Tomato Zonate Spot Virus Research Articles

Clustering and cellular distribution characteristics of virus particles of Tomato spotted wilt virus and Tomato zonate spot virus in different plant hosts.

BackgroundTomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are the two dominant species of thrip-transmitted tospoviruses, cause significant losses in crop yield in Yunnan and its neighboring provinces in China. TSWV and TZSV belong to different serogroup of tospoviruses but induce similar symptoms in the same host plant species, which makes diagnostic difficult. We used different electron microscopy preparing methods to investigate clustering and cellular distribution of TSWV and TZSV in the host plant species.ResultsNegative staining of samples infected with TSWV and TZSV revealed that particles usually clustered in the vesicles, including single particle (SP), double particles clustering (DPC), triple particles clustering (TPC). In the immunogold labeling negative staining against proteins of TZSV, the antibodies against Gn protein were stained more strongly than the N protein. Ultrathin section and high pressure freeze (HPF)-electron microscopy preparations revealed that TSWV particles were distributed in the cisternae of endoplasmic reticulum (ER), filamentous inclusions (FI) and Golgi bodies in the mesophyll cells. The TSWV particles clustered as multiple particles clustering (MPC) and distributed in globular viroplasm or cisternae of ER in the top leaf cell. TZSV particles were distributed more abundantly in the swollen membrane of ER in the mesophyll cell than those in the phloem parenchyma cells and were not observed in the top leaf cell. However, TZSV virions were mainly present as single particle in the cytoplasm, with few clustering as MPC.ConclusionIn this study, we identified TSWV and TZSV particles had the distinct cellular distribution patterns in the cytoplasm from different tissues and host plants. This is the first report of specific clustering characteristics of tospoviruses particles as well as the cellular distribution of TSWV particles in the FI and globular viroplasm where as TZSV particles inside the membrane of ER. These results indicated that tospoviruses particles possessed specific and similar clustering in the saps of diseased plants. Furthermore, the results of this study will also provide a basis for further study on the tospoviruses assembling, maturation and movement.

Occurrence of Tomato zonate spot virus on Potato in China

Occurrence of <i>Tomato zonate spot virus</i> on Potato in China

First Report of Tomato zonate spot virus in Iris tectorum in China.

Iris tectorum Maxim, a very popular Chinese traditional medicinal perennial herb belonging to the Iridaceae family, is widely grown as a year-round ornamental in China. During May to August 2014, as part of a survey for tospoviruses (family Bunyaviridae) in flue-cured tobacco, symptoms suspected to be caused by tospoviruses were observed on I. tectorum around farmers' fields in Kunming, Yunnan province. Symptoms were chlorotic spots on younger leaves and necrosis on older leaves. Since Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are two common tospoviruses in flue-cured tobacco fields in Yunnan, ELISA with monoclonal TSWV antibody (provided by J. X. Wu, Zhejiang University, China) and polyclonal TZSV antiserum (provided by J. H. Dong, Yunnan Academy of Agriculture Science, China) was performed to identify the presence of virus. Positive extinction values (ODλ405nm 0.835 ± 0.121 and 1.024 ± 0.193, as compared with the negative 0.153 ± 0.076 and the positive control 0.510 ± 0.109 at a confidence interval of P ≤ 0.05) were obtained from two symptomatic samples with TZSV antibody but not with TSWV. The absence of TSWV was confirmed with a commercially available immune-strip (Agdia, Elkhart, IN), following the manufacturer's instructions. To further verify the causal agent of these symptoms, total RNA was isolated from two symptomatic and one asymptomatic samples and reverse transcribed using degenerate primer J13 (1). These cDNAs were then used as a template in a universal PCR assay using specific primers TZSVNF (5'-ATGTCTAACGTCCGGAGTTTAACAC-3') and TZSVNR (5'-TTAAAAAGACAGATCATTGCTG-3'), which amplify the complete nucleocapsid (N) protein. The PCR was carried out for denaturation at 94°C for 3 min, and subsequently 30 cycles were carried out, with each cycle consisting of 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min, followed by a final extension step at 72°C for 10 min. An 0.8-Kb DNA fragment was amplified from symptomatic samples and cloned into a pGEM-T Easy (Promega, Madison, WI) vector. Three clones of each sample were selected and sequenced. BLAST analysis of the obtained sequences (Accession Nos. KM452916 and KM452917) revealed that the N sequences of these isolates have 96 to 99% nucleotide identity and 99 to 100% amino acid identity with the deposit TZSV sequence in NCBI from Yunnan (JN116580 to JN116583 and EF552433) (2). These combined results provide further confirmation of TZSV infection. It is known that perennial herb or ornamental plants may act as reservoirs for tospoviruses that can infect cultivated crops because tospoviruses have a very broad host range. Therefore, elaborate inspections for tospoviruses and appropriate management strategies to limit virus spread are necessary for production of crops. To our knowledge, this is the first report of TZSV in I. tectorum Maxim.

