甘肃省中西部地区镰孢菌的分类研究
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摘要
镰孢菌属(Fusarium Link)是真菌中的一个重要类群,普遍存在于土壤和动植物有机体中。其中许多种类是重要的植物病原菌,可侵染多种经济作物,导致作物减产和引起巨大的经济损失。甘肃省位于我国西部,其镰孢菌种类资源丰富。虽然过去曾有许多学者对甘肃省内的镰孢菌进行过不同程度的分类学和病理学研究,但以现代的镰孢菌分类系统观点对该属真菌开展的研究还很有限。因此,作者根据现代镰孢菌分类系统,在传统形态分类学基础上,对甘肃省内的镰孢菌进行了系统研究,目的是明确甘肃省发生的镰孢菌属真菌的种类和多样性分布特点,为镰孢菌病害的正确病原鉴定及科学防治提供理论依据。
在2006~2007年间,在甘肃省包括高寒草地在内的23个市、县、镇的主要作物产区,采集不同植物及其根际土壤标样,采用常规的方法对各个样品进行真菌分离,共得到862个镰孢菌菌株,根据培养和形态学特征鉴定出16种镰孢菌。采用rDNA ITS和EF-1α序列分析的方法对疑难镰孢菌株进行了比较研究,进一步明确了这些镰孢菌的分类地位。另外,采用ISSR-PCR分子标记技术对来自甘肃省的89株木贼镰孢菌进行了遗传多样性分析。研究结果如下:
(1)根据培养和形态学特征鉴定出的16种镰孢菌为木贼镰孢(F. equiseti)、尖镰孢(F. oxysporum)、腐皮镰孢(F. solani)、锐顶镰孢(F. acuminatum)、芬芳镰孢(F. redolens)、轮枝镰孢(F. verticillioides)、大刀镰孢(F. culmorum)、层出镰孢(F. proliferatum)、半裸镰孢(F. semitectum)、弯角镰孢(F. camptoceras)、砖红镰孢(F. lateritium)、三线镰孢(F. tricinctum)、燕麦镰孢(F. avenaceum )、接骨木镰孢(F. sambucinum)、胶孢镰孢(F. subglutinans)和克鲁克威尔镰孢(F. crookwellense)。
(2)在分离获得的镰孢菌中,分离频率最高的4个种依次是木贼镰孢(F. equiseti)、尖镰孢(F. oxysporum)、腐皮镰孢(F. solani)以及锐顶镰孢(F. acuminatum),分离频率分别占总鉴定菌株总数的29.93%、28.54%、12.18%和10.09%。其他分离鉴定的镰孢菌包括芬芳镰孢(F. redolens)、轮枝镰孢(F. verticillioides)、大刀镰孢(F. culmorum)、层出镰孢(F. proliferatum)、半裸镰孢(F. semitectum)和弯角镰孢(F. camptoceras),这些镰孢菌的分离频率低于10%,但高于1%。分离频率低于1%的镰孢菌包括砖红镰孢(F. lateritium)、三线镰孢(F. tricinctum)、燕麦镰孢(F. avenaceum )、接骨木镰孢(F. sambucinum)、胶孢镰孢(F. subglutinans)和克鲁克威尔镰孢(F. crookwellense)。
(3)16种镰孢菌中的优势种为木贼镰孢和尖镰孢,其分布几乎覆盖在甘肃省的所有市、县;其次是锐顶镰孢和腐皮镰孢,它们分别分布于12个和10个市、县;芬芳镰孢和大刀镰孢分布于8个和5个市、县,其余10个种的分布地区较少,均在5个市、县以下,其中胶孢镰孢和克鲁克威尔镰孢两个种只在兰州1个市发现,而接骨木镰孢只在酒泉市分离得到2株。
(4)采用EF-1α和rDNA ITS序列分析方法对6株疑难镰孢菌株进行了研究,经与GenBank中已知菌株序列对比,最后分别确定为层出镰孢(F. proliferatum)1株,芬芳镰孢(F. redolens)1株,尖镰孢(F. oxysporum)1株,锐顶镰孢(F. acuminatum)1株,腐皮镰孢(F. solani)2株。
(5)采用ISSR指纹图谱分析技术,对来自甘肃不同地区的89株木贼镰孢菌的基因组DNA的遗传多样性进行了研究。从供试21个引物中筛选出3个引物,并进行扩增和分析。采用NYSTS2.10e软件对矩阵进行遗传分析,形成聚类图。在相似系数为0.675时,可将试验菌株分为6个类群。图谱分析结果表明,ISSR图谱多样性与其菌株的培养性状之间存在一定的相关性,但与寄主和地理来源之间未表现出相关性。
The genus Fusarium Link is one of the most important groups of fungi, which is widely distributed in soil and organisms of animals and plants. Many species of Fusarium are well-known pathogenic fungi of some economic crops, and they often cause serious diseases and significantly reduce crop production. Gansu province is located in the western China and it is rich in Fusarium species. Over the past decades, some mycologists had investigated the Fusarium species and the diseases caused by these fungi in Gansu province. However, it is very limited in the taxonomic study of the genus Fusarium, especially in view of modern classification system. Therefore, the paper was designed to conduct the investigation of Fusarium species in Gansu province based on the classical morphological characteristics combined with molecular analysis. The current study will provide a theoretical basis for the correct identification of Fusarium species and scientific control of Fusarium disease.
