关键词: 茶树病毒, 病原鉴定, 多重PCR, 分子检测, 福建省

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