Abstract:
Strict quarantine measures for the export of South African citrus fruit to European and
US markets require the development of sensitive and accurate detection methods for the
pathogen Phyllosticta citricarpa – a fungus causing citrus black spot disease. Because of the
presence of other, non-pathogenic Phyllosticta species, rapid and accurate verification of the
Phyllosticta species present on exported citrus fruit is important to producers, exporters and
regulatory authorities to prevent unnecessary losses. We have analysed over 800 samples
collected over 7 years and have compared sample preparation and detection protocols applied
in different environments: nurseries, production systems including phytosanitary inspections
in orchards, pack houses and export terminals in order to compile protocols for the detection
of P. citricarpa. Standard procedures of sample preparation and DNA extraction were adapted
to suit diverse inoculum sources. Low pathogen numbers in symptomless green leaves, for
example, obliged the use of a wet-dry enrichment technique constituting the stimulation
of fungal growth for easier detection. Physical maceration was adapted for sturdy material
using liquid nitrogen or bead beating. The use of a two-step polymerase chain reaction (PCR)
with nested primers significantly increased both the sensitivity and the specificity of the PCR
performed on soil samples, overcoming problems with relatively impure DNA extracts and
low pathogen numbers. The assays have proven to be highly consistent, thereby providing a
reliable, reproducible and highly sensitive detection and diagnostic service to the southern
African citrus industries in order to sustain market access.
