Rapid Diagnosis of Major Phytoplasma Infected Trees Using the Loop-Mediated Isothermal Amplification Method in South Korea

Tree diseases associated with phytoplasma infections predominantly affecting nine host trees have serious impacts on tree growth and cause significant economic losses in South Korea. Loop-mediated isothermal amplification (LAMP)-based primers for early detection were developed to evaluate their accuracy. First, the 16S rRNA gene of phytoplasma was successfully amplified from the extracted DNA of various infected tree species using the polymerase chain reaction method. Two types diagnostic kits developed for phytoplasma detection were evaluated. The first kit detected phytoplasma infection within 30 min under isothermal conditions at 65°C, while the second kit did so within 40 min. Both kits could detect the nine different species of host trees infected with phytoplasma. When tested with 10 ng of the synthetic target gene, the FAM value became detectable at 10 min and remained consistent until 40 min. The lowest detection concentration was 0.01 pg/µL, and the limit of detection was 100 copies/µL. All of the phytoplasmas from nine diseased hosts were early detected. Furthermore, phytoplasma was not detected in healthy specimens, confirming the diagnostic kits' accuracy in distinguishing between healthy and infected strains. The LAMP method confirmed rapid, accurate, and visually assessable detection of phytoplasma, suggesting it will enable early diagnosis of phytoplasma infections in South Korea.