广东省马铃薯块茎软腐病病原菌鉴定
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摘要
为明确广东省惠州市马铃薯块茎软腐病的病原菌,采用常规病组织分离法获得4株菌株,通过生理生化特征和分子生物学特性对其进行鉴定,并测定了该病原菌对20个马铃薯品种的致病力。结果显示,该病原菌为革兰氏阳性菌,在LB平板上菌落有透明和不透明2种形态;除葡萄糖发酵阳性、对链霉素和青霉素具有抗性等特征不同外,BP-hd-1、BP-hd-2、BP-hd-3和BP-hd-4菌株与短小芽胞杆菌Bacillus pumilus菌株的其它生理生化特征均相同。系统进化分析结果表明,4株菌株的16S rDNA序列分别与短小芽胞杆菌AI-Khrj18(KY123871)、ML270(KC692158)、NCTC10337(LT906438)和ARD21(KX023236)的相似性为100.0%;4株菌株的gyrB基因序列均与短小芽胞杆菌AUEC29菌株(HM585095)的gyrB基因相似性最高,为99.7%～99.8%。生理生化特征及分子生物学鉴定结果表明,引起广东省马铃薯块茎软腐病的病原菌为短小芽胞杆菌。致病力测定结果显示,接种菌株BP-1后20个马铃薯品种的发病率均为100.0%,表明该病原菌对马铃薯有强致病力。
In order to confirm the pathogen of potato tuber soft rot in Huizhou City, Guangdong Province, four isolates of the pathogen were isolated with diseased tissue isolation method. Four isolates were identified with physiological and biochemical characteristics and molecular biological identification. Twenty potato varieties were used for pathogenicity test of the pathogen. The results showed that the pathogen was a gram-positive bacterium and possessed transparent colonies and opaque colonies on LB plates. The physiological and biochemical characteristics of BP-hd-1, BP-hd-2, BP-hd-3 and BP-hd-4 same as those of Bacillus pumilus except for glucose fermentation positive reaction, streptomycin resistance and penicillin resistance. The results of phylogenetic analysis showed that 16S rDNA gene sequences of the four isolates were highly similar to those of B. pumilus AI-Khrj18(KY123871), ML270(KC692158), NCTC10337(LT906438) and ARD21(KX023236), with an identity of 100.0%, respectively. The gyrB gene sequences of the four isolates were highly similar to that of B. pumilus AUEC29(HM585095), with an identity of 99.7%-99.8%. The pathogen of potato soft rot was identified as B. pumilus based on physiological and biochemical characteristics and molecular biological identification. The results of the pathogenicity tests showed that the disease incidence of 20 potato varieties artificially inoculated by BP-1 were 100.0%, indicating that the pathogen was highly pathogenic to potato.
In order to confirm the pathogen of potato tuber soft rot in Huizhou City, Guangdong Province, four isolates of the pathogen were isolated with diseased tissue isolation method. Four isolates were identified with physiological and biochemical characteristics and molecular biological identification. Twenty potato varieties were used for pathogenicity test of the pathogen. The results showed that the pathogen was a gram-positive bacterium and possessed transparent colonies and opaque colonies on LB plates. The physiological and biochemical characteristics of BP-hd-1, BP-hd-2, BP-hd-3 and BP-hd-4 same as those of Bacillus pumilus except for glucose fermentation positive reaction, streptomycin resistance and penicillin resistance. The results of phylogenetic analysis showed that 16S rDNA gene sequences of the four isolates were highly similar to those of B. pumilus AI-Khrj18(KY123871), ML270(KC692158), NCTC10337(LT906438) and ARD21(KX023236), with an identity of 100.0%, respectively. The gyrB gene sequences of the four isolates were highly similar to that of B. pumilus AUEC29(HM585095), with an identity of 99.7%-99.8%. The pathogen of potato soft rot was identified as B. pumilus based on physiological and biochemical characteristics and molecular biological identification. The results of the pathogenicity tests showed that the disease incidence of 20 potato varieties artificially inoculated by BP-1 were 100.0%, indicating that the pathogen was highly pathogenic to potato.
