Development of a Multiplex PCR Assay to Detect Neofusicoccum parvum and Botryosphaeria dothidea in Walnut

In walnut orchards, frequent symptoms of cankers and dieback (fruit blight, twig and branch cankers up to tree death) are caused by different agents, in particular by Botryosphaeriaceae, primarily Neofusicoccum parvum and Botryosphaeria dothidea. This study aimed at developing a sensitive, rapid, specific and internally controlled multiplex PCR assay for the detection of these species. The ability of the multiplex PCR, with an internal inhibition control (i.e. E. coli DNA), to specifically and successfully detect members of the two targeted species was validated using 11 different isolates for each fungal target (inclusivity) and 20 tree-associated fungal species different from B. dothidea or N. parvum (exclusivity). Applicability to plant materials was further investigated and showed an absence of amplification for asymptomatic husk or twig samples while the amplification profiles of symptomatic tissues ranged from no amplification to amplification of both species, in correlation with the observed fungal contamination level. In conclusion, we developed a rapid diagnostic tool for simultaneous detection of two major fungal pathogens of walnut. Although this protocol was tailored for walnut husks and twigs, applicability to any plant sample contaminated by these pathogens can be considered.