黑龙江省链格孢属病原菌遗传多样性及杀菌剂抗药性分析
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摘要
2007～2009年在黑龙江省各地区不同寄主植物上分离了109株链格孢属病原菌,其中Alternaria solani 73株、Alternari brassicola 15株、Alternaria porri 21株。从分离到的A. solani,A. brassicola及A. porri菌株中各挑选一株,并利用ITS1和ITS2通用引物扩增了3种链格孢属病原菌的基因组DNA。扩增结果显示所测定菌株ks09-7的碱基序列与NCBI上注册的A. solani ITS序列之间的同源性为98.35%;所测定菌株gl09-2的碱基序列与NCBI上注册的A. brassicicola ITS序列之间的同源性为97.81%;所测定菌株zb09-10的碱基序列与NCBI上注册的A. porri ITS序列之间的同源性为98.71%。
本研究利用生长速率法测定了马铃薯早疫病菌对恶醚唑、腐霉利和戊唑醇的敏感性。恶醚唑的敏感性测定结果表明,不同年份和不同地区分离的马铃薯早疫病菌对恶醚唑的敏感性之间存在着差异,EC_(50)值在0.0643~9.0666μg·mL~(-1),最不敏感菌株(nh09-5)的EC_(50)值是最敏感菌株(hg08-20)的141.00倍,平均值为2.5196±2.2025μg·mL~(-1),抗性指数低于3的马铃薯早疫病菌株有47个,抗性指数在3~10之间的马铃薯早疫病病菌有3个,说明齐齐哈尔地区讷河市分离的马铃薯早疫病菌已有对恶醚唑产生抗药性的趋势;腐霉利的敏感性测定结果表明,不同年份和不同地区分离的茄链格孢菌对腐霉利的敏感性无显著差异,其敏感性呈连续的单峰曲线分布,EC_(50)值在0.3472~8.1799μg·mL~(-1)之间,最不敏感菌株的EC_(50)值是最敏感菌株的23.56倍,平均值为4.4457±0.6019μg·mL~(-1),未出现敏感性下降的抗药性群体,因此可作为马铃薯早疫病菌对腐霉利的敏感性基线;戊唑醇的敏感性测定结果表明,不同年份分离的马铃薯早疫病菌对戊唑醇的敏感性之间无显著差异,但不同地区分离的马铃薯早疫病菌有一定的差异,其敏感性呈连续的单峰曲线分布,EC_(50)值在3.0903~23.5613μg·mL~(-1)之间,最不敏感菌株的EC_(50)值是最敏感菌株的7.62倍,平均值为10.3584±1.6331μg·mL~(-1),未出现敏感性下降的抗药性群体,因此可作为马铃薯早疫病菌对戊唑醇的敏感性基线。总之,腐霉利和戊唑醇可以使用在马铃薯早疫病的防治上,但应注意监测其抗药性的动态变化。
利用RAPD技术对分离自黑龙江省哈尔滨市、齐齐哈尔市、牡丹江市、鹤岗市、佳木斯市和加格达奇市等6个地区的90株链格孢属病原菌进行了遗传多样性分析。从20个随机引物中筛选出一条扩增条带清晰,多态性好的引物,并利用NTSYS 2.0进行链格孢属病原菌的聚类分析。结果表明引物OPE-19扩增后产生的各个群体间相似性系数分布在0.60~1.00。在60.8%相似性水平上可划分为3个主要RAPD组群和8个次要RAPD组群。牡丹江市和齐齐哈尔市分离的马铃薯早疫病菌分别划分为8个RAPD组群。以上结果说明黑龙江省链格孢属病原菌RAPD组群与寄主植物和分离地区之间没有相关性。
利用AFLP技术对分离自黑龙江省哈尔滨、齐齐哈尔、牡丹江、鹤岗、佳木斯、加格达奇等6个地区分离的91株链格孢属病原菌进行了遗传多样性分析。在相似性系数为0.66时91个链格孢属病原菌被分为1个主要组群和3个次要组群。在这四个组群中,第一(AG1_(0.66))、第二(AG2_(0.66))和第四(AG4_(0.66))组群属于由大孢子种组成的链格孢属病原菌,而第三组群(AG3_(0.66))绝大多数属于由小孢子种组成的链格孢病原菌。综合分析上述聚类结果可得出链格孢属病原菌的AFLP组群与孢子类型有一定相关性,而链格孢属病原菌的AFLP组群与分离地区之间没有相关性。在相似性系数为0.72时91个链格孢属病原菌被分为2个主要组群和10个次要组群。在这12个组群中,第一(AG1_(0.72))、第二(AG2_(0.72))和第三组群(AG3_(0.72))主要由葱链格孢菌组成,第四(AG4_(0.72))、第五(AG5_(0.72))、第六(AG6_(0.72))、第七(AG7_(0.72))、第八(AG8_(0.72))和第十二组群(AG12_(0.72))全部由茄链格孢菌组成,而第九(AG9_(0.72))、第十(AG10_(0.72))和第十一组群(AG11_(0.72))主要由芸薹链格孢菌组成。链格孢属病原菌的AFLP组群与寄主植物之间有一定的相关性,而与分离地区之间没有相关性。
A total of 109 Alternaria Pathogens isolates were isolated from different host plants cultivated in various locations of Heilongjiang province in 2007~2009. Of which, Alternaria solani isolates were 73, Alternaria brassicola isolates were 15, Alternaria porri isolates were 21. One isolate were chosed from 3 speceis Altenaria Pathogens isolated, respectively. The genomic DNA extracted from 3 speceis Alternaria Pathogens isolates were amplified by using ITS1 and ITS2 primer combination. The results indicated that the homology between the base sequences of ks09-7 determined and ITS sequences of Alternaria solani registered in NCBI was 98.35%; the homology between the base sequences of gl09-2 determined and ITS sequences of Alternaria brassicicola registered in NCBI was 97.81%; the homology between the base sequences of zb09-10 determined and ITS sequences of Alternaria porri registered in NCBI was 98.71%.
