DNA POLYMORPHISM AND VIRULENCE VARIATION OF A PHYTOPHTHORA POPULATION ISOLATED FROM CASSAVA
International Center for Tropical Agriculture, AA 6713, Cali, Colombia
Background and objectives
Cassava (Manihot esculenta) has great social importance as the principal carbohydrate source in developing countries. A major limiting factor in cassava-producing areas in Latin America is root rot, a destructive and widespread disease. However, a major constraint in cassava is the lack of understanding of the root rot-causing pathogen. In addition to breeding for resistance to root rot, it is essential to obtain information on the variability of virulence in Phytophthora.
Materials and methods
In this study, 193 samples were obtained from cassava-infected tissue and rhizosphere. The 122 Phytophthora isolates were recovered by a direct-plating method and by a baiting technique using in vitro cassava propagated fragments as a bait. The 122 isolates were characterized by morphology, pathogenicity, PCR-RAPD analysis and DNA hybridization using a Phytophthora-specific probe . Reference isolates of different Phytophthora species were also included in the molecular analysis. Virulence variation was determined on 10 cassava genotypes by inoculating 30 selected isolates from different Colombian regions into cassava sprouts by the wound-inoculation method.
Results and conclusions
The majority of isolates were not clearly differentiated by colony type or growth on PDA modified with antibiotics. Identification of the isolates by morphology was difficult because production of fungal structures was not possible with all isolates.
Amplification of the internal transcribed spacer (ITS) region of the rDNA was obtained with template DNA from 12 isolates using extracted DNA. The amplified product for the ITS region of all species was 900 bp. Restriction digestion with AluI, MspI and HindI of the product amplified for the ITS region showed different restriction patterns, which corresponded to the species tested. Five random 10-mer primers (Operon Technologies) gave reproducible bands. Polymorphisms with these single primers differentiated the species.
The Colombian Phytophthora population showed a high degree of diversity and the isolates were grouped into 12 clusters. By DNA hybridization, all the isolates were detected by the probe. This method was more sensitive than ELISA for detection of Phytophthora. Although symptom severity differed among isolates, no correlation between virulence variation and DNA polymorphism was observed.
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