Identification of environmental and methodological factors driving variability of Pepper Mild Mottle Virus (PMMoV) across three wastewater treatment plants in the City of Toronto.

PMMoV has been widely used to normalize the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, influenza, and respiratory syncytial virus (RSV) to account for variations in the fecal content of wastewater. PMMoV is also used as an internal RNA recovery control for wastewater-based epidemiology (WBE) tests. While potentially useful for the interpretation of WBE data, previous studies have suggested that PMMoV concentration can be affected by various physico-chemical characteristics of wastewater. There is also the possibility that laboratory methods, particularly the variability in centrifugation steps to remove supernatant from pellets can cause PMMoV variability. The goal of this study is to improve our understanding of the main drivers of PMMoV variability by assessing the relationship between PMMoV concentration, the physico-chemical characteristics of wastewater, and the methodological approach for concentrating wastewater samples. We analyzed 24-hour composite wastewater samples collected from the influent stream of three wastewater treatment plants (WWTPs) located in the City of Toronto, Ontario, Canada. Samples were collected 3 to 5 times per week starting from the beginning of March 2021 to mid-July 2023. The influent flow rate was used to partition the data into wet and dry weather conditions. Physico-chemical characteristics (e.g., total suspended solids (TSS), biological oxygen demand (BOD), alkalinity, electrical conductivity (EC), and ammonia (NH3)) of the raw wastewater were measured, and PMMoV was quantified. Spatial and temporal variability of PMMoV was observed throughout the study period. PMMoV concentration was significantly higher during dry weather conditions. Multiple linear regression analysis demonstrates that the number and type of physico-chemical parameters that drive PMMoV variability are site-specific, but overall BOD and alkalinity were the most important predictors. Differences in PMMoV concentration for a single WWTP between two different laboratory methods, along with a weak correlation between pellet mass and TSS using one method may indicate that differences in sample concentration and subjective subsampling bias could alter viral recovery and introduce variability to the data.

Pepper mild mottle virus intended for use as a process indicator for drinking water treatment: Present forms and quantitative relations to norovirus and rotavirus in surface water.

Pepper mild mottle virus (PMMoV) has been proposed as a potential indicator of human enteric viruses in environmental water and for viral removal during drinking water treatment. To investigate the occurrence and present forms of PMMoV and quantitative relations to norovirus GII and rotavirus A (RVA) in surface waters, 147 source water samples were collected from 21 drinking water treatment plants (DWTPs) in Japan between January 2018 and January 2021, and the concentrations of viruses in suspended and dissolved fractions were measured using real-time RT-PCR. PMMoV was detected in 81-100 % of samples in each sample month and observed concentrations ranged from 3.0 to 7.0 log10 copies/L. The concentrations of PMMoV were higher in dissolved fraction compared to suspended fractions, while different partitioning was observed for NoV GII depending on seasons. The concentrations of PMMoV were basically higher than those of norovirus GII (1.9-5.3 log10 copies/L) and RVA (1.9-6.6 log10 copies/L), while in 18 samples, RVA presented higher concentrations than PMMoV. Partial regions of VP7, VP4, and VP6 of the RVA in the 18 samples were amplified using nested PCR, and the genotypes were determined using an amplicon-based next-generation sequencing approach. We found that these source water samples included not only human RVA but also various animal RVA and high genetic diversity due to the existence of animal RVA was associated with a higher RVA concentration than PMMoV. Our findings suggest that PMMoV can be used as an indicator of norovirus GII and human RVA in drinking water sources and that the indicator performance should be evaluated by comparing to zoonotic viruses as well as human viruses.

Plant Immunity against Tobamoviruses.

Tobamoviruses are a group of plant viruses that pose a significant threat to agricultural crops worldwide. In this review, we focus on plant immunity against tobamoviruses, including pattern-triggered immunity (PTI), effector-triggered immunity (ETI), the RNA-targeting pathway, phytohormones, reactive oxygen species (ROS), and autophagy. Further, we highlight the genetic resources for resistance against tobamoviruses in plant breeding and discuss future directions on plant protection against tobamoviruses.

