Establishment of Agrobacterium tumefaciens-mediated transformation system for Rhizoctonia solani AG-1 I A, the causal agent of rice sheath blight.

In order to construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1ⅠA,the virulent isolate GD118 of this pathogen was selected as the initial isolate for transformation.The conditions for the transformation of GD118 were optimized in 5aspects,i.e.pre-induction time,coculture time,acetosyringone concentration at coculture stage,coculture temperature and pH values of solid induction medium(SIM)at coculture phase.Finally,an Agrobacterium tumefaciens-mediated transformation(ATMT)system for R.solani AG-1ⅠA was established successfully.The optimal conditions for this ATMT system are as follows:the concentration of hygromycin B at 30μg/mL for transformant screening,8hof pre-induction time,20hof coculture time,200μmol/L of acetosyringone in SIM at coculture stage,coculture temperature at 25℃and pH 5.6to 5.8of SIM at coculture phase.The transformants still showed high resistance to hygromycin B after 5generations'subcultures.Ten transformants were randomly picked out for PCR verification using the specific primers designed from the hphgene,and the results revealed that an expected band of 500bp was amplified from all of the 10transformants.Meantime,PCR amplification for these 10transformants was carried out using specific primers designed from the Vir gene of A.tumefaciens,with 4strains of A.tumefaciens as positive controls for eliminating the false-positive caused by A.tumefaciens's contamination,and the results showed that an expected band of 730bp could be amplified from the 4strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10transformants.The results of above two PCR amplifications clearly showed that T-DNA was indeed inserted into the targeted isolate GD118.