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 had been developed into a registered bioherbicide. This position changed in 2014, when the US Environmental Protection Agency granted an unrestricted Section 3 registration for tobacco mild green mosaic virus (TMGMV) strain U2 as a herbicide active ingredient 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 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. © 2023 Society of Chemical Industry.

African eggplant-associated virus: Characterization of a novel tobamovirus identified from Solanum macrocarpon and assessment of its potential impact on tomato and pepper crops.

A novel tobamovirus was identified in a fruit of Solanum macrocarpon imported into the Netherlands in 2018. This virus was further characterized in terms of host range, pathotype and genomic properties, because many tobamoviruses have the potential to cause severe damage in important crops. In the original fruit, two different genotypes of the novel virus were present. The virus was able to infect multiple plant species from the Solanaceae family after mechanical inoculation, as well as a member of the Apiaceae family. These species included economically important crops such as tomato and pepper, as well as eggplant and petunia. Both tomato and pepper germplasm were shown to harbor resistance against the novel virus. Since most commercial tomato and pepper varieties grown in European greenhouses harbor these relevant resistances, the risk of infection and subsequent impact on these crops is likely to be low in Europe. Assessment of the potential threat to eggplant, petunia, and other susceptible species needs further work. In conclusion, this study provides a first assessment of the potential phytosanitary risks of a newly discovered tobamovirus, which was tentatively named African eggplant-associated virus.

Solanum elaeagnifolium and S. rostratum as potential hosts of the tomato brown rugose fruit virus.

Invasive weeds cause significant crop yield and economic losses in agriculture. The highest indirect impact may be attributed to the role of invasive weeds as virus reservoirs within commercial growing areas. The new tobamovirus tomato brown rugose fruit virus (ToBRFV), first identified in the Middle East, overcame the Tm-22 resistance allele of cultivated tomato varieties and caused severe damage to crops. In this study, we determined the role of invasive weed species as potential hosts of ToBRFV and a mild strain of pepino mosaic virus (PepMV-IL). Of newly tested weed species, only the invasive species Solanum elaeagnifolium and S. rostratum, sap inoculated with ToBRFV, were susceptible to ToBRFV infection. S. rostratum was also susceptible to PepMV-IL infection. No phenotype was observed on ToBRFV-infected S. elaeagnifolium grown in the wild or following ToBRFV sap inoculation. S. rostratum plants inoculated with ToBRFV contained a high ToBRFV titer compared to ToBRFV-infected S. elaeagnifolium plants. Mixed infection with ToBRFV and PepMV-IL of S. rostratum plants, as well as S. nigrum plants (a known host of ToBRFV and PepMV), displayed synergism between the two viruses, manifested by increasing PepMV-IL levels. Additionally, when inoculated with either ToBRFV or PepMV-IL, disease symptoms were apparent in S. rostratum plants and the symptoms were exacerbated upon mixed infections with both viruses. In a bioassay, ToBRFV-inoculated S. elaeagnifolium, S. rostratum and S. nigrum plants infected tomato plants harboring the Tm-22 resistant allele with ToBRFV. The distribution and abundance of these Solanaceae species increase the risks of virus transmission between species.

Effect of Populus nigra spring and autumn leaves extract on Capsicum annuum infected with pepper mild mottle virus.

Capsicum annuum is one of the main vegetable crops for the local market and exportation in Egypt. In this concern, pepper mild mottle virus (PMMoV) infection caused a significant decrease in Capsicum sp. leading to large economic losses. An isolate of PMMoV was obtained from naturally infected pepper plants, exhibiting different patterns of mottling, leaf distortion, yellowing, and stunting of leaves. The virus was identified. The molecular detection of PMMoV was done using RT-PCR with specific primers designed for coat protein genes. An RT-PCR product (474) bp of the coat protein gene of (PMMoV) was cloned. The target of the investigation was the effect of spring and autumn ethanol extracts of Populus nigra leaves on C. annuum seedling growth and infected C. annuum with (PMMoV) under greenhouse conditions. The experimental data showed that treated spring leaf extract of P. nigra enhanced infected C. annuum seedling growth parameters and fruit quality compared to uninfected seedlings. P. nigra spring leaf extract containing some allo-chemicals had a negative effect on uninfected seedlings. P. nigra autumn leaf extract significantly improved the growth and fruit quality of infected C. annuum seedlings compared to the control.

