| In this study, we investigated some biological characteristics, the genome structure and identity of Botrytis cinerea-debilitation related RNA virus (BcDRV) and the mechanisms responsible for pathogenicity debilitation of strain CanBc-1 of Botrytis cinerea, the causal agent of plant grey mould disease. Meanwhile, we performed the expression profiling analysis for the hypovirulent strain CanBc-1 and the virulent strain CanBc-1c-66 (a single-conidium isolate of strain CanBc-1) of B. cinerea. Main results are summarized below.Firstly, twenty-three strains of B. cinerea were isolated from 14 species of plants grown from different places, and were compared for cultural characteristics and pathogenicity on oilseed rape (Brassica napus). Results showed that strains CanBc-1 and CanBc-3 of B. cinerea was severely debilitated in pathogenicity and in mycelial growth, compared with the other virulent strains of B. cinerea. A species of double-stranded (ds) RNA of approximately 3.0 kb in length was detected in strain CanBc-1, but was not detected in strain CanBc-3 and the other 21 strains of B. cinerea. Single-conidium (SC) isolates of strain CanBc-1 differed greatly in pathogenicity. Some SC isolates of strain CanBc-1 (such as CanBc-1c-66) became virulent on leaves of oilseed rape and the 3.0-kb dsRNA was eliminated from these SC isolates. Strains CanBc-1 and CanBc-1c-66 were found to be resistant to fungicides Carbendazim and dimmethachlon.Secondly, the full-length cDNA sequence of Botrytis cinerea debilitation-related virus (BcDRV,3.0-kb dsRNA) was obtanined. It is 2806 bp in length and AU-rich (66.7%). Based on sequence analysis of BcDRV, we found the BcDRV belongs to genus Mitovirus (Narnaviridae). TEM observation showed that strain CanBc-1 shows degenerative symptoms in hyphal cytoplasma and contained numerous malformed mitochondria without formation of cristae inside. It appears that mitochondria of Botrytis cinerea may be the targets of BcDRV.Thirdly, we also analyszed the mechanisms responsible for debilitation of hypovirulent strain CanBc-1 of B. cinerea. Strain CanBc-1 was compared with the virulent strains CanBc-1c-66 and CanBc-2 of B. cinerea for formation of infection cushions on onion scales and leaves of oilseed rape and tomato as well as production of pectinases, toxic metabolites, oxalic acid and laccase in different growth media. Results showed that the three investigated strains of B. cinerea could produce polygalacturonases, toxic metabolites, oxalic acid and laccase in cultures, and the genes coding for endopolygalacturonases (Bcpg1-6), laccases (Bclcc1 and Bclcc2) and botrydial were detected to express normally. Formation of infection cushions was common on epidermis of onion scales and oilseed-rape leaves which inoculated with strains CanBc-1c-66 and CanBc-2, but was rare on the plant tissues inoculated with strain CanBc-1. It is indicated that rare formation of infection cushions is the main cause for the failure of infection of onion and oilseed-rape by the hypovirulent strain CanBc-1 of B. cinerea.Finally, expression profile analysis was performed on hypovirulent for strains CanBc-1 and CanBc-1c-66 of B. cinerea. A total of 2288 differentially expressed genes (DEGs) were identified. Among these genes,1392 were up-regulated and 896 were down-regulated in strain CanBc-1. The 2288 DEGs were then grouped into 19 KEGG functional categories. They include carbohydrate metabolism (8%), lipid metabolism (5%), nucleotide metabolism (2%), amino acid metabolism (4%), glycan biosynthesis and metabolism (1%), metabolism of Cofactors and vitamins (2%), biosynthesis of polyketides and terpenoids (1%), energy metabolism (3%), xenobiotics biodegradation and metabolism (1%), enzyme family (10%), transcription (2%), translation (2%), folding (2%), sorting and degradation (2%), replication and repair (2%), membrane transport (5%), Signal Transduction, Signaling Moleculars and Interaction, Cellular Process and unclassified function (43%). We found 12 DEGs which had been confirmed as the pathogenicity-related factors in B. cinerea or in other plant pathogens. These genes may be involved in the pathogenesis process of B. cinerea.
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