引文
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Kong GF.2014.Isolation and identification of Bacillus pumilus and optimization of its fermentation conditions.Master's Thesis.Hangzhou:Zhejiang Sci-Tech University(in Chinese)[孔高飞.2014.一种短小芽胞杆菌分离鉴定及培养条件优化.硕士学位论文.杭州:浙江理工大学]
La Duc MT,Satomi M,Agata N,Venkateswaran K.2004.gyrB as a phylogenetic discriminator for members of the Bacillus anthraciscereus-thuringiensis group.Journal of Microbiological Methods,56(3):383-394
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Osborne CA,Galic M,Sangwan P,Janssen PH.2005.PCR-generated artefact from 16S r RNA gene-specific primers.FEMS Microbiology Letters,248(2):183-187
Peng HN.2013.Screening and molecular identification of bacteriostatic Bacillus pumilus 4D-14,probiotics development and application research.Ph.D Thesis.Qingdao:Ocean University of China(in Chinese)[彭虹旎.2013.短小芽胞杆菌(Bacillus pumilus 4D-14)的分离与分子鉴定、抑菌性及微生态制剂应用研究.博士学位论文.青岛:中国海洋大学]
Schaad NW,Jones JB,Chun W.2011.Laboratory guide for identification of plant pathogenic bacteria(3rd edition).Zhao TC translated.Beijing:China Agricultural Science and Technology Press(in Chinese)[Schaad NW,Jones JB,Chun W.2011.植物病原细菌鉴定试验指导(第3版).赵廷昌译.北京:中国农业科学技术出版社]
Tamura K,Stecher G,Peterson D,Filipski A,Kumar S.2013.MEGA6:molecular evolutionary genetics analysis version 6.0.Molecular Biology and Evolution,30(12):2725-2729
Wang JS,Wei ZM,Fang ZD.1985.Identification of the soft rot Erwinia from infested potato tubers.Acta Phytopathologica sinica,15(1):25-30(in Chinese)[王金生,韦忠民,方中达.1985.马铃薯软腐病细菌的鉴定.植物病理学报,15(1):25-30]
Wang L,Li XB,Suo HC,An K,Luo HM,Liu XJ.2017.Soft rot of potatoes caused by Bacillus amyloliquefaciens in Guangdong Province,China.Canadian Journal of Plant Pathology,39(4):533-539
Yamamoto S,Harayama S.1996.Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrB genes and on the amino acid sequences of their products.International Journal of Systematic Bacteriology,46(2):506-511
You CP,Xiao AP,Li XM,Tang XN.2001 Screening and identification of antagonistic microorganisms against Pyricularia grisea.Acta Agriculturae Universitatis Jiangxiensis,23(4):519-521(in Chinese)[游春平,肖爱萍,李湘明,唐祥宁.2001.稻瘟病菌拮抗微生物的筛选及鉴定.江西农业大学学报,23(4):519-521]
Zhang R,Li JH,Hu JD,Wang JB,Wang D.2006.Isolation of the pathogenic bacteria on potato during storage by 16S rDNA sequence analysis.Journal of Gansu Agricultural University,41(5):71-75(in Chinese)[张茹,李金花,扈进冬,王晋渤,王蒂.2006.应用16S rDNA序列分析对马铃薯贮藏期三个病原细菌菌株的鉴定.甘肃农业大学学报,41(5):71-75]
Bathily H,Babana AH,SamakéF.2010.Bacillus pumilus,a new pathogen on potato tubers in storage in Mali.African Journal of Microbiology Research,4(20):2067-2071
Buchanan RE.Gibbems NE.1984.Bergey’s manual of determinative bacteriology.Beijing:Science Press,pp.729-751(in Chinese)[布坎南RE,吉本斯NE.1984.伯杰细菌鉴定手册.北京:科学出版社,pp.729-751]
Dong XZ,Cai MY.2001.Manual of familiar bacterium identification.Beijing:Science Press,pp.62-65(in Chinese)[东秀珠,蔡妙英.2001.常见细菌系统鉴定手册.北京:科学出版社,pp.62-65]
