黑龙江省瓜类白粉病菌生理小种鉴定及其多样性分析
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摘要
白粉病是瓜类作物广泛发生的一种世界性病害,温室及露地均可发生,由于为害瓜类作物的白粉病菌生理小种分化快,所以明确当地白粉病菌生理小种及其地理分布对引进抗源材料,开展抗病育种及防治等方面均具有重要意义。应用分子标记辅助育种可提高选择效率,加快新抗病品种的培育。
本实验比较了三种不同纯化保存白粉病菌的方法,结果显示,活体保存法保存时间最长约为3个月,离体打孔法保存时间最短约为1个月,MS添加激素IAA叶碟保存法可大约保存1.5个月,最适合子叶生根的激素IAA浓度为1.5mg/l。三种方法各有利弊,应根据需要选择合适的保存方法。
2010年采集黑龙省不同生态区不同设施内的甜瓜、黄瓜、南瓜、西瓜等瓜类白粉病病菌17份,利用国际通用的瓜类白粉病鉴定标准对其进行生理小种鉴定,根据13个鉴别寄主的抗感反应,初步确定黑龙江省瓜类白粉病菌存在3个生理小种,即单囊壳白粉菌(Podosphaera xanthii)的生理小种1和生理小种N1,还有一个新生理小种,其中优势小种是生理小种1。对其中部分白粉病菌进行RAPD分析,从119个随机引物中筛选出10个条带清晰重复性好的引物,扩增得到157个位点,其中多态性位点为138个,多态性位点频率为97.89%,说明黑龙江省葫芦科作物白粉菌有丰富的遗传多样性。利用NTSYS-PC软件进行数据分析,结果表明:供试菌株之间遗传相似系数的变化幅度为0.52-0.75,根据遗传相似系数用类平均法(UPGMA)对其聚类,以遗传相似系数0.60为阈值,供试菌株可区分为4个类群。同是生理小种1的菌株部分聚到了同一类,新生理小种与部分生理小种1菌株聚到同一类,同是生理小种N1的两个菌株未聚到同一类;相同地理来源或相同寄主来源的白粉菌也未聚到一类。初步确定瓜类白粉病菌群体基因型与致病性之间不形成对应关系,与菌株地理来源、寄主来源及设施类型亦无明显直接关系。
应用通用引物对部分黑龙江省瓜类白粉病菌进行转录单位间隔区(IGS)分析,结果表明,不同菌株的IGS区存在长度异质性和数量多态性。应用MspⅠ, AsuⅠ, Hinp1Ⅰ3个限制性内切酶对IGS扩增产物进行酶切(IGS-RFLP),不同菌株的酶切位点不同,电泳后产生多样性丰富的图谱,成为菌株特有的DNA指纹,实验所用的3个限制性内切酶都可以有效地将供试菌株加以鉴别,但未发现不同小种间的特异性标记。利用NTSYS-PC软件对IGS1-RFLP酶切图谱进行数据分析,各菌株之间遗传相似系数的变化幅度为0.46-0.80,说明白粉菌间有丰富的遗传多样性,以遗传相似系数0.60为阈值,供试菌株可区分为3个类群,初步确定瓜类白粉病菌群体基因型与致病性之间不形成对应关系,与菌株地理来源、寄主来源及设施类型亦无明显直接关系。
Powdery mildew is one of the most important disease attacking cucurbits in greenhouse and open-field. The powdery mildew physiological races infect cucurbits change quickly.Consequently, knowledge on the prevalence and geographic distribution of these races has great significances for the choice of appropriate strains, resistance breeding and prevention, using molecular-maker assisted breeding can enhance the selection efficiency and accelerate breeding of resistant variety.
Compared three different methods for purifing and preserving pathogen, the result showed that preservation time was the longest by live plants, it was about three months; preservation time was the shortest by detached leaves, it was about one month; preservation time was one and half months by detached cotyledon MS medium adding hormone IAA, the best concentration of hormone IAA for cotyledon rooting was 1.5mg/l. Three methods have their advantage and dis advantage respectively, according to requirement selecting proper preservation method.