During 2006 and 2007 the samples of different plants and their rhizosphere soil were collected from the crop-growing areas of 23 cities or counties in Gansu province, including the alpine grassland. A total of 862 Fusarium isolates were acquired by conventional isolation technique, and from which 16 Fusarium species were identified according to their morphological characteristics. Some suspected isolates were analyzed by methods of rDNA ITS and EF-1αsequence analysis in order to confirm their taxonomic status. On the other hand, 89 strains of Fusarium equiseti from Gansu province were investigated for genetic diversity by means of ISSR-PCR analysis. The results are as follows.
The 16 species of Fusarium identified on the basis of morphological characteristics were F. acuminatum, F. avenaceum, F. camptoceras, F. culmorum, F. equiseti, F. lateritium, F. oxysporum, F. proliferatum, F. redolens, F. sambucinum, F. semitectum, F. solani, F. subglutinans, F. tricinctum, F. verticillioides and F. crookwellense.
Among the Fusarium species the most frequently isolated species were F. equiseti, F. oxysporum, F. solani and F. acuminatum, with the isolation rate of 29.93%, 28.54%, 12.18% and 10.09% respectively. The species with isolation rate of between 1% and 10% include F. redolens, F. verticillioides, F. culmorum, F. proliferatum, F. semitectum and F. camptoceras. The less isolated Fusarium species were F. lateritium, F. tricinctum, F. avenaceum, F. sambucinum, F. subglutinans and F. crookwellense, their isolation rates were under 1%.
The predominant species of the 16 Fusarium species were F. equiseti and F. oxysporum, They were distributed in almost all cities or counties in Gansu province. The relatively frequently distributed species of Fusarium included F. acuminatum and F. solani, they were isolated from 12 and 10 cities or counties respectively. F. redolens and F. culmorum were generated from eight and five cities or counties respectively. The other ten Fusarium species were less geographically distributed in Gansu province. For example, F. subglutinans and F. crookwellense were only found in Lanzhou, and only two strains of F. sambucinum were isolated in Jiuquan in the study.
Six suspected Fusarium isolates were further analyzed by rDNA ITS and EF-1αgene sequencing, and they were compared with those sequenced strains in GenBank. Finally, one isolate was identified as F. proliferatum, one islated as F. redolens, one isolate as F. oxysporum, one isolate as F. acuminatum and two isolates as F. solani.