Fang ZD.1998.Plant pathology research methods(3rd edition).Beijing:China Agriculture Press,pp.180(in Chinese)[方中达.1998.植病研究方法(第3版).北京:中国农业出版社,pp.180]
Huang FL,Zhang L,Li XD,Cai DX.2017.Development situation and countermeasures of China’s potato industry.Agricultural Outlook,13(1):25-31(in Chinese)[黄凤玲,张琳,李先德,蔡典雄.2017.中国马铃薯产业发展现状及对策.农业展望,13(1):25-31]
Kong GF.2014.Isolation and identification of Bacillus pumilus and optimization of its fermentation conditions.Master's Thesis.Hangzhou:Zhejiang Sci-Tech University(in Chinese)[孔高飞.2014.一种短小芽胞杆菌分离鉴定及培养条件优化.硕士学位论文.杭州:浙江理工大学]
La Duc MT,Satomi M,Agata N,Venkateswaran K.2004.gyrB as a phylogenetic discriminator for members of the Bacillus anthraciscereus-thuringiensis group.Journal of Microbiological Methods,56(3):383-394
Li FD,Wang LL,Wang P,Guo TS,Yang C,Dou JH,Qi XD,Luo ZX,Jie WQ,Zhao ZL,et al.2017.The influence of Bacillus pumilus strain BPS48 on potato growth.Gansu Agricultural Science and Techonology,(11):36-37(in Chinese)[李芳弟,王利立,王鹏,郭天顺,杨晨,窦俊焕,齐小东,罗朝霞,颉炜清,赵中梁,等.2017.短小芽胞杆菌BPS48对马铃薯生长的影响.甘肃农业科技,(11):36-37]
Osborne CA,Galic M,Sangwan P,Janssen PH.2005.PCR-generated artefact from 16S r RNA gene-specific primers.FEMS Microbiology Letters,248(2):183-187
Peng HN.2013.Screening and molecular identification of bacteriostatic Bacillus pumilus 4D-14,probiotics development and application research.Ph.D Thesis.Qingdao:Ocean University of China(in Chinese)[彭虹旎.2013.短小芽胞杆菌(Bacillus pumilus 4D-14)的分离与分子鉴定、抑菌性及微生态制剂应用研究.博士学位论文.青岛:中国海洋大学]
Schaad NW,Jones JB,Chun W.2011.Laboratory guide for identification of plant pathogenic bacteria(3rd edition).Zhao TC translated.Beijing:China Agricultural Science and Technology Press(in Chinese)[Schaad NW,Jones JB,Chun W.2011.植物病原细菌鉴定试验指导(第3版).赵廷昌译.北京:中国农业科学技术出版社]
Tamura K,Stecher G,Peterson D,Filipski A,Kumar S.2013.MEGA6:molecular evolutionary genetics analysis version 6.0.Molecular Biology and Evolution,30(12):2725-2729
Wang JS,Wei ZM,Fang ZD.1985.Identification of the soft rot Erwinia from infested potato tubers.Acta Phytopathologica sinica,15(1):25-30(in Chinese)[王金生,韦忠民,方中达.1985.马铃薯软腐病细菌的鉴定.植物病理学报,15(1):25-30]
Wang L,Li XB,Suo HC,An K,Luo HM,Liu XJ.2017.Soft rot of potatoes caused by Bacillus amyloliquefaciens in Guangdong Province,China.Canadian Journal of Plant Pathology,39(4):533-539
Yamamoto S,Harayama S.1996.Phylogenetic analysis of Acinetobacter strains based on the nucleotide sequences of gyrB genes and on the amino acid sequences of their products.International Journal of Systematic Bacteriology,46(2):506-511
You CP,Xiao AP,Li XM,Tang XN.2001 Screening and identification of antagonistic microorganisms against Pyricularia grisea.Acta Agriculturae Universitatis Jiangxiensis,23(4):519-521(in Chinese)[游春平,肖爱萍,李湘明,唐祥宁.2001.稻瘟病菌拮抗微生物的筛选及鉴定.江西农业大学学报,23(4):519-521]
Zhang R,Li JH,Hu JD,Wang JB,Wang D.2006.Isolation of the pathogenic bacteria on potato during storage by 16S rDNA sequence analysis.Journal of Gansu Agricultural University,41(5):71-75(in Chinese)[张茹,李金花,扈进冬,王晋渤,王蒂.2006.应用16S rDNA序列分析对马铃薯贮藏期三个病原细菌菌株的鉴定.甘肃农业大学学报,41(5):71-75]