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On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System
Published on: February 23, 2018
Rapid Detection of a Downy Mildew Pathogen, Peronospora destructor, in Infected Onion Tissues and Soils by
In Seong Lee1, Wonyong Kim1, Gyeongpyo Jo1
1Department of Applied Biology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, South Korea.
Abstract:
Downy mildew of onion caused by a soil-inhabiting water mold, Peronospora destructor, is one of the most devastating diseases that can destroy entire onion fields in a matter of days. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay that allows for rapid detection of P. destructor by visual inspection. The internal transcribed spacer 2 region of P. destructor was used to design primer sets for LAMP reactions. The optimal temperature and incubation time were determined for the most efficient primer set. In the optimized condition, the LAMP assay exhibited at least 100 times more sensitivity than conventional PCR, detecting femtogram levels of P. destructor genomic DNA (gDNA). Detection of the pathogen from a small number of spores without gDNA extraction further confirmed the high sensitivity of the assay. For specificity, the LAMP assay was negative for gDNA of other fungal pathogens that cause various diseases on onion and oomycetes, whereas the assay was positive for gDNA extracted from onion tissues showing the typical downy mildew symptoms. Finally, we examined the efficacy of the LAMP assay in detection of P. destructor in soils. Soils collected from onion fields that had been contaminated with P. destructor were solarized for 60 days. Whereas the LAMP assay was negative for the solarized soils, we were able to detect P. destructor that oversummers in fields. The LAMP assay developed in this study enables rapid detection and diagnosis of downy mildew of onion in infected tissues and in soil.
Insights
A new loop-mediated isothermal amplification (LAMP) assay rapidly detects onion downy mildew pathogen, *Peronospora destructor*. This highly sensitive method aids in early disease diagnosis in plants and soil.
Area of Science:
- Plant Pathology
- Molecular Diagnostics
- Agricultural Science
Background:
- Downy mildew, caused by *Peronospora destructor*, is a destructive onion disease.
- Rapid and accurate detection of *P. destructor* is crucial for disease management.
Purpose of the Study:
- To develop a sensitive and specific loop-mediated isothermal amplification (LAMP) assay for *P. destructor* detection.
- To evaluate the assay's performance in plant tissues and soil samples.
Main Methods:
- Designed LAMP primers targeting the internal transcribed spacer 2 region of *P. destructor*.
- Optimized reaction conditions (temperature, time) for the LAMP assay.
- Assessed sensitivity against conventional PCR and specificity against other pathogens.
- Validated the assay on infected onion tissues and soil samples.
Main Results:
- The LAMP assay demonstrated at least 100-fold higher sensitivity than PCR, detecting femtogram levels of *P. destructor* DNA.
- The assay successfully detected the pathogen from spores without DNA extraction.
- High specificity was confirmed, with no cross-reactivity to other onion pathogens.
- The assay detected *P. destructor* in naturally contaminated soils and differentiated between solarized and non-solarized samples.
Conclusions:
- The developed LAMP assay provides a rapid, sensitive, and specific tool for diagnosing onion downy mildew.
- This assay can be used for early detection in infected plants and for monitoring pathogen presence in soil.
- The visual detection capability of the LAMP assay facilitates its application in field settings.
