青海省马铃薯软腐病病原菌的鉴定
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摘要
为明确引发青海省马铃薯(Solanum tuberosum L.)软腐病的病原菌,通过对病原菌形态、生理生化特性、16SrRNA基因序列以及胡萝卜果胶杆菌胡萝卜亚种(Pectobacterium carotovorum subsp.carotovorum)的pECC2F序列特征分析,对青海省5个地区马铃薯软腐病病样的病原菌进行病原学鉴定。结果表明,分离的5个菌株均能引发马铃薯软腐病,病原菌鉴定为胡萝卜果胶杆菌胡萝卜亚种。生理生化研究表明,该病原菌菌株均可在37℃生长,能使明胶液化,具有耐盐性(50g/L NaCl),对红霉素不敏感,过氧化氢酶反应显示为阳性,蔗糖试验还原反应、氧化酶、磷酸酶活性和吲哚产生试验显示均为阴性,可以利用柠檬酸盐、乳糖、纤维二糖、棉子糖和蜜二糖,但是不能利用D-麦芽糖、山梨醇、D-阿拉伯糖醇和α-甲基葡萄糖。接种马铃薯块茎致病力测定分析表明,亚种内不同地区的菌株间不存在致病力差异。
To identify causal pathogen causing soft rot of potato(Solanum tuberosum L.)in Qinghai province,five strains isolated from five counties in Qinghai were characterized by morphological,biochemical and physiological methods,and analysed 16 SrRNA sequence as well as Pcc subspecies pECC2 Fregion.The results indicated that all of the five strains were pathogenic to potato and caused root rot of potato,so we suggest that Pectobacterium carotovorumsubsp.carotovorum was the soft rot pathogens on potato(Solanum tuberosum L.)in Qinghai.The physiological and biochemical tests showed the bacterium grown at 37℃,caused gelatin liquefaction,represented the tolerance to saltin 50 g/L NaCl solution,and had no sensitivity to erythromycin.The bacterium also gave a positive response to catalase,but negative responses to oxidase,phosphatase activity,indole production,and sucrose reduction reaction.The bacterium utilized some substances including citrate,lactose,cellobiose,raffinose,and melibiose,could not utilize D-maltose,D-sorbitol,D-arabitol,andα-methylglucoside.The pathogenicity tests of all of the bacterial strain collections showed that no significant difference among the same subspecies isolated from different regions was found in Qinghai.
To identify causal pathogen causing soft rot of potato(Solanum tuberosum L.)in Qinghai province,five strains isolated from five counties in Qinghai were characterized by morphological,biochemical and physiological methods,and analysed 16 SrRNA sequence as well as Pcc subspecies pECC2 Fregion.The results indicated that all of the five strains were pathogenic to potato and caused root rot of potato,so we suggest that Pectobacterium carotovorumsubsp.carotovorum was the soft rot pathogens on potato(Solanum tuberosum L.)in Qinghai.The physiological and biochemical tests showed the bacterium grown at 37℃,caused gelatin liquefaction,represented the tolerance to saltin 50 g/L NaCl solution,and had no sensitivity to erythromycin.The bacterium also gave a positive response to catalase,but negative responses to oxidase,phosphatase activity,indole production,and sucrose reduction reaction.The bacterium utilized some substances including citrate,lactose,cellobiose,raffinose,and melibiose,could not utilize D-maltose,D-sorbitol,D-arabitol,andα-methylglucoside.The pathogenicity tests of all of the bacterial strain collections showed that no significant difference among the same subspecies isolated from different regions was found in Qinghai.
引文
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[10]FAQUIHI H,TERTA M,AMDAN M,et al.Phenotypic and genotypic diversity of Pectobacterium carotovorum subsp.carotovorumcausing soft rot disease of potatoes in Morocco[J].European Journal of Plant Pathology,2015,143(4):801-811.
[11]YAHIAOUI-ZAIDI R,JOUAN B,ANDRIVON D.Biochemical and molecular diversity among Erwiniaisolates from potato in Algeria[J].Plant Pathology,2003,52(1):28-40.
[12]GALLELLI A,GALLI M,SIMONEDD,et al.Phenotypic and genetic variability of Pectobacterium carotovorumisolated from artichoke in the Selevalley[J].Journal of Plant Pathology,2009,91(3):757-761.
