黑龙江省黄瓜霜霉病菌生理小种分化的研究
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摘要
由鞭毛菌亚门、卵菌纲、霜霉科、古巴假霜霉菌[Pseudoperonospora cubensisi(Berk.EtCurt)Rost]侵染引起的黄瓜霜霉病,是黄瓜生产中发生最广、造成损失最严重的病害。由于黄瓜霜霉菌是一种活体营养专性寄生菌,无法人工培养,因而限制了对黄瓜霜霉菌生理分化、群体遗传等方面的深入研究。
本试验尝试以ITS区序列分析方法,配合RAPD分析方法和传统的形态(孢子囊)差异分析及致病性差异分析方法,探究黑龙江省黄瓜霜霉病菌的生理小种分化问题,为黄瓜的抗病育种提供依据。
研究结果如下:
对其中的20个菌株致病性进行测定,结果显示不同菌株对同一寄主的致病性不同,证明其毒性存在明显的差异。根据20个菌株在9个鉴别寄主上的反应,可将其划分为5个致病性类型。分别为Ⅰ组:Pc-hb2、Pc-hb3、Pc-qq1、Pc-qq3、Pc-md1、Pc-dq2、Pc-da3、Pc-hh2、Pc-hh3、Pc-hh4、Pc-hh5,Ⅱ组:Pc-im1、Pc-dq1、Pc-hh1;Ⅲ组:Pc-hb1、Pc-qq2;Ⅳ组:Pc-hb6;Ⅴ组:Pc-hb4、Pc-hb5、Pc-hb7。采自同一城市的菌源毒性存在较大差异。病菌毒性和孢子囊大小及地理来源间无相关性。
采用改良的CTAB法成功的提取了来自全省7个城市黄瓜霜霉病菌基因组DNA。并对的黄瓜霜霉病菌RAPD分子标记,结果说明,各菌株间相似性较低,各地由于栽培品种的不同,寄主与病原物之间强烈的相互选择,使黄瓜霜霉病菌这一专性寄生菌产生较为丰富的遗传多样性,存在较大的变异。其中菌株RAPD多态性与地理来源之间存在一定的相关性,多数来自同一城市的菌源间的亲缘关系较近。采自同一城市的菌源在形态、大小及亲缘关系上较为接近。
通过筛选确定了黄瓜霜霉病菌ITS区扩增的优化体系,并采用此体系对31个黄瓜霜霉病菌株进行了扩增,均可得到约为900bp的片段。经序列测定和分析得知:菌株ITS区多态性与地理来源之间存在一定的相关性,多数来自同一城市的菌源间的亲缘关系较近:来自黑河的的菌株Pc-hh1和Pc-hh3与所有菌株遗传距离较远,这和其地理位置也有一定的关系:但不同城市菌源之间菌株ITS区多态性与地理来源的关系无规律性,即并非城市地理位置相距越近,其亲缘关系就越近;来自漠河的菌株Pc-mh1与其他菌株并无明显差异。
Cucumber downy mildew,caused by Pseudoperonospora cubensis(Berk.& M.A.Curtis) Rostovzev,is the most common and destructive disease of cucumber.In the warm and humidity condition,the reproduction and spread of the pathogen are rapidly,and the disease may destroy entire plants of susceptible varieties,cause heavy losses in short periods of time.
The study consisted of these parts:(1)study on the morphological observation and measurement of sporangia of P.cubensis.(2)study on the virulence identification of P.cubensis.(3)RAPD analysis of P.cubensis from different areas.(4)ITS analysis of P.cubensis from different areas.
The 20 isolates could be devided into 5 pathotypes based on virulence on the 9 identified host, they areⅠ:Pc-hb2,Pc-hb3,Pc-qq1,Pc-qq3,Pc-md1,Pc-dq2,Pc-da3,Pc-hh2,Pc-hh3,Pc-hh4,Pc-hh5;Ⅱ: Pc-jm1,Pc-dq1,Pc-hh1;Ⅲ:Pc-hb1,Pc-qq2;Ⅳ:Pc-hb6;Ⅴ:Pc-hb4,Pc-hb5,Pc-hb7.
DNA of P.cubensis from 7 cities of Heilongjiang province were successfully extracted by CTAB method,RAPD analysis showed that the relationship of 25 isolates were not close,and genetically complex.There was relationship between RAPD analysis and their origin.Most of the isolates from the same city were close and genetic similarity.
Using the established system,all the 31 genomic DNA samples could obtain an approximate 900bp,which collected from different regions of Heilongjiang Province.According the sequence measurement and the analysis:The germ stub ITS area many Tais and of geography source relativity with certain existence,the relative relation of the most germ from same city source is nearer;Come from black river of of the germ stub Pc-hh 1 with Pc-hh 3 with all germ stub heredity distance farther,this with its geography position also have a certain relation;But different city germ's relation between many Tais and geography source of the germ stub ITS area of the source have no regulation,namely and not city geography position is apart from more near,it is relative to relate to more near;The germ from Mo river stub Pc-mh1 have no obvious difference with other germ stubs.