Identification of a new tospovirus causing necrotic ringspot on tomato in China.

BackgroundEmerging tospoviruses cause significant yield losses and quality reduction in vegetables, ornamentals, and legumes throughout the world. So far, eight tospoviruses were reported in China. Tomato fruits displaying necrotic and concentric ringspot symptoms were found in Guizhou province of southwest China.FindingELISA experiments showed that crude saps of the diseased tomato fruit samples reacted with antiserum against Tomato zonate spot virus (TZSV). Electron microscopy detected presence of quasi-spherical, enveloped particles of 80–100 nm in such saps. The putative virus isolate was designated 2009-GZT. Mechanical back-inoculation showed that 2009-GZT could infect systemically some solanaceous crop and non-crop plants including Capiscum annuum, Datura stramonium, Nicotiana benthamiana, N. rustica, N. tabacum and Solanum lycopersicum. The 3012 nt full-length sequence of 2009-GZT S RNA shared 68.2% nt identity with that of Calla lily chlorotic spot virus (CCSV), the highest among all compared viruses. This RNA was predicted to encode a non-structural protein (NSs) (459 aa, 51.7 kDa) and a nucleocapsid protein (N) (278 aa, 30.3 kDa). The N protein shared 85.8% amino acid identity with that of CCSV. The NSs protein shared 82.7% amino acid identity with that of Tomato zonate spot virus(TZSV).ConclusionOur results indicate that the isolate 2009-GZT is a new species of Tospovirus, which is named Tomato necrotic spot virus (TNSV). This finding suggests that a detailed survey in China is warranted to further understand the occurrence and distribution of tospoviruses.

Comparative analyses of the toxic effects of imidacloprid and spirotetramat on Frankliniella occidentalis (Pergande), the vector of tomato zonate spot virus: a laboratory assay

The tomato zonate spot virus (TZSV) is responsible for substantial yield losses in vegetables, tobacco and other cash crops in China in recent years. The western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera, Thripidae), is the main vector of TZSV in Yunnan Province. Because controlling the population of insect vectors is the most efficient way to prevent epidemics of viral diseases, we conducted laboratory assays to assess the potential of using spirotetramat as an alternative to imidacloprid in controlling F. occidentalis. Our results demonstrated that spirotetramat was significantly more lethal than imidacloprid to F. occidentalis at different concentrations. The LC30, LC50 and LC90 values of spirotetramat were all much lower than those of imidacloprid after the same time of treatment. Frankliniella occidentalis was more sensitive when treated with spirotetramat than imidacloprid at the concentrations of 500, 250, 125 and 62.5mg/L, and the median lethal concentrations (LC50) were 285.53, 82.24 and 11.19mg/L at 12, 36 and 72 hours after treatment, respectively. Spirotetramat caused 100% mortality of F. occidentalis at 500 and 250 mg/L after 48 hours treatment, and at the lowest concentration 125 mg/L at 72 hours after treatment, respectively. Field experiments carried out elsewhere have confirmed our findings. Together they allow us to conclude that spirotetramat is a promising pesticide for the control F. occidentalis in China.

