Tobacco Mild Green Mosaic Virus Research Articles

Development and Application of Attenuated Plant Viruses as Biological Control Agents in Japan.

In 1929, it was reported that yellowing symptoms caused by a tobacco mosaic virus (TMV) yellow mosaic isolate were suppressed in tobacco plants that were systemically infected with a TMV light green isolate. Similar to vaccination, the phenomenon of cross-protection involves a whole plant being infected with an attenuated virus and involves the same or a closely related virus species. Therefore, attenuated viruses function as biological control agents. In Japan, many studies have been performed on cross-protection. For example, the tomato mosaic virus (ToMV)-L11A strain is an attenuated isolate developed by researchers and shows high control efficiency against wild-type ToMV in commercial tomato crops. Recently, an attenuated isolate of zucchini yellow mosaic virus (ZYMV)-2002 was developed and registered as a biological pesticide to control cucumber mosaic disease. In addition, attenuated isolates of pepper mild mottle virus (PMMoV), cucumber mosaic virus (CMV), tobacco mild green mosaic virus (TMGMV), melon yellow spot virus (MYSV), and watermelon mosaic virus (WMV) have been developed in Japan. These attenuated viruses, sometimes called plant vaccines, can be used not only as single vaccines but also as multiple vaccines. In this review, we provide an overview of studies on attenuated plant viruses developed in Japan. We also discuss the application of the attenuated strains, including the production of vaccinated seedlings.

Inter-coat protein loading of active ingredients into Tobacco mild green mosaic virus through partial dissociation and reassembly of the virion

Chemical pesticide delivery is a fundamental aspect of agriculture. However, the extensive use of pesticides severely endangers the ecosystem because they accumulate on crops, in soil, as well as in drinking and groundwater. New frontiers in nano-engineering have opened the door for precision agriculture. We introduced Tobacco mild green mosaic virus (TMGMV) as a viable delivery platform with a high aspect ratio and favorable soil mobility. In this work, we assess the use of TMGMV as a chemical nanocarrier for agriculturally relevant cargo. While plant viruses are usually portrayed as rigid/solid structures, these are “dynamic materials,” and they “breathe” in solution in response to careful adjustment of pH or bathing media [e.g., addition of solvent such as dimethyl sulfoxide (DMSO)]. Through this process, coat proteins (CPs) partially dissociate leading to swelling of the nucleoprotein complexes—allowing for the infusion of active ingredients (AI), such as pesticides [e.g., fluopyram (FLP), clothianidin (CTD), rifampicin (RIF), and ivermectin (IVM)] into the macromolecular structure. We developed a “breathing” method that facilitates inter-coat protein cargo loading, resulting in up to ~ 1000 AIs per virion. This is of significance since in the agricultural setting, there is a need to develop nanoparticle delivery strategies where the AI is not chemically altered, consequently avoiding the need for regulatory and registration processes of new compounds. This work highlights the potential of TMGMV as a pesticide nanocarrier in precision farming applications; the developed methods likely would be applicable to other protein-based nanoparticle systems.

Phenotypical and molecular characterization of new pepper genotypes resistant to Chili pepper mild mottle virus firstly detected in Europe and other tobamoviruses

The tobamoviruses pose a recent threat to local varieties of Gernika pepper (cv. “Derio”) and Ibarra chili (cv. “Ibarroria”), causing a substantial decrease in both yield and fruit quality. Tobamoviruses such as Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV) or Pepper mild mottle virus (PMMoV) are the viruses that cause the greatest impact in pepper crops of northern Spain. The emergence of a novel Tobamovirus, Chili pepper mild mottle virus (CPMMoV; GenBank MN164455.1), firstly identified in Europe, exacerbates these hazards. This situation has jeopardized the ongoing cultivation in some of the virus affected plots. A molecular marker-assisted backcrossing (MABC) breeding program was thus initiated to introduce the L3 and L4 resistance genes into these susceptible local varieties. MABC consisted of one crossing followed by four backcrosses and two self-fertilizations. After each crossbreeding event, the selection criteria of plants were based on plants resistance and fruits resemblance with the original sensitive varieties. This study allowed the homozygous introgression of resistance genes in the new obtained genotypes, reducing the reliance on hybrid seed production and associated costs. The resulting genotypes demonstrated robust resistance when inoculated with PMMoV 1.2, PMMoV 1.2.3, and CPMMoV. In addition, greenhouse yield trials were performed during two consecutive years to identify and select the most productive genotypes harboring the L3 or L4 gene, with the aim of registering some of them as commercial varieties. The productivity of the new selected genotypes did not display significant differences when compared to the original sensitive varieties. The introduction of genetic resistance in these local pepper varieties not only mitigates the immediate hazard posed by Tobamovirus but also fosters the preservation of local agricultural genetic resources, ensuring their continued cultivation and economic sustainability.