Capsicum chinense Jacq.-derived glutaredoxin (CcGRXS12) alters redox status of the cells to confer resistance against pepper mild mottle virus (PMMoV-I).

KEY MESSAGE: The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.

Transcriptome Analysis of Tomato Leaves Reveals Candidate Genes Responsive to Tomato Brown Rugose Fruit Virus Infection.

Tomato brown rugose fruit virus (ToBRFV) is a newly-emerging tobamovirus which was first reported on tomatoes in Israel and Jordan, and which has now spread rapidly in Asia, Europe, North America, and Africa. ToBRFV can overcome the resistance to other tobamoviruses conferred by tomato Tm-1, Tm-2, and Tm-22 genes, and it has seriously affected global crop production. The rapid and comprehensive transcription reprogramming of host plant cells is the key to resisting virus attack, but there have been no studies of the transcriptome changes induced by ToBRFV in tomatoes. Here, we made a comparative transcriptome analysis between tomato leaves infected with ToBRFV for 21 days and those mock-inoculated as controls. A total of 522 differentially expressed genes were identified after ToBRFV infection, of which 270 were up-regulated and 252 were down-regulated. Functional analysis showed that DEGs were involved in biological processes such as response to wounding, response to stress, protein folding, and defense response. Ten DEGs were selected and verified by qRT-PCR, confirming the reliability of the high-throughput sequencing data. These results provide candidate genes or signal pathways for the response of tomato leaves to ToBRFV infection.

The first detection of mpox virus DNA from wastewater in China.

Wastewater monitoring may be a valuable early surveillance tool for studying mpox virus (MPXV) circulation in China, a country with high population density and very few mpox patients. To evaluate the effectiveness of wastewater monitoring for MPXV in detecting local hidden transmission of the epidemic in the early period, the Chinese Center for Disease Control and Prevention initiated a wastewater monitoring program for MPXV in China in July 2023. To enhance the monitoring sensitivity of the program, an MPXV monitoring point was established in a gathering place of high-risk mpox population. Three different concentration methods, PEG precipitation, ultrafiltration, and magnetic beads method were evaluated and compared. Due to its high recovery efficiency, low limit of detection, and high degree of automation, the magnetic beads method was selected for the daily surveillance of MPXV in wastewater. On September 5, 2023, MPXV DNA was detected at the MPXV monitoring point in Zibo City, marking the first instance of MPXV detection of MPXV in wastewater in China. Next-generation sequencing was conducted on the MPXV genome obtained from the positive wastewater, positive environmental samples, and the single case of mpox in Zibo in September. The results showed that the genotypes of these three genomes were different but all belong to the IIb branch of the C.1 lineage, indicating a probably hidden transmission of mpox. Wastewater monitoring is potentially an effective early surveillance tool for tracking the spread of MPXV in areas with high population density and very few mpox patients.

Molecular characterization and host reaction to tomato mottle mosaic virus isolated from sweet pepper seeds in Japan.

Many countries have identified tomato mottle mosaic virus (ToMMV) as a serious threat to tomato production. Here, we constructed and characterized infectious clones of ToMMV isolated from Japanese sweet pepper seeds. The genome of the Japanese isolate is 6399 nucleotides in length and exhibits the highest identity with previously characterized isolates. For example, it is 99.7% identical to that of the Mauritius isolate, which occurs worldwide. Phylogenetic analysis based on complete genome sequences revealed that the Japanese isolates clustered in the same clade as those from other countries. When homozygous tomato cultivars with tobamovirus resistance genes were inoculated with an infectious cDNA clone of ToMMV, the virus systemically infected tomato plants with symptoms typical of Tm-1-carrying tomato cultivars. In contrast, tomato cultivars carrying Tm-2 or Tm-22 showed symptoms only on the inoculated leaves. Furthermore, when commercial cultivars of Tm-22 heterozygous tomato were inoculated with ToMMV, systemic infections were observed in all cultivars, with infection frequencies ranging from 25 to 100%. Inoculation of heterozygous sweet pepper cultivars with tobamovirus resistance genes (L1, L3, and L4) with ToMMV resulted in an infection frequency of about 70%, but most of the infected L1, L3, and L4 cultivars were symptomless, and 10-20% showed symptoms of necrosis and yellowing. Tomato mosaic virus strain L11A, an attenuated virus, did not provide cross-protection against ToMMV and led to systemic infection with typical symptoms. These results suggest that ToMMV might cause extensive damage to existing tomato and sweet pepper cultivars commonly grown in Japan.

