A Tobamovirus Causing Heavy Losses in Protected Pepper Crops in Spain

During a four-year (1982–1985) survey of plant viruses infecting pepper cultivars grown under plastic in the Southeastern region of Spain, a tobamovirus was found to be the major disease agent of this crop. The virus produces slight or no symptoms on the leaves, but causes chlorotic mottling, malformation and reduction in size with occasional necrosis on the fruits and was able to infect all commercial pepper cultivars tested, including those resistant to other tobamoviruses, causing a catastrophic disease. The biological and serological characterization of the virus showed that it is very similar to pepper mild mottle virus (PMMV) (Wetter et al. 1984) and therefore we have termed it as Spanish strain of PMMV (PMMV-S). The need of grouping all the so-called “pepper strains” of tobacco mosaic virus (TMV) as a new distinct member of the tobamovirus group with the name of PMMV is emphasized.     

Relation Between the Chemical Structure and the Biological Activity of Hydroxystilbenes Against Botrytis cinerea

Abstract A Spanish strain of pepper mild mottle virus (PMMV-S) (A lonso et al. 1989) can be differentiated from the Italian PMMV (W etter et al. 1984) by the responses of Capsicum spp. with resistance genes to tobamoviruses, by radioimmunoassay and by the electrophoretic mobility of their viral particles. Moreover, the analysis of the electrophoretic mobility of the viral particles in agarose gels and that of the viral coat proteins in polyacrylamide-urea gels are reliable and rapid techniques for distinguishing PMMV isolates from other members of the tobamovirus group and thus can be used for diagnostic purposes. These results support the proposal of grouping these pepper viruses, including the "pepper strains" of TMV, as a new tobamovirus subgroup (PARES 1985, BETTI et al. 1987, WETTER et al. 1987, ALONSO et al. 1989).     

A tobamovirus infecting capsicum in Australia

A tobamovirus infection of Capsicum annuum is recorded for the first time in New South Wales, Australia. The causal virus is described and shown to differ from tobacco (TMV) and tomato (ToMV) mosaic viruses in its host reactions and serology. Seventeen capsicum cultivars were tested for reaction to the Australian isolate and ranked according to symptom severity. Field and glasshouse observations indicated that the virus causes a decrease in height and weight of plants, fruit malformation and leaf mosaic symptoms. It is proposed that the capsicum tobamovirus strains are sufficiently distinctive from TMV and ToMV to be grouped together and designated a separate virus: capsicum mosaic virus (CaMV).     

Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana

The complete nucleotide sequence of Chinese rape mosaic virus has been determined. The virus is a member of the tobamovirus genus of plant virus and is able to infect Arabidopsis thaliana (L.) Heynh systemically. The analysis of the sequence shows a gene array that seems to be characteristic of crucifer tobamoviruses and which is slightly different from the one most frequently found in tobamoviruses. Based on gene organization and on comparisons of sequence homologies between members of the tobamoviruses, a clustering of crucifer tobamoviruses is proposed that groups the presently known crucifer tobamovirus into two viruses with two strains each. A name change of Chinese rape mosaic virus to oilseed rape mosaic virus is proposed.Abbreviations 2-ME 2-mercaptoethanol - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - UTR untranslated region - MP movement protein - CP capsid protein - CRMV Chinese rape mosaic virus - TVCV turnip vein clearing virus - PaMMV paprika mild mottle virus - PMMV-I pepper mild mottle virus (Italian isolate) - PMMV-S pepper mild mottle virus (Spanish isolate) - ToMV tomato mosaic virus - TMV tobacco mosaic virus - TMGMV tobacco mild green mosaic virus - ORSV odontoglossum ringspot virus - SHMV sunn hemp mosaic virus - CGMMV cucumber green mottle mosaic virus - ORMV oilseed rape mosaic virus     

Detection of tobacco mosaic virus and tomato mosaic virus in pepper seeds by enzyme linked immunosorbent assay (ELISA)

Pepper seed samples were tested for the infection of tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) by enzyme linked immunosorbent assay (ELISA). Out of 26 pepper seed samples tested, 17 were infected with TMV and ToMV in ELISA. About 34.7% of pepper seed samples were found to be healthy. Infections of TMV or ToMV were recorded to be 61.53% and 11.5%, respectively of the total tested seed samples.     

