福建地区不结球白菜黑腐病病原菌的分离与初步鉴定
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摘要
为鉴定福州地区不结球白菜黑腐病致病菌,以感染黑腐病的不结球白菜植株为材料进行了病原菌分离和鉴定。通过观察所分离病原菌的形态及接种试验发现其形态和发病症状与黑腐病的形态和发病症状相一致。以细菌通用引物16S对该病原菌进行16SrDNA的PCR扩增测序分析,发现该病原菌与野油菜黄单胞菌(Genbank:CP017308.1)16SrDNA序列同源性为99.8%。从接种病原菌植株中重新分离病原菌,其16SrDNA序列与之前接种菌株序列一致。以上结果初步证明分离的病原菌为不结球白菜黑腐病病原菌,且该病原菌为十字花科黑腐病野油菜黄单胞菌野油菜致病种。
Pathogen that caused the black rot disease on non-heading Chinese cabbages in Fuzhou was investigated.Microbial samples were collected from the diseased plants for isolation.Based on the morphology and inoculation test,selected microorganisms were tested further.Their 16 SrDNA were amplified by PCR with the universal primer 16 S.The positive fragment was sequenced and analyzed against NCBI database.A 99.8% homology was found between an isolated bacterium and Xanthomonas campestris pv.campestris(Genbank:CP017308.1).Subsequently,the 16 SrDNA sequence of the strain from the infected cabbage was confirmed to be identical to that of the pathogen for the inoculation.It was,therefore,concluded that the isolated strain was indeed the pathogen that caused the black rot disease on the non-heading cabbages,and was preliminary identified as Xanthomonas campestris pv.campestris.
Pathogen that caused the black rot disease on non-heading Chinese cabbages in Fuzhou was investigated.Microbial samples were collected from the diseased plants for isolation.Based on the morphology and inoculation test,selected microorganisms were tested further.Their 16 SrDNA were amplified by PCR with the universal primer 16 S.The positive fragment was sequenced and analyzed against NCBI database.A 99.8% homology was found between an isolated bacterium and Xanthomonas campestris pv.campestris(Genbank:CP017308.1).Subsequently,the 16 SrDNA sequence of the strain from the infected cabbage was confirmed to be identical to that of the pathogen for the inoculation.It was,therefore,concluded that the isolated strain was indeed the pathogen that caused the black rot disease on the non-heading cabbages,and was preliminary identified as Xanthomonas campestris pv.campestris.
引文
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[14]朱伯华,龙韵阗,李娅玲,等.花椰菜和青花菜主要病虫害无害化防治技术[J].长江蔬菜,2012,(9):47-51.
[15]秦韶梅,冷德训,孙秀丽.十字花科蔬菜黑腐病软腐病的综合防治[J].西北园艺,2006,(9):34.
[16]曹坳程,刘晓漫,郭美霞,等.作物土传病害的危害及防治技术[J].植物保护,2017,43(2):6-16.
[17]杨丽丽,郑伟.设施蔬菜栽培连作障碍分析及综合防治[J].山西农业科学,2012,40(8):917-920,924.
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[19]李焕玲.蔬菜四种细菌性新病害病原菌的鉴定研究[D].北京:中国农业科学院,2013.
[20]BUTTNER D,BONAS U.Getting across-bacterial type III effector proteins on their way to the plant cell[J].EMBO J,2002,21(20):5313-5322.
[21]芦燕.大白菜黑腐病病原菌鉴定和抗病性鉴定方法研究[D].杨凌:西北农林科技大学,2008.
[22]陶文沂.工业微生物生理与遗传育种学[M].北京:中国轻工业出版社,1997.
[23]李广宁,邓小丽,周丽沙,等.一株藤黄单胞菌属细菌HF-1127的分离与鉴定[J].生物技术,2009,19(5):43-46.
[24]焦振泉,刘秀梅.16srRNA序列同源性分析与细菌系统分类鉴定[J].国外医学(卫生学分册),1998,(1):14-18.
[25]SOGIN M L,GUNDERSON J H.Structural diversity of eukaryotic small subunit ribosomal RNAs.Evolutionary implications[J].Acad Sci,1987,503:125-139.
[26]LEE I M,BARTOSZYK I M,GUNDERSEN-RINDAL D E,et al.Phylogeny and classification of bacteria in the genera clavibacter and Rathayibacter on the basis of 16SRNA gene sequence analyses[J].Appl Environ Microbiol,1997,63(7):2631-2636.
[27]戴欣,陈月琴,周慧,等.海洋细菌的分子鉴定分类[J].中山大学学报:自然科学版,2000,39(1):68-71.
[28]FENG Y J,ZHANG H M.Genetic identification and analysis of multiresistance of antibiotic conferred by aplasmid isolated from field bacteria[J].Acta Shanxi University of Science and Technology,2003,21(5):54-58.
[29]陈善义,陶万强,王合,等.北京地区核桃黑斑病病原菌的分离、致病性测定和16SrDNA序列分析[J].果树学报,2011,28(3):469-473,549.
[30]张会敏,冯友军.一株野生细菌的16SrDNA序列分析与系统发育树的构建[J].生物信息学,2005,(1):1-4,18.
