Detection and characterization of an isolate of Tomato mottle mosaic virus infecting tomato in China

1. Introduction

Tomato (Solanum lycopersicum L.) is one of the most economically important vegetable crops throughout the world. The annual tomato production showed an increasing tendency year by year, while the incidence and severity of diseases have limited the yield and quality of tomato causing serious losses. Viral diseases are the major constraints in tomato production among the known pathogen-induced diseases (Hanssen et al. 2010).

There are at least 136 characterized viruses that have been reported infecting tomato in the world which are much greater than the other vegetables (Brunt et al. 1996; Xu et al. 2017). Tobamoviruses are a kind of economically important viruses in the family Virgaviridae, which contains 37 members according to the taxonomy by the International Committee on Taxonomy of Viruses (https://talk.ictvonline.org/taxonomy/). The tobamoviruses were divided into different subgroups according to their genomic structure, host range, the amino acid composition of the coat proteins (CPs), etc. The phylogenetic relationship of the tobamoviruses conducted by Li et al. (2017) revealed that in the Solanaceae-infecting group, Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV), Tomato mottle mosaic virus (ToMMV) and Tomato brown rugose fruit virus (ToBRFV) are the four reported tomato-infecting tobamoviruses and are classi fied into one clade based on the complete genome and amino acid sequences. TMV and ToMV are the most epidemic viruses among the world,ToBRFV is a new species that has been characterized in Jordan (Salem et al. 2016) and Israel (Luria et al.2017), while ToMMV was also a recently characterized species and showed fast spread in the world. ToMMV was first described in 2013 infecting tomato in Mexico (Li et al. 2013) and subsequently was detected on tomato in different parts of the world including the USA (Webster et al. 2014; Fillmer et al. 2015; Padmanabhan et al.2015; Sui et al. 2017), Israel (Turina et al. 2016), Brazil(KT222999) and Spain (Ambrós et al. 2017). Additionally,Pirovano et al. (2014) showed its presence in Cicer arietinum L. in Italy and Li et al. (2014, 2017) established two isolates infecting pepper from Yunnan Province and Tibet Autonomous Region in China.

智能电网是将现代的信息、通信、控制及管理等先进技术与传统电力系统的技术和业务相融合的发展模式，而电力通信网提供了智能电网需要的信息采集、控制和管理的通道，是实现智能电网的基本保证。智能电网在业务技术应用、网络技术、运行模式方面都提出了新的要求，适用于智能电网的通信技术主要具备以下特征：①具备双向性、实时性、可靠性特征，基于安全性考虑，理论上应与公网隔离的电力通信专网；②具备技术先进性，能够承载智能电网现有业务和未来扩展业务；③最好具备自主知识产权，可具有面向电力智能电网业务的定制开发和业务升级能力。

The genome of ToMMV contains four open reading frames expressing four proteins like the other tobamoviruses in the family Virgaviridae, ～126 kDa protein and ～180 kDa read-through protein involved in virus replication, ～30 kDa movement protein and ～18 kDa coat protein. The sequence characteristic of ToMMV genome like the other tobamoviruses represents that the genome contains a Ω fragment in the 5´ untranslated region (UTR) which had no G residues except the most-proximal m7G cap(Richards et al. 1977; Zhang et al. 2008), a conserved region TCCCTCCACTTAAATCGAAGGGTT located in the 3´ UTR with the CCCA ending sequence (Chng et al. 1996)and the ‘4404-50 motif’ reported as the tobamovirusesspecific nucleotide motif which indicated that the 29 sites of the 47 nucleotides (nt) are invariant in all tobamoviruses sequences (Gibbs et al. 2004). Li et al. (2017) showed that the remaining 18 variable sites are also conserved among the previously reported ToMMV isolates and 9 of the 18 variable sites can distinguish ToMMV from ToMV. The ToMMV-specific sequence is as follows:GGTGATGTTACAACTTTCATAGGAAATACTGTTATTATA GCCGCGTG.

In a survey of viral diseases in 2016, a devastating disease infecting tomato crops was observed in the greenhouses and natural fields in Hainan Province in China. The tomato plants showing virus-like symptoms were collected for virus detection. The pathogen has been identified as ToMMV and characterized the full sequence of ToMMV Hainan isolate-infecting tomato.

