Determine fungicide efficacy using qRT-PCR for detection and quantification of Botrytis cinerea inoculum and viability on pear leaves as a substrate, before and after fungicide application

To validate efficacy of fungicides and best application methods, it is important to determine fungal growth after treatment. qPCR has made it possible to accurately and sensitively quantify in planta fungal levels of selected pathogens. The study aims to establish a qPCR fungicide efficacy testing protocol and determine the efficacy of fungicides against Botrytis cinerea.

Preliminary trials were conducted to test the interference of fungicides on DNA extraction and identify the most ideal tissue for DNA extraction. Primer/probe sets for B. cinerea and Pyrus communis were identified, designed and optimised for multiplex qPCR. A preliminary time trial generated qPCR data on

1. cinerea growth. Leaves for qPCR fungicide efficacy trials were harvested, inoculated (1 x 10⁵ conidia/ml), treated (fluopyram and pyrimethanil) and incubated for different time periods prior to qPCR analysis.

The presence of fungicides did not interfere with the DNA extraction method. The B. cinerea and P. communis primer/probe sets worked well and were successfully optimised for duplex qPCR. Although DNA of B. cinerea increased for longer incubated samples, the incubation periods weren’t significantly different. Samples prepared for the molecular fungicide efficacy trial plated onto potato dextrose agar (PDA) revealed little differences. Samples have been submitted for duplex qPCR analysis.

A duplex qPCR protocol was established to test fungicide efficacy and quantify B. cinerea from P. communis. The qPCR method was effective and highly sensitive to detect the presence of B. cinerea from non-symptomatic leaves. Samples for the fungicide efficacy trial have been submitted for qPCR.

B. cinerea and P. communis primer/probe sets are available to accurately and sensitively identify B. cinerea in/on P. communis tissue or other surfaces for preharvest and postharvest purposes.