Isolates of Rhizoctonia solani AG2-2 obtained from turf with symptoms of large-patch disease of warm-season turfgrasses were compared with known AG2-2 isolates belonging to cultural types IIIB and IV. Some isolates that were previously identified as type IV have been separated here and named LP isolates. Comparisons among isolates were based on cultural morphology, hyphal growth rate, pathogenicity and restriction fragment length polymorphism (RFLP) analysis in the nuclear encoded ribosomal DNA (rDNA) genes. The cultural characteristics of LP isolates varied from those of types IIIB and IV. LP isolates did not show distinct sclerotial formation and zonation, and the colour of their mycelia and pigment deposition was dark brown. LP isolates had slower hyphal growth rates than types IIIB and IV, with an optimum temperature of 25ЊC compared with 28ЊC for types IIIB and IV. LP isolates were less virulent on radish but highly virulent on zoysia grass when compared with isolates of types IIIB and IV. Genomic DNA was digested separately with Eco RI, Ban III, Xba I and Sal I, and probed with cloned rDNA from Alternaria alternata in Southern hybridizations. LP isolates had one RFLP pattern, while both IIIB and IV possessed four different patterns each. Cluster analysis of RFLPs showed that R. solani AG2-2 is divided into three genetic subgroups, consisting of the IIIB, IV and LP isolates, respectively. The polymerase chain reaction (PCR) amplified rDNA internally transcribed spacer (ITS) regions of the IIIB, IV and LP isolates had the same length but produced different restriction patterns when digested with Msp I and Taq I. These results indicate that there are three cultural types in R. solani AG2-2, namely IIIB, IV and LP.

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3^10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms. ß

Isolates of an unidentified Rhizoctonia sp. (NP isolates), obtained from creeping bentgrass (Agrostis stolonifera var. palustris) in Japan that exhibited symptoms of a new disease, were compared with isolates of three varieties of Waitea circinata var. oryzae, var. zeae, and var. circinata. NP isolates also were compared with isolates of R. oryzae obtained from creeping bent-grass exhibiting white patch-like symptoms (RW isolates). The color and size of sclerotia, color of mycelia, and pigment deposition of NP isolates was similar to that of RW isolates and W. circinata var. circinata, but distinctly different from W. circinata var. oryzae and W. circinata var. zeae. The optimal temperature for hyphal growth of NP isolates, RW isolates, and W. circinata var. circinata was 28°C, and for W. circinata var. oryzae and W. circinata var. zeae was 30°C. Pathogenicity tests on creeping bentgrass showed that the severity of disease caused by NP isolates, RW isolates, and W. circinata var. circinata was greater than with W. circinata var. oryzae, but lower than with W. circinata var. zeae. No significant differences in symptom expression were apparent among NP isolates, RW isolates, and W. circinata var. circinata. A phylogenic tree, obtained using the results of random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), showed that isolates of W. circinata var. oryzae and W. circinata var. zeae separated into individual clusters, while NP isolates, RW isolates, and W. circinata var. circinata clustered together. The lengths of the rDNA internal transcribed spacer (ITS) region of NP isolates, RW isolates, and W. circinata var. circinata were identical but smaller than those of W. circinata var. oryzae and W. circinata var. zeae. Restriction fragment length polymorphism (RFLP) analysis of the rDNA-ITS region, using three enzymes (HapII, HinfI, and MboI), also showed that NP isolates were the same as RW isolates and W. circinata var. circinata, but different from W. circinata var. oryzae and W. circinata var. zeae. Based on these results, the NP isolates causing a new disease on bentgrass are W. circinata var. circinata, and that RW isolates are also W. circinata var. circinata but not R. oryzae. We propose that the name of the disease on creeping bentgrass caused by W. circinata var. circinata is brown ring patch.