观赏海棠干腐病病原菌鉴定
|
摘要
从观赏海棠干腐病病斑处分离纯化病原菌,采用柯赫氏法则对从病原菌进行验证,通过形态学结合ITS序列的系统发育分析鉴定确定病原菌种名,得到观赏海棠干腐病相关病原菌ZWY0501、ZWY0502(登录号:MG554650、MG554651),分离频率分别为55.36%、42.86%。2株孢子均无色,香蕉形或椭圆形,(3.13～5.51)μm×(1.19～1.89)μm。ITS系统发育树中,2株约590 bp菌株与已登录的苹果腐烂病病株(Valsa mali,登录号为:KP337612)同源性最高,最大相似率达到99%。形态学及ITS序列的系统发育分析鉴定ZWY0501、ZWY0502均为苹果黑皮腐壳菌(Valsa mali),其为观赏海棠树皮腐烂病病原。研究结果为该病害的控制奠定了科学基础。
The objective of the present study was to isolate,purify and identify the pathogen from Malus crabapple valsa canker.The pathogen isolates were obtained from tissue lasions of valsa canker on crabapple tree trunks and confirmed via Koch?s postulates,then they were identified on the basis of anamorphic morphology and rDNA Internal Transcribed Spacer(ITS) sequences.The two isolates,obtained from the Malus crabapple,were numbered as ZWY0501 and ZWY0502(ncbi GenBank ID:MG554650、MG554651),the isolation frequency of which were 55.36% and 42.86%,respectively.Conidia of both isolates were hyaline,banana-shaped or elliptic,with sizes of 3.13-5.51×1.19-1.89 μm.A same ITS sequence of about 590 bp was obtained from the two isolates ZWY0501 and ZWY0502 and it shared a maximum semilarity of 99% with that of Valsa mali.Phylogenic analysis showed that the two isolates were clustered in the same clade with Valsa mali(GenBank ID:KP337612) but were separated in different degrees from other species in the dendrogram.Based on anamorphic morphology and rDNA internal transcribed spacer(ITS) sequences,the pathogen was identified as Valsa mali.The results of the study would provide scientific basis for selecting target fungicides and resistant crabapple varieties for controlling the disease.
The objective of the present study was to isolate,purify and identify the pathogen from Malus crabapple valsa canker.The pathogen isolates were obtained from tissue lasions of valsa canker on crabapple tree trunks and confirmed via Koch?s postulates,then they were identified on the basis of anamorphic morphology and rDNA Internal Transcribed Spacer(ITS) sequences.The two isolates,obtained from the Malus crabapple,were numbered as ZWY0501 and ZWY0502(ncbi GenBank ID:MG554650、MG554651),the isolation frequency of which were 55.36% and 42.86%,respectively.Conidia of both isolates were hyaline,banana-shaped or elliptic,with sizes of 3.13-5.51×1.19-1.89 μm.A same ITS sequence of about 590 bp was obtained from the two isolates ZWY0501 and ZWY0502 and it shared a maximum semilarity of 99% with that of Valsa mali.Phylogenic analysis showed that the two isolates were clustered in the same clade with Valsa mali(GenBank ID:KP337612) but were separated in different degrees from other species in the dendrogram.Based on anamorphic morphology and rDNA internal transcribed spacer(ITS) sequences,the pathogen was identified as Valsa mali.The results of the study would provide scientific basis for selecting target fungicides and resistant crabapple varieties for controlling the disease.
引文
[1] 魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979.
[2] 赵忠仁,王元珪.落叶果树病虫害及其防治[M].南京:江苏科学技术出版社,1991:28-33.
[3] 中国农业科学院果树研究所,中国农业科学院柑橘研究所.中国果树病虫志[M].2版.北京:中国农业出版社,1996:1-6.
[4] 许志刚.普通植物病理学[M].2版.北京:中国农业出版社,1997:46-57.
[5] 侯明生.农业植物病理学[M].北京:科学出版社,2006.
[6] 刘开启,牟惠芳,刘晖,等.苹果树腐烂病侵染来源的研究[J].山东农业大学学报,1996,27(3):281-284.
[7] 贺冰,贺运春,钱江.陕山西太谷、祁县苹果树枝干病害的种类及为害性研究[J].山西农业科学,2011,39(4):356-358.
[8] 樊民周,张王斌,安德荣.陕西苹果树腐烂病病原菌的鉴定[J].西北农业学报,2004,13(3):60-61.