[13]MAISURIA V B,NERURKARA S.Characterization and differentiation of soft rot causing Pectobacterium carotovorum of Indian origin[J].European Journal of Plant Pathology,2013,136(1):87-102.
[14]MA B,HIBBING M E,KIM H S,et al.Host range and molecular phylogenies of the soft rot enterobacterial genera Pectobacterium and Dickeya[J].Phytopathology,2007,97(9):1150-1163.
[15]DUARTE V,BOERS H D,WARD L J,et al.Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil[J].Journal of Applied Microbiology,2004,96(3):535-545.
[16]GARDAN L,GOUY C,CHRISTEN R,et al.Elevation of three subspecies of Pectobacterium carotouorumto species level:Pectobacterium atrosepticum sp nov,Pectobacterium betavasculorum sp nov and Pectobacterium wasabiae sp nov[J].International Journal of Systematic and Evolutionary Microbiology,2003,53(2):381-391.
[17]GALLOIS A,SAMSON R,AGERON E,et al.Erwiniacarotovora subsp.odorifera subsp.nov.,associated with odorous soft rot of chicory(Cichorium intybus L.)[J].International Journal of Systematic&Evolutionary Microbiology,1992,42(4):582-588.
[18]NABHAN S,BOER S H D,MAISSE,et al.Taxonomic relatedness between Pectobacterium carotovorumsubsp.carotovorum,Pectobacterium carotovorum subsp.odoriferum and Pectobacterium carotovorum subsp.brasiliense subsp.nov[J].Journal of Applied Microbiology,2012,113(4):904-913.
[19]MAHMOUDI E,SOLEIMANI M J,TAGHAVI M.Detection of bacterial soft-rot of crown imperial caused byPectobacterium carotovorumsubsp.carotovorumusing specific PCR primers[J].Phytopathologia Mediterranea,2007,46(2):168-176.
[20]NABHAN S,WYDRA K,LINDE M,et al.The use of two complementary DNA assays,AFLP and MLSA,for epidemic and phylogenetic studies of petolytic enterobacterial strains with focus on the heterogeneous species Pectobacterium carotovorum[J].Plant Pathology,2012,61(3):498-508.
[21]ONKENDI E M,MOLELEKI L N.Characterization of Pectobacterium carotovorum subsp.carotovorum and brasiliense from diseased potatoes in Kenya[J].European Journal of Plant Pathology,2014,139(3):557-566.
[22]PITMAN A R,WRIGHT P J,GALBRAITH M D,et al.Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand[J].Australasian Plant Pathology,2008,37(6):559-568.
[23]SEO S T,FURUYA N,LIM C K,et al.Phenotypic and genetic diversity of Erwinia carotovora ssp.carotovora strains from Asia[J].Journal of Phytopathology,2002,150(3):120-127.
[24]TERTA M,EI KARKOURI A,AIT MHANDR,et al.Occurrence of Pectobacterium carotovorum strains isolated from potato soft rot in Morocco[J].Cellular and Molecular Biology,2010,56:1324-1333.
[2]PEROMBELON M C M.Potato disease caused by soft-rot Erwinias:an overview of pathogenesis[J].Plant Pathology,2002,51(1):1-12.
[3]TOTH I K,BELL K S,HOLEVA M C,et al.Soft-rot Erwinae:from genes to genomes[J].Molecular Plant Pathology,2003,4(1):17-30.
[4]CHOI O,KIM J.Pectobacteriumcarotovorum subsp.brasiliense causing soft rot on paprika in Korea[J].Journal of Phytopathology,2013,161(2):125-127.
[5]CZAJLOWSKI R,PROMBELON M C M,UANEEN J A,et al.Control of blackleg and tuber soft rot of potato caused by Pectobacteriumand Dickeyaspecies:a review[J].Plant Pathology,2011,60(6):999-1013.
[6]NGADZE E,BRADY C L,COUTINHO T A,et al.Pectinolytic bacteria associated with potato soft rot and blackleg in South Africa and Zimbabwe[J].European Journal of Plant Pathology,2012,134(3):533-549.
