Differentiation of <I>Rhizoctonia spp.</I>, Causal Agent of Strawberry Root Rot Disease, Using DNA Fingerprint Technique

Abstract

Isolation of plant pathogen from roots of wilted strawberry in 5 plantations located at Ban Borkaew Samoeng district, Mae Ram, Mae Rim district, Inthanon Royal Project Research Station and Angkhang Royal Project Research Station in Chiang Mai province and at Ban Huay Namrin in Chiang Rai province, 70 isolates of Rhizoctonia spp. were found. Number of nuclei in mycelial cells were determined by staining with Geimsa stain. Of all, 68 isolates were binucleate Rhizoctonia spp. (97%) and two were multinucleate Rhizoctonia spp. (3%). After pairing with known 5 tester isolates, only 60 isolates could be classified into various anastomosis grouping (AG) of 3 AG; AG-A, AG-G and AG-P but 10 isolates were unable to identify. The most common AG isolate recovered was AG-A (41.5%) followed by AG-G (37.1%) and AG-P (7.1%). Among unknown, eight isolate were binucleate and did not anastomose with any tester isolates. Frequencies of each AG isolate found were different i.e. AG-A was found from all collection sites whereas AG-G was found from three sites, Mae Rim, Borkaew and Inthanon and AG-P was found from Borkaew only. Fungal DNA was extracted and amplified in the portion of 28S rDNA using the polymerase chain reaction (PCR). These amplified fragments were digested with 4 restriction enzymes (Hhal, Mbol, Mspl and Taql).  It was found that no single restriction enzyme accurately identified all AG. The use of a combination of four restriction enzymes data could group all 75 isolates into 12 groups, which correspond to the AG determination. There was no relation among each group and collection site from using PCR-RFLP technique.