Development and validation of a fast PCR-based detection method for pathogenic isolates of the citrus black spot fungus, Guignardia citricarpa

Abstract

Based on the ITS regions of the ribosomal DNA, specific primer sets were developed for the citrus pathogen Guignardia citricarpa and the common citrus endophyte, G. mangiferae, and tested for their specificity against 37 isolates of G. citricarpa, 29 isolates of G. mangiferae, 10 isolates of related species and other fungi found on citrus. The efficacy of the PCR-detection method for G. citricarpa was approximately 60-70% for lesions without pycnidia, and approximately 90% for lesions with pycnidia. A reliability of 99% can be reached by analysing multiple lesions per sample. An internal control was developed to monitor DNA samples for PCR inhibition; samples with PCR inhibition should be re-examined. Detection by PCR is more rapid than the current five-day incubation method prescribed by the European Union for diagnosis of black spot lesions lacking the diagnostic pycnidia. The latter method had an efficacy of 40-50%, while culturing of suspected lesions had an efficacy of 10%. Species-specific primers and ITS sequence data showed that G. citricarpa can occur as a symptomless endophyte in leaves. This shows that wild and cultivated plants occurring in citrus groves are potential carriers of this quarantine fungus. Application of the presently developed PCR method for the detection of G. citricarpa will enable citrus producing as well as importing countries to prevent further spread of this harmful organism.