奉新县猕猴桃溃疡病病原菌鉴定
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摘要
为明确江西省奉新县猕猴桃溃疡病病原菌种类。从该县7个猕猴桃生产基地采集呈典型症状的发病枝条和叶片,采用稀释分离法对其进行病菌分离并作致病性测定,共获得27个致病性细菌菌株。对各菌株进行培养性状、形态特征观察和生理生化测定,结果与文献中对丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv.actinidiae)的描述相吻合。提取各菌株基因组DNA,分别对其16S rDNA和16S-23S rDNA基因片段进行PCR扩增、测序、序列分析和构建系统发育树,结果各菌株16S rDNA和16S-23S rDNA序列长度均为1 500 bp和280 bp,且菌株间序列完全一致。将获得的两段序列利用Blast程序分别在GenBank中进行同源性搜索,发现其与丁香假单胞菌猕猴桃致病变种同源性均为100%。待鉴定菌株在系统发育树上与该变种处于同一分支。根据实验结果,将奉新县猕猴桃溃疡病的病原鉴定为丁香假单胞菌猕猴桃致病变种。
This paper aims to identify the pathogen of kiwifruit bacterial canker in Fengxin County of Ji?angxi Province.Diseased trunks and leaves with typical canker symptoms were collected from seven kiwifruit production bases in the county.The pathogenic bacteria were isolated from the samples by dilution method and their pathogenicity was tested,and 27 pathogenic bacterial strains were obtained.For each strain,its culture and morphological characteristics were observed and its physiological and biochemical characteristics were de?termined.The results were consistent with the descriptions of Pseudomonas syringae pv.actinidiae(Psa)in the literature.The genomic DNA of each strain was extracted,and the regions of 16 S rDNA and 16 S-23 S rDNA were PCR amplified and sequenced.The sequences were analyzed and used for constructing phylogenetic trees.It showed that the sequence lengths of 16 S rDNA and 16 S-23 S rDNA of all strains were 1 500 bp and 280 bp,and the sequences of the strains were identical.Using blast program,the two segment sequences were searched for homology in GenBank.It was found that they shared 100% homology of either 16 S rDNA sequence or 16 S-23 S rDNA sequence with Psa.In the phylogenetic trees,the representive strain was grouped together with Psa.Based on the results,the pathogen of kiwifruit bacterial canker in Fengxi County was identified as P.syringae pv.actinidiae.
This paper aims to identify the pathogen of kiwifruit bacterial canker in Fengxin County of Ji?angxi Province.Diseased trunks and leaves with typical canker symptoms were collected from seven kiwifruit production bases in the county.The pathogenic bacteria were isolated from the samples by dilution method and their pathogenicity was tested,and 27 pathogenic bacterial strains were obtained.For each strain,its culture and morphological characteristics were observed and its physiological and biochemical characteristics were de?termined.The results were consistent with the descriptions of Pseudomonas syringae pv.actinidiae(Psa)in the literature.The genomic DNA of each strain was extracted,and the regions of 16 S rDNA and 16 S-23 S rDNA were PCR amplified and sequenced.The sequences were analyzed and used for constructing phylogenetic trees.It showed that the sequence lengths of 16 S rDNA and 16 S-23 S rDNA of all strains were 1 500 bp and 280 bp,and the sequences of the strains were identical.Using blast program,the two segment sequences were searched for homology in GenBank.It was found that they shared 100% homology of either 16 S rDNA sequence or 16 S-23 S rDNA sequence with Psa.In the phylogenetic trees,the representive strain was grouped together with Psa.Based on the results,the pathogen of kiwifruit bacterial canker in Fengxi County was identified as P.syringae pv.actinidiae.
