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Identification and Differentiation of Monilinia Species Causing Brown Rot of Pome and Stone Fruit using
Antonios Papavasileiou1, Panagiotis B Madesis1, George S Karaoglanidis1
1First and third authors: Plant Pathology Laboratory, Faculty of Agriculture, Forestry and National Resources, Thessaloniki, Aristotle University of Thessaloniki, Greece; second author: Institute of Applied Biotechnology, CERTH, Thermi, Thessaloniki, Greece.
Abstract:
Brown rot is a devastating disease of stone fruit caused by Monilinia spp. Among these species, Monilinia fructicola is a quarantine pathogen in Europe but has recently been detected in several European countries. Identification of brown rot agents relies on morphological differences or use of molecular methods requiring fungal isolation. The current study was initiated to develop and validate a high-resolution melting (HRM) method for the identification of the Monilinia spp. and for the detection of M. fructicola among other brown rot pathogens. Based on the sequence of the cytb intron from M. laxa, M. fructicola, M. fructigena, M. mumecola, M. linhartiana, and M. yunnanensis isolates originating from several countries, a pair of universal primers for species identification and a pair of primers specific to M. fructicola were designed. The specificity of the primers was verified to ensure against cross-reaction with other fungal species. The melting curve analysis using the universal primers generated six different HRM curve profiles, each one specific for each species. Τhe HRM analysis primers specific to M. fructicola amplified a 120-bp region with a distinct melt profile corresponding to the presence of M. fructicola, regardless of the presence of other species. HRM analysis can be a useful tool for rapid identification and differentiation of the six Monilinia spp. using a single primer pair. This novel assay has the potential for simultaneous identification and differentiation of the closely related Monilinia spp. as well as for the differentiation of M. fructicola from other common pathogens or saprophytes that may occur on the diseased fruit.
Insights
A new high-resolution melting (HRM) method rapidly identifies brown rot pathogens (Monilinia spp.) in stone fruit. This technique accurately detects the quarantine pathogen Monilinia fructicola, crucial for disease management.
Area of Science:
- Plant Pathology
- Molecular Biology
- Mycology
Background:
- Brown rot, caused by Monilinia spp., is a significant threat to stone fruit production.
- Monilinia fructicola is a quarantine pathogen in Europe, necessitating accurate detection methods.
- Current identification methods rely on morphology or molecular techniques requiring fungal isolation.
Purpose of the Study:
- To develop and validate a high-resolution melting (HRM) method for identifying Monilinia species.
- To specifically detect the quarantine pathogen Monilinia fructicola among other brown rot agents.
Main Methods:
- Designed universal primers targeting the cytb intron for Monilinia species identification.
- Designed M. fructicola-specific primers for targeted detection.
- Verified primer specificity against cross-reactions with other fungal species.
- Utilized HRM analysis for melting curve profiling.
Main Results:
- Universal primers generated six distinct HRM curve profiles, each specific to a Monilinia species.
- M. fructicola-specific primers amplified a 120-bp region with a unique melt profile, confirming its presence.
- The HRM assay demonstrated high specificity and accuracy in differentiating Monilinia species.
Conclusions:
- HRM analysis is a rapid and effective tool for identifying and differentiating six Monilinia species.
- This novel assay enables simultaneous identification and differentiation of closely related Monilinia species.
- The method facilitates the differentiation of M. fructicola from other common pathogens and saprophytes on diseased fruit.
