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Detection of Fusarium oxysporum f. sp. fragariae by Using Loop-Mediated Isothermal Amplification
Hiroshi Katoh1, Shuichiro Yamazaki2, Takashi Fukuda2
1Faculty of Agriculture, Takasaki University of Health and Welfare, Takasaki, Gunma 370-0033, Japan.
Abstract:
We developed a loop-mediated isothermal amplification (LAMP) assay for detecting Fusarium oxysporum f. sp. fragariae, the causal agent of wilt in strawberry plants. This assay was based on genomic regions between the portions of transposable elements Han and Skippy of the fungus. The LAMP assay allowed the efficient detection of F. oxysporum f. sp. fragariae DNA by visual inspection, without requiring gel electrophoresis. The detection limit was 100 pg of genomic DNA, which is comparable to that of PCR. The LAMP primers successfully discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and other fungi. The LAMP assay at 63°C, which was found to be the optimal treatment temperature, for 1.5 h successfully detected F. oxysporum f. sp. fragariae California strains GL1270 and GL1385. When the assay was performed using a Genelyzer FIII portable fluorometer, these California strains were successfully detected in 1 h. The assay facilitated the detection of conidia in soil samples after they were precultured on a selective medium for F. oxysporum (FoG2) as well as latent infection in strawberry plants after preculturing. The LAMP assay for visual inspection of DNA required only a heating block and an incubator, reducing the cost of this assay. Thus, it could be suitable for the detection of F. oxysporum f. sp. fragariae strains in centers that store prefoundation and foundation stocks of strawberry, including plant nurseries.
Insights
A new loop-mediated isothermal amplification (LAMP) assay efficiently detects Fusarium oxysporum f. sp. fragariae, the cause of strawberry wilt. This cost-effective method visually identifies the pathogen in plants and soil, aiding nursery disease management.
Area of Science:
- Plant Pathology
- Molecular Biology
- Mycology
Background:
- Fusarium oxysporum f. sp. fragariae causes significant wilt disease in strawberry plants.
- Accurate and rapid detection of this pathogen is crucial for disease management and preventing crop loss.
Purpose of the Study:
- To develop a novel loop-mediated isothermal amplification (LAMP) assay for the specific and sensitive detection of Fusarium oxysporum f. sp. fragariae.
- To evaluate the assay's efficiency, detection limit, and specificity compared to existing methods.
- To assess the applicability of the LAMP assay for detecting the pathogen in environmental and plant samples.
Main Methods:
- Development of LAMP primers targeting specific genomic regions (transposable elements Han and Skippy) of Fusarium oxysporum f. sp. fragariae.
- Optimization of LAMP reaction conditions, including temperature (63°C) and time (1.5 hours).
- Validation of the assay using genomic DNA, pure fungal cultures, soil samples, and infected strawberry plants, with visual inspection and fluorometer detection.
Main Results:
- The developed LAMP assay detected Fusarium oxysporum f. sp. fragariae DNA with a detection limit of 100 pg, comparable to PCR.
- The assay demonstrated high specificity, distinguishing pathogenic strains from nonpathogenic Fusarium oxysporum and other fungi.
- Successful detection of the pathogen in soil samples and latent infections in strawberry plants after appropriate pre-culturing.
Conclusions:
- The visual LAMP assay provides a rapid, cost-effective, and sensitive method for detecting Fusarium oxysporum f. sp. fragariae.
- Its simplicity and minimal equipment requirements make it suitable for use in plant nurseries and diagnostic centers.
- This assay can aid in early disease detection and management strategies for strawberry wilt.
