白菜种传黑斑病菌分子鉴定及抑菌药剂初探
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摘要
本文以来自国内外的白菜黑斑病菌为研究材料，进行了基本培养条件的比对研究和5.8s rDNA及其侧翼ITS区的克隆、测序、序列变异及系统进化关系的分析，建立了白菜黑斑病菌芸薹链格孢(Alternaria brassicae)、甘蓝链格孢(A.brassicicola)及萝卜链格孢(A.japonica)三个种的分子鉴定方法；根据白菜生产中黑斑病防治的需要，进行了14种杀菌剂抑制黑斑病菌菌丝生长效果的比较研究。试验获得到了以下结果。
黑斑病菌及其近缘种真菌核糖体5.8s rDNA及其侧翼ITS区序列比对结果显示，不同种菌株ITS1比ITS2在碱基构成上有更大的变异，而且ITS1的序列长度变异比ITS2的大；而种内虽然各菌株的寄主和地理来源不同，但ITS1和ITS2在长度上均没有变异，碱基构成上存在微小的变异。对该区序列的系统聚类分析表明，白菜黑斑病菌三个种虽然地理来源和寄主不同，但种内的不同菌株均在一个独立的聚类组中，而且和链格孢属内其它种在聚类关系上明显分开，可基于该区进行黑斑病菌的分类鉴定。
设计合成了进行白菜黑斑病菌三个种鉴定三对特异性引物，PCR鉴定检测结果表明：Abre1(AGGCTGAAATCTCTCGAGACGA)和Abre2(AAGGCGAGTCTCCAGCAAACTA)引物对能特异性扩增芸薹链格孢的371bp的片段，Abra1(ACCTCAGCAGCATCTGCTGTTG)和Abra2(GGCTTTATGGATGCTGACCTTG)引物对能特异性扩增甘蓝链格孢的457bp片段，Ajap1(AGCAGTGCATTGCTTTACGGCG)和Ajap2(CCAGTAGGCCGGCTGCCAATT)引物对能特异性扩增萝卜链格孢411bp的片段，而且其它近源种未扩增出目标片段，说明这三对引物可以作为白菜黑斑病菌三个种鉴定的分子特征标记。
检测表明，PDA是白菜黑斑病菌三个种5个菌株营养生长的最适培养基，菌落直径可以作为区分三个种的参考指标。白菜黑斑病菌三个种芸薹链格孢、甘蓝链格孢和萝卜链格孢菌株最适生长温度20-25℃，甘蓝链格孢和萝卜链格孢菌株菌丝生长温度和原有报道相比提高了5℃；芸薹链格孢中国菌株菌丝生长pH范围为4-8，其它所有供试菌株生长pH为3-11；24h光照、12h光暗交替和24h黑暗对芸薹链格孢中国菌株和萝卜链格孢菌株菌丝生长速率影响不大，其它菌株在24h光照和12h光暗交替下的生长速率比24h黑暗处理的显著增大。
14种杀菌剂对白菜黑斑病菌两个优势种芸薹链格孢和甘蓝链格孢国内外株系的生长抑制作用表明：同种但不同来源的病原菌对药剂的敏感性有明显差异，来源相同的不同种菌株对药剂的敏感性也不一致；扑海因、噁醚唑及戊唑醇抑制效果最好，不同菌株对扑海因的敏感性差异不大，EC_(50)为1.2108-2.8863μg／ml；本文检测到具内吸作用的戊唑醇和噁醚唑等三唑类杀菌剂能有效抑制白菜黑斑病菌菌丝生长，各菌株对其敏感性差异明显，噁醚唑EC_(50)为0.1275-7.0968μg／ml，戊唑醇EC_(50)为0.7069-4.3574μg／ml。
Aimed at the regions coding for 5.8s rDNA and the flanking internal transcribed spacers(ITSl and ITS2) from 20 domestic and foreign isolates belonging to three species of Alternarla brasslcae, A.brassicicola, A.japonica and the related species, the study amplified with universal primer pair ITS4 and ITS5, cloned , sequenced ,aligned and analyzed phylogenetic relationship. Using three specific primers pairs designed and synthesized depending on the sequence information identified the three Alternaria species separately. In need of effective control chemicals in cabbage production measured the median effective concentrations (EC50) of 14 fungicides on four Altemaria strains.