Three primers screened from 21 primers were used in ISSR-PCR fingerprint analysis of 89 isolates of F. equiseti from different regions in Gansu province. A dendrogram was produced on the basis of the matrix using UPGMA algorithm by NYSTS2.10e. At similar coefficient of 0.675, the test isolates could be divided into six groups. It was found that there was to some extent relativity between ISSR image diversity and their culture characteristics, but no relationship between their hosts and geographical origins.
在2006~2007年间,在甘肃省包括高寒草地在内的23个市、县、镇的主要作物产区,采集不同植物及其根际土壤标样,采用常规的方法对各个样品进行真菌分离,共得到862个镰孢菌菌株,根据培养和形态学特征鉴定出16种镰孢菌。采用rDNA ITS和EF-1α序列分析的方法对疑难镰孢菌株进行了比较研究,进一步明确了这些镰孢菌的分类地位。另外,采用ISSR-PCR分子标记技术对来自甘肃省的89株木贼镰孢菌进行了遗传多样性分析。研究结果如下:
(1)根据培养和形态学特征鉴定出的16种镰孢菌为木贼镰孢(F. equiseti)、尖镰孢(F. oxysporum)、腐皮镰孢(F. solani)、锐顶镰孢(F. acuminatum)、芬芳镰孢(F. redolens)、轮枝镰孢(F. verticillioides)、大刀镰孢(F. culmorum)、层出镰孢(F. proliferatum)、半裸镰孢(F. semitectum)、弯角镰孢(F. camptoceras)、砖红镰孢(F. lateritium)、三线镰孢(F. tricinctum)、燕麦镰孢(F. avenaceum )、接骨木镰孢(F. sambucinum)、胶孢镰孢(F. subglutinans)和克鲁克威尔镰孢(F. crookwellense)。
(2)在分离获得的镰孢菌中,分离频率最高的4个种依次是木贼镰孢(F. equiseti)、尖镰孢(F. oxysporum)、腐皮镰孢(F. solani)以及锐顶镰孢(F. acuminatum),分离频率分别占总鉴定菌株总数的29.93%、28.54%、12.18%和10.09%。其他分离鉴定的镰孢菌包括芬芳镰孢(F. redolens)、轮枝镰孢(F. verticillioides)、大刀镰孢(F. culmorum)、层出镰孢(F. proliferatum)、半裸镰孢(F. semitectum)和弯角镰孢(F. camptoceras),这些镰孢菌的分离频率低于10%,但高于1%。分离频率低于1%的镰孢菌包括砖红镰孢(F. lateritium)、三线镰孢(F. tricinctum)、燕麦镰孢(F. avenaceum )、接骨木镰孢(F. sambucinum)、胶孢镰孢(F. subglutinans)和克鲁克威尔镰孢(F. crookwellense)。
(3)16种镰孢菌中的优势种为木贼镰孢和尖镰孢,其分布几乎覆盖在甘肃省的所有市、县;其次是锐顶镰孢和腐皮镰孢,它们分别分布于12个和10个市、县;芬芳镰孢和大刀镰孢分布于8个和5个市、县,其余10个种的分布地区较少,均在5个市、县以下,其中胶孢镰孢和克鲁克威尔镰孢两个种只在兰州1个市发现,而接骨木镰孢只在酒泉市分离得到2株。
(4)采用EF-1α和rDNA ITS序列分析方法对6株疑难镰孢菌株进行了研究,经与GenBank中已知菌株序列对比,最后分别确定为层出镰孢(F. proliferatum)1株,芬芳镰孢(F. redolens)1株,尖镰孢(F. oxysporum)1株,锐顶镰孢(F. acuminatum)1株,腐皮镰孢(F. solani)2株。
(5)采用ISSR指纹图谱分析技术,对来自甘肃不同地区的89株木贼镰孢菌的基因组DNA的遗传多样性进行了研究。从供试21个引物中筛选出3个引物,并进行扩增和分析。采用NYSTS2.10e软件对矩阵进行遗传分析,形成聚类图。在相似系数为0.675时,可将试验菌株分为6个类群。图谱分析结果表明,ISSR图谱多样性与其菌株的培养性状之间存在一定的相关性,但与寄主和地理来源之间未表现出相关性。
The genus Fusarium Link is one of the most important groups of fungi, which is widely distributed in soil and organisms of animals and plants. Many species of Fusarium are well-known pathogenic fungi of some economic crops, and they often cause serious diseases and significantly reduce crop production. Gansu province is located in the western China and it is rich in Fusarium species. Over the past decades, some mycologists had investigated the Fusarium species and the diseases caused by these fungi in Gansu province. However, it is very limited in the taxonomic study of the genus Fusarium, especially in view of modern classification system. Therefore, the paper was designed to conduct the investigation of Fusarium species in Gansu province based on the classical morphological characteristics combined with molecular analysis. The current study will provide a theoretical basis for the correct identification of Fusarium species and scientific control of Fusarium disease.
