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Article

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

1
Department of Plant Pathology and MARA Key Laboratory of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing 100193, China
2
Coconut Research Institute/HPDST Hainan Innovation Center of Academician Team, Chinese Academy of Tropical Agricultural Sciences, Wenchang 571339, China
3
Sanya Institute of China Agricultural University, Sanya 572000, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(14), 2144; https://doi.org/10.3390/plants14142144
Submission received: 16 April 2025 / Revised: 6 July 2025 / Accepted: 7 July 2025 / Published: 11 July 2025
(This article belongs to the Section Plant Molecular Biology)

Abstract

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.

1. Introduction

Areca palm (Areca catechu L.) is a perennial evergreen species belonging to the genus Areca within the family Palmaceae. Originally native to Malaysia, it is now widely cultivated throughout South and Southeast Asia, spanning 16 countries and regions, including India and Malaysia [1]. Its primary cultivation areas in China are Hainan and Taiwan Provinces, with smaller-scale production in Yunnan, Guangdong, and Guangxi Provinces [2]. The areca palm is valued for its dietary and medicinal properties, including applications in treating various human ailments [3]. It is recognized as one of Hainan’s key crops, part of the province’s so-called “Six Trees”, and accounts for over 95% of the total area of areca cultivation in China [4]. Currently, the areca palm industry supports the livelihoods of more than 2.3 million farmers in Hainan [5], contributing significantly to regional agricultural development, rural revitalization, and income generation for farming communities.
Yellow leaf disease (YLD) of areca palm, caused by the areca palm yellow leaf phytoplasma (APYL), is a highly destructive condition first identified in 1914 in central Kerala, India [6]. Subsequent occurrences were reported in China in 1981 [7] and in Sri Lanka in 2015 [8]. In China, the disease was initially detected in Tunchang County, Hainan Province, and has since spread to nearly all areca-growing regions across the island, inflicting significant losses on the local areca palm industry [5,9]. Without effective control strategies in cultivation practices, early and accurate pathogen detection remains critical for effective disease management.
In India, three distinct ‘Candidatus Phytoplasma’ species have been identified in association with YLD: ‘Ca. P. asteris’ (16SrI-B), ‘Ca. P. sacchari’ (16SrXI-B), and ‘Ca. P. cynodontis’ (16SrXIV-A) [10,11,12]. However, previous studies in China reported that YLD-associated phytoplasmas primarily belonged to the 16SrI-B and 16SrI-G subgroups [13,14]. More recently, other phytoplasma groups 16SrII and 16SrXXXII have been detected in YLD-affected areca palm samples collected from Hainan [15,16].
To date, a range of detection methods has been developed for identifying APYL, including electron microscopy [17], enzyme-linked immunosorbent assay (ELISA) [18], nested PCR [14,19], loop-mediated isothermal amplification (LAMP) [20], droplet digital PCR (ddPCR) [21], and quantitative PCR (qPCR) [22,23]. While each technique offers specific advantages, they also present significant limitations. For example, electron microscopy is labor-intensive, requires complex sample preparation, and incurs high costs. Serological methods such as ELISA may suffer from challenges in antisera production and risks of cross-reactivity with host antigens. Although LAMP enables isothermal amplification, its complicated primer design [24] and susceptibility to contamination can result in false positives. In comparison, qPCR and ddPCR provide high sensitivity but demand costly reagents, sophisticated instrumentation, and stricter laboratory conditions. Moreover, products generated by ELISA, LAMP, or qPCR are not suitable for sequencing.
Nested PCR using universal phytoplasma primers remains a widely adopted method for APYL detection and classification, primarily because the amplified 16S rDNA fragments can be sequenced. However, this approach has drawbacks, particularly the risk of false-positive results due to non-specific amplification, which can significantly compromise detection accuracy [25]. To address issues related to the low concentration and uneven distribution of APYL in host tissues, a nested PCR primer set (F4/R1 and F2/R2 [20]) was previously developed, showing improved sensitivity over traditional universal primer sets such as P1/P7 and R16mF2/R16mR1 or P1/P7 and R16mR2n/R2 [4,26]. However, studies have shown that this primer set could generate non-specific amplification products.
Recent surveillance in Hainan Province revealed that phytoplasmas of the 16SrI group are prevalent, with increasing reports of 16SrII group detection in regions such as Wenchang and Danzhou. The 16SrXXXII group has been observed in only a single case [16]. Currently, no existing diagnostic method can simultaneously detect both 16SrI and 16SrII APYL strains.
This study developed a novel nested PCR primer pair combination to overcome the challenge of non-specific amplification and enable concurrent detection of both 16SrI and the newly identified 16SrII APYL groups. This new system offers high specificity and sensitivity for APYL detection across both phytoplasma groups in Hainan, thus providing a reliable diagnostic tool to support early identification and control of yellow leaf disease in areca palm seedlings and field-grown plants.

2. Results

2.1. Application of Universal Nested PCR for Phytoplasma Detection

A total of 335 genomic DNA samples from areca palm were screened using the universal nested PCR primer set P1/P7, followed by R16mF2/R16mR1, targeting phytoplasma 16S rDNA (Table A1). Among these, 50 samples produced amplification bands of approximately 1400 bp (Figure 1). The nested PCR products from these 50 putative positives were then sequenced by Sangon Biotech (Shanghai, China). Sequence analysis revealed the following: 16 samples corresponded to areca palm chloroplast DNA, 20 matched the bacterial sequences, only 10 were confirmed as being phytoplasma-specific (Table A1 NO. 1–10), and 4 samples failed to yield any usable sequencing data (Figure 2).

2.2. Nested PCR Primer Test and Combination

Based on multiple sequence alignments of 16S rDNA from APYL, areca chloroplasts, and pathogenic and endophytic bacteria, and guided by established PCR primer design principles, one outer primer pair (HNP-1F/HNP-1R) and three internal primer pairs (HNP-2F/2R, HNP-3F/3R, and HNP-4F/4R) were designed (Table 1). These nested PCR primers were evaluated using genomic DNA from areca palm leaves infected with 16SrI and 16SrII phytoplasma groups and DNA from Burkholderia andropogonis and Pantoea ananatis as reference strains. Among the internal primer sets, HNP-2F/2R and HNP-3F/3R specifically amplified target bands only in the positive control and areca palm samples infected with 16SrI or 16SrII phytoplasmas (Figure 3A,B). However, HNP-4F/4R also produced a ~1500 bp band from the DNA of the bacterial leaf blight pathogen (Figure 3C), indicating non-specific amplification. Therefore, for subsequent nested PCR assays, HNP-1F/1R was paired with either HNP-2F/2R or HNP-3F/3R.

