Detection of Colletotrichum coccodes by Real-Time PCR

Abstract

Colletotrichum coccodes causes dangerous potato and tomato diseases, known as anthracnose and black spot. Morphologically, they are often difficult to distinguish from diseases caused by other microorganisms. On green tomato fruits, the disease may be asymptomatic; it appears only on ripe red fruits. For quick and accurate diagnosis and identification of the pathogen, a test system for real-time PCR is proposed. To develop a test system, the nucleotide sequence of the glycerol-3-phosphate dehydrogenase gene was determined in 45 different strains of C. coccodes. Based on the results obtained and analysis of similar sequences of other species available in the GenBank Database, C. coccodes species-specific primers and a probe were designed. To test the specificity of the created test system, PCR was done with DNA isolated from pure cultures of 15 different species of parasitic and saprotrophic fungi associated with tomato and potato plants (Fusarium oxysporum, Fusarium verticillium, Phomopsis phaseoli, Alternaria alternatа, Helminthosporium solani, Colletotrichum coccodes, Phellinus ferrugineovelutinus, Stemphylium vesicarium, Helminthosporium solani, Phomopsis phaseoli, Neonectria radicicola, Rhizoctonia solani, Penicillium sp., Cladosporium fulvum, and Cladosporium cladosporioides). The presence of Colletotrichum coccodes DNA was determined at a 20‒27 threshold cycle. The remaining types of fungi were determined after 40 cycles or were not detected at all. The test system makes it possible to confidently detect in the analyzed PCR-mixture concentrations of C. coccodes exceeding 0.01 ng/mm³. The test system was used to study the presence of C. coccodes in tomato leaves with symptoms of fungal diseases and potato tubers without external symptoms of disease. Leaves with fungal lesion symptoms were collected from two different fields in Krasnodar krai; tubers, from the fields in Kostroma, Moscow, Kaluga, and Nizhny Novgorod oblasts. A single tomato leaf containing C. coccodes DNA was found, while the presence of C. coccodes DNA tubers grown in Kostroma, Moscow, and Kaluga oblasts was detected in five samples.