Tobacco Mild Green Mosaic Virus Research Articles

First report of Tomato brown rugose fruit virus infecting tomato in Florida, USA

Florida is a leading producer of fresh-market tomatoes (Solanum lycopersicum) in the US, a $262 million industry for the state. In October 2019, eight tomato plants showing chlorotic, cupped leaves and stunted foliage (Fig. 1) were collected from a c. 1.6 ha community garden in Alachua County, Florida. The symptoms observed were characteristic of Tomato yellow leaf curl virus (TYLCV). Samples were tested by PCR using TYLCV-specific primers by Navot et al. (1992). Three samples tested positive for TYLCV. Lateral flow immunoassay tests (“immunostrip assays”; Agdia, USA) for Pepino mosaic virus and Cucumber mosaic virus were negative. However, an immunostrip for Tobacco mosaic virus (TMV) gave a faint positive result for one of three TYLCV-positive sample, suggesting a mixed infection. Tobamovirus-like rigid rod-shaped particles of ∼300 nm in length, were also observed in leaf dips from this plant using an LVEM5 transmission electron microscope (DeLong America, USA). Tests for common tobamoviruses, i.e. TMV, Tomato mosaic virus, Tomato mottle mosaic virus, (Sui et al., 2017), Tobacco mild green mosaic virus (Cohen et al., 2006), and Tomato brown rugose fruit virus (ToBRFV) (Ling et al., 2019) were conducted using specific primers in RT-PCR with total RNA extracted using RNeasy® Plant Mini kits (Qiagen, Germany). Additionally, conventional PCR primers, DPI-782(5859nt-5879nt) = 5′-TAGCGAAGTGTGGAAACCTG-3′ and DPI-783(6154nt-6183nt) = 5′-GGTGCAGAGGACCATTGTAA-3′ (amplicon size of 342 bp), and primers and probes for a real-time RT-PCR Taqman assay DPI-784(6029nt-6049nt) = 5′-CCGAAACGTTAGACGCTACTC-3′, DPI-785(6106nt -6127nt) = ACCTGTTCCTTTGACCAA TTCT-3′, and DPI-787(6060nt-6083nt) TaqMan probe = 5′-ATG CAACGGTGGCTATAAGGA-3’) from the coat protein ∼5800-6130 nt region were developed for ToBRFV. Six of eight samples, including three TYLCV-positive samples, were positive for ToBRFV. The sequenced PCR products (GenBank Accession No. MW322766) were 98 to 99 % identical to ToBRFV isolates from Jordan and Mexico (KT383474, MK319944). The identification of ToBRFV was further confirmed by USDA-APHIS-PPQ-S&T laboratory (Laurel, Maryland, USA) by RT-PCR and sequencing. The Ct values of the real-time assay were in the range of 27 to 31. The weak positive result from the immunostrip, relatively high Ct values and co-infection with TYLCV suggest that ToBRFV was present in a low titre in these samples. ToBRFV was also detected in tomatoes imported from Mexico and for sale in grocery stores in Naples and Gainesville by University of Florida and Florida Department of Agriculture and Consumer Services (FDACS) around the same time (Batuman et al., 2020), Immediately following the survey, all solanaceous plants in the organic garden were destroyed. FDACS is taking all precautionary measures to test tomatoes produced in Florida. Further studies are needed to better understand the risk factors from this virus in Florida. The authors would like to thank Department of Plant Industry (DPI), Florida Department of Agriculture and Consumer Services (FDACS) for all assistance with the project.

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.

First report of tobacco mild green mosaic virus in Turkey

First report of tobacco mild green mosaic virus in Turkey

First Report of Tobacco Mild Green Mosaic Virus and Tomato Brown Rugose Fruit Virus Infecting Capsicum annuum in Jordan