The alignment of the DNA sequences of the internal transcribed spacers ITS1 and ITS2 and 5.8s rDNA showed the base composition of ITS1 is more variable than that of ITS2 among the species and also the length of ITS1 varied more than that of ITS2. In spite of from different regions and different hosts base composition of ITS1 and ITS2 has major variation and not for sequence length for different strains of each species. Phylogenetic analyses of ITS1 and ITS2 aligned sequence reveals all strains of each species from different locations and different hosts formed a clade which can distinguish obviously from the other by the related species.
Three specific primers pairs are designed depending on the sequence information, the primer pairs Abrel (AGGCTGAAATCTCTCGAGACGA) and Abre2 (AAGGCGAGTCTCCAG -CAAACTA) can amplify specifically the 371bp fragment of Alternaria brassicae, Abral (ACCTCAGCAGCATCTGCTGTTG) andAbra2 (GGCTTTATGGATGCTGACCTTG) primer pairs for 457bp fragment of A.brassicicola specifically and AjapK AGCAGTGCATTGCTTTACG -GCG)and Ajap2 (CCAGTAGGCCGGCTGCCAATT) primer pairs for 411bp fragment of A.japonica. the three primers pairs can identify them separately and can been used as a molecular marker .
A comparative study was conducted to evaluate the effect of medium, temperature, pH, and light duration on mycelial growth rate and colony characteristics ofA.brassicicola,A.brassicae and A.japonica. The results indicated that PDA was the optimum medium whereas SNA could be a selective medium for Alternaria. The three species could be differentiated on different media based on colony diameter. The optimum growth temperatures of the three species ranged from 20℃ to 25℃, with those of A.brassicicola and A.japonica 5℃ higher than previous reports. The pH range of mycelial growth was from 4 to 8 for domestic strains of A. brassicae while from 3 to 11 for all others. Light duration had little effect on the growth rate of domestic isolate of A.brassicae and A.japonica. However, the growth rate of the other strains was significantly higher under 24h light or 12h alternate light than that under 24h darkness.
Studies on the inhibitory effect on mycelial growth disclosed that the sensitivity to fungicides of the strains from the same or different regions has notable difference. Among them Iprodion, Difenoconazole and Tebuconazole show best inhibition and the distinction of the sensitivity of all strains on Iprodion is not obvious and the In vitro median effective concentrations (EC50) ranged from 1.2108ug/ml to 2.8863 ug/ml, but it is obvious on Difenoconazole and Tebuconazole, the EC50 of Difenoconazole from 0.1275ug/ml to 7.0968ug/ml., and Tebuconazole from 0.7069ug/ml to 2.8863ug/ml.
黑斑病菌及其近缘种真菌核糖体5.8s rDNA及其侧翼ITS区序列比对结果显示，不同种菌株ITS1比ITS2在碱基构成上有更大的变异，而且ITS1的序列长度变异比ITS2的大；而种内虽然各菌株的寄主和地理来源不同，但ITS1和ITS2在长度上均没有变异，碱基构成上存在微小的变异。对该区序列的系统聚类分析表明，白菜黑斑病菌三个种虽然地理来源和寄主不同，但种内的不同菌株均在一个独立的聚类组中，而且和链格孢属内其它种在聚类关系上明显分开，可基于该区进行黑斑病菌的分类鉴定。
设计合成了进行白菜黑斑病菌三个种鉴定三对特异性引物，PCR鉴定检测结果表明：Abre1(AGGCTGAAATCTCTCGAGACGA)和Abre2(AAGGCGAGTCTCCAGCAAACTA)引物对能特异性扩增芸薹链格孢的371bp的片段，Abra1(ACCTCAGCAGCATCTGCTGTTG)和Abra2(GGCTTTATGGATGCTGACCTTG)引物对能特异性扩增甘蓝链格孢的457bp片段，Ajap1(AGCAGTGCATTGCTTTACGGCG)和Ajap2(CCAGTAGGCCGGCTGCCAATT)引物对能特异性扩增萝卜链格孢411bp的片段，而且其它近源种未扩增出目标片段，说明这三对引物可以作为白菜黑斑病菌三个种鉴定的分子特征标记。
检测表明，PDA是白菜黑斑病菌三个种5个菌株营养生长的最适培养基，菌落直径可以作为区分三个种的参考指标。白菜黑斑病菌三个种芸薹链格孢、甘蓝链格孢和萝卜链格孢菌株最适生长温度20-25℃，甘蓝链格孢和萝卜链格孢菌株菌丝生长温度和原有报道相比提高了5℃；芸薹链格孢中国菌株菌丝生长pH范围为4-8，其它所有供试菌株生长pH为3-11；24h光照、12h光暗交替和24h黑暗对芸薹链格孢中国菌株和萝卜链格孢菌株菌丝生长速率影响不大，其它菌株在24h光照和12h光暗交替下的生长速率比24h黑暗处理的显著增大。
