Development and Validation of a Fast PCR-Based Detection Method for Pathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa

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.

[1]  T. Jung,et al.  Detection and quantification of Phytophthora species which are associated with root‐rot diseases in European deciduous forests by species‐specific polymerase chain reaction , 1999 .

[2]  A. Snowdon.  A colour atlas of post-harvest diseases and disorders of fruits and vegetables. Volume 1: General introduction and fruits. , 1990 .

[3]  T. Kiely.  Preliminary studies on Guignardia citricarpa, n.sp.: the ascigerous stage of Phoma citricarpa McAlp. and its relation to black spot of Citrus. , 1948 .

[4]  L. W. Timmer,et al.  The Role of Colletotrichum spp. in Postharvest Anthracnose of Citrus and Survival of C. acutatum on Fruit. , 1998, Plant disease.

[5]  W. Maccheroni,et al.  Nonpathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa, Identified as a Cosmopolitan Endophyte of Woody Plants, G. mangiferae (Phyllosticta capitalensis). , 2002, Phytopathology.

[6]  F. Niepold,et al.  Application of the one-tube PCR technique in combination with a fast DNA extraction procedure for detecting Phytophthora infestans in infected potato tubers , 1997 .

[7]  Jim Duncan,et al.  Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction , 1997, European Journal of Plant Pathology.

[8]  B. C. Sutton,et al.  Studies in Phyllosticta I , 1974 .

[9]  J. Duncan,et al.  The use of nested PCR in the polymerase chain reaction for the detection of Phytophthora fragariae and P. cactorum in strawberry , 1997 .

[10]  J. Ristaino,et al.  PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora , 1998, Applied and Environmental Microbiology.

[11]  C. Rabie,et al.  Light and temperature effects on symptom development and sporulation of Guignardia citricarpa Kiely, on Citrus sinensis (Linn) Osbeck. , 1970 .

[12]  D. Jobes,et al.  Plant DNA isolation: a method to efficiently remove polyphenolics, polysaccharides, and RNA , 1995 .

[13]  J. Stalpers,et al.  CBS course of mycology. , 1975 .

[14]  P. R. Scott,et al.  Quarantine pests for Europe. Second Edition. Data sheets on quarantine pests for the European Union and for the European and Mediterranean Plant Protection Organization. , 1997 .

[15]  E. A. Henchal,et al.  Development of internal controls for probe-based nucleic acid diagnostic assays. , 1999, Analytical biochemistry.

[16]  S. Sreenivasaprasad,et al.  Molecular characterization of slow-growing orange and key lime anthracnose strains of Colletotrichum from citrus as C. acutatum. , 1996 .

[17]  H. Schüepp.  Untersuchungen über Guignardia citricarpa Kiely, den Erreger der Schwarzfleckenkrankheit auf Citrus , 1960 .

[18]  R. Chiu.  Studies on black spot of citrus. , 1955 .

[19]  X. Li,et al.  Attenuation of PCR inhibition in the presence of plant compounds by addition of BLOTTO , 1995, Nucleic Acids Res..

[20]  P. Scott,et al.  Quarantine Pests for Europe , 1996 .

[21]  J. Böhm,et al.  Real‐time Quantitative PCR: DNA Determination in Isolated Spores of the Mycorrhizal Fungus Glomus mosseae and Monitoring of Phytophthora infestans and Phytophthora citricola in their Respective Host Plants , 1999 .