Pepper (Capsicum annuum L.) is one of the major vegetable crops in Jordan. During the winter growing seasons of 2015 and 2016, virus symptoms including stunting of young plants, puckering, and yellow mottling of leaves in sweet pepper plants grown under plastic houses in Jordan Valley were observed. The most obvious symptoms were the misshapen fruits that affected the market value of the crop, which resembled recent descriptions of tomato brown rugose fruit virus (ToBRFV) on tomato fruits (Salem et al. 2016). Symptoms on mechanically inoculated indicator hosts including Chenopodium quinoa, Datura stramonium, D. metel, Nicotiana glutinosa, and N. tabacum, and serological testing using double-antibody sandwich ELISA (antiserum A128 of the PLAVIT collection at IPSP-CNR, Italy) suggested the presence of a tobamovirus. Total RNA was extracted from fruits and leaves of 39 symptomatic pepper plants, using an SV-Total RNA Extraction kit (Promega, U.S.A.). Samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for the most common tobamoviruses infecting pepper, including tomato mosaic virus, tobacco mosaic virus, tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (Takeuchi et al. 2005). In addition, generic primers for detection of tobamoviruses were also used (Dovas et al. 2004). In RT-PCR, amplicons of the expected product size (400 and 728 bp) using the generic tobamovirus and TMGMV-specific primers, respectively, in all symptomatic plant samples were obtained, but such amplicons were not obtained from healthy plant extracts or water negative controls. RT-PCR products of the RdRp (400 bp) and CP (728 bp) partial regions were purified and ligated into pGEM T-Easy Vector (Promega), and two clones for each PCR product were sequenced and deposited in NCBI GenBank (accession nos. MK816313 to 16). BLASTN analysis showed that these nucleotide sequences had 96 to 99% identity to TMGMV genome sequences (JX534224 and MH730962) in NCBI GenBank. Furthermore, total RNA was extracted with TRIzol reagent (Invitrogen, U.S.A.) according to the manufacturer’s specifications. After ribosomal RNA depletion, the cDNA library was constructed using a TruSeq RNA Sample Prep kit (Illumina, U.S.A.) and sequenced by Illumina HiSeq X-ten platform (Biomarker, China). Raw sequencing data were analyzed using CLC Genomics Workbench 9.5 (Qiagen, Denmark). After raw reads were processed, a total of 80,818,734 paired-end reads of 150 bp were obtained, generating 266,412 contigs (>200 nt) with de novo assembly by CLC Genomics Workbench 9.5. BLASTN analysis of the assembled contigs revealed the presence of two virus-derived contigs: TMGMV (6,414 nt, MK648158) and ToBRFV (6,388 nt, MK648157), which represented a nearly full-length genome. To confirm the ToBRFV identity, all samples were tested by RT-PCR using two pairs of primers: ToBRFV F1 (5′-GTATTTTTGTTTTACAACATATACCAAC-3′) and ToBRFV R1 (5′-AGTGCGAATGTGATTTAAAACTGTGAA-3′), and ToBRFV F7 (5′-GGAAGAAGTCCCGATGTCTGTAAGGCTT-3′) and ToBRFV R7 (5′-GATGCAGGTGCAGAGGACCATTGTAAAC-3′), designed on ToBRFV genome (KT383474; Salem et al. 2016). Twenty-two out of 39 tested samples originated specific amplicons (1,300 and 697 bp for RdRp and CP, respectively). RT-PCR products of two samples were purified and sent for direct sequencing, and results were deposited in NCBI GenBank (MK834288, MK834289, MK834294, and MK834295), revealing nucleotide identity of 99 to 100% to ToBRFV for both RdRp and CP (KT383474 and KX619418). To our knowledge, this is the first report of TMGMV and ToBRFV infecting pepper in Jordan. The occurrence of these tobamoviruses that are transmitted through seeds, in Jordan Valley, the main area for pepper production, may represent a potential threat to other susceptible vegetables and requires careful monitoring to avoid future outbreaks and significant yield losses.

Soil mobility of synthetic and virus-based model nanopesticides.

Large doses of chemical pesticides are required to achieve effective concentrations in the rhizosphere, which results in the accumulation of harmful residues. Precision farming is needed to improve the efficacy of pesticides, but also to avoid environmental pollution, and slow-release formulations based on nanoparticles offer one solution. Here, we tested the mobility of synthetic and virus-based model nanopesticides by combining soil column experiments with computational modelling. We found that the tobacco mild green mosaic virus and cowpea mosaic virus penetrate soil to a depth of at least 30 cm, and could therefore deliver nematicides to the rhizosphere, whereas the Physalis mosaic virus remains in the first 4 cm of soil and would be more useful for the delivery of herbicides. Our experiments confirm that plant viruses are superior to synthetic mesoporous silica nanoparticles and poly(lactic-co-glycolic acid) for the delivery and controlled release of pesticides, and could be developed as the next generation of pesticide delivery systems.