2.3. Specificity Validation of Nested PCR

To evaluate detection specificity, the outer primers HNP-1F/1R were paired with internal primers HNP-2F/2R and HNP-3F/3R and tested against genomic DNA from areca palm samples infected with 16SrI and 16SrII phytoplasmas, as well as DNA from five other pathogens. Results showed that the HNP-2F/2R primer pair consistently amplified a specific band of approximately 429 bp only in the positive control and in areca palm samples infected with 16SrI and 16SrII phytoplasmas (Figure 4A). However, the HNP-3F/3R set also amplified a 652 bp fragment in healthy areca palm samples and those infected with 16SrXXXII group phytoplasmas (Figure 4B), indicating reduced specificity. Therefore, HNP-2F/2R was selected as the internal primer pair for all subsequent assays.

2.4. Optimization of Nested PCR Annealing Temperatures

In the nested PCR assays, annealing temperatures for both outer and internal primer sets were systematically optimized across a gradient from 40 °C to 60 °C. For the outer primer pair HNP-1F/1R, no significant differences in amplification intensity were observed between 40.0 °C and 53.6 °C. However, as the temperature increased from 56.0 °C to 60.0 °C, a marked decline in band intensity was noted (Figure 5A), leading to the selection of 53.6 °C as the optimal annealing temperature.
For the internal primers HNP-2F/2R, a temperature gradient analysis revealed consistent amplification of the target band within the range of 46.4 °C to 51.3 °C, while no detectable amplification occurred at 60.0 °C (Figure 5B). Based on these observations, 51.3 °C was chosen as the optimal annealing temperature for the HNP-2F/2R primer set.

2.5. Sensitivity Determination of Nested PCR

To evaluate the sensitivity of the developed method for detecting phytoplasmas from the 16SrI and 16SrII groups, nested PCR was performed using the outer primers HNP-1F/1R and internal primers HNP-2F/2R on recombinant plasmids harboring 16S rDNA fragments specific to each group.
For the 16SrI group, a distinct and specific amplification band was consistently observed across a plasmid concentration range from 7.5 ng/μL down to 7.5 × 10−7 ng/μL. As the concentration decreased, the intensity of the amplification band gradually weakened and became undetectable at 7.5 × 10−7 ng/μL (Figure 6A). Similarly, in the case of the 16SrII group, a clear target band of 429 bp was stably amplified within the range of 4 ng/μL to 4 × 10−3 ng/μL, with diminishing intensity as the plasmid concentration decreased and no amplification detected at 4 × 10−3 ng/μL (Figure 6B).
These findings demonstrate that the nested PCR assay has a detection limit of 7.5 × 10−7 ng/μL for the 16SrI group and 4 × 10−7 ng/μL for the 16SrII group of phytoplasmas.

2.6. Comparison of the Developed Nested PCR to the Universal Nested PCR

To evaluate the detection efficiency of the newly developed nested PCR method compared to the conventional universal nested PCR approach, 30 areca palm samples collected from the field were tested (Table 2).
The universal nested PCR method using primer sets P1/P7 and R16mF2/R16mR1 detected phytoplasma only in the positive control sample and failed to identify any positive cases among the field samples (Figure 7A). However, the newly developed nested PCR method detected phytoplasmas in 10 of the 30 field samples (Figure 7B). The sequencing analysis of these 10 positive samples confirmed that all amplicons corresponded to phytoplasmas belonging to the 16SrI group. These results underscore the higher sensitivity and reliability of the specific nested PCR method established in this study for detecting APYL phytoplasmas, offering a valuable tool for routine surveillance and early diagnosis in areca palm plantations affected by yellow leaf disease.

2.7. Construction and Analysis of Phylogenetic Trees

Phylogenetic analysis was conducted based on the fragments amplified using the HNP-2F/2R primers. The obtained sequences were compared with 16S rRNA sequences of known phytoplasmas in the GenBank database. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 11, with Acholeplasma laidlawii serving as the out-group. In the resulting tree, phytoplasmas from the 16SrI and 16SrII groups formed distinct clades. This demonstrates that the nested PCR method developed in this study can effectively distinguish and group phytoplasmas associated with yellow leaf disease in areca palm within the 16SrI and 16SrII groups (Figure 8).

3. Discussion

Phytoplasmas, previously referred to as mycoplasma-like organisms (MLOs) [30], are prokaryotes that lack a cell wall [31,32] and remain notoriously difficult to culture in artificial media [33]. In this study, we designed outer and internal primers targeting conserved 16S rDNA regions specific to the APYL phytoplasma. Among these, the primer set HNP-1F/HNP-1R and HNP-2F/HNP-2R was ultimately selected for its specificity in amplifying APYL. The annealing temperatures and nested PCR parameters were systematically optimized to ensure reliable detection.
Universal primers such as P1/P7 and R16mF2/R16mR1 are commonly employed for detecting phytoplasmas across diverse host species. However, in this work it was found that their non-specific amplification was quite notable. Our results indicated that the specificity of the universal primers P1/P7 and R16mF2/R1 for the amplification of APYL is relatively poor, resulting in a high false-positive rate (72% to 80%). This observation underscores their limited specificity in the context of the areca palm. A similar issue was noted in detecting phytoplasma associated with sisal purple leafroll disease [34]. In earlier field tests during 2018 and 2019, screening of 100 leaf samples with these universal primers yielded only one confirmed phytoplasma sequence, identified as ‘Ca. P. asteris’. A previous study introduced a nested PCR approach using primer sets F4/R1 and F2/R2 [20] to improve detection accuracy, targeting a 525 bp fragment. While this approach enhanced detection rates, subsequent analyses revealed non-specific amplification as a limitation.
The nested PCR method developed in the present study demonstrated high specificity, reliably detecting phytoplasmas from both the 16SrI and 16SrII groups currently identified in areca palms, without cross-reactivity to other phytoplasma groups or bacterial pathogens.
In terms of sensitivity, the method achieved a minimum detection threshold of 7.5 × 10−7 ng/μL for the ‘Ca. P. asteris’ strain (Figure 6A) and 4 × 10−3 ng/μL for the 16SrII phytoplasma group (Figure 6B). While qPCR and ddPCR offer lower detection limits, 1.16 copies/μL [23] and 0.07 copies/μL [22], respectively, our nested PCR approach presents a cost-effective alternative that does not compromise specificity or reliability. All amplified fragments obtained in this study were confirmed by sequencing to correspond to APYL phytoplasma.
In conclusion, the nested PCR method developed here provides a robust, economical, and specific tool for detecting and identifying APYL-associated phytoplasmas from the 16SrI and 16SrII groups. This approach offers valuable technical support for routine monitoring, early diagnosis, and effective areca palm yellow leaf disease management.

