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.

Areca palm velarivirus 1 (APV1), the causal agent of yellow leaf disease (YLD), poses a serious threat to the economically important areca palm industry in the Hainan Province, China, yet its evolutionary dynamics remain poorly understood. Here, we performed a large-scale molecular survey by sequencing the coat protein (CP) gene from 364 APV1-infected samples collected across major cultivation regions of Hainan. Population genetic analyses revealed extremely high haplotype diversity (H_d = 0.997) but very low nucleotide diversity (π = 0.017). Neutrality tests (Tajima’s D = −2.266; Fu’s F_S = −23.697) and a unimodal mismatch distribution supported a scenario of recent demographic expansion from a restricted ancestral pool. Evolutionary analyses indicated that the CP gene is subject to strong purifying selection, although eight codons exhibited episodic positive selection, suggesting ongoing viral adaptation. Furthermore, we identified three distinct genetic clusters with significant geographic structuring, indicating that viral dissemination is shaped by local factors. Together, these results reveal a recent explosive invasion of APV1 characterized by rapid island-wide expansion and emerging local differentiation. This work provides novel insights into the evolutionary trajectory of APV1 and establishes a genomic basis for improved surveillance and management of YLD.

Phytoplasmas are a group of wall-less, unculturable prokaryotic pathogenic bacteria that colonize the phloem of plants and are transmitted by piercing–sucking insects. As a typical tropical island province in China, Hainan Island has abundant biodiversity due to its unique geographical location and climatic conditions, which provide a favorable ecological environment for the survival and propagation of phytoplasmas, which infect different hosts, cause different symptoms, or belong to different subgroups. Based on host species, disease symptoms and 16Sr subgroups, 69 representative phytoplasma records from four 16Sr groups have been identified on Hainan Island, showing rich diversity in host range and pathogen species. The diversity of plant hosts and the evolutionary relationship of phytoplasmas not only affect the occurrence and prevalence of phytoplasma-related diseases but also bring great challenges to the epidemic monitoring, detection, diagnosis and prevention management of these diseases. This review summarizes current research progress on host diversity, phylogenetic evolution, mixed infection, diversity of transmission vectors, and geographical isolation differentiation, as well as adaptive evolution of phytoplasmas on Hainan Island. Furthermore, the challenges brought by plant host diversity and phylogenetic evolution to disease monitoring, diagnosis and prevention management are discussed. This review aims to provide a comprehensive theoretical basis for the in-depth study of phytoplasma-related diseases on Hainan Island, and to offer practical guidance for scientific monitoring, early warning and comprehensive prevention and control of these diseases.

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.