[9] 殷辉,周建波,张志斌,等.山西省苹果树腐烂病菌的种群结构分析[J].菌物学报,2016,35(12):1493-1502.YIN H,ZHOU J B,ZHANG Z B,et al.Population structure of Valsa species from Malus pumila in Shanxi Province[J].Mycosystema,2016,35(12):1493-1502.(in Chinese)
[10] KIRK P M,CANNON P F,DACID J C,et al.Dictionary of the Fungi[J].Mycological Research,2002,106(4):507-508.
[11] TOGASHI K.Some studies on a Japanese apple canker and its causal fungus,Valsa mali[J].Jour.Coll.Agr.Hokkaido Imp.Univ.,1924,12 (3):265-321.
[12] 阎应理,荆志刚.苹果腐烂病菌分生孢子田间消失规律研究[J].植物病理学报,1988,18(4):2-8.
[13] 高克祥,刘晓光.苹果树腐烂病研究概况[J].河北林学院学报,1995,10(1):87-91.
[14] GEORGE N A.Plant Pathology[M].San Diego,CA:Academic Press,1978.
[15] 陈策.国外对于苹果树腐烂病、桃树腐烂病以及其他同类病害的研究近况[J].中国果树,1982(Supp.1):68-77.
[16] 刘福昌,陈策,史秀琴,等.苹果树腐烂病菌(Valsa mali Miyabe et Yamada)潜伏侵染研究[J].植物保护学报,1979,6(3):1-8.
[17] 臧睿,黄丽丽,康振生,等.陕西苹果树腐烂病菌(Cytospora spp.)不同分离株的生物学特性与致病性研究[J].植物病理学报,2007,37(4):343-351.ZANG R,HUANG L L,KANG Z S,et al.Biological characteristics and pathogenicity of different isolates of Cytospora spp.isolated from apple trees in Shaanxi Province[J].Acta Phytopathologica Sinica,2007,37(4):343-351.(in Chinese)
[18] 韦洁玲,黄丽丽,郜佐鹏,等.苹果树腐烂病室内快速评价方法的研究[J].植物病理学报,2010,40(1):14-20.WEI J L,HUANG L L,GAO Z P,et al.Laboratory evaluation methods of apple valsa canker disease caused by Valsa ceratosperma sensu kobayashi[J].Acta Phytopathologica Sinica,2010,40(1):14-20.(in Chinese)
[19] 王旭丽.中国苹果树腐烂病菌的种类:rDNA-ITS序列和表型比较研究[D].杨陵:西北农林科技大学,2007.
[20] 郭翎.观赏苹果引种与苹果属(Malus Mill.)植物DNA指纹分析[D].泰安:山东农业大学,2009.
[21] 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 Moutains[J].Journal of Northwest Forestry University,2016,31(5):188-193.(in Chinese)
[22] SAITOU N,NEI M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425.
[23] NEI M.Relative efficiencies of different tree-making methods for molecular data[M]//MIYAMOTO M M,CRACRAFT J L.Phylogenetic analysis of DNA sequences.Oxford:Oxford University Press,1991.
[24] HOLDER M,LEWIS P O.Phylogeny estimation:traditional and bayesian approaches[J].Nature Reviews Genetics,2003,4(4):275-284.
[25] 潘彦平,范鑫磊,田呈明.山西省壳囊孢属真菌分类学[J].北京林业大学学报,2016,38(11):79-88.PAN Y P,FAN X L,TIAN C M.Taxonomy of Cytospora in Shanxi Province,northern China[J].Journal of Beijing Forestry University,2016,38(11):79-88.(in Chinese)
[26] 李美娜,王金友.苹果树腐烂病病原菌来源的探讨[J].北方果树,1990(2):28-30.LI M N,WANG J Y.Astudy on the source of pathogens of apple tree Valsacanker[J].Northern Fruits,1990(2):28-30.(in Chinese)
[27] KOBAYASHI T.Taxonomic study on Japanese fung[J].Journal of the Japanese Plant Pathology,1972,38:170-173.
[28] KANEHIRA T,KAMEOKA Y,SHINOHARA M.Electrophoretic Patterns of the enzymes from Valsa Ceratosperma[J].Transction of the Mycological Society of Japan,1991,32:199-205.