本试验尝试以ITS区序列分析方法,配合RAPD分析方法和传统的形态(孢子囊)差异分析及致病性差异分析方法,探究黑龙江省黄瓜霜霉病菌的生理小种分化问题,为黄瓜的抗病育种提供依据。
研究结果如下:
对其中的20个菌株致病性进行测定,结果显示不同菌株对同一寄主的致病性不同,证明其毒性存在明显的差异。根据20个菌株在9个鉴别寄主上的反应,可将其划分为5个致病性类型。分别为Ⅰ组:Pc-hb2、Pc-hb3、Pc-qq1、Pc-qq3、Pc-md1、Pc-dq2、Pc-da3、Pc-hh2、Pc-hh3、Pc-hh4、Pc-hh5,Ⅱ组:Pc-im1、Pc-dq1、Pc-hh1;Ⅲ组:Pc-hb1、Pc-qq2;Ⅳ组:Pc-hb6;Ⅴ组:Pc-hb4、Pc-hb5、Pc-hb7。采自同一城市的菌源毒性存在较大差异。病菌毒性和孢子囊大小及地理来源间无相关性。
采用改良的CTAB法成功的提取了来自全省7个城市黄瓜霜霉病菌基因组DNA。并对的黄瓜霜霉病菌RAPD分子标记,结果说明,各菌株间相似性较低,各地由于栽培品种的不同,寄主与病原物之间强烈的相互选择,使黄瓜霜霉病菌这一专性寄生菌产生较为丰富的遗传多样性,存在较大的变异。其中菌株RAPD多态性与地理来源之间存在一定的相关性,多数来自同一城市的菌源间的亲缘关系较近。采自同一城市的菌源在形态、大小及亲缘关系上较为接近。
通过筛选确定了黄瓜霜霉病菌ITS区扩增的优化体系,并采用此体系对31个黄瓜霜霉病菌株进行了扩增,均可得到约为900bp的片段。经序列测定和分析得知:菌株ITS区多态性与地理来源之间存在一定的相关性,多数来自同一城市的菌源间的亲缘关系较近:来自黑河的的菌株Pc-hh1和Pc-hh3与所有菌株遗传距离较远,这和其地理位置也有一定的关系:但不同城市菌源之间菌株ITS区多态性与地理来源的关系无规律性,即并非城市地理位置相距越近,其亲缘关系就越近;来自漠河的菌株Pc-mh1与其他菌株并无明显差异。
Cucumber downy mildew,caused by Pseudoperonospora cubensis(Berk.& M.A.Curtis) Rostovzev,is the most common and destructive disease of cucumber.In the warm and humidity condition,the reproduction and spread of the pathogen are rapidly,and the disease may destroy entire plants of susceptible varieties,cause heavy losses in short periods of time.
The study consisted of these parts:(1)study on the morphological observation and measurement of sporangia of P.cubensis.(2)study on the virulence identification of P.cubensis.(3)RAPD analysis of P.cubensis from different areas.(4)ITS analysis of P.cubensis from different areas.
The 20 isolates could be devided into 5 pathotypes based on virulence on the 9 identified host, they areⅠ:Pc-hb2,Pc-hb3,Pc-qq1,Pc-qq3,Pc-md1,Pc-dq2,Pc-da3,Pc-hh2,Pc-hh3,Pc-hh4,Pc-hh5;Ⅱ: Pc-jm1,Pc-dq1,Pc-hh1;Ⅲ:Pc-hb1,Pc-qq2;Ⅳ:Pc-hb6;Ⅴ:Pc-hb4,Pc-hb5,Pc-hb7.
DNA of P.cubensis from 7 cities of Heilongjiang province were successfully extracted by CTAB method,RAPD analysis showed that the relationship of 25 isolates were not close,and genetically complex.There was relationship between RAPD analysis and their origin.Most of the isolates from the same city were close and genetic similarity.
Using the established system,all the 31 genomic DNA samples could obtain an approximate 900bp,which collected from different regions of Heilongjiang Province.According the sequence measurement and the analysis:The germ stub ITS area many Tais and of geography source relativity with certain existence,the relative relation of the most germ from same city source is nearer;Come from black river of of the germ stub Pc-hh 1 with Pc-hh 3 with all germ stub heredity distance farther,this with its geography position also have a certain relation;But different city germ's relation between many Tais and geography source of the germ stub ITS area of the source have no regulation,namely and not city geography position is apart from more near,it is relative to relate to more near;The germ from Mo river stub Pc-mh1 have no obvious difference with other germ stubs.
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