The experiment determined for sensitivity of A. solani isolates to difenoconazole, procymidone and tebuconazole in vitro by using growth rate method. The results of sensitivity to difenoconazole indicated that there were significant differences of sensitivity to difenoconazole between A. solani isolates collected from different locations and various years. The EC_(50) values ranged from 0.0643 to 9.066μg.mL~(-1), the EC_(50) values of most insensitive isolate (nh09-5) was 141 folds of the most sensitive (hg08-20), with a mean of 2.5196±2.2025μg.mL~(-1). A. solani isolates less than resistance index were 47, A. solani isolates between the 3.0 and 10 of resistance index were 3. The above results suggested that A. solani isolates collected from Nehe city of Qiqihar locality have been occurred trend of resistance to difenoconazole. The results of sensitivity to procymidone indicated that there were no significant differences of sensitivity to procymidone between A. solani isolates collected from different locations and various years. Their sensitivities distributed as a unimodal curve, EC_(50) values ranged from 0.3472μg·mL~(-1) to 8.1799μg·mL~(-1), the most insensitive was 23.56 folds of the most sensitive, with a mean of 4.4457±0.6019μg·mL~(-1), so it can be used as baseline-sensitivity of A. solani to procymidone. The results of sensitivity to tebuconazole indicated that there is no significant difference of sensitivity to tebuconazole between A. solani isolates collected from various years, but there is significant difference of sensitivity to tebuconazole between A. solani isolates collected from different locations. Their sensitivities distributed as a unimodal curve, EC_(50) values ranged from 3.0903μg·mL~(-1) to 23.5613μg·mL~(-1), the most insensitive was 7.62 folds of the most sensitive, with a mean of 10.3584±1.6331μg·mL~(-1), so it can be used as baseline-sensitivity of A. solani to procymidone. In conclusion, procymidone and tebuconazole can be used to control potato early blight in Heilongjiang province, but we should pay attention to monitor dynamic changes of resistance of Alternaria solani to procymidone and tebuconazole.
To examine the genetic diversity of 90 Alternaria Pathogens isolates collected from Harbin, Qiqihar, Mudanjiang, Hegang, Jiamusi and Jiagedaqi city of Heilongjiang province, the study was performed the genotypic characteristics by using random amplified polymorphic DNA (RAPD). One primer having a clear amplified bands and good polymorphic fragments was selected from 20 random primers, and carried out the cluster analysis of Alternaria Pathogens by using NTSYs program. The result indicated that the similarity coefficients of the RAPD group amplified by OPE-19 primer ranged from 0.60 to 1.00. According to the RAPD analysis by using OPE-19 primer, 90 isolates contains standard isolates were separated into three major RAPD groups and eight minor groups at similarity level of 60.8%. A. solani isolates collected from Mudanjiang and Qiqihar city were separated into 8 RAPD groups, respectively. The above result suggested that tRAPD groups were not correlated with the host plants and isolated locations.