Tobacco mild green mosaic virus found naturally infecting Nicotiana glauca in Brazil

Nicotiana glauca exhibiting very mild mottling on the leaves was found in the municipality of Piracicaba, state of Sao Paulo, Brazil. Transmission electron microscope analysis of symptomatic tissue, biological assay on virus indicator hosts, and near complete genome sequence indicated infection with the tobamovirus Tobacco mild green mosaic virus. To our knowledge this is the first report of the presence of this tobamovirus in Brazil.

Let There Be Light: Targeted Photodynamic Therapy Using High Aspect Ratio Plant Viral Nanoparticles.

The development of plant viral nanoparticles (VNP) loaded with different molecular versions of a photodynamic drug is described. Specifically, tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV) are developed as drug carriers that encapsulate the monocationic, dicationic, tricationic, and tetracationic versions of a porphyrin-based photosensitizer drug (Zn-Por). While TMV has been extensively explored for various nanotechnology applications, this is the first study investigating TMGMV for medical applications. Light-activated cancer cell killing of Zn-Por-loaded VNPs is studied in vitro using melanoma and cervical cancer models. Native and nucleolin-targeted VNP drug carriers are developed and their efficacy assessed. A fivefold increase in cancer cell killing is observed using nucleolin-targeted TMV loaded with tricationic Zn-Por and displaying the nucleolin-specific F3 peptide.

First Report of a Mixed Infection of Tomato mottle mosaic virus and Tobacco mild green mosaic virus on Eggplants in China

Eggplant (Solanum melongena L.) is an economically important vegetable crop in China. During March 2015 to June 2017, eggplant plants with typical viral symptoms, including dark purple mottle on flowers and slight mosaic and distortion on leaves, were observed at several farms in Shandong, Henan, Liaoning, and Hebei provinces, China. Visual observations estimated the symptom incidence at 20 to 40%. In total, 23 plants were sampled, and four samples (two flowers and two leaves) were randomly selected for total RNA extraction. High-throughput sequencing of small interfering RNA was performed with the pooled extracts using a second-generation sequencing technique by Igenecode Co. Ltd. (Beijing, China), and then the RNA of the virus was screened with bioinformatics methods. A sequence analysis revealed that 52.14 and 47.46% of the total virus sRNA was mapped to tomato mottle mosaic virus (ToMMV) and tobacco mild green mosaic virus (TMGMV) (NCBI reference sequences NC_022230.1 and NC_001556.1), respectively. To validate the presence of ToMMV and TMGMV, total RNA was extracted from each of the four samples, and reverse transcription polymerase chain reaction (RT-PCR) assays were carried out using primer pairs ToMMV-dF (5′-CTGGAGAAGACTGGGTCTAG-3′)/ToMMV-dR (5′-TTCGGTAAGTTCAATGGGACCT-3′) (Li et al. 2017) and 524-F (5′-CGTCATCGAGTACGTTTTAA-3′)/524-R (5′-AGGAAATCTCACAACAATAG-3′) (Cohen et al. 2001). Amplicons of 363-bp ToMMV CP and 500-bp TMGMV CP sequences were obtained from each of the four samples, and the sequences were 100% identical, respectively. Sequences of isolates QZ17091801 and QZ17102625 were deposited in GenBank (MH211033 for CP of ToMMV and MG807477 for CP of TMGMV). BLAST analysis showed that ToMMV CP sequence of the eggplant isolate shared 100% identity with that of a Chinese pepper isolate (KR824951.1) and a U.S. tomato isolate (KT810183.1), whereas the TMGMV CP sequence shared 99% identity with that of a German tobacco isolate (AJ429096.1) and a French trailing petunias isolate (AM262165.1). Field surveys were conducted in several provinces of north China, and 23 flower and leaf samples showing dark purple mottle and chlorotic symptoms were collected and tested by RT-PCR. ToMMV was detected in 20 of the 23 symptomatic samples, whereas TMGMV was detected in all of the 23 samples. To confirm that these eggplant plants were infected by ToMMV and TMGMV, one of the four samples was inoculated back to 10 healthy 3-week-old eggplant plants (cv. Heili) using mechanical inoculation. Ten days later, slight mosaic and distortion symptoms were observed on leaves of the inoculated plants. Total RNA was extracted from the symptomatic leaves and used as template for the ToMMV and TMGMV detection by RT-PCR. Both 363-bp ToMMV CP and 500-bp TMGMV CP sequences obtained were identical to that of the original isolate, confirming the inoculated eggplants were mix infected by ToMMV and TMGMV. To the best of our knowledge, this is the first record of a mixed infection of eggplant by ToMMV and TMGMV in China. Given the economic and nutritional importance of eggplant in China, the occurrence of both viruses on eggplant would be likely to negatively affect yield and farmers’ income. Therefore, ToMMV and TMGMV should be considered in future disease management of susceptible vegetable and ornamental plants.

