New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus.

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

Pepper Mild Mottle Virus as Indicator of Pollution: Assessment of Prevalence and Concentration in Different Water Environments in Italy.

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 106) > treated sewage (2.9 × 105) > river waters (6.1 × 102) > estuarine waters (4.8 × 102) > bathing waters (8.5 × 101) > groundwater (5.9 × 101). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

Tobamoviruses of two new species trigger resistance in pepper plants harbouring functional L alleles.

Tobamoviruses are often referred to as the most notorious viral pathogens of pepper crops. These viruses are not transmitted by invertebrate vectors, but rather by physical contact and seeds. In this study, pepper plants displaying mild mottle and mosaic symptoms were sampled in four different regions of Peru. Upon double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests, seven samples cross-reacted weakly with antibodies against pepper mild mottle virus (PMMoV), suggesting the presence of tobamoviruses. When employing RT-PCR, conserved primers amplified cDNA fragments of viruses from two putative new tobamovirus species in the samples. The complete genome of two representative isolates were, therefore, sequenced and analysed in silico. These viruses, which were tentatively named yellow pepper mild mottle virus (YPMMoV) and chilli pepper mild mottle virus (CPMMoV), shared highest nucleotide genome sequence identities of 83 and 85 % with bell pepper mottle virus (BpeMV), respectively. Mechanical inoculation of indicator plants with YPMMoV and CPMMoV isolates did not show any obvious differences in host ranges. These viruses were also inoculated mechanically on pepper plants harbouring different resistance L alleles to determine their pathotypes. Pepper plants carrying unfunctional L alleles (L0) to tobamoviruses were infected by all isolates and presented differential symptomatology for YPMMoV and CPMMoV. On the other hand, pepper plants carrying L1, L2, L3 and L4 alleles were resistant to all isolates, indicating that these viruses belong to pathotype P0.

Tobamoviruses can be frequently present in the oropharynx and gut of infants during their first year of life.

Plant viruses have been reported to be common in the gut of human adults, presumably as result of food ingestion. In this work, we report that plant viruses can also be found frequently in the gut and oropharynx of children during their first year of life, even when they are exclusively breast-fed. Fecal and oropharynx samples were collected monthly, from birth to 1 year of age, from three apparently healthy children in a semi-rural community and analyzed by next generation sequencing. In 100% of the fecal samples and 65% of the oropharynx samples at least one plant virus was identified. Tobamoviruses in the Virgaviridae family were by far the most frequently detected, with tropical soda apple mosaic virus, pepper mild mottle virus, and opuntia tobamovirus 2 being the most common species. Seventeen complete virus genomes could be assembled, and phylogenetic analyses showed a large diversity of virus strains circulating in the population. These results suggest that children are continuously exposed to an extensive and highly diverse collection of tobamoviruses. Whether the common presence of plant viruses at an early age influences the infant's immune system, either directly or through interaction with other members of the microbiota, remains to be investigated.

Genome characterization of brugmansia latent virus, a novel tobamovirus.

A novel tobamovirus, brugmansia latent virus (BrLV), was discovered during a study of brugmansia (Brugmansia spp.) in the living collections held at the Royal Botanic Gardens, Kew. Here, we report the complete genome sequence of BrLV, which is 6,397 nucleotides long and contains the four open reading frames (RNA-dependent RNA polymerase, methyltransferase/helicase, movement, and coat proteins) typical of tobamoviruses. The complete genome sequence of BrLV shares 69.7% nucleotide sequence identity with brugmansia mild mottle virus (BrMMV) and 66.7 to 68.7% identity with other tobamoviruses naturally infecting members of the Solanaceae plant family. Phylogenetic analysis of the complete genome nucleotide sequence and the deduced amino acid sequences of the four tobamovirus proteins place BrLV in a subcluster with BrMMV within the Solanaceae-infecting tobamovirus subgroup as a new species.

Evaluation of Human- and Animal-Specific Viral Markers and Application of CrAssphage, Pepper Mild Mottle Virus, and Tobacco Mosaic Virus as Potential Fecal Pollution Markers to River Water in Japan.

