reported for the first time. Recombination events were found in the two unique sequence types（T5-2 and T6） which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, S？o Paulo isolates and a few Florida isolates.
基金supported by the Chinese Modern Agricultural Technology Systems(CARS-27)the Special Fundfor Agro-Scientific Research in the Public Interest,China(2010003067)the project of Asian Citrus Psyllid and Huanglongbing Field Research and Outreach by North Carolina State University and United State Department of Agriculture(2010-0195-02)
文摘Citrus Huanglongbing （HLB,yellow shoot disease） was first observed in the coastal Chaoshan Plain of Guangdong Province,China,in the late 19th century based on descriptions of yellow shoot symptoms. “Candidatus Liberibacter asiaticus”has been considered as a putative pathogen associated with HLB since 1994.Information about the curent prevalence of this bacterium is important for HLB control in Guangdong and also provides useful reference for HLB study elsewhere.In 2007,we collected HLB symptomatic citrus samples from 16 cultivars in 12 prefecture cities,mostly in the north and west regions of Guangdong,where major citrus fruits are currently produced.Among the 359 samples collected,241 （67.1%） were positive for “Ca.L.asiaticus”,distributed in 15 out of the 16 cultivars from all 12 cities,indicating the widespread prevalence of “Ca.L.asiaticus” in Guangdong Province.The detection rates varied from 16.7 to 100% depending on location and cultivar.Lower detection rates were found in newer citrus cultivation cities among the previously less popular but now promoted cultivars.In reviewing the citrus management and pest control practice,we believe that infected nursery stocks play a key role in the current spread of “Ca.L.asiaticus”.
基金supported by the earmarked fund for China Agriculture Research System (CARS-27)the Special Fund for Agro-Scientific Research in the Public Interest, China (2010003067)
文摘Candidatus Liberibacter asiaticus （CaLas）, an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing （HLB） in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder （Cuscuta spp.）. To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.
基金supported by the National Key R&D Program of China (2018YFD0201500 and 2018YFD1000300)the National Natural Science Foundation of China (31972393)the China Agriculture Research System of MOF and MARA (CARS-26)
文摘‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘Changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.
文摘Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The disease is caused by Candidatus Liberibacter spp., which is vectored by the psyllids Diaphorina citri Kuwayama and Trioza erytreae. Secretory proteins are important in bacterial pathogenesis and structure components. Some of them are expressed at a high level. To obtain the highly-expressed secretory protein genes (SPGs) for antiserum preparation, six candidate SPGs were chosen from Candidatus Liberibacter asiaticus by bioinformatic analysis and were further tested by qPCR and RT-qPCR methods, respectively. The result showed that two SPGs, 408 and pap (both are Flp pilus assembly protein genes), have relative high amounts of DNA and RNA transcripts of early HLB-infected green orange leaves. The 408 and pap genes were further constructed into the plant expression vector pCAMBIA1300 (GV1300: GFP) and expressed in tobacco leaf epidermal cells for subcellular localization analysis. The transient expression results indicated that the 408 protein is located in the nuclei and cytoplasm of tobacco leaf cells. However, the pap protein is located in the cytoplasm of tobacco leaf cells, which may help the pathogen invade into plant cells. This research is an important foundation for the preparation of the antiserum against Candidatus Liberibacter asiaticus and the early detection of HLB disease.
基金Funded by the National Natural Science Foundation of China(31671992,31871925)the National Key R&D Program of China(2018YFD0201500)the Key R&D Program of Guangxi,China(Gui Ke AA18118046).
文摘An active miniature inverted-repeat transposable element(MITE),MCL as-A,was previously identified from'Candidatus Liberibacter asiaticus'known to be associated with citrus Huanglongbing(HL B,yellow shoot disease).To explore the recent transposition status of MCLas-A,389'Ca.L.asiaticus'strains collected from nine regions in China were amplifed using a specific primer set and three representative'Ca.L.asiaticus'strains were analyzed by next-generation sequencing(NGS)approach.PCR and genomic analysis showed that the entire MCLas A was only present in 1.80%(7/389)and the jumping-out type of the MITE was predominant(8123%)in samples tested,suggesting high frequency transposition occurred in'Ca.L.asiaticus'strains recently collected from China.Biological roles of transposition of the active MITE remain to be determined.
基金supported by the New Zealand Institute for Plant and Food Research Limited Internal Core fundingthe Plant Biosecurity Cooperative Research Centre (Australia) (CRC2079)the Sustainable Farming Fund (New Zealand, 09/143, 12/058, and 404861)Potatoes New Zealand Inc.
文摘The psyllid Bactericera cockerelli was first reported in New Zealand in 2006 and spread quickly throughout all potato growing regions.In 2009,B.cockerelli was associated with the plant pathogenic bacterium Candidatus Liberibacter solanacearum,the putative causal agent of zebra chip disease in potato.Both the psyllid and the bacterium have non-crop host plants which can serve as reservoirs when the crop is not available.Growers apply different management strategies that fit integrated pest management programmes to manage B.cockerelli and subsequently lower incidence of zebra chip disease in potato crops.Despite best management efforts,complete control of B.cockerelli and zero incidence of zebra chip disease are not achievable at the current time.
基金Public service sectors(agriculture)special project(Grant No.201003067)from Ministry of Agriculture of the People's Republic of ChinaNational Natural Science Fund(Grant No.31201480)by National Natural Science Foundation of China(NSFC)
基金supported by grants from the Citrus Research and Development Foundation,USA(Grant No.CRDF#15-010)the New Varieties Development and Management Corporation(NVDMC),on behalf of the Florida citrus industry,USA,the Fundamental Research Funds for the Central Universities,China(Grant No.2022CDJXY-004)from the USDA-NIFA-SCRI,USA(Grant No.2015-70016-2302).
文摘Huanglongbing(HLB)is the most devastating disease for citrus worldwide.Candidatus Liberibacter asiaticus(C Las),vectored by Asian citrus psyllid(ACP,Diaphorina citri Kuwayama),is the most common pathogen causing the disease.Commercial citrus varieties are highly susceptible to HLB,whereas trifoliate orange(Poncirus trifoliata)is considered highly tolerant to HLB.An F1 segregating population and their parent trifoliate orange and sweet orange,which had been exposed to intense HLB pressure for three years,was evaluated for disease symptoms,ACP colonization,C Las titer and tree vigor repeatedly for two to three years.Trifoliate orange and sweet orange showed significant differences for most of the phenotypic traits,and the F1 population exhibited a large variation.A high-density SNP-based genetic map with 1402 markers was constructed for trifoliate orange,which exhibited high synteny and high coverage of its reference genome.A total of 26 quantitative trait locus(QTLs)were identified in four linkage groups LG-t6,LG-t7,LG-t8 and LG-t9,of which four QTL clusters exhibit a clear co-localization of QTLs associated with different traits.Through genome-wide analysis of gene expression in response to C Las infection in‘Flying Dragon’and‘Larger-Flower DPI-50-7’trifoliate orange,85 differentially expressed genes were found located within the QTL clusters.Among them,seven genes were classified as defense or immunity protein which exhibited the highest transcriptional change after C Las infection.Our results indicate a quantitative genetic nature of HLB tolerance and identified candidate genes that should be valuable for searching for genetic solutions to HLB through breeding or genetic engineering.