Tobacco Mild Green Mosaic Virus (TMGMV) Isolates from Different Plant Families Show No Evidence of Differential Adaptation to Their Host of Origin.

The relevance of tobamoviruses to crop production is increasing due to new emergences, which cannot be understood without knowledge of the tobamovirus host range and host specificity. Recent analyses of tobamovirus occurrence in different plant communities have shown unsuspectedly large host ranges. This was the case of the tobacco mild green mosaic virus (TMGMV), which previously was most associated with solanaceous hosts. We addressed two hypotheses concerning TMGMV host range evolution: (i) ecological fitting, rather than genome evolution, determines TMGMV host range, and (ii) isolates are adapted to the host of origin. We obtained TMGMV isolates from non-solanaceous hosts and we tested the capacity of genetically closely related TMGMV isolates from three host families to infect and multiply in 10 hosts of six families. All isolates systemically infected all hosts, with clear disease symptoms apparent only in solanaceous hosts. TMGMV multiplication depended on the assayed host but not on the isolate's host of origin, with all isolates accumulating to the highest levels in Nicotiana tabacum. Thus, results support that TMGMV isolates are adapted to hosts in the genus Nicotiana, consistent with a well-known old virus-host association. In addition, phenotypic plasticity allows Nicotiana-adapted TMGMV genotypes to infect a large range of hosts, as encountered according to plant community composition and transmission dynamics.

Host-virus interaction between tobacco mild green mosaic virus strain U2 and tropical soda apple resulting in systemic hypersensitive necrosis and the host range, survival, spread, and molecular characterization of the virus.

Tobacco mild green mosaic virus strain U2 (TMGMV-U2) is a registered active ingredient in a bioherbicide to control tropical soda apple (TSA), Solanum viarum, an invasive weed. As required for registration, we developed empirical data on the host-virus interaction and the virus's host range, survival, spread, and genomic sequence. TMGMV-U2 killed TSA plants by causing systemic hypersensitive necrosis (SHN). It elicited local lesions in inoculated leaves which was followed by the plant's wilting and death. It moved from inoculated terminal leaves through the vasculature to roots and then to newly developed leaves. Phloem death was implicated in wilting and plant death. The SHN response was attenuated in plants grown at constant 32 °C. TMGMV-U2 titer in TSA was low compared to a systemically susceptible tobacco. The virus remained infective for up to 6 months in infected dead TSA tissues and in soil in which infected plants had grown. Susceptible tobacco and pepper plants grown in soil that previously had infected dead TSA or in soil amended with the virus remained asymptomatic and virus-free. A susceptible pepper crop grown in a field block following two consecutive crops of TMGMV-U2-infected susceptible tobacco grew disease-free and virus-free and without yield loss. Purified TMGMV-U2 was infective for 1 year when stored at -20 °C or 5 °C and for 1 month at room temperature. No virus spread was found in the field. Genomic analyses confirmed the registered isolate to be a U2 strain and free of satellite TMV. The TMGMV-U2-susceptible species preponderantly belonged to the Solanaceae. A few hosts that were killed belonged to this family. Several new hosts to TMGMV-U2 were found. These data enabled registration of TMGMV-U2. TMGMV-U2 can be used safely as a bioherbicide without risks to nontarget plants and the environment. © 2023 Society of Chemical Industry.

Use of plant viruses as bioherbicides: The first virus-based bioherbicide and future opportunities.

