| Sclerotinia sclerotiorum is one of the globally spreading pathogen, which could affect more than 450 species of plants belonging to 75 families. The most common hosts include oil crops, brassica, soybean, sunflower etc. Sclerotinia sclerotiorum could cause severe diseses in brassica and affect the yield and quality of crop to 10-30% and 80% in severe cases. The study of highly efficient and safety measure are important to control this pathogen. Use of decline virulence-associated virus controlling the disease caused by fungi is one of the most important ways of biological control. Many decline virulence-associated viruses have been found in numerous fungi including Sclerotinia sclerotiorum, but the species are limited. The study aimed at finding and identifying more mycoviruses from Sclerotinia sclerotiorum, as well as their interaction to host.83 strains of sclerotinia sclerotiorum were isolated from the lesion of rapeseed plant stem from the field of xiaogan city, Hubei province,China.. Their biological traits of mycelia growth rate, biomass, size and yield of sclerotia, colony morphology and pathogenicity were studied. Significant diversity was found in different aspects such as colony morphology, mycelial growth rate and pathogenicity. The MCG identification of 30 strains suggested that 30 strains were divided into 17 groups of mycelium compatibility, of which 9 mycelium compatibility groups contained only one strain on the basis of compatibility. The other MCGs contain 2-5 strains. Although some strains came from the same stem, non-fusion phenomenon of mycelium can also be found in them, which showed the mixed-infection on the same stem. All of these results indicated that even in a very small range, complex diversity could be found in sclerotinia sclerotiorum.The strain XG36-1 was found significantly different with others in slow growth rate, fan-shaped colonies, developed aerial hyphae, fewer sclerotia were irregularly distributed in the colony. Strain XG36-1 showed very weak pathogenicity pattern on the leaves of brassica. The phenotype of XG36-1 is similar to Ep-1PN which is attenuated and has been reported already. In order to prove the decline mechanism of XG36-1, the following research had been done:(1) Analysis of the biological traits of phenotypes of protoplast regenarants of XG36-1; (2) Analysis of the biological traits of Sexual progeny of hypovirulent strain XG36-1; (3) Experimental infectious on decline traits of XG36-1; (4) Observation of the cells of XG36-1 under electron microscope and seperation of virus particle; (5) Seperation and identification of viral nuclear acid. It was found that phenotypes of protoplast regenarants of XG36-1 could be classed in to three groups, typeâ… , typeâ…¡, typeâ…¢.25.5% of protoplast regenarants belong to typeâ… ,23.8% of protoplast regenarants belong to typeâ…¡, the typeâ…¢have no significant difference to attenuated strain XG36-1 and its seperation rate is 51.7%. 104 isolates were isolated from ascospores. All of them have the identical phenotype of common strain which is different from the XG36-1. It indicates that XG36-1 has the nucleus inheritance factor from common strain. The decline results from non-chromosome inheritance factors and they are in contagious distribution in cell. Agrobacterium tumefaciens mediated transformation was performed to transform the normal strain of Sclerotinia sclerotiorum XG36-1A34 and transformants with normal phenotype and hygromycin resistance were screened. XG36-1 and XG36-1A34R were dual cultured. Isolation with the phenotype of XG36-1 indicated inheritance factors which relate to decline. It indicates that these inheritance factors are removable and affective. Observation with transmission electron microscopy showed that the size of individual particle is about 40 nm in hyphal cells of XG36-1. No virus like particle (VLP) was found in hyphal cells of sexual ascospores of strain. Hyphal contact with strain XG36-1 can transfer the VLP. Sucrose gradient centrifugation method was adopted to separate and purify tiny VLP from mycelium of strain XG36-1. Virus nuclear acid was not seperated from mycelium.Another strain, XG19-2, was found to have no significant difference in Growth, colony morphology, the formation of sclerotia and Pathogenicity with other normal strains of Sclerotinia sclerotiorum. But with CF-11 adsorption, dsRNA of about 4kb length could be separated. cDNA cloning was performed with random primers and two positive clones with the length of about 700 bp was screened. Sequencing analysis suggested that the two clones have incomplete open reading frames. The Blastp analysis of peptide chain suggested that they have high homology with encoding RNA-dependent RNA polymerase from mycovirus Diaporthe ambigua RNA virus1 (DaRV1). The result showed dsRNA from strain of XG19-2 is an unknown chromosome from mycovirus with the given name of Sclerotinia sclerotiorum XG19-2 RNA virus, SsXG19-2RV.The study shows high biodiversity of Sclerotinia sclerotiorum in small filed. Attenuated Strain of XG36-1 was seperated and proved to be closely related to infect by unknown mycovirus. The virus particle from XG36-1 resulted in deeply decline of Virulence. So it is of importance to evaluate the value of a biocontrol factor of it. A new virus which has significant relative relationship with DARV1 was separated from XG19-2 with normal phenotype. Diaporthe ambigua RNA virus 1 could result in Virulence recession of Diaporthe ambigua. But mycovirus of SsXG19-2RV from XG19-2 have no affection with host. This not only indicates mycovirus with the same class can live on different host but also their different interaction with host. So the study of SsXG19-2RV could reveal the relationship between mycovirus and host to deepen the realization of biocontrol of Sclerotinia sclerotiorum and also enrich the theoretical system of mycovirus. |