Seventeen powdery mildew samples were collected and isolated from mildewed melon, cucumber, pumpkin and watermelon in greenhouse or open field of different ecological regions in Heilongjiang Province in 2010, all those samples were detected using the standard identification hosts of powdery mildew. According to performance on thirteen hosts which are universal in the world, three races on Cucurbitaceae were preliminarily confirmed which included a new physiological race, physiological races 1 and physiological race N1 of Podosphaera xanthii in Heilongjiang Province. Race 1 was the dominant race. Partial isolates of powdery mildew DNA were evaluated using Random Amplified Polymorphic DNA (RAPD) with 10 primers selected from 119 random primers. A total of 157 bands were scored, polymorphic bands were 138, the frequency of polymorphic bands was 97.89%, and it showed that high genetic diversity existed among the isolates of P. xanthii on Cucurbitaceae in Heilongjiang Province. On the basis of RAPD data analyzed by software NTSYS-PC, the similarity coefficient varied from 0.52 to 0.57 and a dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method. The tested isolates were clustered into 4 groups at the similarity coefficient of 0.60, the isolates of race 1 were clustered into the same group partly, the new race was clustered into the same group with partial race 1 isolates; two isolates of race N1 were not clustered into the same group with partial race 1 isolates. The isolates of the same geographic or host origin were also not clustered into the same group. So, no obvious association was found between DNA polymorphism and pathogen races differentiations as identified by biological tests on cucurbitaceae. Also, no association was found between the results of RAPD analysis and any other of parameter tested: geographic, greenhouse type or host origin.
Intergenic spacer (IGS) was analyzed for strains of powdery mildew on cucurbits using universal primers, results showed that there were length heterogeneity and polymorphism on IGS. IGS-RFLP digested by MspⅠ, AsuⅠand Hinp1Ⅰshowed that digest sites varied from the strains with profile polymorphism. The IGS-RFLP profile could become distinctive DNA fingerprint for strains. All the tested strains could be effectively identified using restrictive enzymes of MspⅠAsuⅠHinp1Ⅰ. But, no specific RFLP marker was observed between different races. On the basis of IGS1-RFLP data analyzed by software NTSYS-PC, the similarity coefficient varied from 0.46 to 0.80, the isolates were clustered into three groups at the similarity coefficient of 0.60. No obvious association was found between DNA polymorphism and pathogen races differentiations as identified by biological tests on cucurbitaceae, also no association was found between the genetic diversity and any other of parameter tested: geographic, host origin or greenhouse type.
本实验比较了三种不同纯化保存白粉病菌的方法,结果显示,活体保存法保存时间最长约为3个月,离体打孔法保存时间最短约为1个月,MS添加激素IAA叶碟保存法可大约保存1.5个月,最适合子叶生根的激素IAA浓度为1.5mg/l。三种方法各有利弊,应根据需要选择合适的保存方法。
2010年采集黑龙省不同生态区不同设施内的甜瓜、黄瓜、南瓜、西瓜等瓜类白粉病病菌17份,利用国际通用的瓜类白粉病鉴定标准对其进行生理小种鉴定,根据13个鉴别寄主的抗感反应,初步确定黑龙江省瓜类白粉病菌存在3个生理小种,即单囊壳白粉菌(Podosphaera xanthii)的生理小种1和生理小种N1,还有一个新生理小种,其中优势小种是生理小种1。对其中部分白粉病菌进行RAPD分析,从119个随机引物中筛选出10个条带清晰重复性好的引物,扩增得到157个位点,其中多态性位点为138个,多态性位点频率为97.89%,说明黑龙江省葫芦科作物白粉菌有丰富的遗传多样性。利用NTSYS-PC软件进行数据分析,结果表明:供试菌株之间遗传相似系数的变化幅度为0.52-0.75,根据遗传相似系数用类平均法(UPGMA)对其聚类,以遗传相似系数0.60为阈值,供试菌株可区分为4个类群。同是生理小种1的菌株部分聚到了同一类,新生理小种与部分生理小种1菌株聚到同一类,同是生理小种N1的两个菌株未聚到同一类;相同地理来源或相同寄主来源的白粉菌也未聚到一类。初步确定瓜类白粉病菌群体基因型与致病性之间不形成对应关系,与菌株地理来源、寄主来源及设施类型亦无明显直接关系。
应用通用引物对部分黑龙江省瓜类白粉病菌进行转录单位间隔区(IGS)分析,结果表明,不同菌株的IGS区存在长度异质性和数量多态性。应用MspⅠ, AsuⅠ, Hinp1Ⅰ3个限制性内切酶对IGS扩增产物进行酶切(IGS-RFLP),不同菌株的酶切位点不同,电泳后产生多样性丰富的图谱,成为菌株特有的DNA指纹,实验所用的3个限制性内切酶都可以有效地将供试菌株加以鉴别,但未发现不同小种间的特异性标记。利用NTSYS-PC软件对IGS1-RFLP酶切图谱进行数据分析,各菌株之间遗传相似系数的变化幅度为0.46-0.80,说明白粉菌间有丰富的遗传多样性,以遗传相似系数0.60为阈值,供试菌株可区分为3个类群,初步确定瓜类白粉病菌群体基因型与致病性之间不形成对应关系,与菌株地理来源、寄主来源及设施类型亦无明显直接关系。