引文
[1]黄其玲,高小宁,赵志博,等.GFPuv标记猕猴桃溃疡病菌的生物学特性及其在土壤、根系中的定殖[J].中国农业科学,2013,46(2):282-291.Huang Q L,Gao X N,Zhao Z B,et al.Transformed GFPuv into Pseudomonas syringae pv.actinidiae and its biological characteristics and colonization in soil and roots of kiwifruit[J].Scientia Agricultura Sinica,2013,46(2):282-291.
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[3]井赵斌,杨宏勃,杨玉旺,等.秦岭北麓猕猴桃主产区溃疡病病原菌的分离与鉴定[J].西北林学院学报,2016,31(5):188-193.Jing Z B,Yang H B,Yang Y W,et al.Isolation and identification of Pseudomonas syringae pv.actinidiae in northern area of Qinling Mountains[J].Journal of Northwest Forestry University,2016,31(5):188-193.
[4]方炎祖,朱晓湘,王宇道.湖南猕猴桃病害调查研究初报[J].四川果树科技,1990,18(1):28-29.Fang Y Z,Zhu X X,Wang Y D.Preliminary report on the investigation of kiwifruit diseases in Hunan[J].Sichuan Fruit Science and Technology,1990,18(1):28-29.
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[6]承河元,李瑶.安徽省猕猴桃溃疡病菌鉴定[J].安徽农业大学学报,1995,22(3):219-223.Cheng H Y,Li Y.Identification of pathogen of kiwifruit in Anhui Province[J].Journal of Anhui Agricultural University,1995,22(3):219-223.
[7]梁英梅,张星耀,田呈明,等.陕西省猕猴桃枝干溃疡病病原菌鉴定[J].西北林学院学报,2000,15(1):37-39.Liang Y M,Zhang X Y,Tian C M,et al.Pathogenic identification of kiwifruit bacterial canker in Shaanxi Province[J].Journal of Northwest Forestry College,2000,15(1):37-39.
[8]尹春峰.湖南湘西猕猴桃溃疡病分子检测鉴定及其防治药剂的筛选[D].长沙:湖南农业大学,2015.Yin C F.Molecular detection and screening bactericides for control of kiwifruit bacterial canker in wast of Hunan[D].Changsha:Hunan Agricultural University,2015.
[9]Abelleira A,López M M,Pe?alver J,et al.First report of bacterial canker of kiwifruit caused by Pseudomonassyringae pv.actinidiae in Spain[J].Plant Disease,2011,95(12):1583.
[10]Balestra G M,Varvaro L.Pseudomonas syringae pv.syringae causal agent of disease on floralbuds of Actinidia deliciosa(A.Chev)Liang et Ferguson in Italy[J].Journal of Phytopathology,2010,145(8):375-378.
[11]李莎莎.猕猴桃溃疡病相关细菌的鉴定及致病性研究[D].合肥:安徽农业大学,2013.Li S S.Identification and pathogenicity of bacteria associated with kiwifruit canker[D].Hefei:Anhui Agriculture University,2013.
[12]张慧琴,毛雪琴,肖金平,等.猕猴桃溃疡病病原菌分子鉴定与抗性材料初选[J].核农学报,2014,28(7):1181-1187.Zhang H Q,Mao X Q,Xiao J P,et al.Rapid molecular identification of actinidia bacterial canker and preliminary screening of resistant materials in kiwifruit[J].Acta Agriculturae Nucleatae Sinica,2014,28(7):1181-1187.
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.Dong X Z,Cai M Y.Handbook of common bacterial system identification[M].Beijing:Science Press,2001.
[14]方中达.植病研究方法[M].3版.中国农业出版社,1998.Fang Z D.Research method of plant disease[M].3rd ed.Beijing:China Agriculture Press,1998.
[15]Vanneste J L,Poliakoff F,Audusseau C,et al.First report of Pseudomonas syringae pv.actinidiae,the causal agent of bacterial canker of kiwifruit in France[J].Journal of Plant Pathology,2011,96(1):169-175.
