Determination of genetic diversity in Rhizoctonia solani (AGI-IA); causal agent of rice sheath blight in Iran.

In this study isolates of Rhizoctonia were sampled from rice fields of all rice growing provinces in Iran. Two methods were used for identification of isolates: 1) determination of nuclei number in cells 2) determination of anastomosis group affinity. Hyphal anastomosis is a manifestation of somatic or vegetative incompatibility between isolates. Our findngs showed that only the three northern provinces of Iran (Gilan, Mazandaran and Golestan) where more than 80 % of Iran's rice fields are located, are infected with the disease. 182 isolates from Gilan, 138 isolates from Mazandaran and 73 isolates from Golestan were collected. Of these, 146isolates were identified as AGI-IA, based on the number of nuclei per cell and anastomosis tests.The increase in incidence and severity of rice sheath blight is generally linked to the rapid adoption of high _ yielding cultivars and increased nitrogen fertilization. These and other changes in agronomic practices have lead to a micro climate in the rice that is advantageous to the pathogen. R. solani is complex species. Based on hyphal anastomosis, 14 AGs have been identified for R. solani. R. solani AGI-IA causes sheath blight in rice To determine the genetic diversity among Iranian isolates, we used isozyme (pectic enzyme electrophoretic pattern) and DNA based microsattellite markers. The AGI-IA of R. solani was isolated from diseased rices. Pectic zymogram electrophoresis was used as a marker to determine the genetic variation of the isolates. In this regard two loci, one for polygalacturonase and the other for pectinestrase were used. Six electrophoretic patterns (ZP1-ZP6) for the isolates collected from diseased rice in Golestan province 5 electrophoretic patterns (ZP2- ZP6) for Gilan province and 4electrophoretic patterns (ZP2-ZP5) for Mazandaran province were recognised. Since pectic enzyme secretion in R. solani could be correlated with pathogenecity and physiological disoorders, it may have an impact on breeding strategies for tolerance to this disease. To further study the diversity within R. solani AGI-IA and within a distinct field population we used simple sequence repeat (SSR) or microsattellite markers. Two previously published primers; the tandem repeat sequence of the M13 phage protein III(MR) and a simple sequence repeat (GF) primers were used in this study. Samples were washed, surface sterilized and were transferred to acidified PDA and incubated at 25-28C in the dark. Pure cultures were then kept on PDA.The number of nuclei per cells were determined with 5% Safranin in 3% KOH and anastomosis groups were determined as described (Parmeter 1969). Isolates of R. solani AGI-IA were used for SSR-PCR analyses. DNA extraction and PCR amplification were performed as described by Banniza and Rutherford, 2001. Banding patterns of isolates were compared and characteristic fragments were identified. The presence or absence of each fragment was recorded for all isolates. This data was used for generating a similarity matrix. Similarities were calculated using Dice Coefficient. A cluster analysis was performed based on similarity values by COMPLETE method. The NTSYS statistical program was used for data analys. SSR-PCR of field isolates with both primers of R. solani revealed a high degree of polymorphism among isolates. Among 146 isolates, 118 different genotypes were identified with primer MR with similarities ranging from 54 to 98%. Banding patterns obtained from the primer MR were of a complex nature consisting of 35 bands (22 to 32 bands in each isolate). Only small groups of isolates with identical pattern could be identified. Isolates were clustered into four major groups linked at 69% similarity. Amplification of the same isolates with primer GF resulting in 123 different fingerprints. Banding patterns were simpler, consisting of 14 to 24 fragments. Cluster analysis revealed similarities between genotypes ranging from 57 to 97%. A few groups of identical isolates were present. Isolates were clustered into six major groups linked at 74% similarity. This results indicate the presence of significant genetic diversity among Iranian R. solani isolates of rice fields.