Suppression of Cucumber Green Mottle Mosaic Virus Infection by Boron Application: From the Perspective of Nutrient Elements and Carbohydrates.

Cucumber green mottle mosaic virus (CGMMV) infection causes "blood flesh" symptoms in watermelon fruits, which severely reduces yield and edibleness. However, the growth of watermelon fruits is strongly associated with boron (B), a trace element for improving fruit quality. In this study, B-gradient hydroponic experiments (B concentration: 0, 2.86, and 5.72 mg·L-1 H3BO3) and foliar-spray experiments (B concentration: 30 and 300 mg·L-1 H3BO3) were performed. We found that the B-supplement could inhibit CGMMV infection and especially relieve "blood flesh" symptoms in watermelon fruits. The nutrient element, soluble sugar, and cell wall polysaccharide contents and their metabolism- and transport-related gene expressions were determined in leaves and fruits of the watermelons in B-gradient hydroponic and foliar-spray experiments. We found that the accumulation and metabolism of nutrients and carbohydrates in cells were disrupted by CGMMV infection; however, the B-supplement could restore and maintain their homeostasis. Additionally, we uncovered that NIP5;1 and SWEET4, induced by B-application with CGMMV infection, could majorly contribute to the resistance to CGMMV infection by regulating nutrient elements and carbohydrate homeostasis. These results provided a novel insight into the molecular mechanism of B-mediated CGMMV suppression and an efficient method of B-application for the improvement of watermelon quality after CGMMV infection.

Characterization of a ToMV isolate overcoming Tm-22 resistance gene in tomato.

Tomato mosaic virus (ToMV) is easily transmitted in soil and by contact. By these reasons, it is relatively difficult to control ToMV disease in tomato. Incorporation of the Tm-22 gene has been widely used as a control method for ToMV, but ToMV isolates that overcome this resistance gene have been reported worldwide in recent years. In this study, we determined the entire nucleotide sequences of ToMV isolate [named ToMV-KMT (LC650928)], which was isolated from tomato plants showing symptoms of systemic necrosis in Kumamoto prefecture, Japan. We also analyzed the viral gene of ToMV-KMT that overcome the Tm-22 gene by constructing its infectious cDNA clone and by generating chimeric viruses with a non-breaking strain. According to previous research, Tm-22 recognizes the viral movement protein (MP) and exerts resistance by inducing hypersensitive reaction or hypersensitive cell death. We discovered that a mutation in the 240th amino acid (aspartic acid to tyrosine) of the MP of ToMV-KMT, which may stabilize the protein's structure, is responsible for the ability of this isolate to overcome the resistance of Tm-22.

Isolation and molecular characterization of a tomato brown rugose fruit virus mutant breaking the tobamovirus resistance found in wild Solanum species.

A new tobamovirus named tomato brown rugose fruit virus (ToBRFV) overcomes the effect of the Tm-1, Tm-2, and Tm-22 resistance genes introgressed from wild Solanum species into cultivated tomato (Solanum lycopersicum). Here, we report the isolation and molecular characterization of a spontaneous mutant of ToBRFV that breaks resistance in an unknown genetic background, demonstrated recently in Solanum habrochaites and Solanum peruvianum. The wild isolate ToBRFV-Tom2-Jo and the mutant ToBRFV-Tom2M-Jo were fully sequenced and compared to each other and to other ToBRFV sequences available in the NCBI GenBank database. Sequence analysis revealed five nucleotide substitutions in the ToBRFV-Tom2M-Jo genome compared to ToBRFV-Tom2-Jo. Two substitutions were located in the movement protein (MP) gene and resulted in amino acid changes in the 30-kDa MP (Phe22 → Asn and Tyr82 → Lys). These substitutions were not present in any of the previously described ToBRFV isolates. No amino acid changes were found in the 126-kDa and 183-kDa replicase proteins or the 17.5-kDa coat protein. Our data strongly suggest that breaking the newly discovered resistance in wild tomatoes is associated with one or two mutations on the MP gene of ToBRFV.

