RFLP identification of Colletotrichum species isolated from chilli in Thailand
R. Pongpisutta, W. Winyarat, C. Rattanakreetakul
Acta Horticulturae 973: 181-186: 2013.
2013
บทคัดย่อ
Fifty eight isolates of Colletotrichum species were obtained from anthracnose diseased chilli fruits from different plantations at Chiang Rai, Nakhon Si Thammarat, and Sukhothai. The morphological features studied were colony morphology, conidial sizes, and shapes. All isolates were classified into three species as C. acutatum (Ca), C. capsici (Cc), and C. gloesporioides (Cg). Based on colony characteristics, each species was grouped into 5, 8, and 7 phenotypes, respectively. However, the characteristics of Colletotrichum species showed large variation in morphology, so a molecular marker technique was required to distinguish between them. PCR analysis of the internal transcribed spacer region of the ribosomal DNA (ITS1-5.8S-ITS2) was used for identification with universal primers ITS4/ITS5. PCR products of approximately 600 bp were obtained. Three restriction enzymes, AluI, BamHI, and RsaI were used to study polymorphisms in the ITS regions of the isolates. The digested ITS banding pattern of Ca showed one band at 500 bp when digested with BamHI, while AluI and RsaI failed to digest the amplified products. BamHI and RsaI did not cut the DNA fragments of Cc, whereas AluI digested into two bands of 200 and 400 bp. The amplified product of Cg, when digested with AluI, resulted in two bands of 200 and 380 bp, when digested with BamHI resulted in one band of 500 bp, and when digested with RsaI, generated two bands of 200 and 380 bp. Consequently, AluI and RsaI were considered to be useful to distinguish between species of Ca and Cg. Nucleotide sequencing of ITS products was examined and identified the same three species as the morphological identification. This method is proposed for specific identification of the chilli anthracnose causal agents.