AN INTEGRATED STUDY OF MORPHOLOGICAL AND MOLECULAR PATTERNS OF PHYTOPHTHORA SPECIES CAUSING BLACKPOD DISEASE OF COCOA IN INDIA
P CHOWDAPPA and R CHANDRAMOHANAN
Central Plantation Crops Research Institute Regional Station, Vittal - 574 243, India
Background and Objectives
Black pod is a serious disease of cocoa in India. Recent preliminary studies indicated the occurrence of P. capsici and P. citrophthora on cocoa in India besides P. palmivora . The objective of the present study was to characterize the Phytophthora isolates causing black pod disease of cocoa based on morphological and molecular criteria and to find out their virulence on different cocoa accessions.
Materials and methods
One hundred and thirty Phytophthora isolates recovered from major cocoa growing areas of India were examined. Colony morphology was assessed on 5-day-old carrot agar (CA) plates grown at 24+/-1°C in the dark. Sporangia were produced by the solid-agar-plate and agar-disc-in water methods . Dimensions of 100 sporangia mounted in distilled water were measured and the mean values for L/B ratio and pedicle lengths were calculated. Observations on the formation of chlamydospores were made following incubation at 18°C. The mating types were determined by placing 5 mm diatn discs from advancing margins of 3-day-old cultures of opposite mating types at 50 mm apart on CA. Mean sizes of sexual organs were determined from measurements of 100 mature structures. Temperature/growth relationships were studied at temperatures ranging from 6 to 36°C at 30°C intervals. The total DNA isolated from vegetative mycelium was digested with hya restriction enzymes such as Sal I and Hind RI. The resulting DNA fragments were size fractionated on agarose gels. Electrophoresis of native protein preparations was carried out as described previously . Pathogenicity was evaluated on nearly mature, but unripe detached pods of 20 cocoa accessions following wound inoculation method.
Results and conclusions
Of the 130 Phytophthora isolates, 120 were identified as P. paimivora, five as P. capsici and five as P. citrophthora based on morphological criteria. Colonies of P. palmivora were smooth combed where as isolates of P. capsici displayed petaloid pattern of growth . In contrast, P. citrophthora isolates did not show any particular patterns of growth. Sporangia of P. palmivora were ovoid to ellipsoidal in shape and were shed with a short, broad and occluded pedicels, of less than 4 mm in length. Sporangia of P.capsici were ellipsoidal with tapered / round base and carried characteristically long and thin stalks ranging from 71 to 82 mm. Sporangia of P. citrophthora were non-caducous and highly variable in shape. The mean L/B ratio of sporangia of P. palmivora was 1.47 while L/B ratio of P.apsici and P. citrophthora were 1.84 and 1.76, respectively. P. palmivora sporangia were formed on a sympodium while the formation of sporangia in P. capsici were in umbellate pattern. The formation of in sporangia in P. citrophthora were not in definite pattern. P. palmivora isolates produced chlamydospores whereas P. capsici and P. citrophthora isolates did not produce chlamydospores. A1 and A2 mating types occurred in the ratio of 2:9 and 4:1 in P. palmivora and P.capsici respectively. P.citrophthora isolates were found to be sterile. There were no distinct variation between P. palmivora and P. capsici in shape and size of sex organs. Minimum, opthnum and maxnnum temperature for the three species of Phytophthora were 11-12, 24-30 and 32-33°C. The identification of P. palmivora, P. capsici and P. citrophthora based on these morphological criteria was justified by the fact that these three species have characteristic electrophoretic protein profiles and restriction digestion patterns of total DNA digested with Sal I and Hin dIII. P. palmivora is highly virulent followed by P. citrophthora and P. capsici .
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