【作者】 张林巧；

【导师】 王克荣；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2011， 硕士

【摘要】 粟疫病(Cryphonectria parasitica)是世界最著名的森林病害之一,曾造成美洲粟濒临灭绝。栗疫菌细胞中的低毒力病毒是一类没有衣壳的双链RNA病毒,可以引起寄主粟疫菌的菌落形态变化、分生孢子和色素的产量降低、雌性不育和漆酶产量与活性降低。更重要的是,低毒力病毒的侵染会导致栗疫菌毒力的下降,这一发现为栗疫病的生物防治提供了一种全新的思路。CHV1-CN280是来自我国的低毒力病毒,与其它低毒力病毒相比,具有较高的病毒传播能力,低毒力病毒在栗疫菌株之间传播的成功与否,决定了生物防治能否取得成功。很显然,应用传播能力更高的低毒力病毒,将会大大提高对栗疫病的生物防治效果。本文通过对具有较高的病毒传播能力的低毒力病毒CHV1-CN280与另两个已经进行了全基因组测序的低毒力病毒(CHV1-EP713、CHV1-Euro7)对不同菌株生物学性状影响的比较,评估CHV1-CN280的生防潜力和在田间定殖与扩散能力。与来自欧洲的其它两个低毒力病毒相比,CHV1-CN280对寄主的毒性较弱,除了显著降低栗疫菌的致病力外,对菌株的生长速率、分生孢子与色素产量等影响较小,具有良好的生防特性。对CHV1-CN280在田间的定殖能力的评估结果表明,低毒力病毒CHV1-CN280在田间接种后,能够成功定殖并自然扩散至其它营养体亲和群的野生栗疫菌菌株中。研究认为,与其它低毒力病毒相比,CHV1-CN280在中国具有更好的生防潜力。另外,本文通过外源DNA导入法对CHV1-CN280病毒的p29基因在栗疫菌和稻瘟菌中的功能进行了研究。实验克隆了CHV1-CN280病毒的p29基因,将其连接至不同的表达载体并分别导入栗疫菌及稻瘟菌的基因组。结果发现,该基因在栗疫菌中表达并引起寄主色素减少、孢子量下降、生长速率降低以及漆酶活性降低等低毒力特征的表型变化。p29基因在稻瘟菌中表达则引起寄主分生孢子量、漆酶活性、生长速率等生物学性状正向增长的表型改变。该基因在原寄主和非原寄主中表达的结果具有显著差异。该基因在原寄主中表达的结果能引起寄主部分表型发生类似低毒力性状的改变,而另一部分表型却表现出相反的结果,相比Nuss等人的研究结果,研究认为,CHV1-CN280与CHV1-EP713各自p29具有不完全一致的基因功能,其主要原因应归于两种病毒之间的p29的氨基酸序列具有较大差异。另外,p29在栗疫菌之外的寄主中表达,也不能引起该寄主发生类似栗疫菌中低毒力性状的改变。更多还原

【Abstract】 Chestnut blight fungus(Cryphonectria parasitica) is one of the major diseases of chestnut and has caused serious damage in orchards and in forests.The hypovirulence, which is an attenuation of fungal pathogenis, has reduced the impact ofthis disease and has been used as a biological control method in Europe. So study on biological control of the chestnut blight is possible to provide a new method to control and design the new disease control strategy to give the new theory of direction.CHV1-CN280 is a hypovirus strain isolated in China. In this paper we report the colonization capability of CHV1-CN280 in the field, and the biological characteristics of the hypoviruses CHV1-CN280、CHV1-EP713 and CHV1-Euro7 in the laboratory. Compared to the other two hypoviruses, CHV1-CN280 resulted in a white colonial phenotype of C. parasitica (loss of orange pigmentation), but a moderate reduction in asexual sporulation, and less reduction in fungal virulence. Furthermore, CHV1-CN280 was found established in the field two years later of inoculation, and successfully transmitted into wild virulent strains of C. parasitica which belong to different vegetative compatibility group. Accordingly, CHV1-CN280 showed a better biocontrol capacity of chestnut blight in China.Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence.There have reported that the papain-like protease, p29, present in hypovirulent strain EP713, was shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase. Bioinformatics analysis results show that the amino acid sequences of p29 were quite different between EP713 and CN280.We mapped the p29 gene of CHV1-CN280,and transformate it into virus-free virulent strains EP155.The fungal transformation results presented in this report demonstrate that the expression of viral papain-like protease p29 elicits some similar hypovirus symptoms like the increasing growth rate, the reducing fungal sporulation, pigmentation and the production of the enzyme laccase. Besides, there also have obvious different phenotype between transformant of Cryphonectria parasitica and Magnaporthe oryzae. The combined results suggest that papain-like protease p29 plays a different role when it was transformed into different srain.At the same time, papain-like protease p29 of EP713 and CN280 show different characterizations because of the different sequence.更多还原