Biological and Molecular Characterization of a Crucifer Tobamovirus Infecting Oilseed Rape

In China, the tobamovirus that infects oilseed rape has been misdiagnosed as Tobacco mosaic virus (TMV) based on its morphological similarity and serological relatedness. Recently, a tobamovirus has been isolated from oilseed rape in China, which we named Youcai mosaic virus Br (YoMV-Br), according to its biological and molecular characteristics. It had strong infectivity to Cruciferae but less to Solanaceae, Leguminosae, and Cucurbitaceae, and its virion morphology was consistent with that of the tobamoviruses. At high concentrations, it serologically cross reacted with TMV antiserum. The 3′ terminal sequence (2,283 nucleotides) of YoMV-Br was determined, including the 3′ noncoding region, the CP and MP genes, and the C-terminal part of the replicase gene. Between the MP and CP genes, 77 nucleotides overlapped. Compared with homologous regions of 21 recognized species of Tobamovirus, YoMV-Br had a much higher identity to crucifer species than to other tobamoviruses. Phylogenetic analysis demonstrated that YoMV-Br was closely related to the YoMV cluster of tobamoviruses and distantly to TMV, so that they likely belong to different strains of the same species.     

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR

Aims: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. Methods and Results: A multiplex RT–PCR method consisting of one‐tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT–PCR detected up to 10^?6 dilution of total RNA extracted from infected leaves. Multiplex RT–PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. Conclusions: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. Significance and Impact of the Study: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.     

Serological comparison of an Australian isolate of capsicum mosaic virus with capsicum tobamovirus isolates from Europe and America

A New South Wales isolate (Ca) of capsicum mosaic virus was tested against antisera to it and capsicum tobamovirus isolates from the Netherlands (P8, P11), USA (SL), Argentina (FO) and Sicily (PM). The comparison demonstrated that the four viruses Ca, P8, PM and SL are closely related to each other, forming a series of decreasing relationship to Ca in the above order. FO was related to these but insufficiently to be considered part of the group, and P11 was only slightly related to the others. The literature on serology of tobamoviruses in Capsicum spp. was collated and it is suggested that isolates from Sicily (pepper mild mottle), Australia (capsicum mosaic), The Netherlands (P8, P14) and USA (SL) be considered as strains of a virus distinct from both tobacco mosaic and tomato mosaic viruses and that these isolates all be referred to in future as strains of pepper mild mottle virus.     

A hot pepper cDNA encoding a pathogenesis-related protein 4 is induced during the resistance response to tobacco mosaic virus

Hot pepper (Capsicum annuum) plants exhibit a hypersensitive response (HR) against infection by many tobamoviruses. A clone (CaPR-4) encoding a putative pathogenesis-related protein 4 was isolated by differential screening of a cDNA library prepared from resistant pepper plant leaves inoculated with tobacco mosaic virus (TMV) pathotype P0. The predicted amino acid sequence of CaPR-4 is very similar to those of other plant PR-4s. Southern blot analysis showed that small gene families of PR-4-related sequences were present in the pepper genome. Hot pepper cultivar Bugang, resistant to TMV-P0 and susceptible to TMV-P1.2, induced CaPR-4 expression by pathotype P0 inoculation in inoculated and systemic leaves, but not by pathotype P1.2. Effects of exogenously applied abiotic elicitors upon the CaPR-4 expression were also examined. The expression of the CaPR-4 gene was stimulated by methyl jasmonate (MeJA), ethephon and wounding treatment. However, application of salicylic acid (SA) did not trigger the expression. Evidence is emerging that jasmonic acid and ethylene play key roles in the SA-independent pathways of plant-pathogen interaction. Taken together, these results suggest that the CaPR-4 gene is one of the defense-related genes conferring resistance on pepper plants by the SA-independent pathway and the cross-talk between signaling compounds, jasmonic acid and ethylene could have a great regulatory potential in a plant's defense against TMV.     

Detection by ELISA of Two Tobamoviruses in Orchids Using Monoclonal Antibodies

Five monoclonal antibodies (McAbs) were raised to the tobamovirus, odontoglossum ringspot virus (ORSV). All five McAbs reacted with the virus in double antibody sandwich (DAS) ELISA but not in an ELISA using virus-coated plates. All the McAbs recognized a panel of ORSV strains and isolates, although one of the antibodies reacted better with some isolates and another reacted less with certain isolates than with type ORSV. It was possible to use the same McAbs both as coating and as biotinylated antibody in DAS-ELISA. None of the five McAbs was able to bind to orchid strains of tobacco mosaic virus (TMV). In order to detect strains of both viruses, ORSV and TMV, in infected orchids it was necessary to include also McAbs raised against TMV in the immunoassays. The use of a mixed polyclonal-monoclonal antibody DAS-ELISA system is advocated for detecting both tobamoviruses in orchids.     