During 2006 and 2007 the samples of different plants and their rhizosphere soil were collected from the crop-growing areas of 23 cities or counties in Gansu province, including the alpine grassland. A total of 862 Fusarium isolates were acquired by conventional isolation technique, and from which 16 Fusarium species were identified according to their morphological characteristics. Some suspected isolates were analyzed by methods of rDNA ITS and EF-1αsequence analysis in order to confirm their taxonomic status. On the other hand, 89 strains of Fusarium equiseti from Gansu province were investigated for genetic diversity by means of ISSR-PCR analysis. The results are as follows.
The 16 species of Fusarium identified on the basis of morphological characteristics were F. acuminatum, F. avenaceum, F. camptoceras, F. culmorum, F. equiseti, F. lateritium, F. oxysporum, F. proliferatum, F. redolens, F. sambucinum, F. semitectum, F. solani, F. subglutinans, F. tricinctum, F. verticillioides and F. crookwellense.
Among the Fusarium species the most frequently isolated species were F. equiseti, F. oxysporum, F. solani and F. acuminatum, with the isolation rate of 29.93%, 28.54%, 12.18% and 10.09% respectively. The species with isolation rate of between 1% and 10% include F. redolens, F. verticillioides, F. culmorum, F. proliferatum, F. semitectum and F. camptoceras. The less isolated Fusarium species were F. lateritium, F. tricinctum, F. avenaceum, F. sambucinum, F. subglutinans and F. crookwellense, their isolation rates were under 1%.
The predominant species of the 16 Fusarium species were F. equiseti and F. oxysporum, They were distributed in almost all cities or counties in Gansu province. The relatively frequently distributed species of Fusarium included F. acuminatum and F. solani, they were isolated from 12 and 10 cities or counties respectively. F. redolens and F. culmorum were generated from eight and five cities or counties respectively. The other ten Fusarium species were less geographically distributed in Gansu province. For example, F. subglutinans and F. crookwellense were only found in Lanzhou, and only two strains of F. sambucinum were isolated in Jiuquan in the study.
Six suspected Fusarium isolates were further analyzed by rDNA ITS and EF-1αgene sequencing, and they were compared with those sequenced strains in GenBank. Finally, one isolate was identified as F. proliferatum, one islated as F. redolens, one isolate as F. oxysporum, one isolate as F. acuminatum and two isolates as F. solani.
Three primers screened from 21 primers were used in ISSR-PCR fingerprint analysis of 89 isolates of F. equiseti from different regions in Gansu province. A dendrogram was produced on the basis of the matrix using UPGMA algorithm by NYSTS2.10e. At similar coefficient of 0.675, the test isolates could be divided into six groups. It was found that there was to some extent relativity between ISSR image diversity and their culture characteristics, but no relationship between their hosts and geographical origins.
引文
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