Digital PCR application for sensitive detection of grape plant pathogens using crude lysate samples

Authors: LIU, CHENGJING - Thermo Fisher Scientific; WEI, WEI - Thermo Fisher Scientific; JOUN, DAVID - Thermo Fisher Scientific; Burbank, Lindsey

Submitted to: Plant and Animal Genome Conference
Publication Type: Abstract Only
Publication Acceptance Date: 11/25/2023
Publication Date: 1/12/2024
Citation: Liu, C., Wei, W., Joun, D., Burbank, L.P. 2024. Digital PCR application for sensitive detection of grape plant pathogens using crude lysate samples. Plant and Animal Genome Conference.

Interpretive Summary:

Technical Abstract: There is a need for sensitive detection of plant pathogens. Plant diseases caused by plant pathogens cost the global economy $220 Billion annually. Early detection of plant diseases is a crucial factor to prevent or limit the spread of infection that could cause significant economic loss. Traditional methods of detection, such as symptom development or pathogen culturing is time consuming and may be too late to stop crop loss/disease spread. Here a digital PCR (dPCR) application was investigated for early detection of Xylella fastidiosa in crude lysate from grapevines. Xylella fastidiosa (Xf), a pathogenic bacterium affecting 600+ plant species, causes severe crop diseases including Pierce's disease of grapevine. Visible disease symptoms due to Xf can take several months to develop, and the pathogen itself is challenging to culture in a laboratory. Accordingly, molecular detection methods are preferred for disease identification and surveillance. qPCR is commonly used for Xf detection but requires DNA purification due to PCR inhibitors present in plant tissue, and detection can be unreliable at early stages of infection. Crude plant lysate samples from grapevines infected with Xf were tested using QuantStudio™ Absolute Q™ Digital PCR System and compared that with qPCR using purified DNA extracted with a phenol-chloroform extraction protocol. Asymptomatic infection from crude lysates were detected as early as 4 weeks from infection, a stage when qPCR detection may be unreliable due to low pathogen populations in the plant. The Absolute Q™ Digital PCR System could detect Xf from asymptomatic infected grapevines at 4 weeks post infection, using crude lysate samples which eliminates the need for full DNA extraction. The comparative experiment demonstrated a statistically significant similarity between dPCR and qPCR detection and pathogen quantification. A streamlined approach was successfully implemented by setting up the digestion and conducting dPCR on a single plate, simplifying the testing procedure into a one-step process. This testing process is significantly faster than complete DNA extraction and qPCR and will facilitate high throughput testing of plant material. These advances highlight the potential of dPCR in addressing disease detection and monitoring challenges in plant disease research and surveillance efforts.