江苏省棉花黄萎病菌致病力分化和分子检测
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摘要
棉花黄萎病(Verticillium wilt.)是世界性的土传植物病害,是棉花生产上危害最严重的病害之一。在各地,甚至同一棉田生长过程中其棉花黄萎病的症状类型、发病率、病害严重程度等方面常存在较大差异。这些差异是由于同一棉田棉株个体间的抗病性差异引起的,还是由于土壤中病原菌的致病力分化引起,是一个值得研究的理论问题和现实生产问题。为此,本文对棉花黄萎病菌的致病力分化进行了研究,取得了如下结果:
1.病原菌分离培养后的生物学培养性状结果表明江苏省棉花黄萎病菌以菌核型居多,其次为菌丝型,中间型最少。研究结果还表明在江苏省同一棉田中,甚至个别在同一植株上分离培养的黄萎病菌都存在着形态上的差异。
2.通过比较棉选一号培养基、吕金殿培养基及棉叶培养基,筛选用于从土壤中分离棉花黄萎病菌的培养基。结果表明,棉选一号培养基最好,其配方较为简单,并且杂菌少。
3. 2008年采集江苏省大丰市土样,用水筛法分离土壤棉花黄萎病菌微菌核。结果表明,棉花黄萎病菌在土壤中的垂直分布主要在0-40cm的上层中,在重病区0-20cm的土层中棉花黄萎病微菌核占总的70%左右,在20-30cm分布着20%左右的微菌核,在30-40cm处仅分布10%左右的的微菌核。在轻病区1的0-20cm的土层中棉花黄萎病微菌核占总的71.2%,20-30cm处分布18%,在30-40cm分布10.8%。因此,深翻上层或对表层土壤进行处理,是防治棉花黄萎病菌的有效方法之一。
4.采用了PCR技术对江苏省棉花黄萎菌系进行检测,监测江苏棉花黄萎病菌的致病类型。利用落叶型棉花黄萎病菌的的特异引物D1、D2对采自江苏棉区的64个棉花黄萎病菌菌系进行PCR扩增,54个菌系扩增出500 bp的片段,与前人报道的落叶型棉花黄萎病系扩增出的片段大小一致,说明这些菌系为落叶型棉花黄萎病系,而剩余的10个菌系未扩增出任何片段,利用非落叶型黄萎病菌的特异性引物ND1,ND2对这10个菌系进行扩增,扩增出1500 bp的片段。落叶型黄萎病菌占供试菌系的84.38%,表明目前江苏省棉花黄萎病菌依旧以落叶型黄萎病菌为主。
5.选有代表性的棉花黄萎病菌52个菌株及4个对照菌株,各配成一定浓度的孢子悬浮液,于棉花苗期1-2叶期伤根接菌,在温室中对各代表菌株进行致病力测定。结果表明,各菌株致病力强弱差异明显,并发现江苏棉花黄萎病菌有落叶型菌系的存在,可分为致病力强的Ⅰ型,混合类致病力中等的Ⅱ型和弱致病力的III型分别占17.86%,71.43%,10.71%。结果还表明,同一棉田棉花黄萎病存在落叶型和非落叶型两类表现类型,而且棉花黄萎病菌又同时存在强、中、弱三种不同致病类型,这就表明棉花黄萎病菌是一个易变异的混合基因型群体,群体中的不同个体组成的亚群体具有不同的致病性。
Verticillium wilt, one of the serious diseases of cotton, is a worldwide soil-borne disease. In the different area, even in the same cotton field the symptom types, the disease incidence and the disease index of cotton Verticillium wilt are all different Whether the disease resistance difference of plant individuals or the pathogenicity differentiation of the pathogen in the soil made the difference? It is a good theoretical question and an important reality practical problem. Therefore,we carried out the reseach on the pathogenicity differentiation of verticillium dahliae and got the results as followes.
1. The culture characteristics of the strains in jiangsu province showed that the most of them were sclerotium type, then filament type, and the middle type were fewest. The results also showed that the culture characteristics of the strains isolated in the same cotton field even in the same plant were different.
