Rapid and Accurate Detection of the Causal Agent of Chestnut Rot, through an Internally Controlled Multiplex PCR Assay.

The fungus is a significant threat to the production of sweet chestnut () nuts in Australia and worldwide. The pathogen causes nut rot, which leads to substantial production losses. Early and accurate diagnosis of the disease is essential to delineate and implement control strategies. A specific and sensitive multiplex PCR was developed based on the amplification of three barcode sequences of . The assay reliability was enhanced by including the amplification of a host gene as an internal control. Primers were thoroughly evaluated in silico before assessing them in vitro. Primer annealing temperature and concentration were optimised to enhance the assay sensitivity and specificity. The assay detection limit ranged between 0.1 and 1.0 pg (5 and 50 fg/μL) of genomic DNA per reaction. No cross-reactivity was observed with genomic DNA from closely and distantly related fungal species. We also characterised Australian isolates phenotypically and genotypically and found significant differences in morphologic and virulence traits of the isolates. An understanding of the virulence of and the availability of a reliable and accurate diagnostic technique will enable earlier detection of the pathogen, which will contribute to effective control strategies for the disease.