植物病原真菌及重寄生菌的分离鉴定与侵染性研究
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摘要
由白粉菌(Erysiphauceae)导致的白粉病和核盘菌(Sclerotinia sclerotiorum)导致的菌核病是两种危害农作物较为严重的真菌病害。
白粉菌危害严重，且防治困难。白粉寄生孢(Ampelomyces quisqualis)作为白粉菌上较为重要的重寄生菌，能够在大多数白粉菌上寄生，并对用来防治白粉菌的化学药物具有抗药性，因此白粉寄生孢具有防治植物白粉病的作用。但由于来源不同的白粉寄生孢菌株间存在一定的遗传差异，所以在利用白粉寄生孢防治白粉病时具有一定局限性，所以明确白粉寄生孢的资源状况，筛选新的有潜力的生防菌株就显得及其重要。
由核盘菌侵染引起的菌核病是一种严重病害，核盘菌是一种放线状子囊真菌，其寄主范围和地理分布十分广泛，尤其在南方油菜产区发病严重，该病害在发病严重年份病株率可达50-80％，严重制约油菜品质和产量，造成经济上的损失，是危害油菜最严重的病害之一。
本文对周口地区凤仙花白粉菌和分离自凤仙花白粉病上的白粉寄生孢以及感染了菌核病油菜上的核盘菌进行了显微观察、生物学特性、分子生物学以及致病性等研究，并取得了以下主要结果：
1.对周口地区检出的凤仙花白粉菌进行了显微形态分析、核糖体DNA转录间隔区ITS(inter transcribed spacer)进行PCR扩增及进化分析。结果表明：在扫描电子显微镜下病菌分生孢子呈圆柱形或椭圆形，无支链，ITS序列与来自日本番茄上的O.neolycopersici(AB094991)和来自美国番茄上的O.neolycopersici(AB163915)同源性最高聚为一支，序列同源性达到100%，分析结果表明，来自周口地区的番茄白粉菌为Oidiumneo lycopersici。
2.从河南省周口市采集感染白粉病的凤仙花植株，采用单孢分离法分离得到白粉寄生孢，对该菌进行菌落和显微形态分析，初步证明该菌株为A.quisqualis。利用真菌通用引物ITS1和ITS4对所分离的菌株内转录间隔区进行PCR扩增，得到578bp大小的片段，经过克隆测序和在GenBank中进行BLASTn分析，发现与德国和美国白粉寄生孢ITS序列的同源性达到100％,进一步证明该分离菌株为A.quisqualis。并将白粉寄生孢接种在患有白粉病的大叶黄杨叶面上，经一周时间检查发现，白粉菌菌体由无色渐变成浅黄色或浅褐色，并渐渐萎缩至白粉层消失，说明从周口地区凤仙花白粉病病株上分离得到的白粉寄生孢具有防治白粉病的效果。
3.通过探究白粉寄生孢最适的生长环境研究发现，其温度生长范围较广：10-30℃，菌株生长和产孢的最适温度为20℃。白粉寄生孢在pH5-10时均可生长及产孢，其中pH值为7时最适宜其产孢。最适合生长的理想C源为葡萄糖，最适合的N源为蛋白胨，持续光照对菌株的生长及产孢具有明显的促进作用。
4.从河南省周口市采集感染菌核病的油菜，对核盘菌进行分离和鉴定。采用单孢分离法分离得到该菌系，对该菌系进行了菌落和显微形态分析，初步证明该菌株为S.sclerotiorum。利用真菌通用引物ITS1和ITS4对所分离的单菌系ITS序列进行PCR扩增，得到640bp大小的片段，经过克隆测序和在GenBank中进行BLASTn分析，发现与已知核盘菌属菌核病菌ITS序列的同源性达到100％，进一步证明该分离菌株为S.sclerotiorum。
5.通过研究不同温度、pH值、C源和N源、光照、培养基对核盘菌的最适合生长环境的研究表明，核盘菌温度生长范围较广：15-35℃，其中25℃是菌株生长及产孢的最适温度。核盘菌在pH5-10时均可生长及产孢，其中pH值为8时最适宜其产孢。菌落生长较为理想的C源和N源为蔗糖和酵母浸膏，持续光照对菌株的生长及产孢影响不显著。
6.将菌悬液分别接种在耐菌核病型油菜和易感菌核病型油菜上，4天后用台盼兰染色观察，发现耐病型和易感型均可被感染，菌丝侵入的方式两个基因型也没有差别，但菌丝长度不同。用菌核病侵染番茄和拟南芥植物时发现这两种植物也均可被核盘菌侵染。下一步的工作，将研究白粉寄生孢能否对核盘菌具有生物防治作用。
Powdery mildew and Sclerotinia sclerotiorum are two kinds of the most important plant diseases inChina. Ampelomyces quisqualis is an important mycoparasite of powdery mildew fungus with resistanceagainst most used chemical fungicides, which has potential to control powdery mildew. Because differentaccessions of A.quisqualis could show genetic differences, and the potential application of A.quisqualis isaccession dependent. Therefore, screening A.quisqualis resources to obtain potential new accessionssuitable for local application on fungal disease prevention is important. S.sclerotiorum is a filamentousAscomycete fungus with a wide host range, which causes server losses of the rapeseed production,especially in the southern areas. The disease at the year of serious outbreak can infect to50-80%of plants,which is one of the most important disease of rapeseed causing serious damage to rapeseed production bothon yield and on quality.