Powdery mildew is one of the most important disease attacking cucurbits in greenhouse and open-field. The powdery mildew physiological races infect cucurbits change quickly.Consequently, knowledge on the prevalence and geographic distribution of these races has great significances for the choice of appropriate strains, resistance breeding and prevention, using molecular-maker assisted breeding can enhance the selection efficiency and accelerate breeding of resistant variety.
Compared three different methods for purifing and preserving pathogen, the result showed that preservation time was the longest by live plants, it was about three months; preservation time was the shortest by detached leaves, it was about one month; preservation time was one and half months by detached cotyledon MS medium adding hormone IAA, the best concentration of hormone IAA for cotyledon rooting was 1.5mg/l. Three methods have their advantage and dis advantage respectively, according to requirement selecting proper preservation method.
Seventeen powdery mildew samples were collected and isolated from mildewed melon, cucumber, pumpkin and watermelon in greenhouse or open field of different ecological regions in Heilongjiang Province in 2010, all those samples were detected using the standard identification hosts of powdery mildew. According to performance on thirteen hosts which are universal in the world, three races on Cucurbitaceae were preliminarily confirmed which included a new physiological race, physiological races 1 and physiological race N1 of Podosphaera xanthii in Heilongjiang Province. Race 1 was the dominant race. Partial isolates of powdery mildew DNA were evaluated using Random Amplified Polymorphic DNA (RAPD) with 10 primers selected from 119 random primers. A total of 157 bands were scored, polymorphic bands were 138, the frequency of polymorphic bands was 97.89%, and it showed that high genetic diversity existed among the isolates of P. xanthii on Cucurbitaceae in Heilongjiang Province. On the basis of RAPD data analyzed by software NTSYS-PC, the similarity coefficient varied from 0.52 to 0.57 and a dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method. The tested isolates were clustered into 4 groups at the similarity coefficient of 0.60, the isolates of race 1 were clustered into the same group partly, the new race was clustered into the same group with partial race 1 isolates; two isolates of race N1 were not clustered into the same group with partial race 1 isolates. The isolates of the same geographic or host origin were also not clustered into the same group. So, no obvious association was found between DNA polymorphism and pathogen races differentiations as identified by biological tests on cucurbitaceae. Also, no association was found between the results of RAPD analysis and any other of parameter tested: geographic, greenhouse type or host origin.
Intergenic spacer (IGS) was analyzed for strains of powdery mildew on cucurbits using universal primers, results showed that there were length heterogeneity and polymorphism on IGS. IGS-RFLP digested by MspⅠ, AsuⅠand Hinp1Ⅰshowed that digest sites varied from the strains with profile polymorphism. The IGS-RFLP profile could become distinctive DNA fingerprint for strains. All the tested strains could be effectively identified using restrictive enzymes of MspⅠAsuⅠHinp1Ⅰ. But, no specific RFLP marker was observed between different races. On the basis of IGS1-RFLP data analyzed by software NTSYS-PC, the similarity coefficient varied from 0.46 to 0.80, the isolates were clustered into three groups at the similarity coefficient of 0.60. No obvious association was found between DNA polymorphism and pathogen races differentiations as identified by biological tests on cucurbitaceae, also no association was found between the genetic diversity and any other of parameter tested: geographic, host origin or greenhouse type.
引文
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