First Report of a Mixed Infection of Pepper mild mottle virus and Tobacco mild green mosaic virus in Pepper (Capsicum annuum) in the United States

First Report of a Mixed Infection of Pepper mild mottle virus and Tobacco mild green mosaic virus in Pepper (<i>Capsicum annuum</i>) in the United States

Pumpkin: A New Natural Host of Tobacco mild green mosaic virus in China

Pumpkin: A New Natural Host of <i>Tobacco mild green mosaic virus</i> in China

Delivery of Pesticides to Plant Parasitic Nematodes Using Tobacco Mild Green Mosaic Virus as a Nanocarrier.

Plant parasitic nematodes are a major burden to the global agricultural industry, causing a $157 billion loss each year in crop production worldwide. Effective treatment requires large doses of nematicides to be applied, putting the environment and human health at risk. Challenges are to treat nematodes that are located deep within the soil, feeding on the roots of plants. To attack the problem at its roots, we propose the use of tobacco mild green mosaic virus (TMGMV), an EPA-approved herbicide as a carrier to deliver nematicides. TMGMV self-assembles into a 300 × 18 nm soft matter nanorod with a 4 nm-wide hollow channel. This plant virus is comprised of 2130 identical coat protein subunits, each of which displays solvent-exposed carboxylate groups from Glu/Asp as well as Tyr side chains, enabling the functionalization of the carrier with cargo. We report (1) the successful formulation and characterization of TMGMV loaded with ∼1500 copies of the anthelmintic drug crystal violet (CV), (2) the bioavailability and treatment efficacy of CVTMGMV vs CV to nematodes in liquid cultures, and (3) the superior soil mobility of CVTMGMV compared to free CV.

Subcellular Localization and Detection of Tobacco mosaic virus ORF6 Protein by Immunoelectron Microscopy.

Members of the genus Tobamovirus represent one of the best-characterized groups of plant positive, single stranded RNA viruses. Previous studies have shown that genomes of some tobamoviruses contain not only genes coding for coat protein, movement protein, and the cistron coding for different domains of RNA-polymerase, but also a gene, named ORF6, coding for a poorly conserved small protein. The amino acid sequences of ORF6 proteins encoded by different tobamoviruses are highly divergent. The potential role of ORF6 proteins in replication of tobamoviruses still needs to be elucidated. In this study, using biochemical and immunological methods, we have shown that ORF6 peptide is accumulated after infection in case of two isolates of Tobacco mosaic virus strain U1 (TMV-U1 common and TMV-U1 isolate A15). Unlike virus particles accumulating in the cytoplasm, the product of the ORF6 gene is found mainly in nuclei, which correlates with previously published data about transient expression of ORF6 isolated from TMV-U1. Moreover, we present new data showing the presence of ORF6 genes in genomes of several tobamoviruses. For example, in the genomes of other members of the tobamovirus subgroup 1, including Rehmannia mosaic virus, Paprika mild mottle virus, Tobacco mild green mosaic virus, Tomato mosaic virus, Tomato mottle mosaic virus, and Nigerian tobacco latent virus, sequence comparisons revealed the existence of a similar open reading frame like ORF6 of TMV.