Five human-specific markers were detected in 59-74% of 27 human fecal-source samples collected in Yamanashi Prefecture, Japan. Similarly, potential human-specific markers, crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus were detected in 96-100% of samples, with crAssphage showing the maximum concentration of 12.03 log copies/L. However, these markers were detected in 100% (3/3) of pig fecal-source samples, suggesting their applicability as general fecal pollution markers. Microbial source tracking analysis demonstrated that the rivers are contaminated by human and pig fecal sources. CrAssphage showed higher marker concentrations in river water samples than PMMoV, suggesting the preference of crAssphage to PMMoV as a marker of fecal pollution.

Performance Evaluation of Human-Specific Viral Markers and Application of Pepper Mild Mottle Virus and CrAssphage to Environmental Water Samples as Fecal Pollution Markers in the Kathmandu Valley, Nepal.

Monitoring of environmental water is crucial to protecting humans and animals from possible health risks. Although numerous human-specific viral markers have been designed to track the presence of human fecal contamination in water, they lack adequate sensitivity and specificity in different geographical regions. We evaluated the performances of six human-specific viral markers [Aichi virus 1 (AiV-1), human adenoviruses (HAdVs), BK and JC polyomaviruses (BKPyVs and JCPyVs), pepper mild mottle virus (PMMoV), and crAssphage] using 122 fecal-source samples collected from humans and five animal hosts in the Kathmandu Valley, Nepal. PMMoV and crAssphage showed high sensitivity (90-100%) with concentrations of 4.5-9.1 and 6.2-7.0 log10 copies/g wet feces (n = 10), respectively, whereas BKPyVs, JCPyVs, HAdVs, and AiV-1 showed poor performances with sensitivities of 30-40%. PMMoV and crAssphage were detected in 40-100% and 8-90%, respectively, of all types of animal fecal sources and showed no significantly different concentrations among most of the fecal sources (Kruskal-Wallis test, P > 0.05), suggesting their applicability as general fecal pollution markers. Furthermore, a total of 115 environmental water samples were tested for PMMoV and crAssphage to identify fecal pollution. PMMoV and crAssphage were successfully detected in 62% (71/115) and 73% (84/115) of water samples, respectively. The greater abundance and higher mean concentration of crAssphage (4.1 ± 0.9 log10 copies/L) compared with PMMoV (3.3 ± 1.4 log10 copies/L) indicated greater chance of detection of crAssphage in water samples, suggesting that crAssphage could be preferred to PMMoV as a marker of fecal pollution.

The complete genome sequence of wild tomato mosaic virus isolated from Solanum nigrum reveals recombination in the P1 cistron.

The complete genome sequence of a wild tomato mosaic virus (WTMV) isolate (named WTMV-Sn) was determined and identified in Solanum nigrum in China. The complete genome of WTMV-Sn is 9,659 nucleotides in length, excluding the poly(A) tail and encodes a polyprotein of 3,074 amino acids. This is the first report of WTMV infecting S. nigrum. Despite the high degree of sequence similarity between the WTMV-Sn and WTMV-XC-1 isolates, the 349 nucleotides at the 5' terminus of WTMV-Sn appear to have originated by recombination with another isolate. The recombination parent remains unknown, but the recombination region shares 74.57% sequence identity with isolate WTMV-Laichau, which is below the species demarcation threshold for the genus Potyvirus. A pathogenicity test showed that WTMV-Sn can infect tobacco. This suggests that variation in the P1 cistron of WTMV-Sn may contribute to its ability to infect S. nigrum.

Analysis of Fitness Trade-Offs in the Host Range Expansion of an RNA Virus, Tobacco Mild Green Mosaic Virus.