14种杀菌剂对白菜黑斑病菌两个优势种芸薹链格孢和甘蓝链格孢国内外株系的生长抑制作用表明：同种但不同来源的病原菌对药剂的敏感性有明显差异，来源相同的不同种菌株对药剂的敏感性也不一致；扑海因、噁醚唑及戊唑醇抑制效果最好，不同菌株对扑海因的敏感性差异不大，EC_(50)为1.2108-2.8863μg／ml；本文检测到具内吸作用的戊唑醇和噁醚唑等三唑类杀菌剂能有效抑制白菜黑斑病菌菌丝生长，各菌株对其敏感性差异明显，噁醚唑EC_(50)为0.1275-7.0968μg／ml，戊唑醇EC_(50)为0.7069-4.3574μg／ml。
Aimed at the regions coding for 5.8s rDNA and the flanking internal transcribed spacers(ITSl and ITS2) from 20 domestic and foreign isolates belonging to three species of Alternarla brasslcae, A.brassicicola, A.japonica and the related species, the study amplified with universal primer pair ITS4 and ITS5, cloned , sequenced ,aligned and analyzed phylogenetic relationship. Using three specific primers pairs designed and synthesized depending on the sequence information identified the three Alternaria species separately. In need of effective control chemicals in cabbage production measured the median effective concentrations (EC50) of 14 fungicides on four Altemaria strains.
The alignment of the DNA sequences of the internal transcribed spacers ITS1 and ITS2 and 5.8s rDNA showed the base composition of ITS1 is more variable than that of ITS2 among the species and also the length of ITS1 varied more than that of ITS2. In spite of from different regions and different hosts base composition of ITS1 and ITS2 has major variation and not for sequence length for different strains of each species. Phylogenetic analyses of ITS1 and ITS2 aligned sequence reveals all strains of each species from different locations and different hosts formed a clade which can distinguish obviously from the other by the related species.
Three specific primers pairs are designed depending on the sequence information, the primer pairs Abrel (AGGCTGAAATCTCTCGAGACGA) and Abre2 (AAGGCGAGTCTCCAG -CAAACTA) can amplify specifically the 371bp fragment of Alternaria brassicae, Abral (ACCTCAGCAGCATCTGCTGTTG) andAbra2 (GGCTTTATGGATGCTGACCTTG) primer pairs for 457bp fragment of A.brassicicola specifically and AjapK AGCAGTGCATTGCTTTACG -GCG)and Ajap2 (CCAGTAGGCCGGCTGCCAATT) primer pairs for 411bp fragment of A.japonica. the three primers pairs can identify them separately and can been used as a molecular marker .
A comparative study was conducted to evaluate the effect of medium, temperature, pH, and light duration on mycelial growth rate and colony characteristics ofA.brassicicola,A.brassicae and A.japonica. The results indicated that PDA was the optimum medium whereas SNA could be a selective medium for Alternaria. The three species could be differentiated on different media based on colony diameter. The optimum growth temperatures of the three species ranged from 20℃ to 25℃, with those of A.brassicicola and A.japonica 5℃ higher than previous reports. The pH range of mycelial growth was from 4 to 8 for domestic strains of A. brassicae while from 3 to 11 for all others. Light duration had little effect on the growth rate of domestic isolate of A.brassicae and A.japonica. However, the growth rate of the other strains was significantly higher under 24h light or 12h alternate light than that under 24h darkness.
Studies on the inhibitory effect on mycelial growth disclosed that the sensitivity to fungicides of the strains from the same or different regions has notable difference. Among them Iprodion, Difenoconazole and Tebuconazole show best inhibition and the distinction of the sensitivity of all strains on Iprodion is not obvious and the In vitro median effective concentrations (EC50) ranged from 1.2108ug/ml to 2.8863 ug/ml, but it is obvious on Difenoconazole and Tebuconazole, the EC50 of Difenoconazole from 0.1275ug/ml to 7.0968ug/ml., and Tebuconazole from 0.7069ug/ml to 2.8863ug/ml.
引文
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