Autoxidation Products of the Methanolic Extract of the Leaves of Combretum micranthum Exert Antiviral Activity against Tomato Brown Rugose Fruit Virus (ToBRFV).

Tomato brown rugose fruit virus (ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of C. micranthum, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.

A single amino acid in coat protein of Pepper mild mottle virus determines its subcellular localization and the chlorosis symptom on leaves of pepper.

Pepper mild mottle virus (PMMoV) causes serious economic losses in pepper production in China. In a survey for viral diseases on pepper, two PMMoV isolates (named PMMoV-ZJ1 and PMMoV-ZJ2) were identified with different symptoms in Zhejiang province. Sequence alignment analysis suggested there were only four amino acid differences between the isolates: Val262Gly, Ile629Met and Ala1164Thr in the replicase, and Asp20Asn in the coat protein. Infectious cDNA clones of both isolates were constructed and shown to cause distinctive symptoms. Chlorosis symptoms appeared only on PMMoV-ZJ2-infected plants and the Asp20Asn substitution in the CP was shown to be responsible. Confocal assays revealed that the subcellular localization pattern of the two CPs was different, CP20Asp was mainly located at the cell periphery, whereas most CP20Asn located in the chloroplast. Thus, a single amino acid in the CP determined the chlorosis symptom, accompanied by an altered subcellular localization.

Signaling pathway played by salicylic acid, gentisic acid, nitric oxide, polyamines and non-enzymatic antioxidants in compatible and incompatible Solanum-tomato mottle mosaic virus interactions.

Plants are exposed to a vast array of pathogens. The interaction between them may be classified in compatible and incompatible. Polyamines (PAs) are involved in defense responses, as well as salicylic acid (SA), gentisic acid (GA) and nitric oxide (NO), which can increase the content of reactive oxygen species (ROS), creating a harsh environment to the pathogen. ROS can also damage the host cell and they can be controlled by ascorbate and glutathione. Among phytopathogens, one of the major threats to tomato crops is tomato mottle mosaic virus (ToMMV). Resistance against this virus probably involves the Tm-22 gene. This work aimed to analyze signaling and antioxidant molecules in the defense response against ToMMV in Solanum pimpinellifolium and in S. lycopersicum 'VFNT'. In S. pimpinellifolium plants inoculated with ToMMV, an increase in NO, SA, GA, ascorbate and oxidized glutathione and a decrease in the content of PAs were observed. Characteristic symptoms of diseased plants and high absorbance values in PTA-ELISA indicated a compatible interaction. In VFNT-inoculated plants, less significant differences were noticed. Symptoms and viral concentration were not detected, indicating an incompatible interaction, possibly associated with the effector-triggered immunity (ETI) response.

Antiphytoviral toxins of Actinidia chinensis root bark (ACRB) extract: laboratory and semi-field trials.

BACKGROUND: Actinidia chinensis Planch, which is distributed only in China, has been used to treat hepatitis and cancer. The objective of the present work was to identify the antiviral active ingredient of A. chinensis root bark (ACRB). RESULTS: Bioassay-guided isolation of the most active fraction, the EtOAc extract, led to the identification of seven compounds, (+)-catechins-7-phytol (1), 5-methoxy-coumarin-7-ß-D-glycosidase (2), (+)-catechins (3), fupenzic acid (4), spathodic acid-28-O-ß-D-glucopyranoside (5), 3-oxo-9, 12-diene-30-oic acid (6), and 3-ß-(2-carboxy benzoyloxy) oleanolic acid (7). Of these, 5-methoxy-coumarin-7-ß-D-glycosidase (2) possessed the highest antiviral activity, followed by spathodic acid-28-O-ß-D-glucopyranoside (5). Thus, compounds 2 and 5 were the main active constituents, with potential for further development as biological antiviral agents. CONCLUSION: The results suggest that ACRB possesses great potential value for development of an antiviral agent to control phytoviral diseases. © 2018 Society of Chemical Industry.