4. Materials and Methods

4.1. Materials

In this study, genomic DNA was extracted from field-collected areca palm leaf samples. After sequencing and confirmation of identity, the DNA samples were stored at −20 °C for use in primer specificity validation experiments (Table 3). Further leaf samples were also collected from the field, and their genomic DNA was similarly extracted and preserved at −20 °C. These samples were then used to compare the detection efficiency of the newly designed primers with that of conventional universal primers (Table 2).

4.2. Extraction of Total DNA from Areca Plam Leaf Samples Showing Yellow

Approximately 0.1 g of tissue was collected from the upper one-third of areca palm leaves, cut into 1 mm fragments, and ground using a cell disruptor. Genomic DNA was extracted using the Plant Genomic DNA Extraction Kit (TianGen Biotech, Beijing, China), quantified with a NanoDrop 2000 spectrophotometer, and stored at −20 °C for further use.

4.3. Construction of Recombinant Plasmids for 16S rDNA of Areca Palm Yellow Leaf Phytoplasma

In this study, universal nested PCR primers P1/P7 and R16mF2/R16mR1 (Table 1), targeting the 16S rDNA of phytoplasmas, were used to amplify total genomic DNA extracted from areca palm leaves showing YLD symptoms. Each 25 μL PCR reaction contained 2 μL of DNA template, 12.5 μL of 2× Taq PCR Master Mix (Aidlab Biotechnologies, Beijing, China), 1 μL of each primer (10 μM), and nuclease-free water to a final volume.
The PCR protocol involved an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min. The PCR product (from P1/P7) was diluted 20-fold and used as the template for the nested PCR using internal primers R16mF2/R16mR1. This nested PCR followed the same cycling conditions, except for an annealing temperature of 50 °C.
For visualization, 5 μL of ethidium bromide (10 mg/mL; Sangon Biotech, Shanghai, China) was added to a 1% agarose gel prepared with 1× TAE buffer and mixed thoroughly by vortexing for 10 s. PCR products were electrophoresed at 120 V for 35 min and imaged using a gel documentation system (Synoptics, Cambridge, UK).
For recombinant plasmid construction, PCR products were purified using a gel extraction kit (Tiangen Biotech, Beijing, China), then ligated into a T-vector using a rapid ligation kit (Sangon Biotech, Shanghai, China). The ligation products were transformed into DH5α competent cells (Sangon Biotech, Shanghai, China). Positive colonies were screened in LB liquid medium containing 50 ng/μL ampicillin. Plasmid DNA was extracted using a plasmid mini-prep kit (Tiangen Biotech, Beijing, China) and sequenced by Sangon Biotech (Shanghai, China).

4.4. Design and Primary Screening of Specific Primers

In this study, the outer and inner primers were designed and screened to detect the 16S rDNA of phytoplasmas associated with YLD in areca palm. For primer design, previously reported 16S rDNA sequences from the 16SrI group (GenBank accession numbers: FJ998269 and FJ694685) and from the 16SrII group (GenBank accession number: OQ586085) were used. To improve specificity, these phytoplasma sequences were aligned and compared with those from Areca chloroplasts, known areca bacterial pathogens (e.g., Burkholderia andropogonis and Pantoea ananatis), and other bacterial species (Table 4). Based on sequence divergence, multiple nested PCR primers were designed and synthesized by Sangon Biotech (Shanghai, China).
For the initial amplification, genomic DNA extracted from areca palm leaves infected with 16SrI and 16SrII group phytoplasmas, as well as DNA from Burkholderia andropogonis and Pantoea ananatis, was subjected to PCR using the outer primer pair HNP-1F/1R. The ‘Ca. P. asteris’ strain (GenBank accession number: PV760299) was included as a positive control, and sterile water was a negative control. The resulting PCR products were diluted 20-fold and used as templates for nested PCR using three primer sets: HNP-2F/2R, HNP-3F/3R, and HNP-4F/4R.

4.5. Specificity Verification of Nested PCR

To assess the specificity of the newly developed method, the outer primer pair HNP-1F/1R was used to amplify genomic DNA from a range of sources, including healthy areca palm leaves, bacterial leaf spot pathogens of areca palm, pan-genus bacteria from pineapple, YLD-associated phytoplasmas from the 16SrI and 16SrII groups, and Trema tomentosa (Roxb.) Hara was infected with a 16SrXXXII group phytoplasma (GenBank accession number: PV759645). Complementary DNA from areca yellow leaf virus 1 (GenBank accession number: OQ423126) was also tested. The PCR products were diluted 20-fold and used as templates for a nested amplification using the internal primer sets described previously. The reaction system of nested PCR and cycling parameters followed those outlined in Section 4.3.

4.6. Optimization of Annealing Temperatures for Nested PCR with Internal Primers

To determine the optimal annealing temperature for nested PCR using internal primers for phytoplasma detection, the primer set HNP-1F/1R + HNP-2F/2R was employed, targeting the ‘Candidatus Phytoplasma asteris’ strain H-9 (GenBank accession number: PV760299).
For the PCR of amplification with primers HNP-1F/1R, the reaction mixture was identical to that described in Section 4.3. PCR cycling conditions consisted of an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a temperature gradient from 40 °C to 60 °C in 12 increments (each for 30 s), extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min.
Following this, the PCR products were diluted 20-fold and used as templates for nested amplification with primers HNP-2F/2R. The nested PCR reaction composition mirrored the PCR, with the same temperature gradient applied to optimize annealing conditions.

