A Multiyear Survey and Identification of Pepper- and Tomato-Infecting Viruses in Yunnan Province, China

During pepper and tomato production seasons in 2013–2017, large-scale virus disease surveys were conducted in different regions of Yunnan Province, China. A total of 1,267 pepper and tomato samples with various virus-like symptoms were collected and analyzed for virus infections through dot enzyme-linked immunosorbent assay (dot-ELISA), polymerase chain reaction (PCR), and reverse-transcription (RT)-PCR. The detection results showed that 19 different viruses were present in about 50.9% of the assayed samples, and among these viruses, seven viruses were found in both pepper and tomato samples. Mixed infections with two to three of the 15 identified mixed infection types were found in the pepper samples and 10 identified mixed infection types were found in the tomato samples. Among the infected samples, Tomato spotted wilt orthotospovirus (TSWV) was the most common virus, with a detection rate of about 20.0% followed by Pepper vein yellows virus (PeVYV, 13.0%). This survey revealed for the first time that pepper is a natural host of Tobacco vein distorting virus (TVDV) worldwide and tomato is a natural host of Potato leafroll virus (PLRV) in China. PeVYV, Tobacco mild green mosaic virus (TMGMV) and Wild tomato mosaic virus (WTMV) were first time found in pepper and Tomato mottle mosaic virus (ToMMV) and Chilli veinal mottle virus (ChiVMV) were first time found in tomato in Yunnan Province. Finally, the virus incidences were higher in Kunming, Yuxi, Chuxiong, and Honghe region than other regions.


INTRODUCTION
Pepper (Capsicum annuum) and tomato (Solanum lycopersicum) are two economically important solanaceous vegetable crops in China and many other countries. With the rapid increase of production areas and growth of new cultivars in the recent years, outbreaks of virus disease have become common in solanaceous crops, leading to serious yield losses. Approximately 76 and 136 viruses have been reported for pepper and tomato plants throughout the world, respectively (Dong et al., 2008;Hanssen et al., 2010;Kenyon et al., 2014;Li et al., 2014;Feng et al., 2017;Mauricio-Castillo et al., 2017;Olaya et al., 2017;Wang et al., 2017a;Liu et al., 2019). To date, 28 RNA and three DNA viruses have been reported for pepper crops, and 22 RNA and 12 DNA viruses have been reported for tomato crops in China. Many studies have suggested that Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV) were the most predominant viruses in both pepper and tomato fields in China (Dong et al., 2008;Tao and Zhou, 2008;Zhang et al., 2009Zhang et al., , 2017aQing et al., 2010;Wang and Huang, 2010;Zhang, 2010;Ge et al., 2012;Ruan, 2012;Ding et al., 2013;Li et al., 2014;Wang et al., 2014Wang et al., , 2017bZhao et al., 2014;Guo et al., 2015;Padmanabhan et al., 2015;Yuan et al., 2015;Feng et al., 2017;Liu et al., 2019).
Yunnan Province is located at the southwest border of China, neighboring Laos, Myanmar, and Vietnam. The low-latitude and high-elevation geographic features create a multiclimate weather, suitable for many vegetable productions, including many solanaceous vegetable crops. The total vegetable production acreage in Yunnan Province in 2017 had reached about 1.1 million ha and produced approximately 20.78 million tons of vegetables products, valued about 7.15 billion United States dollars. Pepper and tomato are the two major solanaceous vegetable crops in Yunnan Province. Due to virus diseases, some pepper and tomato fields in this province had total yield losses in the recent years, resulting in a quick increase of pesticide applications, insect tolerance to pesticides, and environment pollutions (Chen and Liu, 2013;Bu et al., 2014).
Identification of viruses and their epidemiology in pepper and tomato fields are critical for the development of effective managements for the viruses. Although field surveys of virus diseases in pepper and tomato fields had been reported for other provinces of China (Wen et al., 2009;Wang et al., 2014Wang et al., , 2017bGuo et al., 2015;Wang L. S. et al., 2015;Gao et al., 2016;Zhang et al., 2017b), similar survey has not been done in Yunnan Province. Through this study, we have found that virus infections in these two crops are more common in Yunnan Province than that in other Chinese provinces.