Inhibitory Components from Glycosmis stenocarpa on Pepper Mild Mottle Virus.

The goal of this study was to identify a source of natural plant compounds with inhibitory activity against pepper mild mottle virus (PMMoV). We showed, using a half-leaf assay, that murrayafoline-A (1) and isomahanine (2) isolated from the aerial parts of Glycosmis stenocarpa have inhibitory activity against PMMoV through curative, inactivation, and protection effects. Using a leaf-disk assay, we confirmed that 2 inhibited virus replication in Nicotiana benthamiana. Using electron microscopy, we found that a mixture of the virus with 2 resulted in damage to the rod-shaped virus.

Characterisation and diagnosis of frangipani mosaic virus from India.

Frangipani mosaic virus (FrMV) is known to infect frangipani tree (Plumeria rubra f. acutifolia) in India but the virus has not been characterized at genomic level and diagnosis is not available. In the present study, an isolate of FrMV (FrMV-Ind-1) showing greenish mosaic and vein-banding symptoms in P. rubra f. acutifolia in New Delhi was characterized based on host reactions, serology and genome sequence. The virus isolate induced local symptoms on several new experimental host species: Capsicum annuum (chilli), Nicotiana benthamiana, Solanum lycopersicum and S. melongena. N. benthamiana could be used as an efficient propagation host as it developed systemic mottle mosaic symptoms all round the year. The genome of FrMV-Ind-1 was 6643 (JN555602) nucleotides long with genome organization similar to tobamoviruses. The Indian isolate of FrMV shared a very close genome sequence identity (98.3 %) with the lone isolate of FrMV-P from Australia. FrMV-Ind-1 together with FrMV-P formed a new phylogenetic group i.e. Apocynaceae-infecting tobamovirus. The polyclonal antiserum generated through the purified virus preparation was successfully utilized to detect the virus in field samples of frangipani by ELISA. Of the eight different tobamoviruses tested, FrMV-Ind-1 shared distant serological relationships with only cucumber green mottle mosaic virus, tobacco mosaic virus, bell pepper mottle virus and kyuri green mottle mosaic virus. RT-PCR based on coat protein gene primer successfully detected the virus in frangipani plants. This study is the first comprehensive description of FrMV occurring in India.

High-level systemic expression of conserved influenza epitope in plants on the surface of rod-shaped chimeric particles.

Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV) genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP) gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV) did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV­based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod­shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP) described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

Identification of a movement protein of Mirafiori lettuce big-vein ophiovirus.

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.

Simultaneous detection and identification of four viruses infecting pepino by multiplex RT-PCR.

Potato virus M (PVM), pepino mosaic virus (PepMV), tomato mosaic virus (ToMV), and potato virus S (PVS) infect pepino and cause serious crop losses. In this study, a multiplex RT-PCR method was developed for simultaneous detection and differentiation of PVS, ToMV, PepMV and PVM. The method was highly reliable and sensitive; validation was accomplished by testing pepino samples collected from different regions of China. In this survey, PVM, ToMV and PVS were detected in 37.0 %, 31.0 % and 5.5 % of samples tested, respectively, confirming the widespread occurrence of these three viruses in China. PepMV was not detected in any of the samples, which indicated that this virus may not be prevalent in China. The results suggest that the new multiplex RT-PCR method has potential to be used routinely for surveys of pepino for virus infection.

Microtubule-associated protein AtMPB2C plays a role in organization of cortical microtubules, stomata patterning, and tobamovirus infectivity.