2. In order to get the best medium for iosolated verticillium dahliae from soil, Mian Xuan 1 medium, Lv-jindian medium and cotton leaves mediun were selected. The result indicated Mian Xuan 1 was best. It was the simplest and less contamination.
3. The microsclerotia of Verticillium dahliae were isolated from the soil sample collected in August 2008 in Dafeng county, jiangsu province.The results showed that the amount of the microsclerotia, were significantly correlative with soil layers. In heavy disease area there was about 70% microsclerotia in 0 to 20 cm deep earth, 20% microsclerotina from 20.1 cm to 30cm deep earth and about 10% microsclerotia in 30 cm to 40 cm deep earth.. There was only few that under 40 cm soil layers. In light disease area there was about 71.2% microsclerotia in 0 to 20cm deep earth, 18% microsclerotina from 20.1 cm to 30cm deep earth and about 10.8% microsclerotia in 30.1cm to 40cm deep earth. There was only few that under 40.1cm soil layers. Thus, deep plowing, or the upper surface of the soil treatment, which is one effective methods.to prevention and treatment Verticillium dahliae .
4. 64 strains of verticillium dahliae from jiangsu province were detected by using PCR technology to monitor their pathotypes. The fragment (500bp) was amplified from 54 strains by using the specific primers D1, D2 of defoliation pathotype of verticillium dahliae and the size was accordant with other defoliation pathotype of verticillium dahliae reported in the literature, which indicated that these strains were defoliation pathotype of verticillium dahliae. The fragment (1500bp) was amplified from 10 strains by using the specific primers ND1 and ND2 of non-defoliating pathotype of verticillium dahliae. The defoliation pathotype of verticillium dahliae pathogen occupied 84.38% of the tested strains, these results indicated that the verticillium dahliae in jiangsu province was still mainly belong to defoliation pathotype at present.
5. The pathogenicity tests of 52 strains of Verticillium dahliae isolated from jiangsu province and 4 control strains were carried out in greenhouse. According to their reaction in hosts,56 strains were classified into 3 types of pathogenicity:type I (severe pathogenic type), type II (intermediate pathogenic type), and type III(weak pathogenic type). Type I was 17.86%, Type II was 71.43%, and type III was 10.71%, respectively. The defoliation strains were found in these strains. These results also explained that both non-defoliating and defoliating symptoms appeared in the same cotton field and indicated that Verticillium dahliae was a mix of easy to variant genotype groups, which composed of different sub-groups of individuals with different pathogenicity.
1.病原菌分离培养后的生物学培养性状结果表明江苏省棉花黄萎病菌以菌核型居多,其次为菌丝型,中间型最少。研究结果还表明在江苏省同一棉田中,甚至个别在同一植株上分离培养的黄萎病菌都存在着形态上的差异。
2.通过比较棉选一号培养基、吕金殿培养基及棉叶培养基,筛选用于从土壤中分离棉花黄萎病菌的培养基。结果表明,棉选一号培养基最好,其配方较为简单,并且杂菌少。
3. 2008年采集江苏省大丰市土样,用水筛法分离土壤棉花黄萎病菌微菌核。结果表明,棉花黄萎病菌在土壤中的垂直分布主要在0-40cm的上层中,在重病区0-20cm的土层中棉花黄萎病微菌核占总的70%左右,在20-30cm分布着20%左右的微菌核,在30-40cm处仅分布10%左右的的微菌核。在轻病区1的0-20cm的土层中棉花黄萎病微菌核占总的71.2%,20-30cm处分布18%,在30-40cm分布10.8%。因此,深翻上层或对表层土壤进行处理,是防治棉花黄萎病菌的有效方法之一。
4.采用了PCR技术对江苏省棉花黄萎菌系进行检测,监测江苏棉花黄萎病菌的致病类型。利用落叶型棉花黄萎病菌的的特异引物D1、D2对采自江苏棉区的64个棉花黄萎病菌菌系进行PCR扩增,54个菌系扩增出500 bp的片段,与前人报道的落叶型棉花黄萎病系扩增出的片段大小一致,说明这些菌系为落叶型棉花黄萎病系,而剩余的10个菌系未扩增出任何片段,利用非落叶型黄萎病菌的特异性引物ND1,ND2对这10个菌系进行扩增,扩增出1500 bp的片段。落叶型黄萎病菌占供试菌系的84.38%,表明目前江苏省棉花黄萎病菌依旧以落叶型黄萎病菌为主。
5.选有代表性的棉花黄萎病菌52个菌株及4个对照菌株,各配成一定浓度的孢子悬浮液,于棉花苗期1-2叶期伤根接菌,在温室中对各代表菌株进行致病力测定。结果表明,各菌株致病力强弱差异明显,并发现江苏棉花黄萎病菌有落叶型菌系的存在,可分为致病力强的Ⅰ型,混合类致病力中等的Ⅱ型和弱致病力的III型分别占17.86%,71.43%,10.71%。结果还表明,同一棉田棉花黄萎病存在落叶型和非落叶型两类表现类型,而且棉花黄萎病菌又同时存在强、中、弱三种不同致病类型,这就表明棉花黄萎病菌是一个易变异的混合基因型群体,群体中的不同个体组成的亚群体具有不同的致病性。