[2]DANIELS M J,BARBER C E,TURNER P C,et al.Cloning of genes involved inpathogenicity of Xanthomonas campestris pv.Campestris using the broad host rang cosmid pLAFR1[J].EMBO J,1984,3(13):3323-3328.
[3]NOMURA K,HE S Y.Powerful screens for bacterial virulence proteins[J].Proc Natl Acad Sci,2005,102:3527-3528.
[4]张穗生,姜伟,玉延华,等.十字花科黑腐病菌中GGDEF结构域蛋白差异的in silico分析[J].基因组学与应用生物学,2011,30(6):722-727.
[5]朱平川,卢洁,张昭颖,等.广西十字花科黑腐病菌致病性分化及遗传多样性分析[J].基因组学与应用生物学,2015,34(3):541-547.
[6]张黎黎,刘玉梅,田自华,等.十字花科蔬菜抗黑腐病育种研究进展[J].园艺学报,2012,39(9):1727-1738.
[7]解永梅,张成玲,赵永强,等.山东省大白菜黑腐病病原的鉴定及其生物学特性的研究[J].山东农业科学,2007,(6):68-70.
[8]芦燕,张鲁刚,惠麦侠,等.陕西省大白菜主产区黑腐病菌致病型的研究[J].西北农林科技大学学报:自然科学版,2008,36(10):132-138.
[9]黄德芬,李成琼,司军,等.甘蓝黑腐病生理小种划分及其抗病性鉴定研究进展[J].中国蔬菜,2011,1(18):6-10.
[10]SWINGSJ G,CIVEROLO E L.Xanthomonas[M].London:Chapman&Hal,1993:1-55.
[11]方中达.植病研究方法[M].北京:中国农业出版社,1998.
[12]李昱佳,李茜,张志想,等.拮抗根癌土壤杆菌的桃内生细菌的筛选鉴定[J].中国农业科学,2017,50(20):3918-3929.
[13]王述彬,王元珪.花菜、甘蓝黑腐病菌的致病性、寄主范围及传播途径[J].江苏农业学报,1992,8(4):31.
[14]朱伯华,龙韵阗,李娅玲,等.花椰菜和青花菜主要病虫害无害化防治技术[J].长江蔬菜,2012,(9):47-51.
[15]秦韶梅,冷德训,孙秀丽.十字花科蔬菜黑腐病软腐病的综合防治[J].西北园艺,2006,(9):34.
[16]曹坳程,刘晓漫,郭美霞,等.作物土传病害的危害及防治技术[J].植物保护,2017,43(2):6-16.
[17]杨丽丽,郑伟.设施蔬菜栽培连作障碍分析及综合防治[J].山西农业科学,2012,40(8):917-920,924.
[18]ROSA V,DAISY P,SILVIA V C.Control of black rot caused by Alternaria alternata in yellow pitahaya(Selenicereus megalanthus)through hot water dips[J].LWT-Food Science and Technology,2017:82.
[19]李焕玲.蔬菜四种细菌性新病害病原菌的鉴定研究[D].北京:中国农业科学院,2013.
[20]BUTTNER D,BONAS U.Getting across-bacterial type III effector proteins on their way to the plant cell[J].EMBO J,2002,21(20):5313-5322.
[21]芦燕.大白菜黑腐病病原菌鉴定和抗病性鉴定方法研究[D].杨凌:西北农林科技大学,2008.
[22]陶文沂.工业微生物生理与遗传育种学[M].北京:中国轻工业出版社,1997.
[23]李广宁,邓小丽,周丽沙,等.一株藤黄单胞菌属细菌HF-1127的分离与鉴定[J].生物技术,2009,19(5):43-46.
[24]焦振泉,刘秀梅.16srRNA序列同源性分析与细菌系统分类鉴定[J].国外医学(卫生学分册),1998,(1):14-18.
[25]SOGIN M L,GUNDERSON J H.Structural diversity of eukaryotic small subunit ribosomal RNAs.Evolutionary implications[J].Acad Sci,1987,503:125-139.
[26]LEE I M,BARTOSZYK I M,GUNDERSEN-RINDAL D E,et al.Phylogeny and classification of bacteria in the genera clavibacter and Rathayibacter on the basis of 16SRNA gene sequence analyses[J].Appl Environ Microbiol,1997,63(7):2631-2636.
[27]戴欣,陈月琴,周慧,等.海洋细菌的分子鉴定分类[J].中山大学学报:自然科学版,2000,39(1):68-71.
[28]FENG Y J,ZHANG H M.Genetic identification and analysis of multiresistance of antibiotic conferred by aplasmid isolated from field bacteria[J].Acta Shanxi University of Science and Technology,2003,21(5):54-58.
[29]陈善义,陶万强,王合,等.北京地区核桃黑斑病病原菌的分离、致病性测定和16SrDNA序列分析[J].果树学报,2011,28(3):469-473,549.
[30]张会敏,冯友军.一株野生细菌的16SrDNA序列分析与系统发育树的构建[J].生物信息学,2005,(1):1-4,18.