FIRST REPORT OF A NEW PEPPER MILD MOTTLE VIRUS PATHOTYPE 1,2 ON PEPPER IN SPAIN

During the summer of 2015, leaf deformations and chlorotic mosaic symptoms on leaves and unusual necrotic lesions on leaves, stems and fruits were observed on pepper (Capsicum annuum) plants grown in Almeria (Spain). Symptomatic leaf and fruit samples from eight collected plants, carrying L3 and L4 resistance against tobamovirus, were analyzed by ELISA specific for potyviruses, Cucumber mosaic virus and each of the following possible pepper-infecting viruses: Paprika mild mottle virus, Tobacco mosaic virus, Tomato mosaic virus, Tobacco mild green mosaic virus, and Pepper mild mottle virus (PMMoV). The eight plants tested negative for all viruses except for PMMoV. Mechanically inoculated extracts from two PMMoV-positive, singly infected symptomatic pepper plants systemically infected C. annuum cv Mazo (Unigenia), but not C. chinense PI159236, C. chacoense PI260429, four commercial L4, and two L3 resistant pepper plants, which exhibited local necrosis symptoms. Total RNAs from two plants inoculated with single infections were extracted, analyzed by reverse transcription PCR using primers designed to specifically amplify the coat protein gene (CP) of PMMoV (Antignus et al., 2008) and directly sequenced. Two independent samples yielded identical nt sequences (GenBank accession No. KT962848) and BLAST analysis showed the highest deduced aa identities of 99.4% with P1,2 isolates (M81413, AB000709, EF434393) of PMMoV. However, the predicted aa sequence possessed an Ala→Thr mutation at residue 66 of the CP. To our knowledge, although PMMoV has been shown to have variants that fail to infect L3 and L4 resistant pepper plants, based on unique CP coding sequences, this is the first report of a new P1,2 pathotype in Spain.

The Influence of Crop Habitat and Control Strategies on Pepper Viruses in Andalusia (Spain)

Andalusia, southern Spain, is a major horticultural production region within the Mediterranean, where over 10,000 ha are dedicated to the production of pepper (Capsicum annuum L.). Approximately two-thirds of the area dedicated to this crop is in a greenhouse and the remaining one-third is comprised of open field crops. Using pepper as a model, we identified and compared the major diseases caused by viruses in the different geographic regions and agronomic systems within the region. Symptomatic samples were collected during 2009 and analyzed by ELISA and RT-PCR for the presence of Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Tomato mosaic virus (ToMV), Pepper mild mottle virus (PMMoV), Potato virus Y (PVY), Tobacco mild green mosaic virus (TMGMV), Tomato chlorosis virus (ToCV) and Parietaria mottle virus (PMoV). Contingency table analysis showed a significant relationship between the presence of major diseases caused by viruses in pepper crops and the different agrosystems in terms of location (inland versus coastal), disease control management (chemical versus integrated), cropping system (open field versus greenhouse), and virus-resistant versus susceptible cultivars. Pepper crops in plastic-covered greenhouses were predominantly associated with arthropod-transmitted virus diseases, such as TSWV. CMV was predominant in provinces located inland, and PMoV was found independent of the agrosystem, disease control methods, or geographic location.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TOBACCO MILD GREEN MOSAIC VIRUS IN HUNGARY

Pepper (Capsicum annuum L.), an important crop in Hun- gary, is frequently infected by Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV) and, recently, also by Pepper mild mottle virus (PMMoV) and Tomato spotted wilt virus (TSWV). In the spring of 2015, mild mosaic symptoms were observed on cv. Ho F1. Symptoms on the fruits were more obvious, consisting of reduction in size, mottling and color changes, brown necrotic streaks and spots. To identify the putative pathogen(s), symptomatic fruits were collected and used for host range determination. Symptoms on mechani- cally inoculated Nicotiana tabacum cv. Samsun, N. tabacum cv. Xanthi-nc, Nicotiana benthamiana, Capsicum annuum cv. Albaregia (L+ gene), C. annuum cv. Feherozon (L1) and C. annuum cv. Brendon F1 (L3) suggested the presence of a tobamovirus belonging to pathotype 0 (Boukema, 1980). Moreover, total RNA was extracted from a symptomatic pepper fruit with the RNeasy plant mini kit (Qiagen, Ger- many) and used in RT-PCR using universal tobamovirus primers for the coat protein gene (Kalman et al., 2001). A products of the expected size (700 bp) was obtained, cloned into pGEM-T Easy Vector (Promega, USA) and sequenced (Biomi, Hungary). The sequence was deposited in GenBank under the accession No. KT374283. Blast analysis showed a 99% identity at nucleotide level with a Spanish isolate (P04/17, accession No. FN594859) of Tobacco mild green mosaic virus (TMGMV). To our knowledge, this is the first report of TMGMV on pepper in Hungary.

