Transmission of Pepino mosaic virus by the Fungal Vector Olpidium virulentus

Transmission of Pepino mosaic virus (PepMV) by the fungal vector Olpidium virulentus was studied in two experiments. Two characterized cultures of the fungus were used as stock cultures for the assay: culture A was from lettuce roots collected in Castellón (Spain), and culture B was from tomato roots collected in Murcia (Spain). These fungal cultures were maintained in their original host and irrigated with sterile water. The drainage water collected from irrigating these stock cultures was used for watering PepMV‐infected and non‐infected tomato plants to constitute the acquisition–source plants of the assay, which were divided into six different plots: plants containing fungal culture A (non‐infected and PepMV‐infected); plants containing fungal culture B (non‐infected and PepMV‐infected); PepMV‐infected plants without the fungus; and plants non‐infected either with PepMV and the fungus. Thirty‐six healthy plants grouped into six plots, which constituted the virus acquisition–transmission plants of the assay, were irrigated with different drainage waters obtained by watering the different plots of the acquisition–source plants. PepMV was only transmitted to plants irrigated with the drainage water collected from PepMV‐infected plants whose roots contained the fungal culture B from tomato with a transmission rate of 8%. No infection was detected in plants irrigated with the drainage water collected from plots with only a fungus or virus infection. Both the virus and fungus were detected in water samples collected from the drainage water of the acquisition–source plants of the assay. These transmission assays demonstrated the possibility of PepMV transmission by O. virulentus collected from tomato crops.     

Vectoring of Pepino mosaic virus by bumble‐bees in tomato greenhouses

Pepino mosaic virus (PepMV) has become an important viral disease of greenhouse tomatoes worldwide. The ability of bumble‐bees (Bombus impatiens), used for pollination, to acquire and transmit PepMV was investigated, and the prevalence of PepMV in plants and bumble‐bees in commercial tomato greenhouses was determined. PepMV infection in plants was determined using enzyme‐linked immunosorbent assay, while in bumble‐bees direct real‐time PCR was used. In the first experiment, the bumble‐bees were exposed for 14 days to PepMV‐infected plants. After 14 days, almost all bumble‐bees were PepMV positive both in the hive (78.5 ± 17.5%) and in the flowers (96.3 ± 3.6%). In the second experiment, bumble‐bees were released into a greenhouse with both PepMV‐infected source plants and healthy non‐infected target plants for 14 days. At the end of the experiment, 61.0 ± 19.5% of the bees collected from the hive and 83.3 ± 16.7% of the bees sampled from the flowers were PepMV positive. Bumble‐bees transmitted PepMV from the infected to the healthy non‐infected tomato plants. Two weeks after bumble‐bee release, the virus was detected in leaf, fruit and flower samples of formerly healthy plants. After 6 weeks, the percentage of PepMV positive samples from the target plants increased to 52.8 ± 2.8% of the leaves and 80.6 ± 8.4% of the fruits. In the control greenhouse without bumble‐bees, the target plants did not become infected. Based on the infection levels in flowers, fruits and leaves, the PepMV infection occurred possibly first in the pollinated flowers, and then spread from the fruit that developed from the flowers to other parts of the plant. In commercial greenhouses where PepMV was present, 92–100% of the plants and 88–100% of the bumble‐bees were PepMV positive. No infected plant samples were found in the control commercial greenhouse, but a small number of bumble‐bees (10%) tested PepMV positive.     

A pathogenicity determinant maps to the N‐terminal coat protein region of the Pepino mosaic virus genome

Pepino mosaic virus (PepMV) poses a worldwide threat to the tomato industry. Considerable differences at the genetic level allow for the distinction of four main genotypic clusters; however, the basis of the phenotypic outcome is difficult to elucidate. This work reports the generation of wild‐type PepMV infectious clones of both EU (mild) and CH2 (aggressive) genotypes, from which chimeric infectious clones were created. Phenotypic analysis in three solanaceous hosts, Nicotiana benthamiana, Datura stramonium and Solanum lycopersicum, indicated that a PepMV pathogenicity determinant mapped to the 3′‐terminal region of the genome. Increased aggression was only observed in N. benthamiana, showing that this factor is host specific. The determinant was localized to amino acids 11–26 of the N‐terminal coat protein (CP) region; this is the first report of this region functioning as a virulence factor in PepMV.     