4.7. Sensitivity Testing of Developed Nested PCR

To evaluate the sensitivity of the nested PCR method developed in this study, recombinant plasmids S97 and W4, each harboring the 16S rDNA fragments from the 16SrI and 16SrII groups of APYL, respectively, were subjected to 10-fold serial dilutions. Plasmid S97 was diluted from 7.5 ng/μL to 7.5 × 10−8 ng/μL, and W4 from 4 ng/μL to 400 zg/μL. The nested PCR was performed using the HNP-1F/1R + HNP-2F/2R primer set to amplify the diluted plasmid templates. The reaction mixture and cycling conditions followed those outlined in Section 4.6.

4.8. Comparison of Developed Nested PCR Method with the Universal Nested PCR Method

In this study, 30 areca palm samples showing yellowing symptoms (Table 2) were simultaneously analyzed to compare the performance of two detection methods: one using universal primers and the other employing the newly developed nested PCR primer set. The universal nested PCR method was conducted according to the protocol detailed in Section 4.3, while the newly developed nested PCR assay followed the procedure described in Section 4.6.

4.9. Phylogenetic Analysis of the Fragment Amplified from Primers HNP1F/1R and HNP-2F/2R

The DNA fragments amplified by primers HNP-2F/2R were compared with 16S rRNA sequences of other phytoplasmas in the GenBank database. A phylogenetic tree was constructed using the neighbor-joining method in MEGA 11 software, with Acholeplasma laidlawii serving as the out-group and bootstrap analysis performed with 1000 replicates [34].

Author Contributions

Conceptualization, Q.T. and X.Z.; methodology, H.G., Q.T., and X.Z.; validation, H.G.; formal analysis, H.G.; investigation, X.M., Z.L., W.S., S.J., and W.Q.; data curation, H.G., Q.T., and X.Z.; writing—original draft preparation, H.G.; writing—review and editing, H.G., Q.T., and X.Z.; review, Q.T. and X.Z.; supervision, Q.T. and X.Z.; and project administration, Q.T. and X.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Hainan Key Project. Hainan Provincial Department of Science and Technology: ZDYF2025XDNY118 and ZDYF2022XDNY208) and the Project of Yazhouwan Scientific and Technological Administration of Sanya (Yazhouwan Scientific and Technological Administration of Sanya: SYND-2022-36).

Data Availability Statement

DNA sequences are available in the GenBank database, with the accession numbers listed in the Results. All other relevant data are within the paper and Appendix A.

Acknowledgments

We sincerely thank Y.H. Wang and Y.Y. Liu, H.Q. Wang, C.D. Xu, T. Deng, and X.X. Zheng for participating in partial sample collections and DNA extraction from 2020 to 2022, and scientific research assistants L.P. Ma, P. Ma, C.P. Guo, and S.D. Long for participating in partial DNA extraction during 2019 and 2024. The original manuscript was proofread for grammar by Syed Majid of Rasheed Department of Agriculture Entomology Section, Bacha Khan University, Charsadda, Pakistan. We would like to express our heartfelt thanks to them. We also thank our laboratory partners for their technical assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
YLDyellow leaf disease of areca palm
APYLareca palm yellow leaf phytoplasma
LAMPloop-mediated isothermal amplification
qPCRquantitative PCR
ddPCRdroplet digital PCR