Differential Effects of Viral Coat Protein on Induction of Hypersensitive Response and Systemic Movement of Tobacco Mosaic Virus and Tobacco Mild Green Mosaic Virus in Nicotiana megalosiphon

Tobacco mosaic virus (TMV) and Tobacco mild green mosaic virus (TMGMV) are both known to induce hypersensitive response (HR) local lesions in Nicotiana megalosiphon, a hybrid plant from a cross between N. suaveolens and N. fragrans, but only TMV is capable of systemic movement. Therefore, the determinants of induction of hypersensitive response and systemic movement of TMV and TMGMV in N. megalosiphon were further analyzed. HR was shown to be independent of the temperature indicating that the resistance responses are different from N gene responses induced in tobacco (N. tabacum cv. Xanthi-nc.) to TMV. Comparison of lesion growth between wild-type and transgenic tobacco plants encoding salicylate hydroxylase (nahG) indicated that TMV spread similarly in N. megalosiphon and Xanthi-nc./nahG plants. In addition, exogenous application of SA did not prevent systemic movement of TMV. Coat protein-deficient TMV failed to induce HR and move systemically in N. megalosiphon indicating that CP is the inducer of HR and determinant for systemic movement. However, single epidermal cells expressing TMV-CP did not undergo cell death suggesting that formation of HR cell death requires viral movement out of the initially infected epidermal cells or the presence of intact virus particles. Furthermore, comparison of different TMV strains, including virus vector carrying the CP of TMGMV-U5 in place of TMV-CP, showed no differences in the induction of HR and timing of systemic virus movement suggesting that CP is not the determinant for differential invasion of TMV and TMGMV.

Amino acids in Tobamovirus coat protein controlling pepper L1a gene‐mediated resistance

In pepper plants (genus Capsicum), the resistance against Tobamovirus spp. is conferred by L gene alleles. The recently identified L variant L(1a) can recognize coat proteins (CPs) of Tobacco mild green mosaic virus Japanese strain (TMGMV-J) and Paprika mild mottle virus Japanese strain (PaMMV-J), but not of Pepper mild mottle virus (PMMoV), as the elicitor to induce resistance at 24 °C. Interestingly, L(1a) gene-mediated resistance against TMGMV-J, but not PaMMV-J, is retained at 30 °C. This observation led us to speculate that L(1a) can discriminate between CPs of TMGMV-J and PaMMV-J. In this study, we aimed to determine the region(s) in CP by which L(1a) distinguishes TMGMV-J from PaMMV-J. By using chimeric CPs consisting of TMGMV-J and PaMMV-J, we found that the chimeric TMGMV-J CP, whose residues in the β-sheet domain were replaced by those of PaMMV-J, lost its ability to induce L(1a) gene-mediated resistance at 30 °C. In contrast, the chimeric PaMMV-J CP with the β-sheet domain replaced by TMGMV-J CP was able to induce L(1a) gene-mediated resistance at 30 °C. Furthermore, viral particles were not detected in the leaves inoculated with either chimeric virus. These observations indicated that the amino acids within the β-sheet domain were involved in both the induction of L(1a) gene-mediated resistance and virion formation. Further analyses using chimeric CPs of TMGMV-J and PMMoV indicated that amino acids within the β-sheet domain alone were not sufficient for the induction of L(1a) gene-mediated resistance by TMGMV-J CP. These results suggest that multiple regions in Tobamovirus CP are implicated in the induction of L(1a) gene-mediated resistance.

