Duplex Real-Time PCR Assays for the Simultaneous Detection and Quantification of Botryosphaeriaceae Species Causing Canker Diseases in Woody Crops

Abstract

Woody canker diseases caused by fungi of the Botryosphaeriaceae family are producing increasing losses in many economically important woody crops, including almond. To develop a molecular tool for the detection and quantification of the most aggressive and threatening species is of main importance. This will help to prevent the introduction of these pathogens in new orchards and to conveniently apply the appropriate control measures. Three reliable, sensitive and specific duplex qPCR assays using TaqMan probes have been designed for the detection and quantification of (a) Neofusicoccum parvum and the Neofusicoccum genus, (b) N. parvum and the Botryosphaeriaceae family and (c) Botryosphaeria dothidea and the Botryosphaeriaceae family. The multiplex qPCR protocols have been validated on artificially and naturally infected plants. Direct systems to process plant materials, without DNA purification, allowed high-throughput detection of Botryosphaeriaceae targets even in asymptomatic tissues. These results validate the qPCR using the direct sample preparation method as a valuable tool for Botryosphaeria dieback diagnosis allowing a large-scale analysis and the preventive detection of latent infection.

Current status of Botryosphaeriaceae species in Italy: Impacts on agricultural crops and forest ecosystems

“…These traditional methods are usually time-consuming compared to molecular diagnostic methods. Real-time PCR has been demonstrated to be an important tool for detecting latent infections and to investigate canker pathogen epidemiology (Luo et al, 2017(Luo et al, , 2019(Luo et al, , 2020Romero-Cuadrado et al, 2023). Once Botryosphaeriaceae become established in an orchard, transmission of inoculum (mycelium, pycnidia or perithecia overwintering in old cankers, fruit mummies or within the buds) (Michailides, 1991) can occur through human activities (e.g., pruning, irrigation), and animals such as birds and insects (Michailides and Morgan, 2016), and environmental factors such as rain and wind.…”