The acquisition of new hosts provides a virus with more opportunities for transmission and survival but may be limited by across-host fitness trade-offs. Major causes of across-host trade-offs are antagonistic pleiotropy, that is, host differential phenotypic effects of mutations, a Genotype x Environment interaction, and epistasis, a Genotype x Genotype interaction. Here, we analyze if there are trade-offs, and what are the causes, associated with the acquisition by tobacco mild green mosaic virus (TMGMV) of a new host. For this, the multiplication of sympatric field isolates of TMGMV from its wild reservoir host Nicotiana glauca and from pepper crops was quantified in the original and the heterologous hosts. TMGMV isolates from N. glauca were adapted to their host, but pepper isolates were not adapted to pepper, and the acquisition of this new host was associated with a fitness penalty in the original host. Analyses of the collection of field isolates and of mutant genotypes derived from biologically active cDNA clones showed a role of mutations in the coat protein and the 3' untranslated region in determining within-host virus fitness. Fitness depended on host-specific effects of these mutations, on the genetic background in which they occurred, and on higher-order interactions of the type Genotype x Genotype x Environment. These types of effects had been reported to generate across-host fitness trade-offs under experimental evolution. Our results show they may also operate in heterogeneous natural environments and could explain why pepper isolates were not adapted to pepper and their lower fitness in N. glaucaIMPORTANCE The acquisition of new hosts conditions virus epidemiology and emergence; hence it is important to understand the mechanisms behind host range expansion. Experimental evolution studies have identified antagonistic pleiotropy and epistasis as genetic mechanisms that limit host range expansion, but studies from virus field populations are few. Here, we compare the performance of isolates of tobacco mild green mosaic virus from its reservoir host, Nicotiana glauca, and its new host, pepper, showing that acquisition of a new host was not followed by adaptation to it but was associated with a fitness loss in the original host. Analysis of mutations determining host-specific virus multiplication identified antagonistic pleiotropy, epistasis, and host-specific epistasis as mechanisms generating across-host fitness trade-offs that may prevent adaptation to pepper and cause a loss of fitness in N. glauca Thus, mechanisms determining trade-offs, identified under experimental evolution, could also operate in the heterogeneous environment in which natural plant virus populations occur.

Tobamoviruses as Models for the Study of Virus Evolution.

The study of tobacco mosaic virus and other tobamovirus species has greatly contributed to the development of all areas of virology, including virus evolution. Research with tobamoviruses has been pioneer, or particularly significant, in all major areas of research in this field, including: the characterization of the genetic diversity of virus populations, the mechanisms and rates of generation of genetic diversity, the analysis of the genetic structure of virus populations and of the factors that shape it, the adaptation of viruses to hosts and the evolution of host range, and the evolution of virus taxa and of virus-host interactions. Many of these continue to be hot topics in evolutionary biology, or have been identified recently as such, including (i) host-range evolution, (ii) predicting the overcoming of resistance in crops, (iii) trade-offs between virus life-history traits in virus evolution, and (iv) the codivergence of viruses and hosts at different taxonomical and spatial scales. Tobamoviruses may be particularly appropriate to address these topics with plant viruses, as they provide convenient experimental systems, and as the detailed knowledge on their molecular and structural biology allows the analysis of the mechanisms behind evolutionary processes. Also, the extensive information on parameters related to infection dynamics and population structure may facilitate the development of realistic models to predict virus evolution. Certainly, tobamoviruses will continue to be favorite system for the study of virus evolution.

Occurrence and seasonal dynamics of RNA viral genotypes in three contrasting temperate lakes.

