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 the areca palm yellow leaf phytoplasma (APYL), poses a significant threat to the sustainability of the areca palm industry. Timely and accurate detection is essential for effectively diagnosing and managing this disease. This study developed a novel nested PCR system using primers specifically designed from conserved regions of the phytoplasma 16S rDNA sequence to overcome limitations such as false positives often associated with universal nested PCR primers. The resulting primer pairs HNP-1F/HNP-1R (outer) and HNP-2F/HNP-2R (inner) consistently amplified a distinct 429 bp fragment from APYL strains belonging to the 16SrI and 16SrII groups. The detection sensitivity reached 7.5 × 10−7 ng/μL for 16SrI and 4 × 10−7 ng/μL for 16SrII. Field validation using leaf samples from symptomatic areca palms confirmed the high specificity and reliability of the new primers in detecting APYL. Compared to conventional universal primers (P1/P7 and R16mF2/R16mR1), this newly developed nested PCR system demonstrated higher specificity, sensitivity, and speed, making it a valuable tool for the early diagnosis and management of YLD in areca palms.