Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (4): 698-710.doi: 10.3864/j.issn.0578-1752.2024.04.006

• PLANT PROTECTION • Previous Articles     Next Articles

Identification of Tea Plant Viruses in Fujian Province and Establishment of Multiplex PCR Detection Assay

CHEN XiHong1,2(), CAI Wei3, YU Yun2, LI Min2, WANG NianWu3, DU ZhenGuo1, SHEN JianGuo2(), GAO FangLuan1()   

  1. 1 Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002
    2 Technology Center of Fuzhou Custom District/Fujian Key Laboratory for Technology Research of Inspection and Quarantine, Fuzhou 350001
    3 Comprehensive Technical Service Center of Rongcheng Customs District, Fuqing 350300, Fujian
  • Received:2023-10-07 Accepted:2023-11-05 Online:2024-02-16 Published:2024-02-20
  • Contact: SHEN JianGuo, GAO FangLuan

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Abstract:

【Objective】This study endeavors to explore the species diversity and prevalence of viruses within tea plantations in Fujian Province. Furthermore, it aims to devise a multiplex PCR assay capable of swiftly detecting multiple viruses simultaneously.【Method】From 2019 to 2023, a total of 1 869 samples were gathered from tea plants displaying virus-like symptoms, including chlorosis, blotch, and necrosis, across nine regions, encompassing Fuzhou, Nanping, Ningde, Quanzhou, Zhangzhou, Xiamen, Sanming, Putian, and Longyan cities. High-throughput sequencing technology, combined with PCR and RT-PCR detection methods, was used to identify the pathogens causing viral diseases in tea plants. Specific target fragments amplified by PCR were cloned, sequenced, and subjected to phylogenetic analysis. In addition, specific primers were designed based on reported virus sequences in GenBank, and a multiplex PCR assay was established for detecting major tea plant viruses in Fujian Province by optimizing reaction conditions, including annealing temperature, primer concentration, and cycle number. The assay’s specificity, sensitivity, and practical application were subsequently determined.【Result】Three viruses were detected in tea plant samples from Fujian tea plantations, with detection rates ranked as follows: oil tea associated geminivirus (OTaGV) at 48.90%, tea plant necrotic ring blotch virus (TPNRBV) at 26.75%, and camellia cryptic virus 1 (CCV1) at 17.98%. Among the 1 258 samples that tested positive for viruses, 807 samples were infected with only one of the three viruses (OTaGV, TPNRBV, or CCV1), corresponding to detection rates of 37.20%, 21.38%, and 5.56%, respectively. The remaining 451 samples were co-infected with two or three viruses, resulting in a co-infection rate of 35.85%. The most common co-infection types were OTaGV+CCV1 (17.49%), TPNRBV+CCV1 (0.40%), TPNRBV+OTaGV (14.71%), and OTaGV+CCV1+TPNRBV (3.26%). Geographically, OTaGV was distributed in all nine regions, with the highest detection rate in Zhangzhou at 96.77%. CCV1 was present in eight regions (excluding Xiamen), with the highest detection rate in Sanming at 66.00%. TPNRBV was found in five regions (Fuzhou, Nanping, Ningde, Quanzhou and Zhangzhou), with the highest detection rate in Quanzhou at 79.13%. Among the nine regions, only OTaGV was detected in Xiamen, OTaGV and CCV1 were detected in Sanming, Putian, and Longyan, and all three viruses were detected in the other five regions. The viral co-infection rate was highest in Zhangzhou at 85.00% and lowest in Ningde at 23.03%. Phylogenetic tree construction based on CCV1 and TPNRBV gene sequences indicated that the CCV1 isolate FW obtained in this study had the closest resemblance to the reported Fujian isolate FJ_SH104 (GenBank accession number: ON807095), and the TPNRBV isolate FU had the closest resemblance to the Fujian isolate QZHA92 (GenBank accession number: OQ948454). The established multiplex PCR detection assay exhibited strong specificity, as it only amplified specific target fragments of OTaGV, TPNRBV, and CCV1, while no amplification product was observed in other viruses or healthy tea plant samples. The lowest sensitivity of the multiplex PCR was detecting OTaGV and TPNRBV at dilutions of 10-4 and CCV1 at a dilution of 10-3. The multiplex PCR assay successfully detected all three viruses in 60 disease samples collected from tea plantations, and the results were consistent with those obtained by single PCR detection.【Conclusion】OTaGV, CCV1, and TPNRBV are the major virus species in tea plants in the Fujian, with OTaGV being reported for the first time in this province. This study also revealed that OTaGV can infect tea plants. Among the detected viruses, OTaGV has the widest distribution, followed by CCV1 and TPNRBV. Presently, tea plants in Fujian are still mainly infected by single virus infections. However, viral co-infections are prevalent, showcasing combinations such as TPNRBV+CCV1, TPNRBV+OTaGV, CCV1+OTaGV, and OTaGV+CCV1+TPNRBV. The established multiplex PCR assay exhibits strong specificity and high sensitivity, making it suitable for the rapid detection of the three viruses (OTaGV, CCV1 and TPNRBV) in tea plantations. Taken together, the findings obtained in this study will provide a theoretical basis and technical support for prevention and control of viral diseases in tea plants in Fujian Province.