Effects ofTomato zonate spot virusInfection on the Development and Reproduction of Its VectorFrankliniella occidentalis(Thysanoptera: Thripidae)

The western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is the main vector of Tomato Zonate Spot Virus (TZSV) in Yunnan, China. We investigated the life history parameters of F. occidentalis on tomato and pepper leaves either with or without TZSV infection. The total duration of the immature stages of F. occidentalis reared on TZSV-infected leaves was significantly shorter than the total duration of those reared on uninfected leaves. Also the survival rates of the instars and prepupae on TZSV-infected tomato and pepper leaf disks were significantly higher than those on uninfected tomato and pepper leaf disks. The F. occidentalis populations reared on TZSV-infected tomato and pepper leaf disks respectively increased 11.97- and 10.64-fold in 1 generation, while those reared on uninfected tomato and pepper leaf disks increased only 8.10- and 6.45-fold, respectively. These results demonstrated that TZSV infection improved the fitness and host suitability of its vector, F. occidentalis. Also our findings suggest that TZSV-infection is likely to induce larger field populations of F. occidentalis, thereby increasing the probability of TZSV transmission. Los trips occidental de las flores, Frankliniella occidentalis, (Pergande) (Thysanoptera: Thripidae), es el principal vector del Virus de la Mancha Zonada del Tomate (VMZT) en Yunnan, China. Se investigo los parametros del ciclo de vida de F. occidentalis sobre hojas de tomate y pimiento con o sin infeccion por VMZT. La duracion total de las etapas inmaduras de F. occidentalis criadas en las hojas infectadas por el VMZT fue significativamente mas corta que la duracion total de las criadas en las hojas no infectadas. Tambien, las tasas de sobrevivencia de los instares y prepupas sobre discos de hojas de tomate y pimiento infectadas por VMZT fueron significativamente mas altas que las tasas sobre discos de hojas de tomate y pimiento no infectadas. Las poblaciones del F. occidentalis criadas sobre discos de hojas de tomate y pimienta infectados por VMZT, incrementaron 11.97 y 10.64 veces, respectivamente en 1 generacion, mientras que las criadas sobre discos de hojas de tomate y pimienta no infectadas incrementaron solo 8.10 y 6.45 veces, respectivamente. Estos resultados demostraron que la infeccion por VMZT mejoro el estado fisico e idoneidad de su vector, F. occidentalis. Tambien nuestros hallazgos sugieren que es probable que la infeccion por VMZT induzca poblaciones de campo de F. occidentalis mas grandes y con ello incrementando la probabilidad de transmision de VMZT. View this article in BioOne

Cytopathological Characterization of Tobacco on Co-infection with Tomato zonate spot virus and Potato virus Y

Cytopathological Characterization of Tobacco on Co-infection with Tomato zonate spot virus and <i>Potato virus Y</i>

Development of a real-time fluorescent quantitative PCR assay for detection of Impatiens necrotic spot virus

Impatiens necrotic spot virus (INSV) is an important plant virus that can cause severe disease in various ornamental and agricultural crops. Several species of thrips transmit INSV, of which the western flower thrip (Frankliniella occidentalis) is the most important. In this study, primers and TaqMan probes based on INSV non-structural protein gene sequences were designed, and a technique was developed for detecting INSV using fluorescent quantitative RT-PCR. The reproducibility, specificity and sensitivity for the RT-PCR were evaluated; and the RT-PCR method was developed to detect INSV in the host plants and western flower thrips. A standard curve constructed by a series of diluted plasmid DNA gave a good linear relationship between Ct value and concentration of plasmid DNA, a low coefficient of variation and good reproducibility. The detection method not only measured quantitatively the concentration of INSV in plant hosts and western flower thrips, but also measured accurately low concentrations of the virus. The measurable concentration fell to as low as 100copies/μl, while RT-PCR could detect only 102copies/μl. The method had high specificity and could distinguish INSV from Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV), both from the same genus of viruses. This is the first report of the same method being used to detect INSV in both plant hosts and western flower thrips, and should be helpful in studies of INSV epidemiology.