Decades of research have demonstrated the crucial importance of viruses in freshwater ecosystems. However, few studies have focused on the seasonal dynamics and potential hosts of RNA viruses. We surveyed microbial-sized (i.e. 5-0.2 µm) mixed community plankton transcriptomes for RNA viral genomes and investigated their distribution between microbial and macrobial plankton over a seasonal cycle across three temperate lakes by quantitative reverse transcriptase PCR (qRT-PCR). A total of 30 contigs bearing similarity to RNA viral genomes were recovered from a global assembly of 30 plankton RNA libraries. Of these, only 13 were found in >2 libraries and recruited >100 reads (of 9.13 x 107 total reads), representing several picornaviruses, two tobamoviruses and a reovirus. We quantified the abundance of four picornaviruses and the reovirus monthly from August 2014 to May 2015. Patterns of viral abundance in the >5 µm size fraction and representation in microbial-sized community RNA libraries over time suggest that one picornavirus genotype (TS24835) and the reovirus (TS148892) may infect small (<5 µm) eukaryotic microorganisms, while two other picornaviruses (TS24641 and TS4340) may infect larger (>5 µm) eukaryotic microorganisms or metazoa. Our data also suggest that picornavirus TS152062 may originate from an allochthonous host. All five viral genotypes were present in at least one size fraction across all 3 lakes during the year, suggesting that RNA viruses may easily disperse between adjacent aquatic habitats. Our data therefore demonstrate that RNA viruses are widespread in temperate lacustrine ecosystems, and may provide evidence of viral infection in larger eukaryotes (including metazoa) inhabiting the lakes.

Occurrence of Pepper Mild Mottle Virus (PMMoV) in Groundwater from a Karst Aquifer System in the Yucatan Peninsula, Mexico.

The Yucatan Peninsula of Mexico hosts a karst aquifer system that is the only source of freshwater for the area; however, it is vulnerable to human-mediated contamination. Pepper mild mottle virus (PMMoV) is one of the most abundant RNA viruses associated with human feces, making it a viable indicator for tracking fecal pollution in aquatic environments, including groundwater. In this study, groundwater samples collected from a karst aquifer from fresh and brackish water locations were analyzed for fecal indicator bacteria, somatic and male F+ specific coliphages, and PMMoV during the rainy and dry seasons. Total coliform bacteria were detected at all sites, whereas Escherichia coli were found at relatively low levels <40 MPN/100 ml. The highest average concentrations of somatic and male F+ specific coliphages were 920 and 330 plaque forming units per 100 ml, respectively, detected in freshwater during the rainy season. PMMoV RNA was detected in 85% of the samples with gene sequences sharing 99-100% of nucleotide identity with PMMoV sequences available in GenBank. Quantification of PMMoV genome copies (GC) by quantitative real-time PCR indicated concentrations ranging from 1.7 × 101 to 1.0 × 104 GC/L, with the highest number of GC detected during the rainy season. No significant correlation was observed between PMMoV occurrence by season or water type (p > 0.05). Physicochemical and indicator bacteria were not correlated with PMMoV concentrations. The abundance and prevalence of PMMoV in the karst aquifer may reflect its environmental persistence and its potential as a fecal indicator in this karst aquifer system.

Local and systemic hormonal responses in pepper leaves during compatible and incompatible pepper-tobamovirus interactions.

Phytohormone levels and the expression of genes encoding key enzymes participating in hormone biosynthetic pathways were investigated in pepper leaves inoculated with two different tobamoviruses. Obuda pepper virus (ObPV) inoculation led to the development of hypersensitive reaction (incompatible interaction), while Pepper mild mottle virus (PMMoV) inoculation resulted in a systemic, compatible interaction. ObPV-inoculation markedly increased not only the levels of salicylic acid (SA) (73-fold) and jasmonic acid (8-fold) but also those of abscisic acid, indole-3-acetic acid, indole-3-butyric acid, cis-zeatin, cis-zeatin-9-riboside and trans-zeatin-9-riboside in the inoculated pepper leaves 3 days post inoculation. PMMoV infection increased only the contents of gibberellic acid and SA. Hormone contents did not change significantly after ObPV or PMMoV infection in non-infected upper leaves 20 days post inoculation. Concentrations of some brassinosteroids (BRs) and progesterone increased both in ObPV- and PMMoV inoculated leaves. ObPV inoculation markedly induced the expression of three phenylalanine ammonia-lyase (PAL) and a 1-aminocyclopropane-1-carboxylate oxidase (ACO) genes, while that of an isochorismate synthase (ICS) gene was not modified. PMMoV inoculation did not alter the expression of PAL and ICS genes but induced the transcript abundance of ACO although later than ObPV. Pre-treatment of pepper leaves with exogenous 24-epi-brassinolide (24-epi-BR) prior to ObPV-inoculation strongly mitigated the visible symptoms caused by ObPV. In addition, 24-epi-BR pre-treatment markedly altered the level of several hormones in pepper leaves following ObPV-inoculation. These data indicate that ObPV- and PMMoV-inoculations lead to intricate but well harmonized hormonal responses that are largely determined by the incompatible or compatible nature of plant-virus interactions.