[29] SUZAKI K,YOSHIDA K,ITO T.Pathogenicity to apple branch and phloridzin degrading activity of Valsa ceratosperma isolated from some broad leaf trees including apple tree[J].Annual Report of the Society of Plant Protection of North Japan,1997,48:145-147.
[30] 臧睿.中国苹果树腐烂病菌的种群组成、分子检测及其ISSR遗传分析[D].杨陵:西北农林科技大学,2012.
[31] 曹支敏,梁英梅,马晓.花椒干腐病研究[J].植物保护学报,1992,19(4):58-63.CAO Z M,LIANG Y M,MA X.Study on dry rot of wild pepper[J].Journal of Plant Protection,1992,19(4):58-63.(in Chinese)
[32] 黄世钰,王建国.刺槐干腐病的研究[J].林业科学,1983,19(4):366-370.HUANG S Y,WANG J G.Study on stem rot of black locust[J].Scientia Silvae Sinicae,1983,19(4):366-370.(in Chinese)
[33] 赵晓军,周建波,赵子俊,等.枣树干腐病病原菌的鉴定[J].菌物学报,2009,28(3):332-335.ZHAO X J,ZHOU J B,ZHAO Z J,et al.the pathogen causing stem dry rot of Ziziphus jujuba[J].Mycosystema,2009,28(3):332-335.(in Chinese)
[34] 张璐璐,贾桂民,叶建丰,等.浙江临安山核桃干腐病发生发展规律[J].浙江农林大学学报,2013,30(1):148-152.ZHANG L L,JIA G M,YE J F,et al.Frequency of carya cathayensis canker disease in Lin?an City,Zhejing Province [J].Journal of Zhejiang A & F University,2013,30(1):148-152.(in Chinese)
[2] 赵忠仁,王元珪.落叶果树病虫害及其防治[M].南京:江苏科学技术出版社,1991:28-33.
[3] 中国农业科学院果树研究所,中国农业科学院柑橘研究所.中国果树病虫志[M].2版.北京:中国农业出版社,1996:1-6.
[4] 许志刚.普通植物病理学[M].2版.北京:中国农业出版社,1997:46-57.
[5] 侯明生.农业植物病理学[M].北京:科学出版社,2006.
[6] 刘开启,牟惠芳,刘晖,等.苹果树腐烂病侵染来源的研究[J].山东农业大学学报,1996,27(3):281-284.
[7] 贺冰,贺运春,钱江.陕山西太谷、祁县苹果树枝干病害的种类及为害性研究[J].山西农业科学,2011,39(4):356-358.
[8] 樊民周,张王斌,安德荣.陕西苹果树腐烂病病原菌的鉴定[J].西北农业学报,2004,13(3):60-61.
[9] 殷辉,周建波,张志斌,等.山西省苹果树腐烂病菌的种群结构分析[J].菌物学报,2016,35(12):1493-1502.YIN H,ZHOU J B,ZHANG Z B,et al.Population structure of Valsa species from Malus pumila in Shanxi Province[J].Mycosystema,2016,35(12):1493-1502.(in Chinese)
[10] KIRK P M,CANNON P F,DACID J C,et al.Dictionary of the Fungi[J].Mycological Research,2002,106(4):507-508.
[11] TOGASHI K.Some studies on a Japanese apple canker and its causal fungus,Valsa mali[J].Jour.Coll.Agr.Hokkaido Imp.Univ.,1924,12 (3):265-321.
[12] 阎应理,荆志刚.苹果腐烂病菌分生孢子田间消失规律研究[J].植物病理学报,1988,18(4):2-8.
[13] 高克祥,刘晓光.苹果树腐烂病研究概况[J].河北林学院学报,1995,10(1):87-91.
[14] GEORGE N A.Plant Pathology[M].San Diego,CA:Academic Press,1978.
[15] 陈策.国外对于苹果树腐烂病、桃树腐烂病以及其他同类病害的研究近况[J].中国果树,1982(Supp.1):68-77.
[16] 刘福昌,陈策,史秀琴,等.苹果树腐烂病菌(Valsa mali Miyabe et Yamada)潜伏侵染研究[J].植物保护学报,1979,6(3):1-8.
