A One-Day Sensitive Method to Detect and Distinguish Between the Citrus Black Spot Pathogen <i>Guignardia citricarpa</i> and the Endophyte <i>Guignardia mangiferae</i>

Abstract

If South African citrus exporters wish to retain their competitive edge in the European market and access new markets such as the United States of America, it is of quarantine importance to distinguish between the citrus black spot pathogen, Guignardia citricarpa, and the harmless endophyte, G. mangiferae. The endophyte is not a sanitary or phytosanitary concern. This paper describes the design of species-specific primers that are able to detect and distinguish between these two Guignardia species. Application of the primer set CITRIC1 and CAMEL2 in conjunction with the ITS4 primer yielded polymerase chain reaction (PCR) amplicons of approximately 580 bp and 430 bp for G. citricarpa and G. mangiferae, respectively. Results obtained with these primers are in accordance with sequence data, and repeated tests verified accuracy and sensitivity. A BLAST search revealed no matches other than G. citricarpa and G. mangiferae, and no positive PCR results were obtained with Colletotrichum gloeosporioides, which is the most common contaminant in black spot lesions. We are, therefore, able to distinguish G. citricarpa and G. mangiferae unequivocally using a PCR-based method. This method was further improved to directly isolate DNA from fruit lesions by means of the DNeasy Plant Mini Kit (Qiagen). This eliminates the prior need for culturing the slow-growing organism, thereby shortening the time required to one day to test for and verify the presence or absence of the pathogenic G. citricarpa in export consignments.