Real-Time and Rapid Detection of Phytopythium vexans Using Loop-Mediated Isothermal Amplification Assay

Area of Science:

• Plant Pathology
• Molecular Diagnostics
• Oomycete Research

Background:

• Phytopythium vexans causes significant root and crown rot in diverse plant species, impacting nurseries and forestry.
• Rapid pathogen spread via irrigation systems necessitates efficient detection methods.
• Conventional detection techniques are time-consuming, costly, and often inconclusive.

Purpose of the Study:

• To develop and evaluate a specific, sensitive, and rapid molecular diagnostic method for Phytopythium vexans identification.
• To overcome the limitations of traditional detection methods for this important plant pathogen.

Main Methods:

• Development of loop-mediated isothermal amplification (LAMP) assays for Phytopythium vexans DNA amplification.
• Screening of multiple LAMP primer sets, identifying PVLSU2 as specific to P. vexans.
• Evaluation of real-time and colorimetric LAMP assays for sensitivity and specificity.

Main Results:

• The PVLSU2 primer set demonstrated high specificity, not amplifying DNA from related oomycetes, fungi, or bacteria.
• The developed LAMP assays detected DNA concentrations as low as 102 fg per reaction.
• Real-time LAMP proved more sensitive than traditional PCR and culture-based methods for infected plant samples.
• Both LAMP assays successfully detected as few as 100 zoospores in 100 ml of water.

Conclusions:

• The developed real-time and colorimetric LAMP assays provide a rapid, sensitive, and specific molecular diagnostic tool for Phytopythium vexans.
• These assays can significantly reduce detection time for diagnostic labs and research institutions.
• Early detection facilitates timely disease management and preparedness against Phytopythium vexans outbreaks.