[7]方中达.植病研究方法(第3版)[M].北京:中国农业出版社,1998:12-164.FANG ZH D.Plant Nathology Research Methods(3rd version)[M].Beijing:China Agriculture Press,1998:12-164.
[8]GARRITY G M,BELL J A,LILBURN T G.Bergey's Manual of Systematic Bacteriology[M].Berlin:Springer Verlag,2004.
[9]KANG H,KWON S W,GO S J.PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp.carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band[J].Plant Pathology,2003,52(2):127-133.
[10]FAQUIHI H,TERTA M,AMDAN M,et al.Phenotypic and genotypic diversity of Pectobacterium carotovorum subsp.carotovorumcausing soft rot disease of potatoes in Morocco[J].European Journal of Plant Pathology,2015,143(4):801-811.
[11]YAHIAOUI-ZAIDI R,JOUAN B,ANDRIVON D.Biochemical and molecular diversity among Erwiniaisolates from potato in Algeria[J].Plant Pathology,2003,52(1):28-40.
[12]GALLELLI A,GALLI M,SIMONEDD,et al.Phenotypic and genetic variability of Pectobacterium carotovorumisolated from artichoke in the Selevalley[J].Journal of Plant Pathology,2009,91(3):757-761.
[13]MAISURIA V B,NERURKARA S.Characterization and differentiation of soft rot causing Pectobacterium carotovorum of Indian origin[J].European Journal of Plant Pathology,2013,136(1):87-102.
[14]MA B,HIBBING M E,KIM H S,et al.Host range and molecular phylogenies of the soft rot enterobacterial genera Pectobacterium and Dickeya[J].Phytopathology,2007,97(9):1150-1163.
[15]DUARTE V,BOERS H D,WARD L J,et al.Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil[J].Journal of Applied Microbiology,2004,96(3):535-545.
[16]GARDAN L,GOUY C,CHRISTEN R,et al.Elevation of three subspecies of Pectobacterium carotouorumto species level:Pectobacterium atrosepticum sp nov,Pectobacterium betavasculorum sp nov and Pectobacterium wasabiae sp nov[J].International Journal of Systematic and Evolutionary Microbiology,2003,53(2):381-391.
[17]GALLOIS A,SAMSON R,AGERON E,et al.Erwiniacarotovora subsp.odorifera subsp.nov.,associated with odorous soft rot of chicory(Cichorium intybus L.)[J].International Journal of Systematic&Evolutionary Microbiology,1992,42(4):582-588.
[18]NABHAN S,BOER S H D,MAISSE,et al.Taxonomic relatedness between Pectobacterium carotovorumsubsp.carotovorum,Pectobacterium carotovorum subsp.odoriferum and Pectobacterium carotovorum subsp.brasiliense subsp.nov[J].Journal of Applied Microbiology,2012,113(4):904-913.
[19]MAHMOUDI E,SOLEIMANI M J,TAGHAVI M.Detection of bacterial soft-rot of crown imperial caused byPectobacterium carotovorumsubsp.carotovorumusing specific PCR primers[J].Phytopathologia Mediterranea,2007,46(2):168-176.
[20]NABHAN S,WYDRA K,LINDE M,et al.The use of two complementary DNA assays,AFLP and MLSA,for epidemic and phylogenetic studies of petolytic enterobacterial strains with focus on the heterogeneous species Pectobacterium carotovorum[J].Plant Pathology,2012,61(3):498-508.
[21]ONKENDI E M,MOLELEKI L N.Characterization of Pectobacterium carotovorum subsp.carotovorum and brasiliense from diseased potatoes in Kenya[J].European Journal of Plant Pathology,2014,139(3):557-566.
[22]PITMAN A R,WRIGHT P J,GALBRAITH M D,et al.Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand[J].Australasian Plant Pathology,2008,37(6):559-568.
[23]SEO S T,FURUYA N,LIM C K,et al.Phenotypic and genetic diversity of Erwinia carotovora ssp.carotovora strains from Asia[J].Journal of Phytopathology,2002,150(3):120-127.
[24]TERTA M,EI KARKOURI A,AIT MHANDR,et al.Occurrence of Pectobacterium carotovorum strains isolated from potato soft rot in Morocco[J].Cellular and Molecular Biology,2010,56:1324-1333.