Molecular methods for the detection and quantification of Pestalotiopsis and Neopestalotiopsis inoculum associated with macadamia

Pestalotiopsis blight and leaf spot in macadamia are caused by species of Pestalotiopsis and Neopestalotiopsis. Conidia produced by these pathogens are mostly dispersed by wind and rain splash and are the primary infecting propagules. Rapid detection and quantification of airborne conidia are essential to determine the seasonal dynamics of inoculum and critical infection periods in macadamia orchards. A quantitative PCR (qPCR) using a new primer pair and a hydrolysis probe was developed to detect and quantify airborne conidia of Pestalotiopsis and Neopestalotiopsis species. The amplification efficiency of the qPCR was 96.4% and the assay had a limit of detection of 400 fg of Pestalotiopsis/Neopestalotiopsis gDNA, which corresponds to approximately 10 conidia. The qPCR assay coupled with spore trapping was used to monitor airborne conidia dispersal under field conditions. The results showed that the assay is specific and sensitive for detecting and quantifying Pestalotiopsis/Neopestalotiopsis conidia in macadamia orchards. The detection and quantification of the pathogen inoculum will improve our understanding of disease epidemiology and the ability to manage these diseases in macadamia.