To examine the genetic diversity of 91 Alternaria Pathogens isolates collected from Harbin, Qiqihar, Mudanjiang, Hegang, Jiamusi and Jiagedaqi city of Heilongjiang province, the study was performed the genotypic characteristics by using amplified fragment length polymorphism (AFLP). 91 isolates of Alternaria Pathogens were separated into one major AFLP group and three minor groups at similarity level of 0.66. Among the four groups, the first (AG1_(0.66)), second (AG2_(0.66)) and fourth groups (AG4_(0.66)) were compsed of the large spore Alternaria species, but the third group (AG3_(0.66)) was compsed of the small spores Alternaria species. The comprehensive analysis of above cluster result suggested that AFLP groups were correlated with spore types, but were not correlated with the isolated locations. 91 isolates of Alternaria Pathogens were separated into two major AFLP group and ten minor groups at similarity level of 0.72. Among the 12 groups, the first (AG1_(0.72)), second (AG2_(0.72)) and third groups (AG3_(0.72)) were mainly compsed of the Alternaria porri; fourth (AG4_(0.72)), fifth (AG5_(0.72)), sixth (AG6_(0.72)), seventh (AG7_(0.72)), eighth (AG8_(0.72)) and twelfth (AG12_(0.72)) groups were composed of the Alternaria solani; but the ninth (AG9_(0.72)), tenth (AG10_(0.72)) and eleventh (AG11_(0.72)) is formed by the small spores Alternaria species. The comprehensive analysis of above cluster result suggested that AFLP groups were correlated with host plants, but were not correlated with the isolated locations.
本研究利用生长速率法测定了马铃薯早疫病菌对恶醚唑、腐霉利和戊唑醇的敏感性。恶醚唑的敏感性测定结果表明,不同年份和不同地区分离的马铃薯早疫病菌对恶醚唑的敏感性之间存在着差异,EC_(50)值在0.0643~9.0666μg·mL~(-1),最不敏感菌株(nh09-5)的EC_(50)值是最敏感菌株(hg08-20)的141.00倍,平均值为2.5196±2.2025μg·mL~(-1),抗性指数低于3的马铃薯早疫病菌株有47个,抗性指数在3~10之间的马铃薯早疫病病菌有3个,说明齐齐哈尔地区讷河市分离的马铃薯早疫病菌已有对恶醚唑产生抗药性的趋势;腐霉利的敏感性测定结果表明,不同年份和不同地区分离的茄链格孢菌对腐霉利的敏感性无显著差异,其敏感性呈连续的单峰曲线分布,EC_(50)值在0.3472~8.1799μg·mL~(-1)之间,最不敏感菌株的EC_(50)值是最敏感菌株的23.56倍,平均值为4.4457±0.6019μg·mL~(-1),未出现敏感性下降的抗药性群体,因此可作为马铃薯早疫病菌对腐霉利的敏感性基线;戊唑醇的敏感性测定结果表明,不同年份分离的马铃薯早疫病菌对戊唑醇的敏感性之间无显著差异,但不同地区分离的马铃薯早疫病菌有一定的差异,其敏感性呈连续的单峰曲线分布,EC_(50)值在3.0903~23.5613μg·mL~(-1)之间,最不敏感菌株的EC_(50)值是最敏感菌株的7.62倍,平均值为10.3584±1.6331μg·mL~(-1),未出现敏感性下降的抗药性群体,因此可作为马铃薯早疫病菌对戊唑醇的敏感性基线。总之,腐霉利和戊唑醇可以使用在马铃薯早疫病的防治上,但应注意监测其抗药性的动态变化。
利用RAPD技术对分离自黑龙江省哈尔滨市、齐齐哈尔市、牡丹江市、鹤岗市、佳木斯市和加格达奇市等6个地区的90株链格孢属病原菌进行了遗传多样性分析。从20个随机引物中筛选出一条扩增条带清晰,多态性好的引物,并利用NTSYS 2.0进行链格孢属病原菌的聚类分析。结果表明引物OPE-19扩增后产生的各个群体间相似性系数分布在0.60~1.00。在60.8%相似性水平上可划分为3个主要RAPD组群和8个次要RAPD组群。牡丹江市和齐齐哈尔市分离的马铃薯早疫病菌分别划分为8个RAPD组群。以上结果说明黑龙江省链格孢属病原菌RAPD组群与寄主植物和分离地区之间没有相关性。
利用AFLP技术对分离自黑龙江省哈尔滨、齐齐哈尔、牡丹江、鹤岗、佳木斯、加格达奇等6个地区分离的91株链格孢属病原菌进行了遗传多样性分析。在相似性系数为0.66时91个链格孢属病原菌被分为1个主要组群和3个次要组群。在这四个组群中,第一(AG1_(0.66))、第二(AG2_(0.66))和第四(AG4_(0.66))组群属于由大孢子种组成的链格孢属病原菌,而第三组群(AG3_(0.66))绝大多数属于由小孢子种组成的链格孢病原菌。综合分析上述聚类结果可得出链格孢属病原菌的AFLP组群与孢子类型有一定相关性,而链格孢属病原菌的AFLP组群与分离地区之间没有相关性。在相似性系数为0.72时91个链格孢属病原菌被分为2个主要组群和10个次要组群。在这12个组群中,第一(AG1_(0.72))、第二(AG2_(0.72))和第三组群(AG3_(0.72))主要由葱链格孢菌组成,第四(AG4_(0.72))、第五(AG5_(0.72))、第六(AG6_(0.72))、第七(AG7_(0.72))、第八(AG8_(0.72))和第十二组群(AG12_(0.72))全部由茄链格孢菌组成,而第九(AG9_(0.72))、第十(AG10_(0.72))和第十一组群(AG11_(0.72))主要由芸薹链格孢菌组成。链格孢属病原菌的AFLP组群与寄主植物之间有一定的相关性,而与分离地区之间没有相关性。
A total of 109 Alternaria Pathogens isolates were isolated from different host plants cultivated in various locations of Heilongjiang province in 2007~2009. Of which, Alternaria solani isolates were 73, Alternaria brassicola isolates were 15, Alternaria porri isolates were 21. One isolate were chosed from 3 speceis Altenaria Pathogens isolated, respectively. The genomic DNA extracted from 3 speceis Alternaria Pathogens isolates were amplified by using ITS1 and ITS2 primer combination. The results indicated that the homology between the base sequences of ks09-7 determined and ITS sequences of Alternaria solani registered in NCBI was 98.35%; the homology between the base sequences of gl09-2 determined and ITS sequences of Alternaria brassicicola registered in NCBI was 97.81%; the homology between the base sequences of zb09-10 determined and ITS sequences of Alternaria porri registered in NCBI was 98.71%.