Appendix A

Table A1. Areca palm leaf samples used in the detection assays using universal nested PCR in this study.
Table A1. Areca palm leaf samples used in the detection assays using universal nested PCR in this study.
No.SampleResultCity/CountyYearNo.SampleResultCity/CountyYear
1J069PositiveWenchang2022169ZB1-2NegativeWenchang2022
2A056PositiveWenchang2022170MQR4NegativeLedong2021
3T40PositiveWenchang2022171HL3-4NegativeLedong2021
4W4PositiveWenchang2022172HL3-1NegativeLedong2021
5S97PositiveWenchang2022173HL1-2NegativeLedong2021
6M25PositiveWenchang2023174HZ3-2NegativeLedong2021
7H61PositiveWenchang2023175HZ5-2NegativeLedong2021
8H64PositiveWenchang2023176T52NegativeLedong2021
9R71PositiveWaning2022177NY1-2NegativeWenchang2022
10R72PositiveWaning2022178ZB2-2NegativeWenchang2022
11R61NegativeWaning2022179M22NegativeWenchang2022
12R62NegativeWaning2022180M23NegativeWenchang2022
13R63NegativeWaning2022181M25NegativeWenchang2022
14R64NegativeWaning2022182M26NegativeWenchang2022
15R65NegativeWaning2022183M27NegativeWenchang2022
16R66NegativeWaning2022184M30NegativeWenchang2022
17R67NegativeWaning2022185M31NegativeWenchang2022
18R68NegativeWaning2022186L30NegativeWenchang2021
19R69NegativeWaning2022187L32NegativeWenchang2021
20R70NegativeWaning2022188L33NegativeWenchang2021
21R73NegativeWaning2022189S40NegativeWenchang2023
22R74NegativeWaning2022190S41NegativeWenchang2023
23R75NegativeWaning2022191S42NegativeWenchang2023
24R76NegativeWaning2022192S43NegativeWenchang2023
25R77NegativeWaning2022193S44NegativeWenchang2023
26R78NegativeWaning2022194S45NegativeWenchang2023
27R79NegativeWaning2022195S46NegativeWenchang2023
28R80NegativeWaning2022196S47NegativeWenchang2023
29R81NegativeWaning2022197S50NegativeWenchang2023
30R82NegativeWaning2022198W11NegativeWenchang2023
31R83NegativeWaning2022199W12NegativeWenchang2023
32R84NegativeWaning2022200W13NegativeWenchang2023
33R85NegativeWaning2022201W14NegativeWenchang2023
34R86NegativeWaning2022202W15NegativeWenchang2023
35R87NegativeWaning2022203W16NegativeWenchang2023
36R88NegativeWaning2022204W17NegativeWenchang2023
37R89NegativeWaning2022205W18NegativeWenchang2023
38R90NegativeWaning2022206W19NegativeWenchang2023
39W1NegativeWaning2022207W20NegativeWenchang2023
40W2NegativeWenchang2022208W21NegativeWenchang2023
41W3NegativeWenchang2022209W22NegativeWenchang2023
42W3-4NegativeWenchang2022210W23NegativeWenchang2023
43A09NegativeWenchang2022211W24NegativeWenchang2023
44A12NegativeWenchang2022212W25NegativeWenchang2023
45A13NegativeWenchang2022213W26NegativeWenchang2023
46A14NegativeWenchang2022214W27NegativeWenchang2023
47A16NegativeWenchang2022215W28NegativeWenchang2023
48A19NegativeWenchang2022216W29NegativeWenchang2023
49A22NegativeWenchang2022217W30NegativeWenchang2023
50A24NegativeWenchang2022218W31NegativeWenchang2023
51A27NegativeWenchang2022219W32NegativeWenchang2023
52A30NegativeWenchang2022220W33NegativeWenchang2023
53A31NegativeWenchang2022221W34NegativeWenchang2023
54A32NegativeWenchang2022222W35NegativeWenchang2023
55A34NegativeWenchang2022223W36NegativeWenchang2023
56A36NegativeWenchang2022224W37NegativeWenchang2023
57A37NegativeWenchang2022225W38NegativeWenchang2023
58A39NegativeWenchang2022226W39NegativeWenchang2023
59A49NegativeWenchang2022227B2-1NegativeBaoting2021
60A50NegativeWenchang2022228B2-2NegativeBaoting2021
61A51NegativeWenchang2022229B2-4NegativeBaoting2021
62A55NegativeWenchang2022230B2-5NegativeBaoting2021
63A57NegativeWenchang2022231B2-6NegativeBaoting2021
64A59NegativeWenchang2022232B2-7NegativeBaoting2021
65A60NegativeWenchang2022233B2-9NegativeBaoting2021
66B13NegativeBaoting2020234B4-1NegativeBaoting2021
67B4NegativeBaoting2020235B4-2NegativeBaoting2021
68B17NegativeBaoting2020236B4-3NegativeBaoting2021
69B20NegativeBaoting2020237B4-4NegativeBaoting2021
70B21NegativeBaoting2020238B4-5NegativeBaoting2021
71B23NegativeBaoting2020239B4-6NegativeBaoting2021
72B24NegativeBaoting2020240B4-7NegativeBaoting2021
73B25NegativeBaoting2020241B4-8NegativeBaoting2021
74B27NegativeBaoting2020242B4-9NegativeBaoting2021
75B30NegativeBaoting2020243B4-10NegativeBaoting2021
76B31NegativeBaoting2020244B4-11NegativeBaoting2021
77B32NegativeBaoting2020245B4-12NegativeBaoting2021
78B33NegativeBaoting2020246B4-13NegativeBaoting2021
79B37NegativeBaoting2020247B4-14NegativeBaoting2021
80B41NegativeBaoting2020248B4-15NegativeBaoting2021
81B44NegativeBaoting2020249B4-16NegativeBaoting2021
82B46NegativeBaoting2020250B4-17NegativeBaoting2021
83B47NegativeBaoting2020251B4-18NegativeBaoting2021
84B49NegativeBaoting2020252B4-19NegativeBaoting2021
85B53NegativeBaoting2020253B5-4NegativeBaoting2021
86B54NegativeBaoting2020254B5-5NegativeBaoting2021