Site-specific risk-based threshold (RBT) concentrations for sewage-associated markers in estuarine swimming waters.

This study establishes site-specific risk-based threshold (RBT) concentrations for sewage-associated markers, including Bacteroides HF183 (HF183), Lachnospiraceae Lachno3 (Lachno3), cross-assembly phage (CrAssphage), and pepper mild mottle virus (PMMoV), utilizing quantitative microbial risk assessment (QMRA) for recreational estuarine waters (EW). The QMRA model calculates a RBT concentration corresponding to a selected target illness risk for ingestion of EW contaminated with untreated sewage. RBT concentrations were estimated considering site-specific decay rates and concentrations of markers and reference pathogen (human norovirus; HNoV), aiding in the identification of high-risk days during the swimming season. Results indicated varying RBT concentrations for fresh (Day 0) and aged (Days 1 to 10) sewage contamination scenarios over 10 days. HF183 exhibited the highest RBT concentration (26,600 gene copis (GC)/100 mL) initially but decreased rapidly with aging (2570 to 3120 GC/100 mL on Day 10) depending on the decay rates, while Lachno3 and CrAssphage remained relatively stable. PMMoV, despite lower initial RBT (3920 GC/100 mL), exhibited increased RBT (4700 to 6440 GC/100 mL) with aging due to its slower decay rate compared to HNoV. Sensitivity analysis revealed HNoV concentrations as the most influential parameter. Comparison of marker concentrations in estuarine locations with RBT concentrations showed instances of marker exceedance, suggesting days of potential higher risks. The observed discrepancies between bacterial and viral marker concentrations in EW highlight the need for optimized sample concentration method and simultaneous measurement of multiple markers for enhanced risk predictions. Future research will explore the utility of multiple markers in risk management. Overall, this study contributes to better understanding human health risks in recreational waters, aiding regulators, and water quality managers in effective decision-making for risk prioritization and mitigation strategies.

Surveillance of SARS-CoV-2 in wastewater by quantitative PCR and digital PCR: a case study in Shijiazhuang city, Hebei province, China.

In this study, we reported the first long-term monitoring of SARS-CoV-2 in wastewater in Mainland China from November 2021 to October 2023. The city of Shijiazhuang was employed for this case study. We developed a triple reverse transcription droplet digital PCR (RT-ddPCR) method using triple primer-probes for simultaneous detection of the N1 gene, E gene, and Pepper mild mottle virus (PMMoV) to achieve accurate quantification of SARS-CoV-2 RNA in wastewater. Both the RT-ddPCR method and the commercial multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) method were implemented for the detection of SARS-CoV-2 in wastewater in Shijiazhuang City over a 24-month period. Results showed that SARS-CoV-2 was detected for the first time in the wastewater of Shijiazhuang City on 10 November 2022. The peak of COVID-19 cases occurred in the middle of December 2022, when the concentration of SARS-CoV-2 in the wastewater was highest. The trend of virus concentration increases and decreases forming a "long-tailed" shape in the COVID-19  outbreak and recession cycle. The results indicated that both multiplex RT-ddPCR and RT-qPCR are effective in detecting SARS-CoV-2 in wastewater, but RT-ddPCR is capable of detecting low concentrations of SARS-CoV-2 in wastewater which is more efficient. The SARS-CoV-2 abundance in wastewater is correlated to clinical data, outlining the public health utility of this work.HighlightsFirst long-term monitoring of SARS-CoV-2 in wastewater in Mainland ChinaCOVID-19 outbreak was tracked in Shijiazhuang City from outbreak to containmentWastewater was monitored simultaneously using RT-ddPCR and RT-qPCR methodsTriple primer-probe RT-ddPCR detects N1 and E genes of SARS-CoV-2 and PMMoV.