Detection of pepper mild mottle virus as an indicator for drinking water quality in Hanoi, Vietnam, in large volume of water after household treatment.

The aims of this study were to examine the removal of bacteria and viruses by household point-of-use (POU) treatments and to apply a previously developed large-volume virus concentration method (∼20 L). First, the removal of microbes by household POU treatment was investigated in the laboratory. Second, the prevalence of viruses in drinking water sources for households and the removal efficiency of microbes by POU treatments in two suburban communities in Hanoi, Vietnam, were investigated. Indigenous pepper mild mottle virus (PMMoV) was used as the main target together with adenovirus, Aichi virus, enterovirus, F-specific bacteriophage genogroup 1, and Escherichia coli to investigate the removal efficiency of household treatments. The results from laboratory and field survey were compared. From the laboratory study, ceramic membranes were not effective for removing viruses and bacteria from water; pathogen reduction was less than 1.5 log10. By contrast, reverse osmosis (RO) devices reduced microbes by 3 to > 5 log10. In a field study, PMMoV was found to be the most prevalent waterborne virus. Household sand filtration was ineffective for removing E. coli, total coliforms and PMMoV; the reduction was less than 1 order of magnitude. Boiling the water and then filtering it with a ceramic membrane reduced E. coli by 3 orders of magnitude, but this was not effective for removing PMMoV. RO filtration was one of the promising methods for removing E. coli, total coliforms and PMMoV to below their detection limits in most of the samples studied. The removal of E. coli, total coliforms and PMMoV was >2.3, >4 and >3 log10, respectively. The laboratory results of virus removal efficiency by POU devices agreed with the field study. Due to the prevalence and characteristics of PMMoV, it is a strong candidate for an indigenous indicator to investigate the viral removal efficiency of household POU treatments.

The complete nucleotide sequence and genomic characterization of tropical soda apple mosaic virus.

We report the first complete genome sequence of tropical soda apple mosaic virus (TSAMV), a tobamovirus originally isolated from tropical soda apple (Solanum viarum) collected in Okeechobee, Florida. The complete genome of TSAMV is 6,350 nucleotides long and contains four open reading frames encoding the following proteins: i) 126-kDa methyltransferase/helicase (3354 nt), ii) 183-kDa polymerase (4839 nt), iii) movement protein (771 nt) and iv) coat protein (483 nt). The complete genome sequence of TSAMV shares 80.4 % nucleotide sequence identity with pepper mild mottle virus (PMMoV) and 71.2-74.2 % identity with other tobamoviruses naturally infecting members of the Solanaceae plant family. Phylogenetic analysis of the deduced amino acid sequences of the 126-kDa and 183-kDa proteins and the complete genome sequence place TSAMV in a subcluster with PMMoV within the Solanaceae-infecting subgroup of tobamoviruses.

Molecular characterisation of a novel recombinant Ribgrass mosaic virus strain FSHS.