Until recently, only a few plant viruses had been studied for use as biological control agents for weeds, but none was developed into a registered bioherbicide. This position changed in 2015 when the U.S. Environmental Protection Agency (EPA) granted an unrestricted Section 3 registration for tobacco mild green mosaic virus (TMGMV) strain U2 as an herbicide active ingredient (a.i.) for a commercial bioherbicide (SolviNix LC). It is approved for the control of tropical soda apple (TSA, Solanum viarum), an invasive 'noxious weed' in the United States. TSA is a problematic weed in cattle pastures and natural areas in Florida. The TMGMV-U2 product kills TSA consistently, completely, and within a few weeks after its application. It is part of the TSA integrated best management practice (BMP) in Florida along with approved chemical herbicides and a classical biocontrol agent, Gratiana boliviana (Coleoptera: Chrysomelidae). TMGMV is nonpathogenic and nontoxic to humans, animals, and other fauna, environmentally safe, and as effective as chemical herbicides. Unlike the insect biocontrol agent, TMGMV kills and eliminates the weed from fields and helps recycle the dead biomass in the soil. Here the discovery, proof of concept, mode of action, risk analyses, application methods and tools, field testing, and development of the virus as the commercial product are reviewed. Also reviewed here are the data and scientific justifications advanced to answer the concerns raised about the use of the virus as a herbicide. The prospects for discovery and development of other plant-virus-based bioherbicides are discussed. This article is protected by copyright. All rights reserved.

Biological and molecular interactions between bell pepper endornavirus and two tobamoviruses

Peppers (Capsicum spp.) are native plants to the Americas. They are cultivated worldwide for direct human consumption and industrial purposes. Peppers can be infected by acute plant viruses, which cause a variety of diseases and crop losses. However, many Capsicum species can also be infected by persistent viruses. These are emerging viruses and they do not cause apparent disease and are transmitted only vertically. Using two near-isogenic lines of bell pepper cv. Marengo, biological and molecular interactions between the persistent virus bell pepper endornavirus (BPEV) and two acute viruses, pepper mild mottle virus (PMMoV) and tobacco mild green mosaic virus (TMGMV), were evaluated by symptom expression, enzyme-linked immunosorbent assay, and RT-qPCR. The relative titer of BPEV decreased at least two-fold at 14 days after infection when BPEV-infected plants were single infected with TMGMV or in mixed infection of PMMoV and TMGMV. The presence of BPEV was associated with symptom reduction in pepper plants infected with single and mixed infections of PMMoV and TMGMV. This suggests that the ubiquitous infection of BPEV may trigger the plant immune response, and therefore, BPEV is active when the plant is infected with PMMoV and/or TMGMV.

Rapid and visual field diagnosis of tomato brown rugose fruit virus using reverse transcription recombinase aided amplification (RT RAA) combined with lateral flow strips (LFS)

Tomato brown rugose fruit virus (ToBRFV) causes significant disease outbreaks in tomato and pepper production areas worldwide. A simple, fast, and visual detection method for ToBRFV is critical for controlling the viral disease. Here, a method was constructed to detect ToBRFV by combining reverse transcription recombinase assisted amplification (RT RAA) and lateral flow test strips (LFS). After selecting the primers and probe, the concentration of the modified reverse primer and magnesium acetate in the RT RAA-LFS assay were optimized. The RT RAA could amplify the target sequence in 16 min at a constant temperature of 39 °C, and the amplification products could be visualized by LFS within 5 min. Importantly, there was no cross-reaction with other viruses that can infect tomatoes, such as tobacco mosaic virus, tomato mild mottle virus, pepper mild mottle virus, tobacco mild green mosaic virus, broad bean wilt virus, pepper vein yellows virus, and chilli veinal mottle virus. Furthermore, the RT RAA-LFS assay was highly sensitive, with a detection limit of 2.1 × 101 copies/50-μL reaction. Using 138 field samples and 6 commercial seeds with suspected infection of ToBRFV, the RT RAA-LFS and RT-PCR methods gave identical results, indicating that the RT RAA-LFS method is as reliable as the standard RT-PCR method for detecting ToBRFV. Our RT RAA-LFS assay could be a valuable tool for the rapid and accurate detection of ToBRFV.

Delivery of Nematicides Using TMGMV-Derived Spherical Nanoparticles.

Spherical nanoparticles (SNPs) from tobacco mild green mosaic virus (TMGMV) were developed and characterized, and their application for agrochemical delivery was demonstrated. Specifically, we set out to develop a platform for pesticide delivery targeting nematodes in the rhizosphere. SNPs were obtained by thermal shape-switching of the TMGMV. We demonstrated that cargo can be loaded into the SNPs during thermal shape-switching, enabling the one-pot synthesis of functionalized nanocarriers. Cyanine 5 and ivermectin were encapsulated into SNPs to achieve 10% mass loading. SNPs demonstrated good mobility and soil retention slightly higher than that of TMGMV rods. Ivermectin delivery to Caenorhabditis elegans using SNPs was determined after passing the formulations through soil. Using a gel burrowing assay, we demonstrate the potent efficacy of SNP-delivered ivermectin against nematodes. Like many pesticides, free ivermectin is adsorbed in the soil and did not show efficacy. The SNP nanotechnology offers good soil mobility and a platform technology for pesticide delivery to the rhizosphere.