Persistence of human respiratory viral RNA in wastewater-settled solids.

Wastewater-based epidemiology has emerged as a valuable tool for monitoring respiratory viral diseases within communities by analyzing concentrations of viral nucleic-acids in wastewater. However, little is known about the fate of respiratory virus nucleic-acids in wastewater. Two important fate processes that may modulate their concentrations in wastewater as they move from household drains to the point of collection include sorption or partitioning to wastewater solids and degradation. This study investigated the decay kinetics of genomic nucleic-acids of seven human respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), human coronavirus (HCoV)-OC43, HCoV-229E, HCoV-NL63, human rhinovirus (HRV), and influenza A virus (IAV), as well as pepper mild mottle virus (PMMoV) in wastewater solids. Viruses (except for PMMoV) were spiked into wastewater solids and their concentrations were followed for 50 days at three different temperatures (4°C, 22°C, and 37°C). Viral genomic RNA decayed following first-order kinetics with decay rate constants k from 0 to 0.219 per day. Decay rate constants k were not different from 0 for all targets in solids incubated at 4°C; k values were largest at 37°C and at this temperature, k values were similar across nucleic-acid targets. Regardless of temperature, there was limited viral RNA decay, with an estimated 0% to 20% reduction, over the typical residence times of sewage in the piped systems between input and collection point (<1 day). The k values reported herein can be used directly in fate and transport models to inform the interpretation of measurements made during wastewater surveillance.IMPORTANCEUnderstanding whether or not the RNA targets quantified for wastewater-based epidemiology (WBE) efforts decay during transport between drains and the point of sample collection is critical for data interpretation. Here we show limited decay of viral RNA targets typically measured for respiratory disease WBE.

Quantifying Plant Viruses: Evolution from Bioassay to Infectivity Dilution Curves along the Model of Tobamoviruses.

This review describes the development of the bioassay as a means of quantifying plant viruses, with particular attention to tobamovirus. It delves into various models used to establish a correlation between virus particle concentration and the number of induced local lesions (the infectivity dilution curve), including the Poisson, Furumoto and Mickey, Kleczkowski, Growth curve, and modified Poisson models. The parameters of each model are described, and their application or performance in the context of the tobacco mosaic virus is explored. This overview highlights the enduring value of the infectivity dilution curve in tobamovirus quantification, providing valuable insights for researchers or practitioners of bioassays and theoreticians of modeling.

A Novel Tiled Amplicon Sequencing Assay Targeting the Tomato Brown Rugose Fruit Virus (ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada.

Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important Solanaceae crop species and can dramatically reduce tomato crop yields. The ToBRFV has rapidly spread around the globe due to its ability to escape detection by antiviral host genes which confer resistance to other tobamoviruses in tomato plants. The development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detected ToBRFV in municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution of ToBRFV in WWI throughout Ontario, Canada. To aid in global ToBRFV surveillance efforts, we developed a tiled amplicon approach to sequence and track the evolution of ToBRFV genomes in municipal WWI. Our assay recovers 95.7% of the 6393 bp ToBRFV RefSeq genome, omitting the terminal 5' and 3' ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recovering ToBRFV genomes. Our ToBRFV assay was developed using existing ARTIC Network resources, including primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance of ToBRFV clades in samples.

Assessing the nucleic acid decay of human wastewater markers and enteric viruses in estuarine waters in Sydney, Australia.