2. Materials and methods

2.1. Plant growth conditions and inoculations

The complete genome sequence of ToMMV was divided into six fragments and amplified by RT-PCR using designed primers. Every fragment overlapped with the adjacent one or two fragments. The six pairs of primers listed in Appendix A were designed based on the conserved sequences of all ToMMV sequence from NCBI database. To obtain whole sequence of the genome, we amplified the ～770 bp of the most proximal 5´ regions and the ～550 bp of the 3´ regions(primers listed in Appendix A) of the genome. The whole genome sequence of ToMMV Hainan was assembled and determined to be 6 397 nt (GenBank no. MG171192), the full-length nucleotide sequence of ToMMV Hainan isolate was compared with the other isolates and tobamoviruses.The results showed that ToMMV Hainan shared the highest identity with ToMMV TiLhaLJ (GenBank no. KR824951) of 99.7% and subsequently with ToMMV YYMLJ (GenBank no. KR824950) of 99.55%, ToMMV MX-5 (GenBank no.KF477193) of 99.44%, ToMMV NY-13 (GenBank no.KT810183) of 99.30%, ToMMV 10-100 (GenBank no.KT810183) of 99.28% and ToMMV VLC-1 (GenBank no.KU594507) of 98.81%, respectively. ToMMV Hainan shared the similarity of 85% with ToMV, 81% with TBRFV and 80% with TMV. To investigate genetic similarity between the ToMMV isolates and the other tobamoviruses, the phylogenetic tree was constructed using the neighbor-joining method analysis based on the complete genome, which showed that seven ToMMV isolates differentiated from the other three tobamoviruses. Within the phylogenetic ToMMV cluster, it is observed that ToMMV Hainan isolate is more closely related with ToMMV TiLhaLJ and ToMMV YYMLJ which are isolated from peppers in China, the ToMMV MX-5 from Mexico and ToMMV NY-13 from New York are in one subgroup and are close to ToMMV 10-100 from the USA,while the isolate ToMMV LVC-1 from Spain is in a different clade with the other six ToMMV isolates (Fig. 2-A).

一个穿西装的印度店员上前招呼。店堂虽小，倒也高爽敞亮，只是雪洞似的光塌塌一无所有，靠里设着唯一的短短一只玻璃柜台，陈列着一些“诞辰石”——按照生日月份，戴了运气好的，黄石英之类的“半宝石”，红蓝宝石都是宝石粉制的。

2.2. Dot enzyme-linked immunosorbent assay(dot-ELISA)

These results presented here show that the distortion and crinkling symptoms on tomato plants in Hainan Province in China are associated with the infection of ToMMV. In this study, we characterized a new isolate ToMMV Hainan and described the phylogenetic relationship with the other ToMMV isolates and tobamoviruses.

2.3. RNA extraction, RT-PCR, full-length genome amplification and sequencing

由于信息和相关视频在网络传播存在“时间差”，此事在中国网友间所引发的强烈反应经历了戏剧性的“反转”，许多“围观者”最初普遍同情三名同胞的遭遇，并对瑞典方面的“疑似种族歧视行为”表示强烈不满，但随着各方提供的细节和视频大量涌现，不少人发现3名“受害人”早先散布的消息经过剪裁取舍，许多信息并不一定属实，而他们的某些做法和形象也令人难以认同和接受，开始纷纷表达对酒店、警方的理解和对3人的不满，有些人甚至迁怒于介入进行领事援助的中国使领馆，认为“不该是非不分给这种人帮忙”。

The complete sequence of ToMMV was assembled and analyzed with the software DNAMAN version 5.0 (Lynnon Biosoft, Quebec, QC, Canada). The sequence fragment and the whole genome were compared against the database from the National Center for Biotechnology Information(NCBI) using a BLAST search.

2.4. Sequence assembly and analysis

The 5´- and 3´-terminal sequence of the ToMMV were obtained through 5´- and 3´-rapid amplication of cDNA ends (RACE) using SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. The 5´ and 3´ gene-specific primer (GSP) (both were designed according to the known ToMMV fragment and listed in Appendix A) paired with the Universal Primer Mix (UPM) (the long primer provided by kits) were used in 5´- and 3´-RACE, respectively.

The phylogenetic trees derived from the alignment of the multiple sequences were constructed using the neighborjoining method with 1 000 bootstrap replications by MEGA 6.0. The sequences used for comparison were obtained from the GenBank database.

3. Results

3.1. Virus detection with dot-ELISA and RT-PCR

In December 2016, a severe tomato disease broke out in the greenhouse and field condition in Hainan Province. The diseased tomato plants showed severe virus-like symptoms with leaves distortion, mosaic and mottle of light and dark green, systemic crinkling which seriously reduced the production (Fig. 1-A). The infected tomato samples were collected and Identification of the pathogens was conducted by serological analysis and biological research.