AtMPB2C is the Arabidopsis (Arabidopsis thaliana) homolog of MPB2C, a microtubule-associated host factor of tobacco mosaic virus movement protein that was been previously identified in Nicotiana tabacum. To analyze the endogenous function of AtMPB2C and its role in viral infections, transgenic Arabidopsis plant lines stably overexpressing green fluorescent protein (GFP)-AtMPB2C were established. The GFP-AtMPB2C fusion protein was detectable in various cell types and organs and localized at microtubules in a punctuate pattern or in filaments. To determine whether overexpression impacted on the cortical microtubular cytoskeleton, GFP-AtMPB2C-overexpressing plants were compared to known microtubular marker lines. In rapidly elongated cell types such as vein cells and root cells, GFP-AtMPB2C overexpression caused highly unordered assemblies of cortical microtubules, a disturbed, snake-like microtubular shape, and star-like crossing points of microtubules. Phenotypically, GFP-AtMPB2C transgenic plants showed retarded growth but were viable and fertile. Seedlings of GFP-AtMPB2C transgenic plants were characterized by clockwise twisted leaves, clustered stomata, and enhanced drought tolerance. GFP-AtMPB2C-overexpressing plants showed increased resistance against oilseed rape mosaic virus, a close relative of tobacco mosaic virus, but not against cucumber mosaic virus when compared to Arabidopsis wild-type plants. These results suggest that AtMPB2C is involved in the alignment of cortical microtubules, the patterning of stomata, and restricting tobamoviral infections.

Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses.

Immunocapture (IC) RT-PCR assays were developed for detection of tomato (ToMV) and tobacco mosaic (TMV) tobamoviruses in spruce and pine extracts. When purified viruses were diluted in root or needle extracts of virus-free conifer seedlings, both IC-RT-PCR assays detected their respective target viruses at concentrations of 10-100 fg ml(-1). This compared to ELISA detection sensitivities of 1 ng ml(-1). Primers were designed from regions of high sequence diversity. Specificity of all primer pairs was confirmed by sequencing of PCR products. PCR distinguished more reliably between the two viruses than ELISA. Moreover, a multiplex IC-RT-PCR assay for the simultaneous detection and differentiation of TMV and ToMV was developed. When root extracts were seeded with both viruses simultaneously, the multiplex assay detected each virus at concentrations of 1-10 pg ml(-1). Six TMV and 18 ToMV isolates from various hosts, water samples and a soil sample were amplified and differentiated by multiplex IC-RT-PCR. No amplifications were observed against pepper mild mottle and ribgrass mosaic tobamoviruses and against six viruses belonging to other taxonomic groups.

Characterization of a pepper mild mottle tobamovirus strain capable of overcoming the L3 gene-mediated resistance, distinct from the resistance-breaking Italian isolate.

Green pepper plants with the L3 resistance gene usually develop necrotic lesions on leaves infected with a Japanese strain of pepper mild mottle tobamovirus (PMMoV-J). A recently discovered strain, PMMoV-Ij, has the ability to overcome L3 resistance. Phytopathological responses of a variety of plant species to PMMoV-J and PMMoV-Ij were determined and the coat protein (CP) sequence comparisons revealed both amino acids 43 and 50 of PMMoV-Ij were unique. This led us to believe that substitutions at these residues would enable PMMoV-J to overcome L3 resistance. This was confirmed by Western blot (immunoblot) detection of PMMoV-J containing both point mutations in upper uninoculated leaves of resistant plants. Computer models suggest the critical residues in overcoming resistance lie in CP regions that putatively interact with other subunits. These results contribute to our understanding of the virus's ability to circumvent plant resistance.

Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes.

Microprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block of potato virus X (PVX). A 25 kDa MP-defective full-length cloned PVX genome carrying a beta-glucuronidase (GUS) reporter gene was co-bombarded with 35S promoter constructs containing either the 25 kDa MP gene of wild-type PVX, the MP gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV). When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by co-inoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. The BMV MP construct did not complement movement of the defective PVX. These results show that co-bombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for transcomplementation experiments.