Loop-Mediated Isothermal Amplification and Polymerase Chain Reaction Methods for Specific and Rapid Detection of...

Area of Science:

• Plant Pathology
• Microbiology
• Molecular Diagnostics

Background:

• Rhodococcus fascians causes economically significant leafy gall disease in ornamental plants.
• Traditional methods for R. fascians detection are slow and labor-intensive, hindering effective disease management.

Purpose of the Study:

• To develop and evaluate rapid diagnostic assays for the accurate detection of the phytopathogenic bacterium Rhodococcus fascians.
• To provide efficient tools for nurseries to identify and manage R. fascians infections.

Main Methods:

• Development of a polymerase chain reaction (PCR) assay using primers targeting the essential pathogenicity genes fasA and fasD.
• Development of a loop-mediated isothermal amplification (LAMP) assay targeting the virulence gene regulator fasR.
• Evaluation of both assays for sensitivity and specificity using pure cultures, naturally infected plants, and artificially inoculated plants.

Main Results:

• The PCR assay demonstrated high accuracy, correctly identifying pathogenic R. fascians in 97% of bacterial strains and 95% of infected plants.
• The LAMP assay showed excellent performance, accurately detecting R. fascians in 93% of naturally infected plants and 98% of strains from leafy galls.
• Both assays achieved a detection limit of approximately 10^3 CFU/reaction, with LAMP offering greater convenience for field use.

Conclusions:

• Developed PCR and LAMP assays provide fast, reliable, and accurate detection of Rhodococcus fascians in vitro and in planta.
• The LAMP assay represents a significant advancement for rapid diagnostics, particularly beneficial for laboratories with limited resources.
• These novel diagnostic tools will aid in the timely identification and management of R. fascians, mitigating economic losses in the ornamental plant industry.