87B56NegativeBaoting2020255B5-6NegativeBaoting2021
88B57NegativeBaoting2020256B5-10NegativeBaoting2021
89B61NegativeBaoting2020257B6-1NegativeBaoting2021
90B63NegativeBaoting2020258B6-2NegativeBaoting2021
91B76NegativeBaoting2020259B6-4NegativeBaoting2021
92C21NegativeQionghai2020260B6-5NegativeBaoting2021
93C26NegativeQionghai2020261B6-6NegativeBaoting2021
94C27NegativeQionghai2020262B6-8NegativeBaoting2021
95C41NegativeQionghai2020263B6-10NegativeBaoting2021
96C47NegativeQionghai2020264B6-11NegativeBaoting2021
97C61NegativeQionghai2020265F04-2NegativeTunchang2020
98C68NegativeQionghai2020266F05-2NegativeTunchang2020
99C69NegativeQionghai2020267F05-1NegativeTunchang2020
100C70NegativeQionghai2020268F06-1NegativeTunchang2020
101C73NegativeQionghai2020269F07-1NegativeTunchang2020
102C85NegativeQionghai2020270F022-1NegativeTunchang2020
103C87NegativeQionghai2020271F028-2NegativeTunchang2020
104C89NegativeQionghai2020272F023-2NegativeTunchang2020
105C90NegativeQionghai2020273F035-1NegativeTunchang2020
106C91NegativeQionghai2020274F036-1NegativeTunchang2020
107C95NegativeQionghai2020275F036-2NegativeTunchang2020
108C99NegativeQionghai2020276F037-1NegativeTunchang2020
109C100NegativeQionghai2020277F037-2NegativeTunchang2020
110BSL-1NegativeDing’an2023278F038-1NegativeTunchang2020
111BSL-2NegativeDing’an2023279F038-2NegativeTunchang2020
112BSL-3NegativeDing’an2023280F039-1NegativeTunchang2020
113BSL-4NegativeDing’an2023281F040-1NegativeTunchang2020
114BSL-5NegativeDing’an2023282F040-2NegativeTunchang2020
115BSL-6NegativeDing’an2023283F041-2NegativeTunchang2020
116BSL-7NegativeDing’an2023284F044-1NegativeTunchang2020
117BSL-8NegativeDing’an2023285F044-2NegativeTunchang2020
118BSL-9NegativeDing’an2023286F045-1NegativeTunchang2020
119BSL-10NegativeDing’an2023287F045-2NegativeTunchang2020
120BSL-11NegativeDing’an2023288F049-2NegativeTunchang2020
121BSL-12NegativeDing’an2023289F055-2NegativeTunchang2020
122BSL-13NegativeDing’an2023290F099-1NegativeTunchang2020
123BSL-14NegativeDing’an2023291C002-1NegativeQionghai2020
124BSL-15NegativeDing’an2023292C007-2NegativeQionghai2020
125BSL-16NegativeDing’an2023293C013-2NegativeQionghai2020
126BSL-17NegativeDing’an2023294C018Y-1NegativeQionghai2020
127BSL-18NegativeDing’an2023295C018Y-2NegativeQionghai2020
128BSL-19NegativeDing’an2023296C021-1NegativeQionghai2020
129BSL-20NegativeDing’an2023297C023-1NegativeQionghai2020
130XS-1NegativeDing’an2023298C026-1NegativeQionghai2020
131XS-2NegativeDing’an2023299C026-2NegativeQionghai2020
132XS-3NegativeDing’an2023300C031-2NegativeQionghai2020
133XS-4NegativeDing’an2023301C048-1NegativeQionghai2020
134XS-5NegativeDing’an2023302C064-1NegativeQionghai2020
135XS-6NegativeDing’an2023303C065-2NegativeQionghai2020
136XS-7NegativeDing’an2023304C071-2NegativeQionghai2020
137XS-8NegativeDing’an2023305C072-2NegativeQionghai2020
138XS-9NegativeDing’an2023306C076-2NegativeQionghai2020
139XS-10NegativeDing’an2023307C077-2NegativeQionghai2020
140XS-11NegativeDing’an2023308C078-1NegativeQionghai2020
141LK-1NegativeDing’an2023309C080-2NegativeQionghai2020
142LK-2NegativeDing’an2023310C056-2NegativeQionghai2020
143LK-3NegativeDing’an2023311C065-2NegativeQionghai2020
144LK-4NegativeDing’an2023312D008-2NegativeDing’an2020
145LK-5NegativeDing’an2023313D039-1NegativeDing’an2020
146LK-6NegativeDing’an2023314D062-2NegativeDing’an2020
147LK-7NegativeDing’an2023315D093-2NegativeDing’an2020
148LK-8NegativeDing’an2023316ZY2-1NegativeWenchang2020
149LK-9NegativeDing’an20233177-4-2NegativeWenchang2020
150LK-10NegativeDing’an2023318003-2NegativeWenchang2020
151LK-11NegativeDing’an2023319B021-2NegativeBaoting2020
152LK-12NegativeDing’an2023320B027-2NegativeBaoting2020
153LK-13NegativeDing’an2023321B029-2NegativeBaoting2020
154LK-14NegativeDing’an2023322B030-1NegativeBaoting2020
155LK-15NegativeDing’an2023323B032-1NegativeBaoting2020
156LK-16NegativeDing’an2023324B037-2NegativeBaoting2020
157LK-17NegativeDing’an2023325B038-2NegativeBaoting2020
158LK-18NegativeDing’an2023326B042-1NegativeBaoting2020
159LK-19NegativeDing’an2023327B042-2NegativeBaoting2020
160LK-20NegativeDing’an2023328B055-2NegativeBaoting2020
161I027NegativeQionghai2022329B059-1NegativeBaoting2020
162N01NegativeLingshui2022330B059-2NegativeBaoting2020
163N06NegativeLingshui2022331B056-1NegativeBaoting2020
164N07NegativeLingshui2022332B056-2NegativeBaoting2020
165N012NegativeLingshui2022333B061-1NegativeBaoting2020
166N013NegativeLingshui2022334B069-1NegativeBaoting2020
167ZH-40NegativeLingshui2022335B069-2NegativeBaoting2020
168NLTNegativeLingshui2022
Note: a total of 10 leaf samples were positive in the detection using the developed nested PCR.