As the disease symptoms developed very quickly in the greenhouse and under field condition, the pathogen was speculated as a tobamovirus other than a tospovirus or a geminivirus. The collected samples were subjected to dot-ELISA using TMV monoclonal antibody (provided by Professor Zhou Xueping’s lab in Zhejiang University in China). The sap from leaves of the diseased tomato plants reacted strongly to TMV antibody showing purple colors as the positive control, which suggested that the tobamovirus is likely associated with the disease (Fig. 1-B). Both TMV and ToMV are widely distributed tobamoviruses in China and the two viruses show positive reaction with TMV antibody,to further investigate which virus caused the disease, the total RNA was extracted and RT-PCR was conducted using the TMV- or ToMV-specific primer pairs (Appendix A). The primer pairs TA/TB and TMf1/TMr1 were used as TMV-specific primers, while the primer pairs ToA/ToB and ToMf1/ToMr1 were designed as ToMV-specific primers. RT-PCR results indicated that no bands were obtained with primer pairs TA/TB, TMf1/TMr1 or ToA/ToB, while a specific band of ～500 bp was generated with ToMf1/ToMr1. Sequencing and BLAST analysis revealed that the sequence was 99%identical to ToMMV coat protein gene. For the confirmation of the result, the tobamovirus degenerate primers TobamoF/TobamoR (Li et al. 2014) and the designed ToMMV-specific primers ToMMVF/ToMMVR were used in RT-PCR. The approximately ～1 000 bp products were obtained with the two primer pairs (Fig. 1-C). Sequence analysis showed that the acquired PCR products shared the highest identity with ToMMV isolate YYMLJ (GenBank no. KR824950.1),which indicated that the symptomatic tomatos from Hainan Province were infected by the ToMMV.

Fig. 1 Dot enzyme-linked immunosorbent assay (dot-ELISA) and RT-PCR detection of the diseased tomato plants. A, symptoms on tomato plants infected with Tomato mottle mosaic virus (ToMMV) in Hainan in China. B, detection of ToMMV by dot-ELISA with Tobacco mosaic virus (TMV) monoclonal antibody. C, detection of ToMMV by RT-PCR using tobamovirus degenerate primers and ToMMV-specific primers. M, mock-inoculated plant; 1–6, tomato samples in Hainan.

3.2. Complete sequence amplification and analysis

Tobacco, pepper and tomato plants were grown in a growth room (28°C day and 24°C night, 16 h/8 h photoperiod).Virus-infected leaf tissues were homogenized in 0.01 mol L–1 phosphate buffer (PBS, 0.01 mol L–1 KH2PO4:0.01 mol L–1 Na2HPO4=49:51 (v/v), pH=7.0) at 1:10 ratio (w/v). The crude extracts were rub-inoculated to the newly grown leaves of 4-week-old tobacco, pepper and tomato plants,respectively. The virus-inoculated plants were grown in the same conditions.

ToMMV Hainan isolate contains the specific genomic structure like the other tobamoviruses in the family Virgaviridae. The whole genome included four open reading frames expressing four proteins. The 5´-most open reading frame (ORF) started at 74 nt encoding a 126-kDa protein and its read-through resulted a 180-kDa protein which are involved in virus replication. The ～30-kDa movement protein and ～18-kDa CP were 4 908–5 714 and 5 717–6 196 nt,respectively. Sequence analyses revealed that the 5´ UTR was 73 nt possessing a Ω fragment, a conserved region 6 270TCCCTCCACTTAAATCGAAGGGTT6 293 located in the 3´ UTR and the ToMMV specific ‘4404-50 motif’ located in 4 409–4 455 nt.

3.3. Biological assay and fulfillment of Koch’s postulates

In our dot-ELISA analysis, the ToMMV-infected tomato samples without TMV infection reacted positively with the TMV monoclonal antibody, which was coincided with the previous reports (Webster et al. 2014; Turina et al. 2016).The dot-ELISA results suggested that ToMMV had the serological relation with TMV. Given the high amino acids sequence similarity between TMV, ToMV and ToMMV,the infections being diagnosed as TMV or ToMV using serological methods solely might actually be ToMMV.Before ToMMV was characterized as a new species, many isolates were deposited in GenBank as ToMV, for example,the sequence from Iran (JX112024, JX112025, JX121570,JX121574, JX121575, JX121576, HQ593616), from Brazil (AF411922, AM411425, AM411430) and from China(JX025564). The sequence from Brazil (AF411922) may be the first sequence recorded corresponding to ToMMV(Moreira et al. 2003).