Field Survey and Sample Collection
Leaves and fruits showing virus-like symptoms were first recorded using a Sony camera (DSC-RX100M5, Sony), harvested, and then stored separately in plastic sampling bags till use. This survey focused on pepper and tomato crops in 38 counties belonging to 12 different cities or autonomous regions from April to October in 2013and April in 2017 More tissue samples were collected from the surveyed fields with higher disease incidences and severer disease symptoms, while fewer samples were collected from the fields with lower disease incidences and milder disease symptoms ( Table 1). The collected samples were analyzed for virus infections immediately or stored at −80 • C or −20 • C until use.

Serological Tests
Serological tests were done using the dot enzyme-linked immunosorbent assay (dot-ELISA) as described (Xie et al., 2013) with slight modifications. Briefly, each sample (0.1 g) was placed in a precooled 2-ml tube with five 3 mm and one 6 mm stainless-steel beads. After addition of 1 ml 0.01 M phosphatebuffered saline (PBS, 0.13 M NaCl, 0.003 M KCl, 0.008 M Na 2 HPO 4 , 0.001 M KH 2 PO 4 , pH 7.4), the tube was placed at −80 • C for 5 min, and then ground using a high-throughput tissue homogenizer (Scientz-48, Ningbo Scientz) set at 60 Hz for 2 × 90 s (twice). The tube was centrifuged at 8,000 × g and 4 • C for 5 min, and the supernatant in each tube was blotted onto a nitrocellulose membrane (2 µl per dot, three dots per sample) followed by air-drying. The membrane was incubated for 30 min in 10 ml PBST buffer ( Total Nucleic Acid Extraction, Polymerase Chain Reaction and Reverse Transcription-PCR Total nucleic acid was extracted from tissue samples using a modified CTAB method (Li et al., 2008a). Degenerated and specific primers for polymerase chain reaction (PCR) or reverse transcription (RT)-PCR were as reported (Ha et al., 2006(Ha et al., , 2008Hirota et al., 2010;Zhou et al., 2011;Zhang et al., 2017b;Li et al., 2018aLi et al., ,b,c, 2020Liu et al., 2019) or made in this study (Supplementary Table 1 Each PCR reaction was 10 µl (5 µl Green Taq Mix (Vazyme Biotech. Co., Ltd., China), 0.2 µl each primer (10 µM each), 1 µl cDNA (RT-PCR) or total nucleic acid (PCR), and 3.6 µl nucleasefree H 2 O), and the reaction condition was initial denaturation at 94 • C for 5 min, 35 cycles (for reactions using specific primers) or 40 cycles (for reactions using degenerated primers) of 94 • C for 30 s, 40-55 • C for 30 s, and 72 • C for 1 min, followed by the final extension at 72 • C for 7 min. The PCR or RT-PCR products were visualized in 1% agarose gels through electrophoresis in 0.5 × TBE buffer. The resulting agarose gels were stained with 5 µl Gold View as instructed (Solarbio Science & Technology Co., Ltd., Beijing, China) and examined under an UV illumination GenoSens1850 system.

Field Survey Result
Pepper and tomato fields in 38 counties, belonging to 12 different cities or autonomous regions (refer to as regions thereafter) were surveyed for virus diseases in 2013, 2014, 2015, 2016, and 2017 ( Table 1 and Figure 1A). A total number of 821 pepper samples and 446 tomato samples were collected during the surveys. The most common symptoms on the infected pepper and tomato plants were mottling or mosaic, necrosis, yellowing, and distortion in leaves. The survey results showed that most of the surveyed pepper and tomato fields in Chuxiong, Yuxi, Honghe, and Kunming regions had a near 100% virus disease incidence, resulting in complete production failures (Figures 1B,C). Most surveyed fields in Wenshan, Dehong, Dali, and Baoshan regions had 10-25% virus disease incidences, while diseased plants in most surveyed fields in the Lijiang, Zhaotong, Qujing, and Puer regions were sporadic.
The virus detection result also showed that TSWV was the most common virus (253 out of 1,267 assayed samples) in Yunnan Province, followed by Pepper vein yellows virus (PeVYV) and CMV ( Figure 2B and Table 2). Further assays showed that virus population structures in the pepper and tomato fields in these regions varied each year ( Figure 2C). For instance, unlike other viruses, TSWV, PeVYV, CMV, and PMMoV were detected in each year in the surveyed pepper and tomato fields, and the incidence of TSWV in 2013, 2014, 2015, and 2016 was at 42.1, 30.3, 59.2, and 4.7%, respectively (Table 2 and Figure 2C).

