The areca palm, Areca catechu L., family Arecaceae is an important herbal medicine which has potential for the treatment of parasitic diseases, digestive function disorders and depression (Peng et al. 2015). Yellow leaf disease (YLD), caused by phytoplasma, is a destructive disease of Areca catechu. In 1981, the YLD was first discovered in Tunchang, Hainan, China. According to the investigation in 2020, the occurrence area of YLD was 32 102.38 hm2 in Hainan, China, resulting in 50%-60% yield loss. Previous researchers based on 16S rDNA gene PCR amplification analysis showed that YLD in Hainan was caused by 16SrI group phytoplasma (Che et al. 2010). In August, 2022, yellow leaf symptoms were observed on middle and lower leaves of Areca catechu. Forty symptomatic plants and three asymptomatic samples were collected in Wenchang, Hainan, China (19°33′9″N, 110°48′5″E). Forty-three samples (0.1g each) were used to extract total DNA (TIANGEN plant genomic DNA extraction kit). Phytoplasma universal primers named P1/P7 (Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee 1996) for 16Sr DNA and primers named fTuf1/rTuf1 and fTufu/rTufu (Schneider et al. 1997) for tuf genes were used for amplifying phytoplasma sequences from isolated DNA samples by nested PCR. No fragment was amplified in asymptomatic plants and four out of forty symptomatic samples could amplify target fragment. R16F2n/R16R2 amplicons (1 248 bp) and fTufu/rTufu amplicons (845 bp) from four symptomatic Areca catechu samples were sequenced in BGI (https://genomics.cn/). The 16Sr DNA GenBank accession numbers of four positive strains (named HNWC5, HNDZ1, HNDZ3 and HNDZ6) were OQ586072, OQ586085, OQ586086, OQ586087, respectively and the tuf GenBank accession numbers were OQ595209, OQ595210, OQ595211, OQ595212, respectively. Sequence alignment showed that the 16S rDNA and tuf sequence of HNDZ1, HNDZ3 and HNDZ6 were 100% consistent. 16S rDNA of HNWC5 was 99.96% consistent with HNDZ1 and tuf of HNWC5 was 98.31% consistent with HNDZ1. Interestingly, blast search based on 16S rDNA gene of HNWC5 showed 100% sequence identity with that of 16SrII group phytoplasma such as 'Eclipta prostrata' phytoplasma strain Ep1(MH144204.1), 'Aeschynomene americana' phytoplasma isolate AA1(MH231157.1) and 'Acacia confusa' witches'-broom phytoplasma isolate HK6(ON408364.1). Blast search based on tuf gene of HNWC5 showed 98.7% sequence identity with that of bamboo witches'-broom phytoplasma (FJ853160.1) and 91.02% sequence identity with that of 'podocarpus nagi' fasciation phytoplasma (KR633146) and 90.78% sequence identity with that of 'Musa acuminata' elephantiasis disease phytoplasma (MF983708). The phylogenetic tree was constructed based on 16Sr DNA gene by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value (Kumar et al. 2016). The result indicated that the HNWC5, HNDZ1, HNDZ3 and HNDZ6 phytoplasma strains clustered a subclade in 16SrII group. The virtual RFLP analysis based on the 16Sr DNA gene sequence was performed by the online phytoplasma classification tool iPhyClassifier (Zhao et al. 2009) using restriction endonucleases of AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI. The result indicated that HNWC5 was most similar to the reference pattern of peanut witches'-broom phytoplasma (16SrII-A subgroup, GenBank accession: L33765) and the pattern similarity coefficient of HNWC5 is 1.00. However, the HpaII restriction endonuclease pattern of HNDZ1, HNDZ3 and HNDZ6 was different from L33765 and the similarity coefficient was 0.97, which indicated this strain may represent a new subgroup within the 16SrII group. To our knowledge, this is the first report of 16SrII group related phytoplasma associated with YLD on Areca catechu in China. Our study contributes to understanding the polymorphism of phytoplasma causing YLD and provides an important reference for pathogen specific detection.

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.

Rubus cochinchinensis, an important traditional Chinese medicine in China is used to treat rheumatic arthralgia, bruises and lumbocrural pain (He et al.2005). In January 2022, yellow leaves of R. c...

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.