In this paper, characteristics of A.quisquals in Zhoukou and interaction between the A.quisquals andpowdery mildew fungi were investigated. Characteristics of S. sclerotiorum in Zhoukou and it’s interactionwith the susceptible and tolerant genotypes of rapeseed were studied.
The results are as following:
1. Powdery mildew appearing in colonies of white patches prevalently on leaves of tomato was foundin Zhoukou of Henan Province. The microscopic morphology and molecular identification of the pathogensthat Cause this disease were studied in terms of internal transcribed spacer (ITS) sequence and phylogenetictrees. The conidia which were borne singly, cylindrical, or ellipsoid-cylindrical without branch wereobserved under the scanning electron micriscipy (SEM). The ITS sequence is highly homologous to ITSsequences from O.neolycopersici(AB163916、AB094991), It draws a conclusion that the pathogen oftomato powdery mildew in Zhoukou belongs to O.neolycopersici.
2. An strain of A.quisqualis was isolated from powdery mildew infected rose balsam (Impatiensbalsamina L.) in Zhoukou city of Henan province with the method of single-spore isolation. Themorphology and microscopic analysis indicated that the isolated stain was A.quisqualis. Universal primersITS1and ITS4for fungi were used to amplify the internal transcribed spacer (ITS) region of the isolated strain, which resulted in a578bp band. The PCR product was sequenced and analyzed by using BLAST,which showed100%homology to AQ in Germany and USA. It further proved that the isolated strain was A.quisqualis. A.quisqualis was inoculated on powdery mildew of Euonymus japonicus, after one-weekinoculation, it showed that the powdery mildew gradually become yellow or shallow brown, and thepowdery mildew gradually disappear, indicating that the A. quisqualis isolate of powdery mildew on RoseBlasam from Zhoukou area can be used for the prevention of powdery mildew.
3. The result showed that A.quisqualis can grow at temperatyre of10-30℃and the optimaltemperature was20℃. The pH value of5-10, the optimal pH value for its growth and sporulation were at7.The optimal C source was lactose, and the optimal N source was peptone. Light could effectively stimulateAQ growth and sporulation.
4. A strain of S. sclerotiorum was isolated from rapeseed in Zhoukou city of Henan province with themethod of single-spore isolation. The morphology and microscopic analyses indicated that the isolatedstain was S. sclerotiorum. Universal primers ITS1and ITS4for fungi were used to amplify the ITS regionof the isolated strain, which resulted in a640bp band. The PCR product was sequenced and analyzed byusing BLASTn, which showed100%homology to found S.sclerotiorum. It further proved that the isolatedstrain was S.sclerotiorum.
5. The result showed that S.sclerotiorum can grow at temperatyre of15-35℃and the optimaltemperature was25℃. The pH value of5-10, the optimal pH value for its growth and sporulation were at8.The optimal C source was sugar, and the optimal N source was yeast extract. Light could effectivelystimulate S.sclerotiorum growth and sporulation.
6. Fungal suspension of S. sclerotiorum was used to inoculate susceptible and tolerant phenotypes ofrapeseed. Four days after inoculation, the infected leaves were stained with trypan blue to study themechanism difference of responses to S. sclerotiorum by different rapeseed genotypes. It indicated that noobvious difference between two genotypes, except that the length of hypha on tolerant genotype wereshorter than those on susceptible genotype of rapeseed. The infection processes of S. sclerotiorum ontomato and Arabidopsis leaves were also investigated. It indicated that S. sclerotiorum can infect tomatoand Arabidopsis. Further study will be carried out to test whether AQ can prevent the growth of S.sclerotiorum.