This research investigated the in-situ decay rates of four human wastewater-associated markers (Bacteroides HF183 (HF183), Lachnospiraceae Lachno3 (Lachno3), cross-assembling phage (crAssphage), pepper mild mottle virus (PMMoV) and three enteric viruses (human adenovirus 40/41 (HAdV 40/41), enterovirus (EV) and human norovirus GII (HNoV GII) in two estuarine water environments (Davidson Park (DP) and Hen and Chicken Bay (HCB) in temperate Sydney, NSW, Australia, employing qPCR and RT-qPCR assays. The study also aimed to compare decay rates observed in mesocosms with previously published laboratory microcosms, providing insights into the persistence of markers and viruses in estuarine environments. Results indicated varying decay rates between DP and HCB mesocosms, with HF183 exhibiting relatively faster decay rates compared to other markers and enteric viruses in sunlight and dark mesocosms. In DP mesocosms, HF183 decayed the fastest, contrasting with PMMoV, which exhibited the slowest. Sunlight induced higher decay rates for all markers and viruses in DP mesocosms. In HCB sunlight mesocosms, HF183 nucleic acid decayed most rapidly compared to other markers and enteric viruses. In dark mesocosms, crAssphage showed the fastest decay, while PMMoV decayed at the slowest rate in both sunlight and dark mesocosms. Comparisons with laboratory microcosms revealed faster decay of markers and enteric viruses in laboratory microcosms than the mesocosms, except for crAssphage and HAdV 40/41 in dark, and PMMoV in sunlight mesocosms. The study concludes that decay rates of markers and enteric viruses vary between estuarine mesocosms, emphasizing the impact of sunlight exposure, which was potentially influenced by the elevated turbidity at HCB estuarine waters. The generated decay rates contribute valuable insights for establishing site-specific risk-based thresholds of human wastewater-associated markers.

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.

Molecular Characterization and Pathogenicity of an Infectious cDNA Clone of Youcai Mosaic Virus on Solanum nigrum.

Virus infections cause devastative economic losses for various plant species, and early diagnosis and prevention are the most effective strategies to avoid the losses. Exploring virus genomic evolution and constructing virus infectious cDNA clones is essential to achieve a deeper understanding of the interaction between host plant and virus. Therefore, this work aims to guide people to better prevent, control, and utilize the youcai mosaic virus (YoMV). Here, the YoMV was found to infect the Solanum nigrum under natural conditions. Then, an infectious cDNA clone of YoMV was successfully constructed using triple-shuttling vector-based yeast recombination. Furthermore, we established phylogenetic trees based on the complete genomic sequences, the replicase gene, movement protein gene, and coat protein gene using the corresponding deposited sequences in NCBI. Simultaneously, the evolutionary relationship of the YoMV discovered on S. nigrum to others was determined and analyzed. Moreover, the constructed cDNA infectious clone of YoMV from S. nigrum could systematically infect the Nicotiana benthamiana and S. nigrum by agrobacterium-mediated infiltration. Our investigation supplied a reverse genetic tool for YoMV study, which will also contribute to in-depth study and profound understanding of the interaction between YoMV and host plant.

A modeling pipeline to relate municipal wastewater surveillance and regional public health data.

As COVID-19 becomes endemic, public health departments benefit from improved passive indicators, which are independent of voluntary testing data, to estimate the prevalence of COVID-19 in local communities. Quantification of SARS-CoV-2 RNA from wastewater has the potential to be a powerful passive indicator. However, connecting measured SARS-CoV-2 RNA to community prevalence is challenging due to the high noise typical of environmental samples. We have developed a generalized pipeline using in- and out-of-sample model selection to test the ability of different correction models to reduce the variance in wastewater measurements and applied it to data collected from treatment plants in the Chicago area. We built and compared a set of multi-linear regression models, which incorporate pepper mild mottle virus (PMMoV) as a population biomarker, Bovine coronavirus (BCoV) as a recovery control, and wastewater system flow rate into a corrected estimate for SARS-CoV-2 RNA concentration. For our data, models with BCoV performed better than those with PMMoV, but the pipeline should be used to reevaluate any new data set as the sources of variance may change across locations, lab methods, and disease states. Using our best-fit model, we investigated the utility of RNA measurements in wastewater as a leading indicator of COVID-19 trends. We did this in a rolling manner for corrected wastewater data and for other prevalence indicators and statistically compared the temporal relationship between new increases in the wastewater data and those in other prevalence indicators. We found that wastewater trends often lead other COVID-19 indicators in predicting new surges.

Human adenovirus-associated health risk in the recreational waters of the Yal-ku lagoon in the Mexican Caribbean.