Geographical Distributions of Viruses
To determine the geographical distributions of these viruses in Yunnan Province, we analyzed the field survey and virus detection data. The result showed that the virus detection rates in Kunming, Yuxi, Honghe, and Chuxiong regions were higher, ranging from 49.3 to 69.6% (Figure 3A), and also had much higher virus incidences in fields. Dehong and Dali regions also had high virus detection rates (62.5 and 50.0%, respectively), but the virus incidences in fields were not high; their high virus detection rates were due possibly to the less number of samples. For virus population structures, a total of 13 viruses were detected in the pepper and tomato samples collected from Chuxiong region, followed by 10 viruses in the samples from Yuxi region, eight viruses in the samples from Honghe region, six viruses in the samples from Kunming region, four viruses in the samples from Dehong region, and two viruses in the samples from Dali region ( Figure 3B). Only one virus was detected in the samples from Baoshan, Wenshan, Lijiang, and Zhaotong regions. No virus was found in the samples from Qujing and Puer regions     RNA viruses, TSWV and PeVYV were the most predominant viruses and were found in 20.5 and 20.1% of the assayed pepper samples, respectively ( Figure 4A). In this study, pepper was first time found as a natural host of TVDV (GenBank accession no. MN889854). In addition, PeVYV (GenBank accession no. MN889851), TMGMV (GenBank accession no. MN889852), and WTMV (GenBank accession no. MN889853) were first time found in the pepper fields in Yunnan Province. Among the 49 pepper-infecting CMV isolates, 48 were CMV I subgroup isolates and one was CMV II subgroup isolate. Of the 429 infected pepper samples, 51 samples were coinfected with two or three different viruses (i.e., CMV + TSWV, CMV + PeVYV, CMV + ToMMV, CMV + TVDV, CMV + WTMV, TSWV + PeVYV, TSWV + BBWV2, TMV + ToMV, ToMV + PMMoV, TMGMV + PMMoV, PeVYV + TVDV, CMV + ToMMV + PMMoV, CMV + TSWV + PeVYV, CMV + TVDV + PeVYV, and TMV + ToMV + PeVYV) (Supplementary Table 2). Among these mixed infections, CMV + TSWV and TSWV + PeVYV were most common combinations. The mixed infection of CMV + TSWV occurred mainly in Chuxiong, Honghe, Yuxi, and Kunming regions, while the mixed infection of TSWV + PeVYV occurred only in Honghe and Yuxi regions. Also, among these 15 co-infected types, eight had CMV and four contained TSWV (Supplementary Table 2).

Distributions of Pepper-Infecting Viruses
TSWV was the most common virus in Kunming, Yuxi, and Chuxiong regions, while PeVYV was the most dominant virus in Honghe, Dehong, Baoshan, and Wenshan regions ( Figure 4B). In Dali region, the most common virus was ChiVMV, while only PMMoV or TSWV was found in the pepper fields in Zhaotong and Lijiang regions ( Figure 4B). The average detection rates of viruses in pepper samples collected in 2013, 2014, 2015, and 2016 were 77.7, 56.7, 75.5, and 38.8%, respectively. In general, the detection rate of TSWV in these years showed a downward trend, while the trend of PeVYV was in increase, especially in 2016 ( Figure 4B).