[17] 臧睿,黄丽丽,康振生,等.陕西苹果树腐烂病菌(Cytospora spp.)不同分离株的生物学特性与致病性研究[J].植物病理学报,2007,37(4):343-351.ZANG R,HUANG L L,KANG Z S,et al.Biological characteristics and pathogenicity of different isolates of Cytospora spp.isolated from apple trees in Shaanxi Province[J].Acta Phytopathologica Sinica,2007,37(4):343-351.(in Chinese)
[18] 韦洁玲,黄丽丽,郜佐鹏,等.苹果树腐烂病室内快速评价方法的研究[J].植物病理学报,2010,40(1):14-20.WEI J L,HUANG L L,GAO Z P,et al.Laboratory evaluation methods of apple valsa canker disease caused by Valsa ceratosperma sensu kobayashi[J].Acta Phytopathologica Sinica,2010,40(1):14-20.(in Chinese)
[19] 王旭丽.中国苹果树腐烂病菌的种类:rDNA-ITS序列和表型比较研究[D].杨陵:西北农林科技大学,2007.
[20] 郭翎.观赏苹果引种与苹果属(Malus Mill.)植物DNA指纹分析[D].泰安:山东农业大学,2009.
[21] 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 Moutains[J].Journal of Northwest Forestry University,2016,31(5):188-193.(in Chinese)
[22] SAITOU N,NEI M.The neighbor-joining method:a new method for reconstructing phylogenetic trees[J].Molecular Biology and Evolution,1987,4(4):406-425.
[23] NEI M.Relative efficiencies of different tree-making methods for molecular data[M]//MIYAMOTO M M,CRACRAFT J L.Phylogenetic analysis of DNA sequences.Oxford:Oxford University Press,1991.
[24] HOLDER M,LEWIS P O.Phylogeny estimation:traditional and bayesian approaches[J].Nature Reviews Genetics,2003,4(4):275-284.
[25] 潘彦平,范鑫磊,田呈明.山西省壳囊孢属真菌分类学[J].北京林业大学学报,2016,38(11):79-88.PAN Y P,FAN X L,TIAN C M.Taxonomy of Cytospora in Shanxi Province,northern China[J].Journal of Beijing Forestry University,2016,38(11):79-88.(in Chinese)
[26] 李美娜,王金友.苹果树腐烂病病原菌来源的探讨[J].北方果树,1990(2):28-30.LI M N,WANG J Y.Astudy on the source of pathogens of apple tree Valsacanker[J].Northern Fruits,1990(2):28-30.(in Chinese)
[27] KOBAYASHI T.Taxonomic study on Japanese fung[J].Journal of the Japanese Plant Pathology,1972,38:170-173.
[28] KANEHIRA T,KAMEOKA Y,SHINOHARA M.Electrophoretic Patterns of the enzymes from Valsa Ceratosperma[J].Transction of the Mycological Society of Japan,1991,32:199-205.
[29] SUZAKI K,YOSHIDA K,ITO T.Pathogenicity to apple branch and phloridzin degrading activity of Valsa ceratosperma isolated from some broad leaf trees including apple tree[J].Annual Report of the Society of Plant Protection of North Japan,1997,48:145-147.
[30] 臧睿.中国苹果树腐烂病菌的种群组成、分子检测及其ISSR遗传分析[D].杨陵:西北农林科技大学,2012.
[31] 曹支敏,梁英梅,马晓.花椒干腐病研究[J].植物保护学报,1992,19(4):58-63.CAO Z M,LIANG Y M,MA X.Study on dry rot of wild pepper[J].Journal of Plant Protection,1992,19(4):58-63.(in Chinese)
[32] 黄世钰,王建国.刺槐干腐病的研究[J].林业科学,1983,19(4):366-370.HUANG S Y,WANG J G.Study on stem rot of black locust[J].Scientia Silvae Sinicae,1983,19(4):366-370.(in Chinese)
[33] 赵晓军,周建波,赵子俊,等.枣树干腐病病原菌的鉴定[J].菌物学报,2009,28(3):332-335.ZHAO X J,ZHOU J B,ZHAO Z J,et al.the pathogen causing stem dry rot of Ziziphus jujuba[J].Mycosystema,2009,28(3):332-335.(in Chinese)
[34] 张璐璐,贾桂民,叶建丰,等.浙江临安山核桃干腐病发生发展规律[J].浙江农林大学学报,2013,30(1):148-152.ZHANG L L,JIA G M,YE J F,et al.Frequency of carya cathayensis canker disease in Lin?an City,Zhejing Province [J].Journal of Zhejiang A & F University,2013,30(1):148-152.(in Chinese)