The study objective was to evaluate human faecal contamination impacts in the Yal-ku lagoon in the Mexican Caribbean and to estimate adenovirus infection and illness risks associated with recreational exposure during water activities. A total of 20 water samples (10 from each site × two sites) (50 L) were collected monthly over a period of 12 months from two selected sampling sites in the swimming area of the Yal-ku lagoon. The occurrence of faecal-associated viruses was explored, and human adenovirus (HAdV) and pepper mild mottle virus (PMMoV) concentrations were quantified. A quantitative microbial risk assessment (QMRA) model was used to estimate exposure and subsequent adenovirus infection and illness risk for 1 h of swimming or snorkelling. Somatic and F + -specific coliphages occurred in 100% of the samples. Both HAdV and PMMoV were detected at a 60% frequency thereby indicating persistent faecal inputs. PMMoV concentrations (44-370 GC/L) were relatively lower than the concentrations of HAdV (64-1,000 GC/L). Estimated mean adenovirus risks were greater for snorkelling than for swimming by roughly one to two orders of magnitude and estimated mean illness risks for snorkelling were >32/1,000. Human faecal contamination is frequent in the Yal-ku lagoon, which is associated with human gastrointestinal illness.

Fine mapping and identification of two NtTOM2A homeologs responsible for tobacco mosaic virus replication in tobacco (Nicotiana tabacum L.).

BACKGROUND: Tobacco mosaic virus (TMV) is a widely distributed viral disease that threatens many vegetables and horticultural species. Using the resistance gene N which induces a hypersensitivity reaction, is a common strategy for controlling this disease in tobacco (Nicotiana tabacum L.). However, N gene-mediated resistance has its limitations, consequently, identifying resistance genes from resistant germplasms and developing resistant cultivars is an ideal strategy for controlling the damage caused by TMV. RESULTS: Here, we identified highly TMV-resistant tobacco germplasm, JT88, with markedly reduced viral accumulation following TMV infection. We mapped and cloned two tobamovirus multiplication protein 2A (TOM2A) homeologs responsible for TMV replication using an F2 population derived from a cross between the TMV-susceptible cultivar K326 and the TMV-resistant cultivar JT88. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated loss-of-function mutations of two NtTOM2A homeologs almost completely suppressed TMV replication; however, the single gene mutants showed symptoms similar to those of the wild type. Moreover, NtTOM2A natural mutations were rarely detected in 577 tobacco germplasms, and CRISPR/Cas9-mediated variation of NtTOM2A led to shortened plant height, these results indicating that the natural variations in NtTOM2A were rarely applied in tobacco breeding and the NtTOM2A maybe has an impact on growth and development. CONCLUSIONS: The two NtTOM2A homeologs are functionally redundant and negatively regulate TMV resistance. These results deepen our understanding of the molecular mechanisms underlying TMV resistance in tobacco and provide important information for the potential application of NtTOM2A in TMV resistance breeding.

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.

Development and application of monoclonal antibody-based dot-ELISA and colloidal gold immunochromatographic strip for rapid, specific, and sensitive detection of tomato brown rugose fruit virus.

Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus that has become a great concern to tomato production industry. Due to the lack of resistant cultivars, precise detection of ToBRFV is essential to prevent the spread of ToBRFV. In this study, we produced highly sensitive and specific monoclonal antibodies against ToBRFV and established dot-enzyme-linked immunosorbent assay (dot-ELISA) and colloidal gold immunochromatographic strip (CGICS)-based methods for ToBRFV detection. These two methods could specifically detect ToBRFV without cross-reaction with seven tested tobamoviruses and three frequently occurring tomato-infecting viruses. Sensitivity analysis showed that the limit of detection of the established dot-ELISA and CGICS methods reached up to 1:6400 and 1:10,000 (w/v, g/mL) dilution of ToBRFV-infected tomato tissue, respectively. Further analyses using field-collected tomato foliar and fruit samples showed that the results obtained by dot-ELISA and CGICS were consistent with those obtained by reverse transcription polymerase chain reaction. The established methods here allow for specific, sensitive, and robust detection of ToBRFV, and will be helpful for precise monitoring and early warning of ToBRFV.