A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana is up-regulated in response to Ralstonia solanacearum infection, pathogen associated molecular patterns and effector molecules and involved in plant immunity

To elucidate the molecular mechanisms of plant immune responses, we isolated genes whose expression was regulated by inoculation with Ralstonia solanacearum. Here, we report the characterization of Nicotiana benthamiana belonging to the SEC14-gene superfamily designated as Nicotiana benthamiana SEC14 (NbSEC14). NbSEC14 rescued growth defects and impaired invertase secretion associated with the yeast sec14p temperature-sensitive mutant, while recombinant NbSec14 protein had phospholipids transfer activity. NbSEC14 expression was up-regulated in N. benthamiana leaves after inoculation with virulent or avirulent R. solanacearum. Expression of NbSEC14 was induced by treatment with chitin, flg22, and by Agrobacterium-mediated transient expression of INF1 elicitin, AvrA from R. solanacearum, and co-expression of the capsid protein from Tobacco mild green mosaic virus with its cognate resistance L1 protein. NbSEC14-silenced plants showed accelerated growth of both the virulent and avirulent R. solanacearum as well as acceleration of disease development. This study may provide useful information for the further analysis of the function of plant Sec14 protein homologs in the regulation of plant immune responses.

고추와 토마토 종자에서 종자전염 세균 및 바이러스의 동시 검출을 위한 One-step Multiplex RT-PCR 방법

우리나라에서 주로 재배되는 가지과작물의 종자 전염 병원균 중에서 세균성 병원균 Xanthomonns campestris pv. vesicatoria(Xcv), Clavibacter michiganensis subsp. michiganensis (Cmm), Erwinia carotovora subsp. carotovora (Ecc)와 바이러스 병원균 Pepper mild mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV)를 종자로부터 동시검출하기 위한 One-step Multiplex RT-PCR을 개발하였다. 각각의 병원균을 특이적으로 증폭시키는 병원균 검출용 프라이머 15세트를 primer-blast 프로그램을 이용하여 제작하였고 각각의 병원균을 특이적으로 검출할 수 있는 5세트의 프라이머 세트(Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMVF/R)를 선발하였다. 이들 프라이머 세트는 프라이머간의 또는 병원균 cDNA간의 간섭없이 타겟 병원균만을 검출하였다. 가지과 작물 중에서 유통 중인 고추종자 20품종과 토마토종자 10품종에 대한 종자 감염 병원균 검출을 위한 PCR을 수행한 결과, 2개 품종의 토마토종자에서 Ecc가 검출되었고 4개 품종의 고추종자에서도 Ecc가 검출되었다. 특히 고추 종자에선 Ecc가 검출된 4개 품종 외에 1개 품종에서 PMMoV, TMGMV가 동시 검출되었다. 이로 인해 개발된 One-step multiplex RT-PCR은 서로 간섭 현상 없이 병원균을 효율적으로 검출할 수 있음을 보여 주었다. The aim of this study was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant seed infection pathogenic bacteria and virus, Xanthomonns campestris pv. vesicatoria (Xcv), Clavibacter michiganensis subsp. michiganensis (Cmm), Erwinia carotovora subsp. carotovora (Ecc), Pepper mild mottle virus (PMMoV) and Tobacco mild green mosaic virus (TMGMV) in pepper and tomato seeds. Most of pepper and tomato bacterial and virus diseases are responsible for germination and growth obstruction. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcv, Cmm, Ecc, PMMoV and TMGMV in pepper and tomato seeds, five pairs (Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMV-F/R) of specific primer were synthesized by primer-blast program. The multiplex PCR for the five pathogens in pepper and tomato seeds could detect specially without interference among primers and/or cDNA of plant seeds and other plant pathogens. The PCR result for pathogen detection using 20 commercial pepper and 10 tomato seed samples, Ecc was detected from 4 pepper and 2 tomato seed samples, PMMoV was detected from 1 pepper seed sample, and PMMoV and TMGMV were simultaneously detected from 1 pepper seed sample.

First report of Tobacco mild green mosaic virus infecting tomato in Iran

Iran is amongst the seven top producers of tomato ( Solanum lycopersicum ) in the world with an approximate production of 4,826,396 tonnes yearly (Faostat, 2009). A number of plant viruses have been previously identified from tomato fields…