Verticillium wilt, one of the serious diseases of cotton, is a worldwide soil-borne disease. In the different area, even in the same cotton field the symptom types, the disease incidence and the disease index of cotton Verticillium wilt are all different Whether the disease resistance difference of plant individuals or the pathogenicity differentiation of the pathogen in the soil made the difference? It is a good theoretical question and an important reality practical problem. Therefore,we carried out the reseach on the pathogenicity differentiation of verticillium dahliae and got the results as followes.
1. The culture characteristics of the strains in jiangsu province showed that the most of them were sclerotium type, then filament type, and the middle type were fewest. The results also showed that the culture characteristics of the strains isolated in the same cotton field even in the same plant were different.
2. In order to get the best medium for iosolated verticillium dahliae from soil, Mian Xuan 1 medium, Lv-jindian medium and cotton leaves mediun were selected. The result indicated Mian Xuan 1 was best. It was the simplest and less contamination.
3. The microsclerotia of Verticillium dahliae were isolated from the soil sample collected in August 2008 in Dafeng county, jiangsu province.The results showed that the amount of the microsclerotia, were significantly correlative with soil layers. In heavy disease area there was about 70% microsclerotia in 0 to 20 cm deep earth, 20% microsclerotina from 20.1 cm to 30cm deep earth and about 10% microsclerotia in 30 cm to 40 cm deep earth.. There was only few that under 40 cm soil layers. In light disease area there was about 71.2% microsclerotia in 0 to 20cm deep earth, 18% microsclerotina from 20.1 cm to 30cm deep earth and about 10.8% microsclerotia in 30.1cm to 40cm deep earth. There was only few that under 40.1cm soil layers. Thus, deep plowing, or the upper surface of the soil treatment, which is one effective methods.to prevention and treatment Verticillium dahliae .
4. 64 strains of verticillium dahliae from jiangsu province were detected by using PCR technology to monitor their pathotypes. The fragment (500bp) was amplified from 54 strains by using the specific primers D1, D2 of defoliation pathotype of verticillium dahliae and the size was accordant with other defoliation pathotype of verticillium dahliae reported in the literature, which indicated that these strains were defoliation pathotype of verticillium dahliae. The fragment (1500bp) was amplified from 10 strains by using the specific primers ND1 and ND2 of non-defoliating pathotype of verticillium dahliae. The defoliation pathotype of verticillium dahliae pathogen occupied 84.38% of the tested strains, these results indicated that the verticillium dahliae in jiangsu province was still mainly belong to defoliation pathotype at present.
5. The pathogenicity tests of 52 strains of Verticillium dahliae isolated from jiangsu province and 4 control strains were carried out in greenhouse. According to their reaction in hosts,56 strains were classified into 3 types of pathogenicity:type I (severe pathogenic type), type II (intermediate pathogenic type), and type III(weak pathogenic type). Type I was 17.86%, Type II was 71.43%, and type III was 10.71%, respectively. The defoliation strains were found in these strains. These results also explained that both non-defoliating and defoliating symptoms appeared in the same cotton field and indicated that Verticillium dahliae was a mix of easy to variant genotype groups, which composed of different sub-groups of individuals with different pathogenicity.
引文
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