Scientific Opinion on the pest categorisation of the tospoviruses

The European Commission requested EFSA's Panel on Plant Health to perform the pest categorisation for the 24 viruses of the Tospovirus genus for the EU territory. The following tospoviruses were analysed: Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), Polygonum ringspot virus (PolRSV), Groundnut ringspot virus (GRSV), Tomato chlorotic spot virus (TCSV), Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Melon severe mosaic virus (MSMV), Tomato yellow (fruit) ring virus (TYRV), Tomato zonate spot virus (TZSV), Groundnut yellow spot virus (GYSV), Groundnut chlorotic fan-spot virus (GCFSV), Groundnut bud necrosis virus (GBNV), Zucchini lethal chlorosis virus (ZLCV), Capsicum chlorosis virus (CaCV), Watermelon bud necrosis virus (WBNV), Watermelon silver mottle virus (WSMoV), Tomato necrotic ringspot virus (TNRV), Calla lily chlorotic spot virus (CCSV), Melon yellow spot virus (MYSV), Soybean vein necrosis associated virus (SVNaV), Bean necrotic mosaic virus (BeNMV) and Pepper necrotic spot virus (PNSV). In reaching its conclusions, the Panel considered four parameters to be of critical importance in the risk assessment area: (i) the presence of a tospovirus, (ii) the existence of host plants, (iii) the existence of thrips vector species and (iv) the potential for damage to crops grown in Europe. Based on its analysis, the Panel concluded that the 24 viruses analysed could be allocated to four different risk groups. Seven viruses (GRSV, TCSV, ANSV, CSNV, MSMV, TYRV, TZSV) for which both thrips species vectors and natural or experimental hosts crops are present in the EU territory were considered by the Panel to represent the highest risk to the EU territory. In contrast, three viruses (INSV, IYSV and PolRSV) already present in the risk assessment area were not considered by the Panel to pose a risk justifying the development of full risk assessments.

Molecular Identification and Characterization of Tomato zonate spot virus in Tobacco in Guangxi, China.

In Guangxi Province of southwest China, diseases caused by Tospoviruses (family Bunyaviridae) pose a serious threat to tobacco (Nicotiana tobacum) production. During surveys conducted annually at Xinrong Village in Jingxi County from 2008 to 2010, more than 130 ha of fields were found to have 10 to 50% of plants exhibiting symptoms similar to spotted wilt caused by Tomato spotted wilt virus (TSWV). During this period, disease symptoms at similar prevalence and incidence were also found at Fushan, Debao County in most of the cultivars produced in these areas, including Yunyan 85, 87, 92, 97, and K326. Symptoms on tobacco varied but commonly included dwarfing, midrib browning, distorted apical buds, and concentric ringspots that coalesced to form large areas of dead leaf tissue. Mechanical inoculation from diseased tobacco leaves with concentric ringspots back to tobacco cv. Yunyan 85 or 87, resulted in 12 of 16 plants with symptoms similar to those observed in the field. No symptoms on plants developed following inoculation with buffer only. Symptoms found in the field resembled those caused by TSWV. However, testing using TSWV-specific antiserum was shown to be negative by double-antibody sandwich-ELISA (Agdia, Elkhart, IN). Total RNA was extracted from 27 diseased tobacco plants collected from different regions in Guangxi using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA extracts were amplified by reverse transcription (RT)-PCR using the degenerate primers T2740 (ATGGGDATNTTTGATTTCATG) and T3920c (TCATGCTCATSAGRTAAATYTCTCT) designed to target the partial RNA-dependent RNA polymerase (RdRp) sequence of members in the genus Tospovirus (3). Amplification was performed at 42°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 7-min final extension at 72°C. A PCR product of approximately 1.2 kb was amplified from 21 diseased plants. RT-PCR amplicons were cloned into the pUC19-T Simple Vector (TaKaRa, Dalian, China) and sequenced in both directions. Sequences were assembled and analyzed by DNAStar 5.01 (DNASTAR, Madison, WI). Sequences of representative isolates were deposited in GenBank (Accession Nos. JN020022 to JN020027). The 1.2-kb partial RdRp sequences of these isolates were shown to have 94.4 to 95.3% nucleotide identity and 96.5 to 97.5% amino acids identity to Tomato zonate spot virus (TZSV) (GenBank Accession No. NC_010491) (1). Among these TZSV isolates from Guangxi, the partial RdRp sequences have 98.0 to 99.4% nucleotide identity and 98.8 to 100% amino acids identity with each other. The presence of TZSV was further confirmed in diseased tobacco plants by indirect ELISA using antiserum of TZSV (provided by Prof. Zhongkai Zhang, Agricultural Academy of Yunnan, China). TZSV has been characterized as a novel tospovirus on various hosts including tobacco in Yunnan province (1,2). To our knowledge, this is the first report of TZSV-associated disease on tobacco in Guangxi Province, southwest China. Further work is necessary to study the epidemiology and management of the disease.