[16]Reesgeorge J,Vanneste J L,Cornish D A,et al.Detection of Pseudomonas syringae pv.actinidiae using polymerase chain reaction(PCR)primers based on the 16S-23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions[J].Plant Pathology,2010,59(3):453-464.
[17]Takikawa Y,Serizawa S,Ichikawa T,et al.Pseudomonas syringae pv.actinidiae pv.nov.:the causal bacterium of canker of kiwifruit in Japan[J].Annals of the Phytopathological Society of Japan,1989,55(4):437-444.
[18]Balestra G M,Mazzaglia A,Quattrucci A,et al.Occurrence of Pseudomonas syringae pv.actinidiae in Jin Tao kiwi plants in Italy[J].Phytopathologia Mediterranea,2009,48(1):299-301.
[19]Balestra G M,Renzi M,Mazzaglia A.First report of bacterial canker of Actinidiadeliciosa caused by Pseudomonas syringae pv.actinidiae in Portugal[J].New Disease Reports,2010,22(11):2510-2513.
[20]Bastas K K,Karakaya A.First report of bacterial canker of kiwifruit caused by Pseudomonassyringae pv.actinidiae in Turkey[J].Plant Disease,2012,96(3):452.
[21]王忠肃,唐显富,刘绍基.猕猴桃细菌溃疡病病原细菌鉴定[J].西南大学学报(自然科学版),1992,14(6):500-503.Wang Z S,Tang X F,Liu S J.Identification of pathogenic bacteria of bacterial canker of actinidia[J].Journal of Southwestern Agricltural University(Natural Science Edition),1992,14(6):500-503.
[22]赵志博.猕猴桃细菌性溃疡病病菌群体结构与致病机制[D].杨凌:西北农林科技大学,2016.Zhao Z B.Population composition and pathogenic mechanism in Pseudomonas syringae pv.actinidiae[D].Yangling:North West Agriculture and Forestry University,2016.
[2]Opgenorth D,Lai M,Sorrell M,et al.Pseudomonas canker of kiwifruit[J].Plant Disease,1983,67(11):1283-1284.
[3]井赵斌,杨宏勃,杨玉旺,等.秦岭北麓猕猴桃主产区溃疡病病原菌的分离与鉴定[J].西北林学院学报,2016,31(5):188-193.Jing Z B,Yang H B,Yang Y W,et al.Isolation and identification of Pseudomonas syringae pv.actinidiae in northern area of Qinling Mountains[J].Journal of Northwest Forestry University,2016,31(5):188-193.
[4]方炎祖,朱晓湘,王宇道.湖南猕猴桃病害调查研究初报[J].四川果树科技,1990,18(1):28-29.Fang Y Z,Zhu X X,Wang Y D.Preliminary report on the investigation of kiwifruit diseases in Hunan[J].Sichuan Fruit Science and Technology,1990,18(1):28-29.
[5]高小宁,赵志博,黄其玲,等.猕猴桃细菌性溃疡病研究进展[J].果树学报,2012,29(2):262-268.Gao X N,Zhao Z B,Huang Q L,et al.Advances in research on bacterial canker of kiwifruit[J].Journal of Fruit Science,2012,29(2):262-268.
[6]承河元,李瑶.安徽省猕猴桃溃疡病菌鉴定[J].安徽农业大学学报,1995,22(3):219-223.Cheng H Y,Li Y.Identification of pathogen of kiwifruit in Anhui Province[J].Journal of Anhui Agricultural University,1995,22(3):219-223.
[7]梁英梅,张星耀,田呈明,等.陕西省猕猴桃枝干溃疡病病原菌鉴定[J].西北林学院学报,2000,15(1):37-39.Liang Y M,Zhang X Y,Tian C M,et al.Pathogenic identification of kiwifruit bacterial canker in Shaanxi Province[J].Journal of Northwest Forestry College,2000,15(1):37-39.