Key words: tea plant virus, pathogen identification, multiplex PCR, molecular detection, Fujian Province

Table 1

Primer used for detection of tea plant virus in Fujian Province"

病毒
Virus		 引物序列
Primer sequence (5′-3′)		 扩增区域
Region		 目的片段大小
Expected size (bp)		 退火温度
Tm (℃)		 参考文献
Reference
OTaGV F: ATATTTGGCACGTGGATCGCAAA MP/NSP 738 57 [16]
R: AGGGGCACAGAAATAGACCGTTT
CCV1 F: CATCTACAGAAGCTCCTGAAGG mCP 707 54 /
R: CCATGCAGATCCAGATTTCACAC
TPNRBV F: CCTTATGTCGACAGTTGCTAC P29/P24 315 54 /
R: CTAAGTCATCCATATGTGTGG
TPLPV F: AAGGTGGCGAGGTCAGTTTCAGTTCA Mtr-Hel 513 52 [8]
R: TCCCCATAGGTTCATCTTGTAGCAGTCG
GLRaV-7 F: GGTTTGAAATGGAAAACATGATAC P61 518 53 [17]
R: CACGTTTAGTTGAATTGGTTAATC
TOCaTV1 F: AGTGTGGTGAGCCTGAGTTATC CP 725 55 [18]
R: GAAGCAAGGGACATTGACCACA
CLGV F: AGCAATCAGTCCACATGTATCGC Polyprotein 662 55 [19]
R: ACATGGGTCCACCACTACTTCC

Fig. 1

Main symptoms caused by tea plant viruses"

Fig. 2

PCR/RT-PCR amplification results of three types of tea plant viruses"

Fig. 3

The detection results of OTaGV, CCV1 and TPNRBV in some susceptible samples of tea plant from Fujian Province"

Table 2

Detection results of tea plant virus in each region of Fujian Province"

病毒种类
Virus species		 检出率Detection rate (%)
福州
Fuzhou		 南平
Nanping		 宁德
Ningde		 泉州
Quanzhou		 漳州
Zhangzhou		 厦门
Xiamen		 三明
Sanming		 莆田
Putian		 龙岩
Longyan		 合计
Total
OTaGV 57.25 25.00 96.73 55.36 96.77 90.77 94.00 87.65 5.12 48.90
CCV1 31.40 10.37 24.18 7.54 59.68 0 66.00 32.10 3.50 17.98
TPNRBV 1.69 57.32 7.19 79.13 33.87 0 0 0 0 26.75
TPLPV 0 0 0 0 0 0 0 0 0 0
GLRaV-7 0 0 0 0 0 0 0 0 0 0

Table 3

Occurrence of single infection and co-infection by tea plant viruses in each region of Fujian Province"

地区
Area		 检出率Detection rate (%)
OTaGV CCV1 TPNRBV OTaGV+
CCV1		 TPNRBV+
CCV1		 TPNRBV+
OTaGV		 OTaGV+CCV1
+TPNRBV
福州Fuzhou 51.76 15.14 1.41 30.63 0 1.06 0
南平Nanping 6.61 2.20 59.47 8.37 2.20 18.94 2.20
宁德Ningde 74.34 1.97 0.66 16.45 0 0.66 5.92
泉州Quanzhou 14.42 1.84 39.57 0 0 38.04 6.13
漳州Zhangzhou 15.00 0 0 50.00 0 23.33 11.67
厦门Xiamen 100.00 0 0 0 0 0 0
三明Sanming 29.79 0 0 70.21 0 0 0
莆田Putian 63.38 0 0 36.62 0 0 0
龙岩Longyan 59.38 40.63 0 0 0 0 0
合计Total 37.20 5.56 21.38 17.49 0.40 14.71 3.26

Fig. 4

Phylogenetic tree of CCV1 and TPNRBV based on maximum likelihood (ML) algorithm Numbers above branches on the left indicated Ultrafast bootstrap values (only shown>75%). The viral isolates from current study were marked with black solid circle"

Fig. 5

Optimization and specificity results of multiplex PCR"

Fig. 6

Sensitivity of multiplex PCR"

Table 4

Multiplex PCR detection results of tea plantation samples"

病毒
Virus		 阳性样品数
Number of positive samples		 检出率
Detection rate
(%)
OTaGV 16 26.7
CCV1 2 3.3
TPNRBV 1 1.7
OTaGV+CCV1 27 45.0
TPNRBV+CCV1 1 1.7
TPNRBV+OTaGV 8 13.3
OTaGV+CCV1+TPNRBV 3 5.0
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