Development of a Rapid Visual Detection Method for Walnut Anthracnose Based on RPA-LFD

Area of Science:

• Plant Pathology
• Molecular Diagnostics
• Mycology

Background:

• Walnut anthracnose, caused by Colletotrichum fungi, is difficult to detect accurately using current single-pathogen assays.
• Complex Colletotrichum populations in the field often lead to false negatives, hindering effective disease management.

Purpose of the Study:

• To develop a rapid, broad-spectrum detection method for Colletotrichum species responsible for walnut anthracnose.
• To overcome the limitations of existing assays in identifying diverse fungal populations.

Main Methods:

• Development of a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay.
• Targeting the conserved internal transcribed spacer (ITS) region with specifically designed primers and probes.
• Systematic screening to select the optimal primer and probe set for specificity and sensitivity.

Main Results:

• The optimized RPA-LFD assay detected multiple Colletotrichum species within 10 minutes at 39°C.
• The assay demonstrated high specificity, with no cross-reactivity with other walnut-associated fungi.
• Sensitivity thresholds varied by species, detecting some at 10 pg/μL and others at 100 pg/μL.

Conclusions:

• The RPA-LFD system offers a simple, sensitive, and specific method for detecting Colletotrichum species.
• Visual result interpretation and rapid turnaround time make it suitable for field applications.
• This technology has significant potential for improving walnut anthracnose management strategies.