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Figure 1. Profile of 16S rDNA sequences amplified by nested PCR with universal primer P1/P7 followed by R16mF2/R16mR1, from areca samples. M: DL2000 DNA Marker; 1–19: areca samples; NC1: blank control; NC2: blank control of nested PCR; PC: ‘Candidatus Phytoplasma asteris’-related strain H-9.
Figure 1. Profile of 16S rDNA sequences amplified by nested PCR with universal primer P1/P7 followed by R16mF2/R16mR1, from areca samples. M: DL2000 DNA Marker; 1–19: areca samples; NC1: blank control; NC2: blank control of nested PCR; PC: ‘Candidatus Phytoplasma asteris’-related strain H-9.
Plants 14 02144 g001
Figure 2. Ratios of different sequences in 50 samples.
Figure 2. Ratios of different sequences in 50 samples.
Plants 14 02144 g002
Figure 3. This study conducted a preliminary screening of three pairs of internal primers. (A) 16S rDNA sequences amplified by nested PCR with primer HNP-1F/1R followed by HNP-2F/2R; (B) 16S rDNA sequences amplified by nested PCR with primer HNP-1F/1R followed by HNP-3F/3R; (C) 16S rDNA sequences amplified by nested PCR with primer HNP-1F/1R followed by HNP-4F/4R. M: marker DL2000; NC1: blank control; NC2: blank control for nested PCR; PC: ‘Candidatus Phytoplasma asteris’-related strain H-9.
Figure 3. This study conducted a preliminary screening of three pairs of internal primers. (A) 16S rDNA sequences amplified by nested PCR with primer HNP-1F/1R followed by HNP-2F/2R; (B) 16S rDNA sequences amplified by nested PCR with primer HNP-1F/1R followed by HNP-3F/3R; (C) 16S rDNA sequences amplified by nested PCR with primer HNP-1F/1R followed by HNP-4F/4R. M: marker DL2000; NC1: blank control; NC2: blank control for nested PCR; PC: ‘Candidatus Phytoplasma asteris’-related strain H-9.
Plants 14 02144 g003
Figure 4. Profile of 16S rDNA sequences amplified by nested PCR with primer HNP-1F/1R followed by HNP-2F/2R and HNP-3F/3R, respectively, for the specificity validation. (A) HNP-2F/2R, (B) HNP-3F/3R. M: marker DL2000; NC1: blank control; NC2: blank control for nested PCR; PC: ‘Candidatus Phytoplasma asteris’-related strain H-9; Lane 5: Burkholderia andropogonis (Robbsia andropogonis); Lane 6: Pantoea ananatis; Lane 7: healthy areca leaf sample; Lane 8: Areca palm velarivirus 1; Lane 9: APYL of 16SrI group; Lane 10: APYL of 16SrII group; Lane 11: ‘Candidatus Phytoplasma malaysianum’ strain SHM-1.
Figure 4. Profile of 16S rDNA sequences amplified by nested PCR with primer HNP-1F/1R followed by HNP-2F/2R and HNP-3F/3R, respectively, for the specificity validation. (A) HNP-2F/2R, (B) HNP-3F/3R. M: marker DL2000; NC1: blank control; NC2: blank control for nested PCR; PC: ‘Candidatus Phytoplasma asteris’-related strain H-9; Lane 5: Burkholderia andropogonis (Robbsia andropogonis); Lane 6: Pantoea ananatis; Lane 7: healthy areca leaf sample; Lane 8: Areca palm velarivirus 1; Lane 9: APYL of 16SrI group; Lane 10: APYL of 16SrII group; Lane 11: ‘Candidatus Phytoplasma malaysianum’ strain SHM-1.
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Figure 5. The HNP-1F/1R + HNP-2F/2R primer set was used, targeting the ‘Candidatus Phytoplasma asteris’-related strain H-9. (A) Annealing temperature optimized for the outer primer HNP-1F/1R. M: marker DL2000; NC: blank control. M: marker DL2000; NC: blank control. (B) Annealing temperature optimized for the inner primer HNP-2F/2R. M: marker DL2000; NC: blank control.
Figure 5. The HNP-1F/1R + HNP-2F/2R primer set was used, targeting the ‘Candidatus Phytoplasma asteris’-related strain H-9. (A) Annealing temperature optimized for the outer primer HNP-1F/1R. M: marker DL2000; NC: blank control. M: marker DL2000; NC: blank control. (B) Annealing temperature optimized for the inner primer HNP-2F/2R. M: marker DL2000; NC: blank control.
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Figure 6. Sensitivity test for nested PCR primer set of HNP-1F/1R and HNP-2F/2R. (A) Gel electrophoresis for the sensitivity of 16SrI phytoplasma using a developed nested PCR. M: marker DL2000; NC1: blank control; NC2: blank control for nested PCR. (B) Gel electrophoresis results for the sensitivity of 16SrII phytoplasma using developed nested PCR. M: marker DL2000; NC: blank control.
Figure 6. Sensitivity test for nested PCR primer set of HNP-1F/1R and HNP-2F/2R. (A) Gel electrophoresis for the sensitivity of 16SrI phytoplasma using a developed nested PCR. M: marker DL2000; NC1: blank control; NC2: blank control for nested PCR. (B) Gel electrophoresis results for the sensitivity of 16SrII phytoplasma using developed nested PCR. M: marker DL2000; NC: blank control.
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Figure 7. Comparison of bands amplified using universal primers (P1/P7 and R16mF2/R16mR1) with the primers developed in this study. (A) universal primers; (B) the primers developed in this study; M: marker DL2000; 1: blank control; 2: blank control for nested PCR; 3: healthy areca leaf sample; 4: ‘Candidatus Phytoplasma asteris’-related strain H-9; 5–34: areca leaf samples collected in the fields.
Figure 7. Comparison of bands amplified using universal primers (P1/P7 and R16mF2/R16mR1) with the primers developed in this study. (A) universal primers; (B) the primers developed in this study; M: marker DL2000; 1: blank control; 2: blank control for nested PCR; 3: healthy areca leaf sample; 4: ‘Candidatus Phytoplasma asteris’-related strain H-9; 5–34: areca leaf samples collected in the fields.
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Figure 8. This is a phylogenetic tree constructed by the neighbor-joining method based on partial fragments of the phytoplasma 16S rRNA gene amplified by HNP-2F/2R. The scale bar length represents the inferred changes in character states. Branch lengths are proportional to the inferred number of character state transitions. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.
Figure 8. This is a phylogenetic tree constructed by the neighbor-joining method based on partial fragments of the phytoplasma 16S rRNA gene amplified by HNP-2F/2R. The scale bar length represents the inferred changes in character states. Branch lengths are proportional to the inferred number of character state transitions. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.
