Members of the family Botryosphaeriaceae are known to be plurivorous phytopathogens,having a wide geographical distribution.Among all commodities affected by these species, grapevine is of significant relevance.However, factors causing pathogenicity in Botryosphaeria spp.and their infection mechanisms are still unclear.In this study, Lasiodiplodiatheobromae (asexual Botryosphaeriarhodina)was analyzed for its pathogenicity.Lasiodiplodiatheobromae strain CSS-01 was used for restriction enzyme mediated integration (REMI) transformation and a library of 6000 L.theobromae mutants was generated with HindⅢ, KpnⅠ, SamⅠ and SacⅠ restriction enzymes.Fifty (50) randomly selected mutants were used for variances in their pathogenicity with detached shoot assay.According to Duncan significance analysis, virulence of 42% of the mutants significantly increased, while 52% showed significantly reduced virulence.Among 14 randomly selected mutants, 50% accounted for having a single integration site.Seven (7) of these single locus insertion mutants (4 virulence enhanced and 3 virulence reduced) were selected and analyzed their flanking sequences by plasmid rescue.All 4 virulence enhanced mutants showed a sequence having a length of 759bp.Softberry analysis predicted a similar sequence encoding Ltcan1 gene in Neurosporacrassa and Schizosaccharomyecspombe.It can be predicted that the removed gene region of virulence reduced mutants may have an association with pathogenicity.This study provides the research material which can be used in functional genomics research to discover new virulence genes of Botryosphaeria spp.and provide a better understanding of their regulatory pathways and infection mechanisms.