Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.

Genetic Diversity and Pathogenicity Variation in Rhizoctonia solani Isolates from Rice in Sichuan Province, China

Fifty-five representative samples of Rhizoctonia solani isolates, which were collected and isolated from five different ecological regions in Sichuan Province, China, were purified and analyzed for the pathogenicity and molecular genetic variation. The hyphal fusion test revealed that almost all the isolates belonged to the AG-IIA group except the isolate D42. In addition, some of the isolates were ＇bridging isolates＇, which could fuse with several groups simultaneously. The pathogenicity analysis on in vitro leaves confirmed a significant pathogenicity variation in the tested isolates. The 55 isolates were then classified into 8 groups by further RAPD （randomly amplified polymorphic DNA） cluster analysis at the similarity coefficient of 0.941. The results suggest that under the certain ecological conditions in Sichuan Province, China, most of the R. solanistrains were genetically stable, but a few changed drastically.