Simple detection of Pythium irregulare using loop-mediated isothermal amplification assay

Area of Science:

• Agricultural Science
• Molecular Biology
• Plant Pathology

Background:

• Pythium irregulare is a significant soil-borne pathogen responsible for damaging crop diseases like damping-off and root rot.
• Accurate and rapid detection of P. irregulare is crucial for effective disease management in agriculture.

Purpose of the Study:

• To develop a rapid, reliable, and practical method for detecting Pythium irregulare.
• To design specific primers for Pythium irregulare based on its ribosomal DNA internal transcribed spacer region.

Main Methods:

• Loop-mediated isothermal amplification (LAMP) assay using primers targeting the P. irregulare ribosomal DNA internal transcribed spacer region.
• Specificity testing against 40 Pythium species, 11 Phytophthora isolates, and 8 other soil-borne pathogens.
• Development of 'Plant-LAMP' and 'Bait-LAMP' methods for direct detection in plant tissues and soil samples.

Main Results:

• The LAMP assay demonstrated high specificity for P. irregulare.
• The limit of sensitivity was 100 fg of pure DNA, comparable to polymerase chain reaction (PCR).
• Successful detection of P. irregulare was achieved from culture medium, rotted root suspensions, and soil samples using the developed LAMP methods.

Conclusions:

• The developed LAMP assay is a simple, rapid, and effective tool for the practical detection of Pythium irregulare.
• The 'Plant-LAMP' and 'Bait-LAMP' variations offer versatile applications for pathogen detection in various agricultural contexts.
• This method provides a valuable alternative for early and accurate diagnosis of P. irregulare infections.