Population structure and genetic diversity of tobacco mild green mosaic virus variants in Western Anatolia of Turkey

Tobamoviruses have been pioneers in understanding the population genetic structure, host-vector interactions, and evolutionary processes of viruses. To better understand the population genetic structure and molecular evolutionary relationships of tobacco mild green mosaic virus (TMGMV), a significant species of tobamoviruses, extensive analyses were performed using bioinformatics tools in this study. 300 samples were collected from plants exhibiting viruses and virus-like symptoms from Turkey's largest tobacco cultivation areas during the plant vegetation period between 2019 and 2020. Samples were tested using conventional molecular techniques for tobamoviruses, including ToBRFV, TMV, TMGMV, ToMV, and ToMMV species. Single and double tobamovirus infections were determined in 258 of 300 samples. Single infections were 225 (75%), 10 (3.33%), and 1 (0.33%) for TMGMV, TMV, and ToMV, respectively. Double infections were 21 (7.0%) and 1 (0.33%) for TMGMV + TMV and TMV + ToMV, respectively. TMGMV-infected samples from each region were chosen, and their complete genomes were revealed. Detailed analyses were carried out from Turkey, with global variants available in GenBank. Molecular evolutionary analyses revealed three main lineages (Clades I, II, and III) at the p126, p183, MP, and complete genome levels and two main lineages at the CP gene (Clades I and II). Variants from Turkey were distributed in two different branches in other phylogenetic trees, except for the CP gene. High haplotype diversity and low nucleotide diversity were determined in each gene region, indicating consistent genetic stability. In addition, purifying selection pressures were determined in specific gene regions. With AMOVA (analysis of molecular variance), it was determined that the source of genetic variation came from within the main lineages, including various hosts and geographies. The differences in lineages were confirmed by independent test statistics. While neutrality tests revealed population expansions in the CP and MP genes, other p183 and p126 genes revealed bottlenecks or balancing selection. The fact that TMGMV was more common than TMV in Turkey strongly supported the phenomenon called “mutational melting” or “Müller latch,” which presumably causes TMV to disappear from its niche.

Türkiye'de Biberde Enfeksiyon Oluşturan Tobacco mild green mosaic virus İzolatlarının Belirlenmesi ve Moleküler Karakterizasyonu

In this study, Tobacco mild green mosaic virus (TMGMV) infection was investigated in pepper crops from two regions having high economical importance in Turkey. A total of 397 leaf and fruit samples showing mosaic, yellowing, mottling, and pitting symptoms were collected to test by double antibody sandwich ELISA (DAS-ELISA) using polyclonal antiserum. DAS-ELISA results indicated that 97 out of all tested samples (24.4%) were found to be infected with TMGMV. Samples resulting positive for TMGMV infection were used to amplify the virus coat protein (CP) gene sequences with specific primers by RT-PCR for further molecular characterization of the virus isolates. A comparison of the CP sequences of the virus isolates revealed that the identity of nucleotides ranged between 97.2 and 100%, and the amino acid similarity ranged between 96.8 and 100% among themselves. To the best of our knowledge, this is the first report of TMGMV infection in Capsicum annuum in Turkey.

The 'Candidatus Phytoplasma tritici' effector SWP12 degrades the transcription factor TaWRKY74 to suppress wheat resistance.