BACKGROUND: The genus Tobamovirus (Virgaviridae) comprises 33 accepted species with the recent addition of eight new viruses and is divided in to three subgroups based on the origin of assembly of the virion and host range. Within the subgroup 1 tobamoviruses the orchid-associated tobamovirus was hypothesized to be a chimeric derivative of recombinations between genome fragments from subgroup 3 and 1. Recombination events involving RdRp, movement and coat protein genes are recorded within subgroup 1 and 2. However natural recombinations have not previously been reported between subgroup 3 tobamoviruses. FINDINGS: The organization and phylogenetic analyses of the complete genome and the different ORFs placed the new isolate within the Ribgrass mosaic virus clade of subgroup 3 tobamoviruses. Recombination detection analyses indicated that the isolate was a chimeric genome with fragments of high similarity to Ribgrass mosaic virus (RMV) strains NZ-439 (HQ667978) and Actinidia-AC (GQ401365.1) infecting herbaceous Plantago sp. and woody Actinidia spp., respectively. The recombinant differed across the whole genome by 3-8 % from other published RMV genomes. CONCLUSION: In this investigation we report an intra-specific recombination between RMV strains NZ-439 (HQ667978) and Actinidia-AC (GQ401365.1), in the replicase component between viral-methyltransferase and viral-helicase regions, resulting in a novel RMV strain FSHS (JQ319720.1) that represents the first described natural recombinant within the RMV cluster of subgroup 3 tobamoviruses.

A new tobamovirus infecting tomato crops in Jordan.

In this study, we completed the whole genome sequence of a new tobamovirus isolated from tomato plants grown in greenhouses in Jordan during the spring of 2015. The 6393-nt single-stranded RNA (ssRNA) genome encodes four proteins, as do other tobamoviruses: two replication-related proteins of 126 kDa and 183 kDa, a 30-kDa movement protein (MP) and a 17.5-kDa coat protein (CP). Phylogenetic analysis showed that this virus does not group with either the tomato mosaic virus (ToMV) or the tobacco mosaic virus (TMV) clades. Instead, it stems from a branch leading to the TMV clade. Analysis of possible recombination events between this virus and representative isolates of closely related tomato-infecting tobamoviruses showed that at least one region originated by recombination. We provide evidence that we have identified a new tobamovirus, for which we propose the name "tomato brown rugose fruit virus".

Simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus in orchids using multiplex RT-PCR.

A system for simultaneous detection of two orchid-infecting viruses was developed and applied to several orchid species. The detection system involved multiplex reverse transcription-polymerase chain reaction (RT-PCR) and could simultaneously identify Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) from the orchid species studied. Multiplex RT-PCR was conducted using two virus-specific primer pairs and an internal control pair of primers to amplify the CymMV and ORSV coat protein regions, and orchid 18S rDNA, respectively. For optimization of multiplex RT-PCR conditions, serial dilutions of total RNA and cDNA were performed and the detection limit of the system was evaluated. The optimized multiplex detection system for CymMV and ORSV was applied to various orchid species, including several cultivars of Doritaenopsis, Cymbidium, Dendrobium, and Phalaenopsis to test the efficacy of this method. Our results indicate that the multiplex RT-PCR detection system will be a rapid, simple, and precise diagnosis tool in a range of orchid species.

Development of an agroinoculation system for full-length and GFP-tagged cDNA clones of cucumber green mottle mosaic virus.

The complete 6243-nucleotide sequence of a cucumber green mottle mosaic virus (CGMMV) isolate from bottle gourd in Zhejiang province, China, was determined. A full-length cDNA clone of this isolate was constructed by inserting the cDNA between the 35S promoter and the ribozyme in the binary plasmid pCB301-CH. A suspension of an Agrobacterium tumefaciens EHA105 clone carrying this construct was highly infectious in Nicotiana benthamiana and bottle gourd. Another infectious clone containing the green fluorescence protein (GFP) reporter gene was also successfully constructed. This study is the first report of the efficient use of agroinoculation for generating CGMMV infections.

Detection of pepper mild mottle virus in pepper sauce in China.

Pepper mild mottle virus (PMMoV) was detected by RT-PCR in all 42 pepper sauce samples from the 10 main manufacturing provinces in China at concentrations ranging from 3.8 to 8.8 (Log10 copies/mL). Their coat protein nucleotide sequences had 97.4 to 100 % identity to each other and 92.4 to 100 % to other published isolates. The samples remained infectious to N. benthamiana, indicating that commercial trade in sauce could contribute to the natural spread of PMMoV.