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DNA-based detection of grapevine trunk-disease pathogens from environmental spore samples
Phillip T Fujiyoshi1, Daniel P Lawrence2, Renaud Travadon2
1United States Department of Agriculture-Agricultural Research Service, Crops Pathology and Genetics Research Unit, Davis, CA 95616, U.S.A.
Methodsx
|November 10, 2021
Summary
New DNA-based methods improve detection of grapevine trunk disease pathogens. Species-specific PCR primers accurately identify Neofusicoccum parvum and Eutypa lata, enabling better timing of vineyard disease prevention strategies.
Area of Science:
- Plant Pathology
- Molecular Biology
- Agricultural Science
Background:
- Grapevine trunk diseases, including Botryosphaeria dieback and Eutypa dieback, are significant threats to vineyards.
- Spore dispersal of causal fungi like Neofusicoccum parvum and Eutypa lata occurs during winter rains, infecting vines through pruning wounds.
- Effective management relies on timely preventative practices, necessitating accurate and rapid pathogen detection.
Purpose of the Study:
- To develop and validate species-specific PCR primers and protocols for rapid detection of N. parvum and E. lata.
- To compare the efficacy of a new DNA-based detection method against traditional culture-based methods for vineyard spore samples.
- To optimize detection protocols for different storage conditions and sample types.
Main Methods:
- Development and extensive testing of species-specific PCR primers for N. parvum and E. lata against related and unrelated fungi.
- Comparison of PCR-based detection with traditional culture methods using environmental spore samples.
- Evaluation of sample storage conditions (fresh vs. frozen at -20°C) and implementation of a novel spore-lysis method for E. lata.
Main Results:
- Species-specific PCR primers for N. parvum and E. lata demonstrated high accuracy, distinguishing them from other vineyard fungi.
- N. parvum spores were consistently detected by PCR whether fresh or frozen, and also by culture.
- E. lata spores were only consistently detected by PCR, requiring a new spore-lysis method and fresh samples; freezing rendered detection inconsistent.
Conclusions:
- Species-specific PCR offers a faster and more reliable method for detecting N. parvum and E. lata spores compared to traditional culturing.
- The developed DNA-based method, particularly with the new lysis protocol for E. lata, enhances pathogen monitoring in vineyards.
- Improved pathogen detection facilitates better timing of preventative strategies against grapevine trunk diseases, potentially reducing crop loss.