'Candidatus Phytoplasma tritici' ('Ca. P. tritici') is an insect-borne obligate pathogen that infects wheat (Triticum aestivum) causing wheat blue dwarf disease, and leads to yield losses. SWP12 is a potential effector secreted by 'Ca. P. tritici' that manipulates host processes to create an environment conducive to phytoplasma colonization, but the detailed mechanism of action remains to be investigated. In this study, the expression of SWP12 weakened the basal immunity of Nicotiana benthamiana and promoted leaf colonization by Phytophthora parasitica, Sclerotinia sclerotiorum, and tobacco mild green mosaic virus. Moreover, the expression of SWP12 in wheat plants promoted phytoplasma colonization. Triticum aestivum WRKY74 and N. benthamiana WRKY17 were identified as host targets of SWP12. The expression of TaWRKY74 triggered reactive oxygen species bursts, upregulated defense-related genes, and decreased TaCRR6 transcription, leading to reductions in NADH dehydrogenase complex (NDH) activity. Expression of TaWRKY74 in wheat increased plant resistance to 'Ca. P. tritici', and silencing of TaWRKY74 enhanced plant susceptibility, which indicates that TaWRKY74 is a positive regulator of wheat resistance to 'Ca. P. tritici'. We showed that SWP12 weakens plant resistance and promotes 'Ca. P. tritici' colonization by destabilizing TaWRKY74.

Sequencing and Variant Detection of Eight Abundant Plant-Infecting Tobamoviruses across Southern California Wastewater.

Tobamoviruses are agriculturally relevant viruses that cause crop losses and have infected plants in many regions of the world. These viruses are frequently found in municipal wastewater, likely coming from human diet and industrial waste across wastewater catchment areas. As part of a large wastewater-based epidemiology study across Southern California, we analyzed RNA sequence data from 275 influent wastewater samples obtained from eight wastewater treatment plants with a catchment area of approximately 16 million people from July 2020 to August 2021. We assembled 1,083 high-quality genomes, enumerated viral sequencing reads, and detected thousands of single nucleotide variants from eight common tobamoviruses: bell pepper mottle virus, cucumber green mottle mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, tomato brown rugose fruit virus, tomato mosaic virus, tomato mottle mosaic virus, and tropical soda apple mosaic virus. We show that single nucleotide variants had amino acid-altering consequences along with synonymous mutations, which represents potential evolution with functional consequences in genomes of these viruses. Our study shows the importance of wastewater sequencing to monitor the genomic diversity of these plant-infecting viruses, and we suggest that our data could be used to continue tracking the genomic variability of such pathogens. IMPORTANCE Diseases caused by viruses in the genus Tobamovirus cause crop losses around the world. As with other viruses, mutation occurring in the virus's genomes can have functional consequences and may alter viral infectivity. Many of these plant-infecting viruses have been found in wastewater, likely coming from human consumption of infected plants and produce. By sequencing RNA extracted from influent wastewater obtained from eight wastewater treatment plants in Southern California, we assembled high-quality viral genomes and detected thousands of single nucleotide variants from eight tobamoviruses. Our study shows that Tobamovirus genomes vary at many positions, which may have important consequences when designing assays for the detection of these viruses by agricultural or environmental scientists.

First Report of Tobacco Mild Green Mosaic Virus Infecting Rehmannia glutinosa in China.

