Validation of Quantitative and Digital Polymerase Chain Reaction Assays Targeting the Mating Types of Phyllosticta citricarpa, the Causal Agent of Citrus Black Spot

Abstract

Citrus black spot (CBS) is a disease caused by the ascomycetous fungus Phyllosticta (formerly Guignardia) citricarpa (Botryosphaeriales, Pezizomycotina) currently present in citrus groves in five counties in southwest Florida. Within Florida, P. citricarpa shows limited reproduction via asexual sporulation due to the presence of only one (MAT1-2-1) of the two required mating types for sexual reproduction. Here, we present two novel PCR assays standardized in quantitative PCR (qPCR) and digital PCR (dPCR) platforms to distinguish both mating types (MAT1-1-1 and MAT1-2-1) of P. citricarpa to monitor for the potential introduction of the MAT1-1-1 mating type into Florida and a novel protocol for DNA extraction from asymptomatic leaves. During citrus harvesting season 2018 − 19, fruit lesions as well as asymptomatic leaves adjacent to symptomatic fruit and asymptomatic trees in CBS-infected groves were surveyed for P. citricarpa presence and mating types. Results support the presence of only the MAT1-2-1 mating type in Florida, after surveying more than 1,145 citrus fruit lesions. We also confirmed the limited dispersal ability of the asexual state of P. citricarpa in Florida in 10 groves using the enhanced capabilities of the dPCR platform in the detection of P. citricarpa directly from asymptomatic leaves with low pathogen inoculum. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .