The pectic enzymes of Rhizoctonia solani AG-8 strains

Isolates of Rhizoctonia solcmi anastomosis group 8 (AG-8) cause bare patch disease in cereal and legume crops. Infection of root tissue by the fungus leads to maceration of the roots and inhibits development of the aerial parts of the plant. There is evidence to suggest that pectic enzymes may be an important factor in the pathogenicity of R. solani. The aim of this study is to investigate the role of pectolytic enzymes produced by R. solani (AG-8) isolates in pathogenicity. Pectic enzyme production by highly virulent (HV) and weakly virulent (WV) isolates from wheat bare patches was analyzed. The predominant type of pectolytic activity produced by both types of strains was polygalacturonase (PG). Staining of isoelectric focusing gels for PG activity revealed differences in the PG isoenzymes produced by HV and WV strains. The differences were more clearly observed in single spore progeny derived from one of the HV strains. Analysis of enzymatic breakdown products of HV and WV enzymes with polygalacturonic acid suggested that the enzymes are endopectinases. Four isoenzyme forms of PCs, from both HV and WV strains, were separated and purified by ion exchange chromatography. Their characteristics; including isoelectric point, molecular masses, and pH optimum; were determined. All, except one isozyme from HV strain degrade polygalacturonic acid by producing oligosaccharide intermediates. One of the isozymes from HV strain does not produce these intermediates. Bioassay of the degradation products showed that these oligosaccharide products induced synthesis of Phenylalanine Ammonia Lyase (PAL), a key enzyme in the defence of the plant. Genomic DNA of R. solani and Aspergillus niger was amplified by PCR using two degenerate primers designed from conserved regions in the amino acid sequences of Aspergillus tiiger PG genes. A single product was obtained from A. niger, whilst multiple products were obtained from R. solani. A southern blot of the amplified products was probed with the product from A. niger. The probe hybridized to single band of the same size as the A niger product. The sequence of this Rhizoclonia band showed that it is a pectinestrase gene. A number of positive plaques were detected when the genomic library (RMZ1) was screened with this gene