Rehmannia glutinosa (family Scrophulariaceae) is an important traditional medicinal plant, whose root is used to treat anemia, hemoptysis, and gynecological diseases in China (Matsumoto et al. 1989). This plant is native to China and cultivated in China, Korea, Japan, and northern Vietnam (Kwak et al. 2020). Viral diseases caused remarkable loss in the yield and quality of R. glutinosa (Ling et al. 2009). To date, ten viruses have been identified globally to infect R. glutinosa and seven of these viruses reported in China (Liu et al. 2018; Zhang et al. 2021). Most plants of R. glutinosa are infected with one or more of these viruses (Kwak et al. 2018; Zhang et al. 2004). In July 2020, a survey of the viral disease infecting R. glutinosa was conducted in commercial plantations of Wenxian, Wuzhi, Mengzhou, and Yuzhou counties in Henan Province, China. The disease symptoms included mosaic, chlorosis, leaf distortion, and the percentage of symptomatic plants was over 70% in the surveyed fields (n=9). Sixty leaf samples of symptomatic R. glutinosa plants were collected from nine cultivation fields in Wenxian, Wuzhi, Mengzhou, and Yuzhou counties (five to seven plants for each field). Total RNA was extracted from one pooled sample containing a portion of all above-mentioned leaf samples using RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China) and analyzed by high-throughput sequencing (HTS) to identify viral pathogens. A transcriptome library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), and sequenced on an Illumina NovaSeq6000 sequencing system at Berry Genomics Corporation (Beijing, China). A total of 27,664,949 high-quality clean reads were obtained after trimming and used for contig assembly. The assembled contigs (n=109,180) were searched using Basic Local Alignment Search Tool (BLAST) at GenBank. BLASTn analysis showed that the R. glutinosa plants were infected with known viruses, including broad bean wilt virus, rehmannia mosaic virus, youcai mosaic virus, and cucurbit chlorotic yellows virus. In addition, one contig (6,418 nt in length) had a nucleotide sequence identity of 99.64% with the TN29 isolate of tobacco mild green mosaic virus (TMGMV, GenBank accession no. MF139550). To confirm the presence of this virus, sixty above-mentioned samples were screened by reverse transcription-polymerase chain reaction (RT-PCR) using the specific primer pairs (Supplementary Table1) TMGMG-CPF/TMGMG-CPR targeting a 545-nt fragment within the CP gene. Amplicons with expected sizes were detected from 47 of 60 samples but not from the negative control (virus-free healthy plant through the tip meristem culture). Seventeen amplicons (11#, 13#, 14#, 21#, 22#, 23#, 25#, 26#, 27#, 31#, 32#, 33#, 37#, 52#, 57#, 59#, and 60#) of TMGMV-CP were selected, and purified. The PCR products were cloned into the pMD19-T vector (TAKARA Biotech, Dalian, China) and sequenced. The sequences were deposited into the GenBank (accession nos. MZ395944 to MZ395960). The near-full-length genomic sequence of TMGMV-Rg14 isolate was obtained from one positive sample (sample no. 14) by RT-PCR amplification of two overlapping fragments using the following primer pairs: TMGMV-40F/TMGMV-3570R and TMGMV-3220F/TMGMV-6400R. The near-full-length genomic sequence of the TMGMV-Rg14 isolate was 6 304 nucleotides (nt) in length and deposited into GenBank (accession no. MZ395975). BLASTn analysis demonstrated that the TMGMV-Rg14 isolate shared a sequence identity ranging from 96.89% (AB078435) to 99.60% (MF139550) with the other TMGMV isolates. Furthermore, the virus-free healthy R. glutinosa plants were inoculated with sap from the positive sample (14#) to confirm the infection of TMGMV. Mosaic symptoms were induced on the systemically infected leaves of the inoculated plants 14 days post inoculation. The systemically infected leaves of inoculated plants were assayed by RT-PCR using the primer pairs TMGMV-CPF/CPR. Amplicons of expected size were detected from the inoculated plants but not from non-inoculated plants. To our knowledge, this is the first report of TMGMV infection on R. glutinosa. Further studies are necessary to select a suitable indicator plant for this TMGMV, its host range, and the symptoms it induces in single infection. Since R. glutinosa is cultivated by vegetative propagation, production of virus-free healthy plants is necessary. This study will help to generate virus-free healthy plants and prevent viral disease on R. glutinosa. Further study is needed to determine its pathological implications and economic impact on R. glutinosa in China.

Bioconjugation Strategies for Tobacco Mild Green Mosaic Virus.

Tobacco mild green mosaic virus (TMGMV) is a plant virus closely related to Tobacco mosaic virus (TMV), sharing many of its structural and chemical features. These rod-shaped viruses, comprised of 2130 identical coat protein subunits, have been utilized as nanotechnological platforms for a myriad of applications, ranging from drug delivery to precision agriculture. This versatility for functionalization is due to their chemically active external and internal surfaces. While both viruses are similar, they do exhibit some key differences in their surface chemistry, suggesting the reactive residue distribution on TMGMV should not overlap with TMV. In this work, we focused on the establishment and refinement of chemical bioconjugation strategies to load molecules into or onto TMGMV for targeted delivery. A combination of NHS, EDC, and diazo coupling reactions in combination with click chemistry were used to modify the N-terminus, glutamic/aspartic acid residues, and tyrosines in TMGMV. We report loading with over 600 moieties per TMGMV via diazo-coupling, which is a >3-fold increase compared to previous studies. We also report that cargo can be loaded to the solvent-exposed N-terminus and carboxylates on the exterior/interior surfaces. Mass spectrometry revealed the most reactive sites to be Y12 and Y72, both tyrosine side chains are located on the exterior surface. For the carboxylates, interior E106 (66.53 %) was the most reactive for EDC-propargylamine coupled reactions, with the exterior E145 accounting for >15 % reactivity, overturning previous assumptions that only interior glutamic acid residues are accessible. A deeper understanding of the chemical properties of TMGMV further enables its functionalization and use as a multifunctional nanocarrier platform for applications in medicine and precision farming.

