Related Experiment Video
Updated: Aug 2, 2025
A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci
Published on: October 29, 2018
A Loop-Mediated Isothermal Amplification Assay for the Identification of Botrytis fragariae in Strawberry
Alejandra Vielba-Fernández1,2, Madeline Dowling3, Guido Schnabel3
1Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain.
Abstract:
Gray mold in strawberry is caused by multiple species of Botrytis, including Botrytis cinerea, B. pseudocinerea, B. fragariae, and B. mali. The species B. cinerea and B. fragariae are widespread in production regions of the eastern United States and Germany, and their distinction is important for disease management strategies. Currently, the only way to differentiate these species in field samples is by PCR, which is time consuming, labor intensive, and costly. In this study, a loop-mediated isothermal amplification (LAMP) technique was developed based on species-specific NEP2 gene nucleotide sequences. The designed primer set specifically amplified B. fragariae DNA and no other Botrytis spp. (B. cinerea, B. mali, and B. pseudocinerea) or plant pathogens. The LAMP assay was able to amplify fragments from DNA extracted from infected fruit using a rapid DNA extraction protocol, confirming its ability to detect low amounts of B. fragaria DNA from field-infected fruit. In addition, a blind test was performed to identify B. fragariae in 51 samples collected from strawberry fields in the eastern United States using the LAMP technique. The B. fragariae samples were identified with a reliability of 93.5% (29 of 32), and none of the B. cinerea, B. pseudocinerea, or B. mali samples included in the test were amplified in 10 min. Our results show that the LAMP technique is a specific and reliable method for the detection of B. fragariae from infected fruit tissue and can help to control this important disease in the field.
Insights
A new loop-mediated isothermal amplification (LAMP) technique accurately detects Botrytis fragariae, a cause of strawberry gray mold. This rapid method offers a cost-effective alternative to PCR for disease management.
Area of Science:
- Plant Pathology
- Molecular Diagnostics
- Agricultural Microbiology
Background:
- Gray mold in strawberries is caused by multiple Botrytis species, including B. cinerea and B. fragariae.
- Distinguishing between B. cinerea and B. fragariae is crucial for effective disease management in strawberry production.
- Current differentiation methods like PCR are time-consuming, labor-intensive, and expensive for field diagnostics.
Purpose of the Study:
- To develop a rapid, specific, and reliable diagnostic method for identifying B. fragariae in strawberry fruit.
- To establish a loop-mediated isothermal amplification (LAMP) assay for detecting B. fragariae based on NEP2 gene sequences.
Main Methods:
- Development of a species-specific primer set targeting the NEP2 gene of B. fragariae.
- Application of the loop-mediated isothermal amplification (LAMP) technique for DNA amplification.
- Validation of the LAMP assay using DNA extracted from infected strawberry fruit and a blind test on field samples.
Main Results:
- The developed LAMP assay specifically amplified B. fragariae DNA, showing no cross-reactivity with other Botrytis species or plant pathogens.
- The assay successfully detected low amounts of B. fragariae DNA from infected fruit using a rapid DNA extraction protocol.
- In a blind test of 51 field samples, the LAMP technique identified B. fragariae with 93.5% reliability (29/32), with no false positives from other species within 10 minutes.
Conclusions:
- The loop-mediated isothermal amplification (LAMP) technique provides a specific, reliable, and rapid method for detecting B. fragariae in infected strawberry tissues.
- This LAMP assay can aid in timely and accurate disease management strategies for gray mold in strawberry cultivation.
- The method offers a cost-effective and efficient alternative to traditional PCR for field-based diagnostics of B. fragariae.