Genomic characterization of Calla lily chlorotic spot virus and design of broad‐spectrum primers for detection of tospoviruses

Calla lily chlorotic spot virus (CCSV) collected from calla lily plants showing symptoms in Taiwan has been identified as a tentative species of the genus Tospovirus based on the comparison of S RNA sequences. In this investigation, the complete sequences of its L and M RNAs were determined. The L RNA contains 8911 nucleotides (nt) and encodes a putative RNA‐dependent RNA polymerase of 2882 amino acids (aa) (332 kDa) in the viral complementary (vc) sense. The M RNA was shown to be 4704 nt in length, encoding a nonstructural NSm protein of 309 aa (35 kDa) in the viral sense and a Gn/Gc glycoprotein precursor (GP) of 1123 aa (128 kDa) in the vc sense. Phylogenetic analysis of the individual tospoviral proteins indicated that CCSV is closely related to Tomato zonate spot virus, a new tentative tospovirus isolated from tomato in southern China. Two degenerate primer pairs gL2740/gL3920c and gM410/gM870c, designed, after sequence comparison, from the highly conserved regions of the L and NSm genes, respectively, were successfully used to detect 12 greenhouse‐cultured tospoviruses, including six formal species and six tentative species, by reverse transcription‐polymerase chain reaction. They were also used to detect Melon yellow spot virus and Watermelon silver mottle virus from field cucurbit samples in Taiwan. The results indicate that the two degenerate primer pairs can be used for prompt detection of most tospoviruses and exploration of unknown tospovirus species.

Characterization of tomato zonate spot virus, a new tospovirus in China

An isolate of a new tospovirus species, causing concentric zoned ringspots on fruits and necrotic lesions on leaves of infected plants, was characterised based on particle morphology, host range and serological properties. The complete nucleotide sequences of large (L), medium (M), and small (S) RNAs of this virus were found to contain 8919, 4945, and 3279 nts respectively. The L RNA encoded the RNA-dependent RNA polymerase (RdRp) (2885 aa, 332.7 kDa). The M RNA encoded a non-structural (NSm) protein (309 aa, 34.4 kDa) and a viral glycoprotein precursor (Gn/Gc) (1122 aa, 127.4 kDa). The S RNA encoded a non-structural protein (NSs) (459 aa, 51.9 kDa) and the nucleocapsid (N) protein (278 aa, 30.6 kDa). This N protein shared amino acid identities of 80.9% with those of calla lily chlorotic spot virus. Our results suggest that the virus studied here belongs to a new tospovirus species, for which the name tomato zonate spot virus is proposed.