Metagenomic analysis for detection and discovery of plant viruses in wild Solanum spp. in South Africa

AbstractWild plant species play an important role in plant virus epidemiology, mainly by harbouring viruses pathogenic to cultivated plant species. The genus Solanum includes important crop species as well as some highly abundant and widely distributed wild plant species. The aim of the present study was to profile the viromes of 11 wild Solanum spp. found in five provinces of South Africa. Metatranscriptomic analysis of pooled RNA samples from each of the Solanum spp. detected viruses belonging to 13–19 families. Reverse transcription PCR confirmed the detection of potato virus Y (PVY), potato leaf roll virus (PLRV), tomato torrado virus (ToTV) and tomato chlorosis virus (ToCV) for the first time in some of the Solanum spp. studied. Phylogenetic analyses indicated that the PVY and PLRV strains from the wild Solanum spp. were divergent from those reported in cultivated crops, while there was limited divergence among ToTV and ToCV isolates. Viruses detected for the first time in South Africa included tobacco mild green mosaic virus, tomato matilda virus, a pepper enamovirus from Rwanda and a cytorhabdovirus from potato in Kenya. Potentially novel viruses were also detected in S. lichtensteinii, S. mauritianum and S. viarum. This study demonstrates the role of Solanum spp. in harbouring viruses pathogenic to solanaceous crops, and in the emergence of new virus strains. It may therefore inform strategies for virus disease management by control of wild solanums that harbour viruses pathogenic to important Solanum crops.

A Survey of Main Pepper Crop Viruses in Different Cultivation Systems for the Selection of the Most Appropriate Resistance Genes in Sensitive Local Cultivars in Northern Spain.

Viral diseases have become one of the main phytosanitary problems for pepper growers in the Basque Country (northern Spain). In 2014, a survey was carried out to determine the prevalence of the most common viruses found in Gernika pepper and Ibarra chili pepper landraces. A total of 97 plots were surveyed and classified according to the crop system. Within these plots, 1107 plants were sampled and tested for tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), tobacco mild green mosaic virus (TMGMV), pepper mild mottle virus (PMMoV), paprika mild mottle virus (PaMMV), potato virus Y (PVY) and tomato spotted wilt virus (TSWV) applying a DAS-ELISA test. PaMMV was verified by the non-radioactive molecular hybridization technique and it was found to be negative. All viruses were detected, but the most prevalent viruses were PVY and TMGMV (19.8% and 10.6% of tested plants, respectively). Differences among cultivation systems were found for most of the tested viruses. PVY had a higher level of infection under open field conditions (27.3%) than under greenhouse conditions (12.3%). Inversely, the viruses belonging to the Tobamovirus genus and TSWV prevailed under greenhouse conditions (28.9% and 5.2%) when compared to open field (11.2% and 1.1%), respectively. Single (28%) and multiple infections (8.9%) were found. All PMMoV isolates were classified as pathotype P1.2. Survey results indicated that tobamovirus and PVY resistance genes would be the most appropriate to be included in breeding programs with these sensitive pepper landraces.

Development of a reverse-transcription loop-mediated isothermal amplification assay for the detection of Tobacco mild green mosaic virus (TMGMV)

Tobacco mild green mosaic virus (TMGMV), a member species of the genus Tobamovirus, infects pepper (Capsicum annuum) and a number of other economically important species in the Solanaceae family. TMGMV infections had seriously impacted pepper production worldwide, including China. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed to detect TMGMV in pepper field samples and seed. This assay was based on four primers that matched to six sequences in the C-terminal region of the TMGMV genome. RT-LAMP assay could detect the presence of the virus in 3.0 × 10−7 µg of total RNA extract from pepper leaves, which was ten times more sensitive than the corresponding reverse-transcription polymerase chain reaction (RT-PCR) assay. This method specifically detected TMGMV but not the closely related species of the same genus Pepper mild mottle virus, Cucumber green mottle mosaic virus and Tomato mosaic virus. In addition, the use of SYBR Green I facilitated the detection of the TMGMV RT-LAMP products by the naked eye. These results indicated that the RT-LAMP assay was a simple, sensitive, specific and affordable diagnostic tool that has the potential to detect and monitor TMGMV infection in field samples.

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.