The experiment determined for sensitivity of A. solani isolates to difenoconazole, procymidone and tebuconazole in vitro by using growth rate method. The results of sensitivity to difenoconazole indicated that there were significant differences of sensitivity to difenoconazole between A. solani isolates collected from different locations and various years. The EC_(50) values ranged from 0.0643 to 9.066μg.mL~(-1), the EC_(50) values of most insensitive isolate (nh09-5) was 141 folds of the most sensitive (hg08-20), with a mean of 2.5196±2.2025μg.mL~(-1). A. solani isolates less than resistance index were 47, A. solani isolates between the 3.0 and 10 of resistance index were 3. The above results suggested that A. solani isolates collected from Nehe city of Qiqihar locality have been occurred trend of resistance to difenoconazole. The results of sensitivity to procymidone indicated that there were no significant differences of sensitivity to procymidone between A. solani isolates collected from different locations and various years. Their sensitivities distributed as a unimodal curve, EC_(50) values ranged from 0.3472μg·mL~(-1) to 8.1799μg·mL~(-1), the most insensitive was 23.56 folds of the most sensitive, with a mean of 4.4457±0.6019μg·mL~(-1), so it can be used as baseline-sensitivity of A. solani to procymidone. The results of sensitivity to tebuconazole indicated that there is no significant difference of sensitivity to tebuconazole between A. solani isolates collected from various years, but there is significant difference of sensitivity to tebuconazole between A. solani isolates collected from different locations. Their sensitivities distributed as a unimodal curve, EC_(50) values ranged from 3.0903μg·mL~(-1) to 23.5613μg·mL~(-1), the most insensitive was 7.62 folds of the most sensitive, with a mean of 10.3584±1.6331μg·mL~(-1), so it can be used as baseline-sensitivity of A. solani to procymidone. In conclusion, procymidone and tebuconazole can be used to control potato early blight in Heilongjiang province, but we should pay attention to monitor dynamic changes of resistance of Alternaria solani to procymidone and tebuconazole.
To examine the genetic diversity of 90 Alternaria Pathogens isolates collected from Harbin, Qiqihar, Mudanjiang, Hegang, Jiamusi and Jiagedaqi city of Heilongjiang province, the study was performed the genotypic characteristics by using random amplified polymorphic DNA (RAPD). One primer having a clear amplified bands and good polymorphic fragments was selected from 20 random primers, and carried out the cluster analysis of Alternaria Pathogens by using NTSYs program. The result indicated that the similarity coefficients of the RAPD group amplified by OPE-19 primer ranged from 0.60 to 1.00. According to the RAPD analysis by using OPE-19 primer, 90 isolates contains standard isolates were separated into three major RAPD groups and eight minor groups at similarity level of 60.8%. A. solani isolates collected from Mudanjiang and Qiqihar city were separated into 8 RAPD groups, respectively. The above result suggested that tRAPD groups were not correlated with the host plants and isolated locations.