白粉菌危害严重，且防治困难。白粉寄生孢(Ampelomyces quisqualis)作为白粉菌上较为重要的重寄生菌，能够在大多数白粉菌上寄生，并对用来防治白粉菌的化学药物具有抗药性，因此白粉寄生孢具有防治植物白粉病的作用。但由于来源不同的白粉寄生孢菌株间存在一定的遗传差异，所以在利用白粉寄生孢防治白粉病时具有一定局限性，所以明确白粉寄生孢的资源状况，筛选新的有潜力的生防菌株就显得及其重要。
由核盘菌侵染引起的菌核病是一种严重病害，核盘菌是一种放线状子囊真菌，其寄主范围和地理分布十分广泛，尤其在南方油菜产区发病严重，该病害在发病严重年份病株率可达50-80％，严重制约油菜品质和产量，造成经济上的损失，是危害油菜最严重的病害之一。
本文对周口地区凤仙花白粉菌和分离自凤仙花白粉病上的白粉寄生孢以及感染了菌核病油菜上的核盘菌进行了显微观察、生物学特性、分子生物学以及致病性等研究，并取得了以下主要结果：
1.对周口地区检出的凤仙花白粉菌进行了显微形态分析、核糖体DNA转录间隔区ITS(inter transcribed spacer)进行PCR扩增及进化分析。结果表明：在扫描电子显微镜下病菌分生孢子呈圆柱形或椭圆形，无支链，ITS序列与来自日本番茄上的O.neolycopersici(AB094991)和来自美国番茄上的O.neolycopersici(AB163915)同源性最高聚为一支，序列同源性达到100%，分析结果表明，来自周口地区的番茄白粉菌为Oidiumneo lycopersici。
2.从河南省周口市采集感染白粉病的凤仙花植株，采用单孢分离法分离得到白粉寄生孢，对该菌进行菌落和显微形态分析，初步证明该菌株为A.quisqualis。利用真菌通用引物ITS1和ITS4对所分离的菌株内转录间隔区进行PCR扩增，得到578bp大小的片段，经过克隆测序和在GenBank中进行BLASTn分析，发现与德国和美国白粉寄生孢ITS序列的同源性达到100％,进一步证明该分离菌株为A.quisqualis。并将白粉寄生孢接种在患有白粉病的大叶黄杨叶面上，经一周时间检查发现，白粉菌菌体由无色渐变成浅黄色或浅褐色，并渐渐萎缩至白粉层消失，说明从周口地区凤仙花白粉病病株上分离得到的白粉寄生孢具有防治白粉病的效果。
3.通过探究白粉寄生孢最适的生长环境研究发现，其温度生长范围较广：10-30℃，菌株生长和产孢的最适温度为20℃。白粉寄生孢在pH5-10时均可生长及产孢，其中pH值为7时最适宜其产孢。最适合生长的理想C源为葡萄糖，最适合的N源为蛋白胨，持续光照对菌株的生长及产孢具有明显的促进作用。
4.从河南省周口市采集感染菌核病的油菜，对核盘菌进行分离和鉴定。采用单孢分离法分离得到该菌系，对该菌系进行了菌落和显微形态分析，初步证明该菌株为S.sclerotiorum。利用真菌通用引物ITS1和ITS4对所分离的单菌系ITS序列进行PCR扩增，得到640bp大小的片段，经过克隆测序和在GenBank中进行BLASTn分析，发现与已知核盘菌属菌核病菌ITS序列的同源性达到100％，进一步证明该分离菌株为S.sclerotiorum。
5.通过研究不同温度、pH值、C源和N源、光照、培养基对核盘菌的最适合生长环境的研究表明，核盘菌温度生长范围较广：15-35℃，其中25℃是菌株生长及产孢的最适温度。核盘菌在pH5-10时均可生长及产孢，其中pH值为8时最适宜其产孢。菌落生长较为理想的C源和N源为蔗糖和酵母浸膏，持续光照对菌株的生长及产孢影响不显著。
6.将菌悬液分别接种在耐菌核病型油菜和易感菌核病型油菜上，4天后用台盼兰染色观察，发现耐病型和易感型均可被感染，菌丝侵入的方式两个基因型也没有差别，但菌丝长度不同。用菌核病侵染番茄和拟南芥植物时发现这两种植物也均可被核盘菌侵染。下一步的工作，将研究白粉寄生孢能否对核盘菌具有生物防治作用。
Powdery mildew and Sclerotinia sclerotiorum are two kinds of the most important plant diseases inChina. Ampelomyces quisqualis is an important mycoparasite of powdery mildew fungus with resistanceagainst most used chemical fungicides, which has potential to control powdery mildew. Because differentaccessions of A.quisqualis could show genetic differences, and the potential application of A.quisqualis isaccession dependent. Therefore, screening A.quisqualis resources to obtain potential new accessionssuitable for local application on fungal disease prevention is important. S.sclerotiorum is a filamentousAscomycete fungus with a wide host range, which causes server losses of the rapeseed production,especially in the southern areas. The disease at the year of serious outbreak can infect to50-80%of plants,which is one of the most important disease of rapeseed causing serious damage to rapeseed production bothon yield and on quality.