Limited degree of serological variability in pepper strains of potato virus Y as revealed by analysis with monoclonal antibodies

The degree of serological variability among pepper strains of potato virus Y (PVY) was assessed through the analysis of samples of infected pepper collected in three main pepper producing regions of Spain. Samples corresponding to the period 1980–1991 were analysed by ELISA with five different monoclonal antibodies (MAbs) produced against potato strains of the virus. The results obtained show a limited degree of epitope variability among pepper PVY-isolates, since only eight out of 32 possible serological profiles were found. Most isolates are not recognised by a MAb directed towards an epitope reported to be present in all potato-PVY isolates. The overall serological behaviour of pepper isolates with these MAbs places them as closer to the group O, of the three groups into which the potato isolates of PVY have been subdivided.     

Knockout of SlTOM1 and SlTOM3 results in differential resistance to tobamovirus in tomato

During tobamovirus–host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant‐encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9‐mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516‐ and 25‐fold lower, respectively, than in wild‐type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three‐fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development.     

Detection of Infectious Tobamoviruses in Forest Soils

Our objectives were to evaluate elution and bait plant methods to detect infectious tobamoviruses in forest soils in New York State. Soils were collected from two forest sites: Whiteface Mountain (WF) and Heiberg Forest (HF). The effectiveness of four buffers to elute tomato mosaic tobamovirus (ToMV) from organic and mineral fractions of WF soil amended with ToMV was tested, and virus content was assessed by enzyme-linked immunosorbent assay (ELISA). The effectiveness of Chenopodium quinoa (Willd.) bait plants to detect the virus also was tested. Both methods then were utilized to detect tobamoviruses in 11 WF and 2 HF soil samples. A phosphate buffer (100 mM, pH 7.0) eluted more ToMV from soil than the other buffers tested. Mineral soil bound more virus than organic soil. Virus recoveries from virus-amended organic and mineral soils were 3 and 10%, respectively, and the detection sensitivity was 10 to 20 ng/g of soil. Roots of bait plants grown in all virus-amended soils tested positive by ELISA, and virus concentrations averaged 10 ng/g. Both ToMV and tobacco mosaic tobamovirus (TMV) were transmitted to C. quinoa by elution from one of two HF soil samples but not from the WF soil samples. A tobamovirus was detected by bait planting in 12 of 73 (16%) root extracts representing 5 of 13 soil samples (38%). Tobamovirus-like particles were seen by transmission electron microscopy in 6 of 12 infected root extracts. Tobamoviruses occur in forest soils in New York State. Abiotic soil transmission to trees may permit localized spread and persistence of these viruses in forest ecosystems.     

Type I J-Domain NbMIP1 Proteins Are Required for Both Tobacco Mosaic Virus Infection and Plant Innate Immunity

Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by recognizing the viral movement protein (MP). Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.     

The symptom difference induced by Tobacco mosaic virus and Tomato mosaic virus in tobacco plants containing the N gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaicvirus (ToMV) are members of the genus Tobamoviruswith a world-wide distribution, and cause severe dis-eases on many economically important crops. TMVand ToMV have very close relationship and both havessRNA genome with a length of about 6400 nucleo-tides, encoding at least three nonstructural proteinsand a 17.6 kD coat protein (CP). Both 126 kD and 183kD proteins function as components of replicase, andthe 30 kD protein is involved in viral ce…     

Strain typing of polish Leptosphaeria maculans isolates supports at the genomic level the multi-species concept of aggressive and non-aggressive strains

47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared with eight well-defined reference strains from Germany, France, Denmark, Australia and one Polish isolate of Phoma nigrificans. The isolates were tested (i) for growth characteristics, (ii) for their ability to form sirodesmins, (iii) for cellulolytic enzymes, and (iv) for pathotype-differentiating molecular markers generated by RAPD-PCR, PCR analysis with pathotype-specific primer pairs and PFGE. With two exceptions all Polish isolates do not form sirodesmins. grow rapidly without penetrating into the substrate and form in most cases yellow or brown pigments in Czapek-Dox liquid cultures. With respect to cellulase secretion and molecular fingerprinting Polish A strains (aggressive) fit into the general picture of the aggressive pathotype group, whereas the NA isolates (non-aggressive) display a higher degree of heterogeneity. This matches with inoculation tests on rape seedlings, which revealed a considerable number of isolates ranging in aggressivity between the conventional A and NA pathotype group. Molecular fingerprinting techniques unequivocally sorted intermediately aggressive isolates into the NA pathotype group. Isolate Ph Bial, which produces sirodesmin but groups within NA isolates according to molecular and physiological markers, may represent a novel third group besides A and NA strains with intermediate aggressivity (IA). We hybridized Southern blots of electrophoretically separated chromosomes with radioactively labelled PCR fragments used for differentiation between A and NA isolates. The specificity of diagnostic PCR amplicons is reflected at the genomic level. The A probe reveals a single hybridizing chromosome exclusively in A strains. The NA probe reveals several chromosomes and is specific for the NA pathotype group. Chromosomes from intermediately aggressive strains are equally well recognized by the NA probe as are Polish isolates with low aggressivity and give no signal with the A probe. Both diagnostic DNA sequences are highly specific for the pathotype group they were derived from. The lack of correspondence of both genetic elements between A and NA strains strongly supports the idea of ascribing the pathotype groups to different species. Whereas the A pathotype group is genetically homogeneous and congruent with the species Leptosphaeria maculans, the NA group needs to be revised taxonomically. NA isolates will presumably have to be split into several independent species.     

