Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass.
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.
Toda
Takeshi
T
The United Graduated School of Agricultural Science, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.
Mushika
Tomoyuki
T
Hyakumachi
Mitsuro
M
D016000:Cluster Analysis; D016172:DNA Fingerprinting; D017931:DNA Primers; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D016533:Mycological Typing Techniques; D010935:Plant Diseases / Q000382:microbiology*; D018515:Plant Leaves / Q000382:microbiology*; D006109:Poaceae / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D019105:Random Amplified Polymorphic DNA Technique; D012232:Rhizoctonia / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004271:DNA, Fungal