湖北省灰霉病菌致病力分化及菌株HBstr-470中真菌病毒的研究

发布时间：2018-05-03 07:01

本文选题：灰霉病菌 + 致病力分化　；参考：《华中农业大学》2015年硕士论文

【摘要】：灰葡萄孢(Botrytis cinerea)引起的灰霉病是一类广泛分布的重要病害,而湖北省的地理位置和气候条件也有利于灰霉病的发生。本论文对湖北省灰霉病菌致病力分化情况以及灰霉菌株HBstr-470中的真菌病毒进行了研究,取得了如下的研究成果:首先,本研究对湖北省各地区保护地果蔬灰霉病的发病情况进行了调查采样,发现由灰霉病菌引起的果蔬灰霉病在全省范围内发生十分普遍。从湖北省除神农架林区外的17个城市采集的灰霉病样品中共分离到1140株灰霉病菌。通过菌丝块接种本氏烟离体叶片的方法测定了其中942株灰霉病菌的致病力,发现中强致病力(病斑直径大于10 mm)灰霉病菌的比例高达93.94%,而弱致病力菌株(病斑直径小于等于10 mm)的比例约为6.06%。通过进一步对弱致病力菌株致病力的重复测定,我们最终获得了15株致病力较弱或无致病力的灰霉病菌。其次,对上述15株弱致病力灰霉菌株中的一株弱致病力菌株HBstr-470进行了生物学特性研究。结果表明,该菌株生长速率十分缓慢,菌落形态异常,长期培养不产菌核,但有较多的气生菌丝并产生丰富的分生孢子,且分生孢子的萌发率较高。通过孢子悬浮液接种本氏烟活体叶片,发现菌株HBstr-470的分生孢子致病力也较弱,并且在与强致病力菌株B05.10的孢子液混合接种时产生比B05.10孢子液单独接种更小的病斑。再次,从弱致病力灰霉菌株HBstr-470的菌丝中检测到了5条ds RNA条带,分别命名为条带A、B、C、D和E,并对这5条ds RNA进行了序列的克隆和分析。结果表明,ds RNA-A的大小约为10,000 bp,与Sclerotinia sclerotiorum hypovirus 1的同源关系较高。ds RNA-B、ds RNA-C和ds RNA-D分别与病毒Rosellinia necatrix quadrivirus 1的片段1、片段3和片段4同源,因此,这三条ds RNA可能属于同一真菌病毒不同基因组片段,其中ds RNA-C编码病毒Rd Rp。此外,基于病毒Rd Rp的系统进化分析也表明,该病毒与Quadriviridae科病毒成员Rn QV-W1075和Rn QV-W1118,还有Totiviridae科病毒成员Sc V-L-A和Sc V-L-BC的亲缘关系较近。ds RNA-E大小约为2,000 bp,与Rosellinia necatrix partitivirus 5和Sclerotinia sclerotiorum partitivirus S有较高的同源关系。因此,弱致病力灰霉菌株HBstr-470可能被多种病毒复合侵染,分别属于Hypoviridae、Quadriviridae和Partitiviridae这三个科。最后,通过原生质体融合技术获得了一株灰霉菌株HBstr-470与灰霉菌株B05.10(01868)原生质体融合后的子代菌株B05.10(W1),该菌株培养形态异常,产生大量气生菌丝和分生孢子,且生长十分缓慢,这与灰霉菌株HBstr-470的培养形态较为一致。
[Abstract]:Botrytis cinerea) is a kind of important disease which is widely distributed, and the geographical location and climatic conditions of Hubei Province are also conducive to the occurrence of gray mold disease. In this paper, the pathogenicity differentiation of cinerea in Hubei Province and the fungal virus in HBstr-470 were studied. The results are as follows: first of all, In this study, the incidence of grey mold of fruits and vegetables in protected areas of Hubei Province was investigated and sampled, and it was found that the occurrence of grey mold of fruits and vegetables caused by cinerea cinerea was very common in the whole province. A total of 1140 strains of Botrytis cinerea were isolated from 17 cities in Hubei Province except Shennongjia forest area. The pathogenicity of 942 strains of Botrytis cinerea was determined by inoculating the leaves of Bensi tobacco in vitro. It was found that the proportion of moderately virulent (disease spot diameter > 10 mm) was 93.94, while that of weakly virulent strain (less than 10 mm) was about 6.06 mm. Through the repeated determination of the pathogenicity of the weakly pathogenic strains, we finally obtained 15 strains of grey mold with weak pathogenicity or no pathogenicity. Secondly, the biological characteristics of one of the 15 weakly virulent strains of Aspergillus virulence (HBstr-470) were studied. The results showed that the growth rate of the strain was very slow, the colony morphology was abnormal, and no sclerotia was produced in long-term culture, but there were more airborne hyphae and abundant conidia, and the germination rate of conidia was higher. It was found that the virulence of conidia of strain HBstr-470 was also weak when inoculated with the spore suspension of B05.10, and the disease spot was smaller than that of B05.10 when inoculated with the spore liquid of strong virulent strain B05.10. Thirdly, five DS RNA bands were detected from the hyphae of the weakly virulent Aspergillus strain HBstr-470, named as the band Acarinobium C (D) and the band E, respectively, and the five DS RNA were cloned and analyzed. The results showed that the size of DS RNA-A was about 10000 BP, and the homology with Sclerotinia sclerotiorum hypovirus 1 was higher. DS RNA-Bds RNA-C and DS RNA-D were homologous to Rosellinia necatrix quadrivirus 1 fragment 1, fragment 3 and fragment 4, respectively. These three DS RNA may belong to different genome fragments of the same fungal virus, among which DS RNA-C encodes Rd Rp. In addition, phylogenetic analysis based on virus RRd p also showed that, The relationship between this virus and Rn QV-W1075 and rn QV-W1118 of Quadriviridae family, and that of SC V-L-A and SC V-L-BC of Totiviridae family is about 2000 BP, which has high homology with Rosellinia necatrix partitivirus 5 and Sclerotinia sclerotiorum partitivirus S. Therefore, HBstr-470 may be infected by multiple viruses, belonging to three families: Hypoviridaeae Quadriviridae and Partitiviridae. Finally, the protoplast fusion technique was used to obtain a strain of Aspergillus cinerea (HBstr-470) fused with Ash strain B05.10101868). The strain B05.10W1 was cultured in abnormal morphology and produced a large number of airborne hyphae and conidia, and the growth was very slow. This was consistent with the culture morphology of HBstr-470.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.44

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