Development of a Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca palm in Hainan, China

Yellow leaf disease (YLD), caused by areca palm yellow leaf phytoplasma (AYLP), is a devastating disease that severely impacts the sustainable development of the areca palm industry. Efficient and accurate detection methods are considered crucial for the diagnosis and management of YLD. To address issues like false positive results using universal nested PCR primers, this study designed specific outer and internal primers based on the conserved regions of the 16S rDNA sequence of phytoplasmas, resulting in a nested PCR primer set, HNP-1F/HNP-1R and HNP-2F/HNP-2R. This set consistently amplified a single specific target band of 429 bp from 16SrI and 16SrII groups of AYLP. The sensitivity threshold for the 16SrI group of AYLP was 7.5×10-7 ng/μL, while that for the 16SrII group of AYLP was 4 ×10-3 ng/μL. To verify and evaluate the efficiency, the developed nested PCR system was used to detect leaf samples collected from trees showing leaf yellowing symptoms in areca palm plantations, and the results showed that the primer set could specifically detect AYLP. This primer set is characterized by rapid and accurate detection, high specificity, and high sensitivity compared to the traditional universal primer set of P1/P7 and R16mF2/R16mR1 or R16mF2/R16mR1 and R16mF2n/R2, providing a scientific basis for the specific diagnosis and early control of yellow leaf disease of areca palm.