|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1997 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥900,000 (Direct Cost : ¥900,000)
Biochemical methods consisting of fatty acid analysis and restriction fragment length polymorphism (RFLP) analysis were performed for the objective indicator for identification and characterization of Rhizoctonia spp.and R.solani AGs.
Percentage composition of whole-cellular fatty acids of 5 Rhizoctonia spp. (R.solani, R.oryzae, R.oryzae-sativae, R.fumigata and R.candida) and 7 AGs including 13 intraspecific groups (ISGs) of R.solani were characterized by gas-liquid chromatography. The major fatty acids identified were palmitic, palmitoleic, oleic and linoleic acids. Principal componet analysis based on the percentage composition of whole-cellular fatty acids revealed consistently low variability among isolates of a single species and significant differences among 5 Rhizoctonia spp.Similar results by principal component analysis were presented low variability among isolates of an identical AG and differences among seven AGs of R.solani. Average linkage cluster analysis also showed that
the isolates in the same species of Rhizoctonia spp.and an identical AG of R.solani formed individual clusters on the dendrogram, and each species of Rhizoctonia and different AGs R.solani couls be distinguished based on the dendrogram. However, all of the ISGs belonging to the AG-1, AG-2 and Ag-4 isolates were differentiated by the average linkage cluster analysis. Based on percentage composition of fatty acids, 3ISGs in AG-1,5 ISGs in AG-2, and 2 ISGs in AG-4, were presented respectively. Isolates of R.solani AG-1 IA showed specific and significant composition of whole-cellular fatty acids from other AGs and ISGs of R.solani.
Isolates of 5 Rhizoctonia spp.and 7 AGs representing 13 ISGs of R.solani were characterized by the RFLPs analysis of a 28S rDNA gene amplified by the polymerase chain reaction (PCR). Amplification products by PCR reaction were 1.4-and 1.8-kirobase pair fragments. RFLP profiles obtained after digestion of 28S rDNA with the restriction enzymes were different depending on the 4enzymes, MspI,HhaI,Sau3AI and HaeIII,used. The RFLP profiles by the digestion with MspI and Hhal showed specific restriction fragments polymorphism among 5 Rhizoctonia spp. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with 4 restriction enzymes were also observed among 7 AGs of R.solani. These differences were maily conseved among ISGs of AG-1 and AG-2. A dendrogram derived from the RFLP profiles showed that ISGs from AG-1 and AG-2 can each be subdivided into distinct groups, that are distantly related to the majority isolates of the other AGs. Isolates of R.solani AG-1 IA showed specific RFLP profiles and phylogenetic differences from the other ISGs and AGs of R.solani.
These two nethods, fatty acid analusis and RFLP analysis, clearly concluded the significant difference among isolates of Rhizoctonia spp. Less