Plants 14 02144 g008
Table 1. Primers for amplification of the 16S rDNA gene using nested PCR.
Table 1. Primers for amplification of the 16S rDNA gene using nested PCR.
PrimerPrimer Sequence (5′-3′)Annealing Temperature (°C)Target Fragment Length (bp)References
P1AAGAATTTGATCCTGGCTCAGGATT551800[27]
P7CGTCCTTCATCGGCTCTT[28]
R16mF2CATGCAAGTCGAACGGA501500[29]
R16mR1CTTAACCCCAATCATCGA
HNP-1FTTCTTGTTTTTAAAAGACCT441072This study
HNP-1RAAACTTGCGCTTCAGCT
HNP-2FTGTGGTCTAAGTGCAAT48429This study
HNP-2RCTGATAACCTCCACTGTGTT
HNP-3FTTCTTGTTTTTAAAAGACCT50837This study
HNP-3RATAACCTCCACTGTGTTTCT
HNP-4FAATGCTCAACATTGTGATGCT48652This study
HNP-4RAAACTTGCGCTTCAGCT
Table 2. Areca leaf samples that were used for comparison between the two methods.
Table 2. Areca leaf samples that were used for comparison between the two methods.
NoSampleCity/CountyYearNoSampleCity/CountyYear
1C002-1Tunchang202016D093-2Tunchang2020
2C007-2Tunchang202017ZY2-1Tunchang2020
3C013-2Tunchang2020187-4-2Tunchang2020
4C018Y-1Tunchang202019003-2Tunchang2020
5C018Y-2Tunchang202020B021-2Tunchang2020
6C021-1Tunchang202021B027-2Tunchang2020
7C023-1Tunchang202022B029-2Tunchang2020
8C026-1Tunchang202023B030-1Tunchang2020
9C026-2Tunchang202024B032-1Tunchang2020
10C031-2Tunchang202025B037-2Tunchang2020
11C042-1Tunchang202026B038-2Tunchang2020
12C064-1Tunchang202027B042-1Tunchang2020
1312-3-2Tunchang202028B042-2Tunchang2020
14D039-1Tunchang202029B055-2Tunchang2020
15D062-2Tunchang202030B059-1Tunchang2020
Table 3. Plant leaf samples used in specificity verification of nested PCR.
Table 3. Plant leaf samples used in specificity verification of nested PCR.
StrainPhytoplasma IdentifiedGenBank Accession NoHost PlantLocation
J069Ca. P. asteris’ 16SrIPV760294Areca palm Areca catechuWenchang, Hainan
S97Ca. P. asteris’ 16SrIPV760296Areca palm A. catechuWenchang, Hainan
T40Ca. P. asteris’ 16SrIPV760293Areca palm A. catechuWenchang, Hainan
H-9Ca. P. asteris’ 16SrIPV760299Periwinkle Catharanthus roseusWenchang, Hainan
A056Ca. P. australasiae’ 16SrIIPV760298Areca palm A. catechuWenchang, Hainan
W4Ca. P. australasiaticum’ 16SrIIPV760297Areca palm A. catechuWenchang, Hainan
SHM-1Ca. P. malaysianum’ 16SrXXXIIPV759645Trema tomentosaQionghai, Hainan
MW1-1Burkholderia andropogonisPV759631Areca palm A. catechuWenchang, Hainan
I027Areca palm velarivirus 1OQ423126Areca palm A. catechuWenchang, Hainan
TC-1Pantoea ananatisPV759632Areca palm A. catechuTunchang, Hainan
Table 4. Reference sequences used in this study.
Table 4. Reference sequences used in this study.
Reference StrainGenBank Accession NumberApplication
Ca. P. asteris’ 16SrIFJ998269Primer Design/Phylogenetic analysis
Ca. P. asteris’ 16SrIFJ694685Primer Design/Phylogenetic analysis
Ca. P. australasaticum’ 16SrIIOQ586085Primer Design
Areca catechu chloroplastNC_050163Primer Design
Burkholderia andropogonisNR_104960Primer Design
Pantoea ananatisMW174802Primer Design
Chrysophyllum albidumLC110196Primer Design
Curtobacterium citreumMF319766Primer Design
Curtobacterium luteumJX437941Primer Design
Sphingomonas yantingensisMF101149Primer Design
Bacillus cereus
(Robbsia andropogonis)
HQ833025Primer Design
Staphylococcus epidermidisCP040883Primer Design
Xanthomonas sacchariMN889285Primer Design
Xanthomonas campestrisJX415480Primer Design
Ca. P. aurantifolia’ WBDL 16SrIIU15442Primer Design
Ca. P. australasiae’ PpYC 16SrIIY10097Primer Design
Ca. P. pruni’ PX11CT1 rrnA 16SrIIIJQ044393Primer Design
Ca. P. ziziphi’ JWB-G1 16SrVAB052876Primer Design
Ca. P. ulmi’ EY1 16SrVAY197655Primer Design
Ca. P. rubi’ RuS 16SrVAY197648Primer Design
Ca. P. trifolii’ CP 16SrVIAY390261Primer Design
Ca. P. sudamericanum’ PassWB-Br3 16SrVIGU292081Primer Design
Ca. P. fraxini’ AshY1 16SrVIIAF092209Primer Design
Ca. P. luffae’ LfWB rrnA 16SrVIIIAF248956Primer Design
Ca. P. luffae’ LfWB rrnB 16SrVIIIAF353090Primer Design
Ca. P. phoenicium’ AlmWB 16SrIXAF515636Primer Design
Ca. P. pyri’ PD1 16SrXAJ542543Primer Design
Ca. P. spartii’ SpaWB 16SrXX92869Primer Design
Ca. P. mali’ AP15 16 SrXAJ542541Primer Design
Ca. P. oryzae’ RYD-Th 16SrXIAB052873Primer Design
Ca. P. cirsii’ CirYS 16SrXIKR869146Primer Design
Ca. P. solani’ STOL11 16SrXIIAF248959Primer Design
Ca. P. japonicum’ JPH 16SrXIIAB010425Primer Design
Ca. P. convolvuli’ BY-S5711 16SrXIIJN833705Primer Design
Ca. P. hispanicum’ MPV 16SrXIIIAF248960Primer Design
Ca. P. meliae’ ChTY-Mo3 16SrXIIIKU850940Primer Design
Ca. P. cynodontis’ BGWL-C1 16SrXIVAJ550984Primer Design
Ca. P. brasiliense’ HibWB26 16SrXVAF147708Primer Design
Ca. P. graminis’ SCYLP 16SrXVIAY725228Primer Design
Ca. P. caricae’ PAY 16SrXVIIAY725234Primer Design
Ca. P. castaneae’ CnWB 16SrXIXAB054986Primer Design
Ca. P. omanense’ IM-1 16SrXXIXEF666051Primer Design
Ca. P. costaricanum’ SoyST1c1 16SrXXXIHQ225630Primer Design
Ca. P. malaysianum’ MaPV 16SrXXXIIOP237525Primer Design
Ca. P. malaysianum’ MaPV 16SrXXXIIEU371934Primer Design
Ca. P. wodyetiae’ FPYD Bangi-2 16SrXXXVIKC844879Primer Design
Acholeplasma laidlawiiCP000896Phylogenetic analysis
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MDPI and ACS Style

Ge, H.; Meng, X.; Lin, Z.; Jan, S.; Song, W.; Qin, W.; Tang, Q.; Zhu, X. Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China. Plants 2025, 14, 2144. https://doi.org/10.3390/plants14142144

AMA Style

Ge H, Meng X, Lin Z, Jan S, Song W, Qin W, Tang Q, Zhu X. Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China. Plants. 2025; 14(14):2144. https://doi.org/10.3390/plants14142144

Chicago/Turabian Style

Ge, Huiyuan, Xiuli Meng, Zhaowei Lin, Saad Jan, Weiwei Song, Weiquan Qin, Qinghua Tang, and Xiaoqiong Zhu. 2025. "Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China" Plants 14, no. 14: 2144. https://doi.org/10.3390/plants14142144

APA Style

Ge, H., Meng, X., Lin, Z., Jan, S., Song, W., Qin, W., Tang, Q., & Zhu, X. (2025). Specific Nested PCR for the Detection of 16SrI and 16SrII Group Phytoplasmas Associated with Yellow Leaf Disease of Areca Palm in Hainan, China. Plants, 14(14), 2144. https://doi.org/10.3390/plants14142144

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