[8]尹春峰.湖南湘西猕猴桃溃疡病分子检测鉴定及其防治药剂的筛选[D].长沙:湖南农业大学,2015.Yin C F.Molecular detection and screening bactericides for control of kiwifruit bacterial canker in wast of Hunan[D].Changsha:Hunan Agricultural University,2015.
[9]Abelleira A,López M M,Pe?alver J,et al.First report of bacterial canker of kiwifruit caused by Pseudomonassyringae pv.actinidiae in Spain[J].Plant Disease,2011,95(12):1583.
[10]Balestra G M,Varvaro L.Pseudomonas syringae pv.syringae causal agent of disease on floralbuds of Actinidia deliciosa(A.Chev)Liang et Ferguson in Italy[J].Journal of Phytopathology,2010,145(8):375-378.
[11]李莎莎.猕猴桃溃疡病相关细菌的鉴定及致病性研究[D].合肥:安徽农业大学,2013.Li S S.Identification and pathogenicity of bacteria associated with kiwifruit canker[D].Hefei:Anhui Agriculture University,2013.
[12]张慧琴,毛雪琴,肖金平,等.猕猴桃溃疡病病原菌分子鉴定与抗性材料初选[J].核农学报,2014,28(7):1181-1187.Zhang H Q,Mao X Q,Xiao J P,et al.Rapid molecular identification of actinidia bacterial canker and preliminary screening of resistant materials in kiwifruit[J].Acta Agriculturae Nucleatae Sinica,2014,28(7):1181-1187.
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.Dong X Z,Cai M Y.Handbook of common bacterial system identification[M].Beijing:Science Press,2001.
[14]方中达.植病研究方法[M].3版.中国农业出版社,1998.Fang Z D.Research method of plant disease[M].3rd ed.Beijing:China Agriculture Press,1998.
[15]Vanneste J L,Poliakoff F,Audusseau C,et al.First report of Pseudomonas syringae pv.actinidiae,the causal agent of bacterial canker of kiwifruit in France[J].Journal of Plant Pathology,2011,96(1):169-175.
[16]Reesgeorge J,Vanneste J L,Cornish D A,et al.Detection of Pseudomonas syringae pv.actinidiae using polymerase chain reaction(PCR)primers based on the 16S-23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions[J].Plant Pathology,2010,59(3):453-464.
[17]Takikawa Y,Serizawa S,Ichikawa T,et al.Pseudomonas syringae pv.actinidiae pv.nov.:the causal bacterium of canker of kiwifruit in Japan[J].Annals of the Phytopathological Society of Japan,1989,55(4):437-444.
[18]Balestra G M,Mazzaglia A,Quattrucci A,et al.Occurrence of Pseudomonas syringae pv.actinidiae in Jin Tao kiwi plants in Italy[J].Phytopathologia Mediterranea,2009,48(1):299-301.
[19]Balestra G M,Renzi M,Mazzaglia A.First report of bacterial canker of Actinidiadeliciosa caused by Pseudomonas syringae pv.actinidiae in Portugal[J].New Disease Reports,2010,22(11):2510-2513.
[20]Bastas K K,Karakaya A.First report of bacterial canker of kiwifruit caused by Pseudomonassyringae pv.actinidiae in Turkey[J].Plant Disease,2012,96(3):452.
[21]王忠肃,唐显富,刘绍基.猕猴桃细菌溃疡病病原细菌鉴定[J].西南大学学报(自然科学版),1992,14(6):500-503.Wang Z S,Tang X F,Liu S J.Identification of pathogenic bacteria of bacterial canker of actinidia[J].Journal of Southwestern Agricltural University(Natural Science Edition),1992,14(6):500-503.
[22]赵志博.猕猴桃细菌性溃疡病病菌群体结构与致病机制[D].杨凌:西北农林科技大学,2016.Zhao Z B.Population composition and pathogenic mechanism in Pseudomonas syringae pv.actinidiae[D].Yangling:North West Agriculture and Forestry University,2016.