又在播放那首《月光》。听着琴声，敦礼仿佛置身在广袤无垠的旷野，皓月当空，月光笼罩下的一切都宁静而神秘；想到月光，他就能听到光与影组成的和谐的旋律，那是灵动的，富于变化的。

Nif、CsA因价格低廉、临床疗效好，已成为临床上治疗高血压、心绞痛和器官移植术后常规服用药物。有研究显示，Nif、CsA均会引起牙龈增生，尤其当两种药物联合应用时，牙龈增生也明显加重[5]，影响患者的咀嚼功能和牙周组织的健康。目前，药物性牙龈增生的具体发病机制尚不清楚，多认为是成纤维细胞增殖活性增强和(或)凋亡受到抑制，进而促进成纤维细胞的活性[12],导致胶原纤维合成与分解代谢失衡，引起以胶原纤维为主的细胞外基质(Extracellular matrix，ECM)的大量聚积[8]，从而导致了牙龈的纤维性增生。

4. Discussion

The dot-ELISA procedures were conducted as described previously with some modification (Li et al. 2015). Briefly, the 3 μL plant sample supernatants in 0.01 mol L–1 PBS (pH 7.4)were spotted onto the nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). The TMV monoclonal antibody solution was diluted by 1:5 000-fold with blocking buffer (5% skimmed milk in PBS with 0.5% Tween-20). The membranes were developed with the substrate nitro-blue tetrazolium (NBT, 0.083 mg mL–1) and 5-bromo-4-chloro-3-indolyl phosphate salt (BCIP, 0.05 mg mL–1).

The total RNA was extracted from tomato leaves with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with DNaseI. A total of 1 μg of total RNA, random primer, and M-MLV reverse transcriptase were used for synthesis of first-strand cDNA using Prime-ScriptTM RT Reagent Kit(TaKaRa Bio. Inc., Dalian, China). RT-PCR was performed according to the manufacturer’s protocols. The primer pairs used in the RT-PCR experiments were listed in Appendix A. The RT-PCR products were puri fied with E.Z.N.A. Gel Extraction Kit (Omega, Norcross, GA, USA) and cloned into pGEM-T vector (Promega, Madison, WI, USA), then transfected into DH5α competent cells. The recombinant clones were sequenced with universal primer pairs M13F/M13R via Sanger sequencing using ABI 3730XL DNA Analyzers (Applied Biosystems, Foster City, CA, USA). At least three independent clones were sequenced.

To investigate the biological characterization of the new isolate ToMMV Hainan, we used the crude sap extracts of tomato sample infected by ToMMV Hainan to inoculate tomato plants, pepper and N. benthamiana plants. The virus caused mosaic and mottle symptoms in the infected tomato plants at 7 days post inoculation (dpi) and later the leaves began narrowing and crinkling, the whole plants showed stunting compared with the uninfected control (Fig. 2-B).On inoculated seedlings of pepper, there were crinkling and mosaic at 7 dpi in the upper systematic leaves, later the necrotic lesions appeared in the growth point (Fig. 2-B). The N. benthamiana was systematically infected and showed crinkling and yellow on upper leaves at 4 dpi. At 6 dpi,the apical shoot of the N. benthamiana seedlings started showing serious necrosis symptoms (Fig. 2-B).

ToMMV Hainan isolate has the highest identity and the closest evolutionary relationship to ToMMV TiLhaLJ and ToMMV YYMLJ which were isolated from peppers in China,while are in a different subgroup with the isolates from Mexico and USA or the isolate from Spain.

Fig. 2 Phylogenetic tree and infectivity of the Tomato mottle mosaic virus (ToMMV) Hainan isolate. A, neighbor-joining phylogenetic tree showing the relationship between ToMMV isolates and other tobamoviruses. B, infectivity of ToMMV Hainan isolate in tomato,pepper and tobacco plants. Left, non-infected plants; middle, ToMMV-infected plants; right, details of the leaf or apical shoot from the infected plants.

ToMMV was firstly identified as a novel tobamovirus species infecting tomatoes in 2013 in Mexico (Li et al. 2013),and now it has been identified in several countries and areas in the world. In China, ToMMV was firstly reported infecting peppers in Yunnan Province and Tibet Autonomous Region in 2014 (Li et al. 2014). Here, we are reporting the first case of ToMMV infection on tomato in Hainan Province of China and characterized the whole sequence. The ToMMV need more attention because of its rapid spread and devastating damage to tomato industry. Therefore, further studies need to be performed to identify the real occurrence and epidemiology of the ToMMV.

5. Conclusion

A severe disease on tomatoes broke out in Hainan Province in China. We identified that Tomato mottle mosaic virus was the pathogen causing the mottle symptoms by dot-ELISA and RT-PCR. The full-length sequence of the new ToMMV Hainan isolate was determined and the biological characterization has been investigated. This was the first Identification and characterization of ToMMV-infecting tomato in Hainan in China.

Acknowledgements

This research was supported by the Agricultural Science and Technology Innovation Program, China (ASTIP). We thank technicians and students from Hainan Runda Modern Agriculture Corporation, China for helping in field surveys and samples collections.

Appendix associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

海兰这才坐下，谦卑道：“在小主面前，妾身不敢不直言。在潜邸时月福晋虽然难免与小主有些龃龉，但从未如此张扬过。事出突然，怕有什么变故。”她抬眼望青樱一眼，低声道，“幸好，小主隐忍。”

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