In this paper, characteristics of A.quisquals in Zhoukou and interaction between the A.quisquals andpowdery mildew fungi were investigated. Characteristics of S. sclerotiorum in Zhoukou and it’s interactionwith the susceptible and tolerant genotypes of rapeseed were studied.
The results are as following:
1. Powdery mildew appearing in colonies of white patches prevalently on leaves of tomato was foundin Zhoukou of Henan Province. The microscopic morphology and molecular identification of the pathogensthat Cause this disease were studied in terms of internal transcribed spacer (ITS) sequence and phylogenetictrees. The conidia which were borne singly, cylindrical, or ellipsoid-cylindrical without branch wereobserved under the scanning electron micriscipy (SEM). The ITS sequence is highly homologous to ITSsequences from O.neolycopersici(AB163916、AB094991), It draws a conclusion that the pathogen oftomato powdery mildew in Zhoukou belongs to O.neolycopersici.
2. An strain of A.quisqualis was isolated from powdery mildew infected rose balsam (Impatiensbalsamina L.) in Zhoukou city of Henan province with the method of single-spore isolation. Themorphology and microscopic analysis indicated that the isolated stain was A.quisqualis. Universal primersITS1and ITS4for fungi were used to amplify the internal transcribed spacer (ITS) region of the isolated strain, which resulted in a578bp band. The PCR product was sequenced and analyzed by using BLAST,which showed100%homology to AQ in Germany and USA. It further proved that the isolated strain was A.quisqualis. A.quisqualis was inoculated on powdery mildew of Euonymus japonicus, after one-weekinoculation, it showed that the powdery mildew gradually become yellow or shallow brown, and thepowdery mildew gradually disappear, indicating that the A. quisqualis isolate of powdery mildew on RoseBlasam from Zhoukou area can be used for the prevention of powdery mildew.
3. The result showed that A.quisqualis can grow at temperatyre of10-30℃and the optimaltemperature was20℃. The pH value of5-10, the optimal pH value for its growth and sporulation were at7.The optimal C source was lactose, and the optimal N source was peptone. Light could effectively stimulateAQ growth and sporulation.
4. A strain of S. sclerotiorum was isolated from rapeseed in Zhoukou city of Henan province with themethod of single-spore isolation. The morphology and microscopic analyses indicated that the isolatedstain was S. sclerotiorum. Universal primers ITS1and ITS4for fungi were used to amplify the ITS regionof the isolated strain, which resulted in a640bp band. The PCR product was sequenced and analyzed byusing BLASTn, which showed100%homology to found S.sclerotiorum. It further proved that the isolatedstrain was S.sclerotiorum.
5. The result showed that S.sclerotiorum can grow at temperatyre of15-35℃and the optimaltemperature was25℃. The pH value of5-10, the optimal pH value for its growth and sporulation were at8.The optimal C source was sugar, and the optimal N source was yeast extract. Light could effectivelystimulate S.sclerotiorum growth and sporulation.
6. Fungal suspension of S. sclerotiorum was used to inoculate susceptible and tolerant phenotypes ofrapeseed. Four days after inoculation, the infected leaves were stained with trypan blue to study themechanism difference of responses to S. sclerotiorum by different rapeseed genotypes. It indicated that noobvious difference between two genotypes, except that the length of hypha on tolerant genotype wereshorter than those on susceptible genotype of rapeseed. The infection processes of S. sclerotiorum ontomato and Arabidopsis leaves were also investigated. It indicated that S. sclerotiorum can infect tomatoand Arabidopsis. Further study will be carried out to test whether AQ can prevent the growth of S.sclerotiorum.
引文
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