Vegetative compatibility of an isolate ofVerticillium dahliae pathogenic to both tomato and pepper

An isolate ofVerticillum dahliae Vdp-4, pathogenic to both tomato and pepper (tomato-pepper pathotype), was examined for its vegetative compatibility with testers of the Japanese vegetative compatibility group (subgroups J1, J2, and J3). Seven isolates ofV. dahliae from the same field as Vdp-4 in Misato, Nagano Pref. and two isolates from Hokkaido were separately determined as either tomato pathotype (B) or pepper pathotype (C). Isolate 5922 previously reported as tomato-pepper pathotype was also examined. Compatiblenit1 and NitM mutants were obtained from all isolates except for isolates Vdp-3 and Vdt-10. The isolate of tomato-pepper pathotype Vdp-4 showed a strong reaction with VCGJ1 and J3 and was thus assigned to J3. Seven of these isolates showed compatibility and were assigned into three provisional subgroups. The isolate 5922 was self-incompatible.     

Activation of Tm-22 resistance is mediated by a conserved cysteine essential for tobacco mosaic virus movement

The tomato Tm-2² gene was considered to be one of the most durable resistance genes in agriculture, protecting against viruses of the Tobamovirus genus, such as tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV). However, an emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), has overcome Tm-2², damaging tomato production worldwide. Tm-2² encodes a nucleotide-binding leucine-rich repeat (NLR) class immune receptor that recognizes its effector, the tobamovirus movement protein (MP). Previously, we found that ToBRFV MP (MP^ToBRFV) enabled the virus to overcome Tm-2²-mediated resistance. Yet, it was unknown how Tm-2² remained durable against other tobamoviruses, such as TMV and ToMV, for over 60 years. Here, we show that a conserved cysteine (C68) in the MP of TMV (MP^TMV) plays a dual role in Tm-2² activation and viral movement. Substitution of MP^ToBRFV amino acid H67 with the corresponding amino acid in MP^TMV (C68) activated Tm-2²-mediated resistance. However, replacement of C68 in TMV and ToMV disabled the infectivity of both viruses. Phylogenetic and structural prediction analysis revealed that C68 is conserved among all Solanaceae-infecting tobamoviruses except ToBRFV and localizes to a predicted jelly-roll fold common to various MPs. Cell-to-cell and subcellular movement analysis showed that C68 is required for the movement of TMV by regulating the MP interaction with the endoplasmic reticulum and targeting it to plasmodesmata. The dual role of C68 in viral movement and Tm-2² immune activation could explain how TMV was unable to overcome this resistance for such a long period.     

A century of tobamovirus evolution in an Australian population of Nicotiana glauca.

The evolution over the past century of two tobamoviruses infecting populations of the immigrant plant Nicotiana glauca in New South Wales (NSW), Australia, has been studied. This plant species probably entered Australia in the 1870s. Isolates of the viruses were obtained from N. glauca specimens deposited in the NSW Herbarium between 1899 and 1972, and others were obtained from living plants in 1985 and 1993. It was found that the NSW N. glauca population was infected with tobacco mosaic tobamovirus (TMV) and tobacco mild green mosaic tobamovirus (TMGMV) before 1950 but only with TMGMV after that date. Half the pre-1950 infections were mixtures of the two viruses, and one was a recombinant. Remarkably, sequence analyses showed no increase in the genetic diversity among the TMGMV isolates over the period. However, for TMV, the genetic diversity of synonymous (but not of nonsynonymous) differences between isolates varied and was correlated with their time of isolation. TMV accumulated to smaller concentrations than TMGMV in N. glauca plants, and in mixed experimental infections, the accumulation of TMV, but not of TMGMV, was around 1/10 that in single infections. However, no evidence was found of isolate-specific interaction between the viruses. We conclude that although TMV may have colonized N. glauca in NSW earlier or faster than TMGMV, the latter virus caused a decrease of the TMV population below a threshold at which deleterious mutations were eliminated. This phenomenon, called Muller's ratchet, or a "mutational meltdown," probably caused the disappearance of TMV from the niche.