矿物油室温保藏棉花黄萎菌的存活率及AFLP分析
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摘要
棉花黄萎病是由大丽轮枝菌引起的土传病害,在世界各棉花产区均有分布,是制约棉花生产的重要因素之一。多年来,我们采用矿物油室温保藏的方法保存了包括大丽轮枝菌(Verticillium dahliae)、变黑轮枝菌(V. nigrescens)、尖孢镰刀菌(Fusarium oxysporum f.sp. vasinfectum)等约2000余株。本文对矿物油室温保藏的261株植物病原真菌进行了活化,以大丽轮枝菌为试材,测定了菌落直径、产孢量和致病性等基本生物学参数,并采用AFLP分子标记技术进行了遗传变异特性分析,取得了以下主要结果。
1.采用PDA平板法,活化了261株1951-1985年间矿物油室温保藏的植物病原真菌菌株。结果表明,随着保藏时间的延长,植物病原真菌的存活率呈下降趋势,菌落直径、产孢量与保藏年限间无相关性。
2.棉花黄萎菌致病性测定结果表明,矿物油室温保藏了17-52年的19个供测试菌株中,45、416、193、196、164和165号菌株仍具有不同程度的致病性,特别是45和416号菌株在保藏了50多年后,仍然具有较强的致病性,这是至今报道保藏时间最长的棉花黄萎菌。
3.利用真核生物核糖体DNA内转录间隔区通用引物ITS1和ITS4,对45个供试菌株的ITS1-5.8S-ITS2区域进行PCR扩增和测序,经BLAST比对分析,菌株3、4、5、8、13、45、164、165、181、193、196、414、416、0-3、0-9、0-11、0-12、0-18、0-21、0-25、1-4、1-5、1-9、1-11、3-4、3-5、3-6、3-12、T-9、t13、V23、1991、2008、AY、JS、HT、JY为大丽轮枝菌(V. dahliae),菌株43、55、152、286、287、288、289、394为变黑轮枝菌(V. nigrescens)。用DNAMAN软件对ITS序列比对结果显示,394号菌株ITS序列的一个碱基与其它变黑轮枝菌不同。
4.使用18对引物对45个菌株进行AFLP分析结果表明,这些菌株呈现丰富的AFLP分子标记多态性。依据UPGMA方法的SAHN聚类分析结果,可将这些菌株分为2组,第一组为变黑轮枝菌,第二组是大丽轮枝菌,该结果与ITS测序结果一致。AFLP分子标记多态性与大丽轮枝菌的致病性有一定的相关性。
Verticillium wilt of cotton, caused by Verticillium dahliae, is one of soilborne diseases. The disease distributed all over the world is one of the most important factors limit the cotton productuction. In our lab, almost 2000 strains of plant pathogenic fungi, including V. dahliae, V. nigrescens, and Fusarium oxysporum f.sp. vasinfectum, were preserved in mineral oil at room temperature. In this thesis, 261 strains of plant pathogenic fungi preserved in mineral oil were recovered to test the viability through PDA plate method. The basic biological parameters of V. dahliae including colony diameter, sporulation, and pathogenicity were tested. The genetic variation characteristics were analyzed by AFLP molecular marker. The main results were shown as below.
1. Two hundred and sixty one strains of plant pathogenic fungi preserved in mineral oil from 1951 to 1985 were recovered by PDA plate method. Results indicated that the survival percentage of plant pathogenic fungi decreased with the preserved time extension, and colony diameter and sporulation had no correlation with preserved years.
2. Pathogenicity of V. dahliae showed that strain 45, 416, 193, 196, 164, and 165 among 19 testing strains preserved in mineral oil at room temperature for 17 to 52 years still had a certain pathogenicity to cotton. The strain 45 and 416 preserved over 50 years still had high pathogenicity. Up to now, they are the longest V. dahliae preserved in mineral oil at room temperature.
3. The region of ITS (Internal transcribed spacer)1-5.8S-ITS2 sequences of 45 strains were amplified and sequenced using primer ITS1 and ITS4. The Basic Local Alignment Search Tool (BLAST) results showed that strain 3, 4, 5, 8, 13, 45, 164, 165, 181, 193, 196, 414, 416, 0-3, 0-9, 0-11, 0-12, 0-18, 0-21, 0-25, 1-4, 1-5, 1-9, 1-11, 3-4, 3-5, 3-6, 3-12, T-9, t13, V23, 1991, 2008, AY, JS, HT, and JY are V. dahliae, and strain 43, 55, 152, 286, 287, 288, 289, and 394 are V. nigrescens. One nucleotide of ITS sequence of strain 394 was different from that of other V. nigrescens.
