Pepper mild mottle virus (PMMoV) belongs to the species Tobamovirus capsicai in the genus Tobamovirus (family Virgaviridae), has emerged as an important pathogen, significantly impacting pepper yield and quality. The objectives of this study are to investigate its phylogeographic history and evolutionary dynamics, and to lay the foundation for accurate monitoring and scientific prevention and control

A specific primer pair flanking the PMMoV coat protein (CP) gene was designed. The CP gene sequences of 28 randomly selected PMMoV isolates from Fujian Province were subsequently amplified and cloned. In addition to the newly obtained sequences, all publicly available CP sequences from GenBank with known collection timestamps and geographical origins were assembled into a final dataset of 255 sequences. After confirming a sufficient temporal signal via a date-randomized test (DRT), a structured coalescent-based Bayesian phylodynamic framework (MultiTypeTree) was employed to reconstruct the evolutionary history and spatial dissemination of the virus.

A target fragment with the expected size was obtained from all 28 PMMoV-positive samples selected in this study. Their CP sequences share＞98% nucleotide identity with known PMMoV isolates. The DRT showed no overlap in the 95% confidence intervals of substitution rates between the real and randomized datasets, confirming a reliable temporal signal for Bayesian molecular dating. Phylogenetic analysis estimated a mean substitution rate of 9.24×10^-4 substitutions/site/year (95% CI: 6.20×10^-4-1.01×10^-3 substitutions/site/year) for the PMMoV CP, which is comparable to rates observed in animal RNA viruses, indicating its rapid evolutionary dynamics. The time to the most recent common ancestor (tMRCA) was dated to 1941 (95% CI: 1921-1957). The root of the maximum clade credibility (MCC) tree was placed in Europe, identifying this region as the most probable origin of contemporary global PMMoV isolates. Temporal migration analysis revealed multiple dispersal routes from Europe to other regions, underscoring its role as a central hub in the virus’s global dissemination. In addition to cross-regional transmission, frequent local spread of PMMoV was observed within regions. Bayesian skyline plot (BSP) analysis further revealed distinct historical population dynamics: the South American population underwent a significant expansion before stabilizing, whereas those in most other regions remained relatively stable over the long term.

Europe served as the most likely source and a critical dissemination hub for the global spread of PMMoV. This study provides a foundation for understanding the molecular epidemiology of PMMoV and informs the development of future disease management strategies.

The study aims to establish a novel visual detection technique for prunus necrotic ringspot virus (PNRSV) by combining reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR/Cas12a system (RT-RAA-CRISPR/Cas12a).

Primers with high amplification efficiency and strong specificity were designed and selected based on the conserved regions of the coat protein (CP) gene of PNRSV. The detection conditions, including primer, probe concentration, temperature, and reaction time were optimized to develop a visual detection method for PNRSV by RT-RAA-CRISPR/Cas12a technology. The specificity of this method was evaluated by detecting PNRSV and common Prunus viruses, including plum pox virus (PPV), apple mosaic virus (ApMV), cucumber mosaic virus (CMV), potato virus X (PVX), and potato virus Y (PVY). The total RNAs from PNRSV-infected fruit were diluted in 10-fold gradients, then RT-PCR, RT-RAA and RT-RAA-CRISPR/Cas12a were performed to compare the sensitivity of the three methods. The RT-RAA-CRISPR/Cas12a and RT-PCR methods were used to detect 31 peach fruit test samples suspected to be infected with the virus collected at the port to verify the practicability of the visual detection method.

The RT-RAA-CRISPR/Cas12a-based visual detection method for PNRSV was successfully established. The optimized working concentrations were as follows: RT-RAA-PNRSV-F2/R2 primers at 0.4 μmol·L^-1, fluorescent reporter (FQ) at 800 nmol·L^-1, CRISPR-Cas12a at 200 nmol·L^-1, and PNRSV-crRNA (CRISPR RNA) at 240 nmol·L^-1, the reaction conditions were performed at 41 ℃ for 45 min. This method showed high specificity for PNRSV and had no cross-reaction with other common Prunus viruses. The limit of detection for PNRSV RNA in peach fruit samples reached 3.06 pg·μL^-1 and 306 fg·μL^-1 using RT-RAA and RT-RAA-CRISPR/Cas12a methods, respectively, showing the sensitivity of RT-RAA-CRISPR/Cas12a was 10 times higher than that of RT-RAA and RT-PCR. Among the 31 tested peach fruit samples at the port, 14 positive samples were identified by RT-PCR, while 15 positive samples were found by RT-RAA-CRISPR/Cas12a, indicating a high level of consistency between the two methods.

The RT-RAA-CRISPR/Cas12a visual detection method for PNRSV has been established. It is characterized by simplicity, rapidity, high sensitivity, high specificity, and visual readability, making it well-suited for rapid on-site detection of PNRSV.

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.

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.

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.

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.