Distributions of Tomato-Infection Viruses
A total of 10 RNA viruses and three DNA viruses were detected in 216 of the 446 assayed tomato samples (Figure 5A). The identified RNA viruses were TSWV, Tomato chlorosis virus (ToCV), Tomato zonate spot orthotospovirus (TZSV), CMV, ToMV, TBTV, ToMMV, ChiVMV, PMMoV, and Potato leafroll virus (PLRV), and the identified DNA viruses were Tomato yellow leaf curl virus (TYLCV), Tomato yellow leaf curl Thailand virus (TYLCTHV), and Papaya leaf curl China virus (PaLCuCNV). Among these viruses, TSWV and TYLCV were most common viruses found in the tomato samples. In this survey, PLRV (GenBank accession no. MN894826) was first time found in the infected tomato samples in China, while ToMMV (GenBank accession no. MN894824) and ChiVMV (GenBank accession no. MN894825) were first time found in the tomato samples in Yunnan Province. It is noteworthy that all the CMV isolates detected in the tomato samples belonged to the CMV II subgroup.
The dominant tomato-infecting viruses in different regions varied. For instance, TSWV was the most common tomatoinfecting virus in Kunming region from 2013 to 2016, whereas CMV and TYLCV were the most common viruses in Yuxi and Chuxiong regions, respectively ( Figure 5B). In 2013 and 2014, virus diseases were more common in Kunming, Yuxi, and Chuxiong regions, Honghe region in 2013, and Dehong region in 2016 ( Figure 5B).

Correlation Between Disease Symptoms and the Causal Viruses
In this study, the most common disease symptoms observed on the infected pepper and tomato plants were leaf mottling or mosaic, distortion, necrosis and yellowing, and plant stunting. To investigate the potential correlations between disease symptoms and the 19 identified viruses, we compared disease symptom and the virus identification data obtained in this study. The result indicated that the correlations were complex. Same virus often caused different disease symptoms in these two host plants of different varieties at different growth stages and in different infection seasons. In contrast, some different viruses caused similar disease symptoms in different infected pepper or tomato plants of the same variety. Furthermore, disease symptoms caused by different combinations of viruses in the pepper and tomato plants also varied and were influenced by the environmental conditions. Therefore, disease symptoms shown on infected plants cannot be directly linked to the causal virus(es). However, disease symptoms caused by a single certain virus in a particular host plant were similar, including blistering and ring spots in the TSWV-infected leaves or leaf distortion (i.e., shoestring) in CMV-infected leaves. Disease symptoms caused by viruses belonging to the same genus (i.e., TMV, ToMV, ToMMV, PMMoV, and TMGMV) were similar (i.e., mottling and mosaic in leaves).