4. Fourty five strains were analyzed using amplified fragment length polymorphism (AFLP) technique. Results showed that these strains had rich polymorphism of AFLP molecular marker. The phylogenetic tree of SAHN was constructed using UPGMA method based on AFLP data. These strains were clustered into two groups. One was V. dahliae group, the other was V. nigrescens group. It was agreed with the result of ITS sequence. Cluster analysis revealed that there was certain correlation between V. dahliae pathogenicity and polymorphism of AFLP molecular markers.
1.采用PDA平板法,活化了261株1951-1985年间矿物油室温保藏的植物病原真菌菌株。结果表明,随着保藏时间的延长,植物病原真菌的存活率呈下降趋势,菌落直径、产孢量与保藏年限间无相关性。
2.棉花黄萎菌致病性测定结果表明,矿物油室温保藏了17-52年的19个供测试菌株中,45、416、193、196、164和165号菌株仍具有不同程度的致病性,特别是45和416号菌株在保藏了50多年后,仍然具有较强的致病性,这是至今报道保藏时间最长的棉花黄萎菌。
3.利用真核生物核糖体DNA内转录间隔区通用引物ITS1和ITS4,对45个供试菌株的ITS1-5.8S-ITS2区域进行PCR扩增和测序,经BLAST比对分析,菌株3、4、5、8、13、45、164、165、181、193、196、414、416、0-3、0-9、0-11、0-12、0-18、0-21、0-25、1-4、1-5、1-9、1-11、3-4、3-5、3-6、3-12、T-9、t13、V23、1991、2008、AY、JS、HT、JY为大丽轮枝菌(V. dahliae),菌株43、55、152、286、287、288、289、394为变黑轮枝菌(V. nigrescens)。用DNAMAN软件对ITS序列比对结果显示,394号菌株ITS序列的一个碱基与其它变黑轮枝菌不同。
4.使用18对引物对45个菌株进行AFLP分析结果表明,这些菌株呈现丰富的AFLP分子标记多态性。依据UPGMA方法的SAHN聚类分析结果,可将这些菌株分为2组,第一组为变黑轮枝菌,第二组是大丽轮枝菌,该结果与ITS测序结果一致。AFLP分子标记多态性与大丽轮枝菌的致病性有一定的相关性。
Verticillium wilt of cotton, caused by Verticillium dahliae, is one of soilborne diseases. The disease distributed all over the world is one of the most important factors limit the cotton productuction. In our lab, almost 2000 strains of plant pathogenic fungi, including V. dahliae, V. nigrescens, and Fusarium oxysporum f.sp. vasinfectum, were preserved in mineral oil at room temperature. In this thesis, 261 strains of plant pathogenic fungi preserved in mineral oil were recovered to test the viability through PDA plate method. The basic biological parameters of V. dahliae including colony diameter, sporulation, and pathogenicity were tested. The genetic variation characteristics were analyzed by AFLP molecular marker. The main results were shown as below.
1. Two hundred and sixty one strains of plant pathogenic fungi preserved in mineral oil from 1951 to 1985 were recovered by PDA plate method. Results indicated that the survival percentage of plant pathogenic fungi decreased with the preserved time extension, and colony diameter and sporulation had no correlation with preserved years.
2. Pathogenicity of V. dahliae showed that strain 45, 416, 193, 196, 164, and 165 among 19 testing strains preserved in mineral oil at room temperature for 17 to 52 years still had a certain pathogenicity to cotton. The strain 45 and 416 preserved over 50 years still had high pathogenicity. Up to now, they are the longest V. dahliae preserved in mineral oil at room temperature.
3. The region of ITS (Internal transcribed spacer)1-5.8S-ITS2 sequences of 45 strains were amplified and sequenced using primer ITS1 and ITS4. The Basic Local Alignment Search Tool (BLAST) results showed that strain 3, 4, 5, 8, 13, 45, 164, 165, 181, 193, 196, 414, 416, 0-3, 0-9, 0-11, 0-12, 0-18, 0-21, 0-25, 1-4, 1-5, 1-9, 1-11, 3-4, 3-5, 3-6, 3-12, T-9, t13, V23, 1991, 2008, AY, JS, HT, and JY are V. dahliae, and strain 43, 55, 152, 286, 287, 288, 289, and 394 are V. nigrescens. One nucleotide of ITS sequence of strain 394 was different from that of other V. nigrescens.
4. Fourty five strains were analyzed using amplified fragment length polymorphism (AFLP) technique. Results showed that these strains had rich polymorphism of AFLP molecular marker. The phylogenetic tree of SAHN was constructed using UPGMA method based on AFLP data. These strains were clustered into two groups. One was V. dahliae group, the other was V. nigrescens group. It was agreed with the result of ITS sequence. Cluster analysis revealed that there was certain correlation between V. dahliae pathogenicity and polymorphism of AFLP molecular markers.
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