Development of a Rapid Molecular Detection Marker for Colletotrichum species with AFLP

Sweet persimmons have been increasingly cultivated in the southern part of Korea. However, anthracnose disease caused by Colletotrichum species is one of the major hindrances in cultivation and productions. In this study, we used polymerase chain reaction(PCR) to detect Colletotrichum species with the AFLP(amplified fragment length polymorphism) method. In AFLP, we used E3(5'-GACTGCGTACCAATTCTA-3') and M1(5'-GATGAGTCCTGAGTAACAG-3') primer combination and, as a result, 262 bp segment was observed in Colletotrichum species only. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless sweet persimmon plants. Based on sequence data for specific segments, Co.B1(5'-GAGAGAGTAGAATTGCGCTG-3') and Co.B2(5'-CTACCATTCTTCTA GGTGGG-3') were designed to detect Colletotrichum species. The 220 bp segment was observed in Colletotrichum species only, but not in other fungal and bacterial isolates.

Identification of DNA Markers Linked to Metalaxyl Insensitivity Loci in Phytophthora infestans

A total of 24 isolates of Phytophthora infestans were tested and analyzed for their resistance to metalaxyl fungicides. Sensitivity to metalaxyl was determined by growing isolates on 20% V8 medium amended with 0, 5, and 100 microg/ml metalaxyl. Four isolates among the 24 tested were resistant to metalaxyl. Eleven isolates were intermediate and nine isolates were sensitive. Amplified fragment length polymorphism (AFLP) assay was used to identify the amplification products of resistant isolates. As a result, selected fragments were cloned, sequences and primer pairs were developed which linked to metalaxyl insensitivity in P. infestans using competitive PCR.