DISCUSSION
In this study, we surveyed pepper and tomato fields in different regions in Yunnan Province, China ( Figure 1A). The result showed that the disease severities in different regions varied significantly. A total of 19 viruses were identified in assayed pepper and tomato samples collected in this study (Figures 2A,B). This number was higher than that previously reported for other regions in China (Wen et al., 2009;Wang et al., 2014Wang et al., , 2017bGuo et al., 2015;Wang L. S. et al., 2015;Gao et al., 2016;Zhang et al., 2017b) and indicated that the virus population structures in the pepper and tomato fields in this region were more complex than that in the other regions of China. Our result showed that TSWV, PeVYV, CMV, and PMMoV occurred every year in the pepper and tomato fields in this province from 2013 to 2016 ( Figure 2C). Among these four viruses, the occurrence of TSWV and CMV showed a decline trend, while the occurrence of PeVYV was gradually increasing. The occurrence of PMMoV maintained relatively stable from 2013 to 2016. Other viruses occurred sporadically in this period. During this survey, 13 viruses were detected in 2016 (Figure 2C), which can be useful for the design and development of pepper and tomato virus disease management strategy in this region.
The occurrence of TSWV in the pepper and tomato fields in 2013-2016 showed a downward trend (Figures 2C, 4B, 5B), probably due to the new control strategy designed for this virus in this area since 2014 , which indicated that timely prevention and control was effective for virus disease management. The virus distributions in the pepper and tomato fields observed in the study (Figures 4B, 5B) revealed that virus diseases were more common in Chuxiong, Yuxi, Kunming, and Honghe regions than other regions, and the virus population structures in different surveyed regions varied (Figures 4B, 5B). For example, TSWV was the most common virus in Kunming and Yuxi regions, while PeVYV was a more common virus in Honghe, Baoshan, and Wenshan regions. Because different viruses are transmitted via different ways, management strategies for different regions should also vary accordingly. For example, applications of insect pesticides and resistant pepper and tomato varieties should be used in Kunming, Dehong, Honghe, Dali, and Baoshan regions because most dominant viruses in these regions are transmitted by insect vectors (Figure 3B). In contrast, a more integrated management measure should be used in Chuxiong region because this region has multiple viruses but none of them is prominent (Figure 3B).
Several recent studies have shown that CMV, tobamoviruses (i.e., TMV, ToMV, and/or PMMoV), and TYLCV were the major viruses in pepper and tomato fields in different provinces of China (Wen et al., 2009;Wu et al., 2013;Wang et al., 2014Wang et al., , 2017bGuo et al., 2015;Wang L. S. et al., 2015;Gao et al., 2016;Liu et al., 2019). The results presented in this study showed that the virus population structures in the pepper and tomato fields in Yunnan Province changed in these years compared with an earlier report (Zhang et al., 1998). In that report, Zhang et al. (1998) had indicated that the main viruses in the pepper and tomato fields in this province were CMV and TMV. Due to the limitation of detection technology in that time, we cannot rule out the possibility of existence of other viruses in that study. TSWV is currently the most important virus in both pepper and tomato fields in Yunnan Province (Figures 4A, 5A) and has severe pathogenicity (Figures 1B,C) and very wide host range Xiao et al., 2015). The infection rates of CMV, TMV, ToMV, PMMoV, and TYLCV in Yunnan Province have declined in recent years. This reduction may be caused by the fact that the corresponding control measures such as virusresistant varieties were made in the management of these viruses based on the previous reports in the province (Zhang et al., 1998;Zhang, 2010). This report indicated that regular survey of the virus diseases was very important in management of viral diseases. Currently, CMV II subgroup isolates are more common in Yunnan Province than in other areas of China. Although both CMV I and II subgroup isolates are present in Yunnan Province, CMV I subgroup isolates are more common in the pepper fields, while CMV II subgroup isolates are more common in the tomato fields. The dominance of CMV I subgroup isolates in the pepper fields is consistent with the report published previously (Li et al., 2008b). The dominance of CMV II subgroup isolates in tomato fields has not been reported previously in other regions of China. It was reported that CMV I subgroup isolates were common in other vegetable crops in China (Chen et al., 2016) and CMV II subgroup isolates were common in the tobacco, lily, and tomato in Yunnan Province (Li et al., 2000;Zhao et al., 2007).
TBTV and TVDV were known to infect tobacco to cause bushy top disease (Wang D. Y. et al., 2015), it may be a challenge for the prevention and control of tobacco bush top disease, since these two viruses are newly identified in pepper and/or tomato fields in Yunnan Province (Li et al., 2018a). PeVYV is a new virus in the pepper fields in this province and is gradually becoming a dominant virus (Figure 4A). Its infection rate in pepper fields in 2016 has reached 19.8% (Figure 2C). The first report of ToMMV in the pepper fields in Yunnan Province was in 2014 (Li et al., 2014) and is now in the pepper, tomato, and eggplant in other parts of China (Chai et al., 2018;Li et al., 2020). Because ToMMV can cause severe diseases in many crops and its infection rate in vegetable crops is gradually increasing in China (Sui et al., 2017;Nagai et al., 2019;Li et al., 2020), it may become a devastating virus in many vegetable crops in China. We consider that more investigations are needed to identify new viruses so that specific virus resistance can be integrated in the pepper and tomato breeding programs.
In this survey, no virus was detected in some samples with virus-like symptoms. It is possible that these virus-like symptoms were induced by other factors, including fungi, insects, abiotic stresses, environmental conditions, and/or pesticides. It is also possible that the causal virus(es) were not covered by the detection methods. As the sample sizes from some locations were small, the disease incidence in these locations needs further validation. Nevertheless, the information presented here should allow a better management of virus diseases in Yunnan Province.

CONCLUSION
We have conducted a comprehensive survey of virus diseases in the pepper and tomato fields in Yunnan Province, one of the main vegetable production provinces in China. Our result indicates that the virus population structure in the pepper fields is more complex than that in the tomato fields. This information is useful for a better management of virus diseases in these two vegetable fields in this region.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
YLi, GT, and FL conceived and designed the experiments. YLi, LX, and WZ performed the experiments. YLi and GT collected the samples and analyzed the data. PL, XC, and YLiu contributed reagents, materials, and analysis tools. YLi, RL, and FL wrote the manuscript. All authors read and approved the final manuscript.