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河北省苹果再植病害致病镰刀菌种类的鉴定及其致病机制初探

标题: 河北省苹果再植病害致病镰刀菌种类的鉴定及其致病机制初探
英文标题: The Identification of the Pathogenic Fusarium Spp. and the Preliminary Study on Their Pathogenic Mechanisms in Apple Replant Disease
作者: 邹庆甲
出版时间: 2012-01-01
所在大学: 河北农业大学
关键词: 镰刀菌,苹果再植病害,致病机制,果胶酶基因
英文关键词: Fusarium,Apple replant diease,Pathogenic mechanism,Polygalacturonase gene
论文级别: 硕士
学位: 学位论文
导师: 曹克强%王树桐
专业: 植物病理学
提交时间: 2012
摘要: 苹果再植病害在世界各地苹果产区都有发生,是影响果园更新换代的主要障碍,严重威胁了苹果产业的可持续发展.本研究在对河北省苹果再植病害病原菌进行病原分离的基础上,以分离频率最高的镰刀菌作为研究对象,对21株供试镰刀菌进行了形态学鉴定和分子生物学鉴定;并测试了供试镰刀菌对海棠幼苗及胚根的致病力;明确了胚根测试镰刀菌致病力的方法;测定了不同致病力的供试镰刀菌半乳糖醛酸酶(PG)、纤维素酶(Cx)、果胶甲基半乳糖醛酸酶(PMG)、多聚半乳糖醛酸反式消除酶(PGTE)和果胶甲基反式消除酶(PMTE)5种胞壁降解酶的活性;利用CODEHOP程序化设计简并引物对控制镰刀菌相关胞壁降解酶的基因进行了初步克隆与分析,结果如下:1.本研究明确了河北省苹果再植病害的致病镰刀菌包括尖孢镰刀菌Fusarium oxysporum,木贼镰刀菌Fusarium equiseti,锐顶镰刀菌Fusarium acuminatum,层出镰刀菌Fusarium proliferatum和茄腐镰刀菌Fusarium solani.其中,层出镰刀菌(F. proliferatum),木贼镰刀菌(F. equiseti),锐顶镰刀菌(F. acuminatum)作为苹果再植病害的病原菌尚属首次报道.2.对海棠幼苗的致病力测定结果表明,供试镰刀菌对海棠幼苗有不同程度的致病力,致病力最强的菌株对海棠幼苗的校正死亡率为50%,而致病力最弱的菌株对海棠幼苗的矫正死亡率仅为3.3%,镰刀菌的致病力与地理来源和种类没有明显的相关性.3.用海棠胚根测试了供试镰刀菌的致病力,结果表明镰刀菌能够使胚根发生褐变,并能抑制胚根的伸长,胚根的褐变率与幼苗的死亡率呈正相关,而胚根长度与死亡率呈负相关,从而确定了胚根作为材料测试镰刀菌致病力的方法.4.具有致病力的菌株在改良的Marcus培养液中能产生多聚半乳糖醛酸酶(PG)、纤维素酶(Cx)、果胶甲基半乳糖醛酸酶(PMG)、多聚半乳糖醛酸反式消除酶(PGTE)和果胶甲基反式消除酶(PMTE)5种胞壁降解酶,明确了镰刀菌的致病力与多聚半乳糖醛酸酶(PG)、纤维素酶(Cx)、果胶甲基半乳糖醛酸酶(PMG)的活性呈正相关.5.利用CODEHOP程序化设计简并引物扩增出了供试菌的果胶酶基因片段,其结果经过BLAST比对,与已发现的尖孢镰刀菌的PG5基因同源性最高,从而证实了所扩增的片段即为PG5基因片段.
英文摘要: Apple Replant Disease (ARD), distributed in all over the apple producing areas around the world, is the main problem that affects the replacement of the apple orchards. It is also a serious threat to the sustainable development of apple industry.Based on the isolation of the pathogen of the ARD, the Fusarium which had a highest isolation frequency were tested. In this study, 21 isolates were identified by observation of the morphological characteristics and determination of molecular features. Begonia seedlings were used to test the pathogenicity of the strains; the radicle of the Begonia were also used to test the pathogenicity of the strains, and this maybe a new method to test the pathogenicity of the isolates. Five Extracellular wall degrading enzymes (PG, CX, PMG, PGTE and PMTE) were tested in this study, with the help of the CODEHOP. Degenerate primers were designed for cloning the fungal polygalacturonase gene fragment and the polygalacturonase gene fragments were cloned and analysed. The results are as follows:1. In this study, five different species of pathogenic Fusarium include F. oxysporum, F. equiseti, F. acuminatum, F. proliferatum and F. solani were identified. Among them, F. proliferatum, F. equiseti, F. acuminatum were the first report as the pathogens of the ARD.2. The results of the pathogenicity to the Begonia seedlings showed that different strains had different pathogenicity, and the pathogenicity had no correlation with the species and the geographic origins of the strains. The corrected mortality of the seedlings treated by the strongest virulent isolate was 50% while it was just 3.3% treated by the lowest virulent isolate.3. After infected by the test fungi the radicles became brown and short. The result showed that it had a positive correlation between the browning rate and the mortality of the seedlings, and it had negative correlation between the radicle length and the mortality of the seedlings. Thus, using radicles maybe a new method to test the pathogenicity of the isolates.4. 5 Extracellular wall degrading enzymes (PG, CX, PMG, PGTE and PMTE) could be produced by the strong virulent isolates and the low virulent isolates, the activities of PG, CX, and PMG produced by these strains showed a postive correlation with the pathogenicity.5. With the help of CODEHOP, degenerate primers were designed for cloning the fungal polygalacturonase gene fragment. The DNA fragments had high similarity with the PG5 from GenBank, This indicated that the product was the fragment of the PG5.