To examine the genetic diversity of 91 Alternaria Pathogens isolates collected from Harbin, Qiqihar, Mudanjiang, Hegang, Jiamusi and Jiagedaqi city of Heilongjiang province, the study was performed the genotypic characteristics by using amplified fragment length polymorphism (AFLP). 91 isolates of Alternaria Pathogens were separated into one major AFLP group and three minor groups at similarity level of 0.66. Among the four groups, the first (AG1_(0.66)), second (AG2_(0.66)) and fourth groups (AG4_(0.66)) were compsed of the large spore Alternaria species, but the third group (AG3_(0.66)) was compsed of the small spores Alternaria species. The comprehensive analysis of above cluster result suggested that AFLP groups were correlated with spore types, but were not correlated with the isolated locations. 91 isolates of Alternaria Pathogens were separated into two major AFLP group and ten minor groups at similarity level of 0.72. Among the 12 groups, the first (AG1_(0.72)), second (AG2_(0.72)) and third groups (AG3_(0.72)) were mainly compsed of the Alternaria porri; fourth (AG4_(0.72)), fifth (AG5_(0.72)), sixth (AG6_(0.72)), seventh (AG7_(0.72)), eighth (AG8_(0.72)) and twelfth (AG12_(0.72)) groups were composed of the Alternaria solani; but the ninth (AG9_(0.72)), tenth (AG10_(0.72)) and eleventh (AG11_(0.72)) is formed by the small spores Alternaria species. The comprehensive analysis of above cluster result suggested that AFLP groups were correlated with host plants, but were not correlated with the isolated locations.
引文
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Bell A A,Wheeler M H. 1986. Biosynthesis and functions of fungal melanins[J]. Annu. Rev.Phytopathol. 24:411-451.
Bottalico A, Logrieco A. 1998. Toxigenic Alternaria species of economic importance. In: Sinha K K , Bhatnager D, Eds, Mycotoxins in Agriculture and Food Safety, Marcel Dekker[M], New York, 65-108.
Caldas E D, Jones A D ,Ward B. et al. 1994. Structural characterization of three new AAL toxins produced by Alternaria alternata .sp.lycopersici[J]. J.Agric. Food Chem, 42: 327- 333 .
Campbell R. 1969. An electron microscope study of spore structure and development in Alternaria brassicicolaj[J]. Microbiol. Gen, 54: 381-392.
Chen X M,, Line R F, Leung H. 1993. Relationship between virulence variation and DNA polymorphism in Puccinia striiformis[J]. Phytopathoology. 83(12):1489-1497.
Cook D E. 1998. Analysis of intraspecific and interspecific variation in the genus Alternaria by zhe use of RAPD-PCR [J].Annals of Applied biology,132(2): 197-209.
Dhiman J S. 1990. Ocurrence of leaf spot and flower blight of marigold.in Punjab[D]. Journal of Research, Punjab Agricultural university,27(2):231-236.
Dong Z, Liu G, Qina Y. et al. 1987. Induction of mutagenesis and transformation by the extract of Alternaria alternata isolated from grains in Linxian,China [J].Carcinogenesis, 8(7):989-991.
Edward J C. 1957. Leaf and infloreseenees blight of Marigold caused by Altenaria zinnia[M]. Sicence and Culturation, 22: 683.
Feng F, Qiu D, Jiang L. 2007. Isolation of cDNA sequences encoding the MAP kinase HOG1 and the MAP kinase kinase PBS2 genes of the fungus Alternaria tenuissima through a genetic approach[J]. Journal of Microbiological Methods, 69(1):188-196.
Goodwin S B, Drenth A, Fry W E. 1992. Cloning and genetic analyses of two highly polymorphic, moderately repetitive nuclear DNAs from Phytophthora infestans [J]. Current Genetics, 22: 107-115.
Halonen M, Stern D A, Wright A L.et al.1997. Alternaria as a major allergen for asthma in children raised in a desert environment [J].Am.J Respir. Crit.Care Med,155(4):1356-1361.
Hiram S , Robert K B. 2001. A review of Alternaria alternata sensitivity[J]. Rev Iberoam Micol, 18: 56-59.
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