Evidence for different,host‐dependent functioning of Rx against both wild‐type and recombinant Pepino mosaic virus

The potato Rx gene provides resistance against Pepino mosaic virus (PepMV) in tomato; however, recent work has suggested that the resistance conferred may not be durable. Resistance breaking can probably be attributed to multiple mutations observed to accumulate in the capsid protein (CP) region of resistance‐breaking isolates, but this has not been confirmed through directed manipulation of an infectious PepMV clone. The present work describes the introduction of two specific mutations, A‐T78 and A‐T114, into the coat protein minimal elicitor region of an Rx‐controlled PepMV isolate of the EU genotype. Enzyme‐linked immunosorbent assay (ELISA) and phenotypic evaluation were conducted in three Rx‐expressing and wild‐type solanaceous hosts: Nicotiana benthamiana, Nicotiana tabacum and Solanum lycopersicum. Mutation A‐T78 alone was sufficient to confer Rx‐breaking activity in N. benthamiana and S. lycopersicum, whereas mutation A‐T114 was found to be associated, in most cases, with a secondary A‐D100 mutation to break Rx‐mediated resistance in S. lycopersicum. These results suggest that the need for a second, fitness‐restoring mutation may be dependent on the PepMV mutant under consideration. Both mutations conferred Rx breaking in S. lycopersicum, whereas neither conferred Rx breaking in N. tabacum and only A‐T78 allowed Rx breaking in N. benthamiana, suggesting that Rx may function in a different manner depending on the genetic background in which it is present.     

Molecular evolution of Pepino mosaic virus during long‐term passaging in different hosts and its impact on virus virulence

In this study the effect of host changes and multiple passages on Pepino mosaic virus (PepMV) evolution was analysed. A population of a mild isolate of PepMV was used to generate five independent evolution lineages on three tomato cultivars, which differ in rate of appearance of symptoms and their severity during viral infection (Beta Lux, Moneymaker and Malinowy O?arowski) and on Datura inoxia. Twenty serial passages were performed over a period of 217–220 days. Symptom severity was monitored along the entire experiment. After the last series of passages total RNAs from each lineage and host were isolated and the triple gene block 3 (TGB3) and coat protein (CP) were amplified, cloned and 10 clones for each gene sequenced. Among the 400 clones for both genes, 143 individual mutations (61 synonymous and 82 nonsynonymous) were identified, with the largest number of nonsynonymous mutations being observed for the tomato cultivars Malinowy O?arowski and Beta Lux. In two of the lineages evolving in the most susceptible variety of tomato (Beta Lux) necrotic changes in leaf blades appeared after 17 passages, leading to death of the plants. In these two lineages the mutation responsible for necrotic symptoms was K67E in TGB3. The appearance of this convergent mutation in independently evolving lineages may suggest that selection in this experimental set up favours more aggressive PepMV variants. We found a positive association between the severity of symptoms and the amount of genetic variability contained on viral populations. Indeed, the severity of symptoms turned out to be a good predictor for several indices of molecular variability. In addition, mapping all observed mutations in CP and TGB3 protein structures revealed that most were located on the surface, indicating a possible implication in viral–viral or viral–host interactions.     

New Necrotic Isolates of Pepino mosaic virus Representing the Ch2 Genotype

New necrotic isolates of Pepino mosaic virus (PepMV) were found in 2007 infecting greenhouse tomato plants in Poland. The isolates differ from previously identified PepMV isolates in host range and symptomatology. They induce severe necrosis on tomato plants ( Solanum lycopersicum ) and local necrotic lesions on Datura inoxia . Phylogenetic analysis, based on three distinct regions, triple gene block 1, the coat protein gene and a part of polymerase gene, revealed that the new necrotic isolates share high nucleotide sequence identity with isolates of the Ch2 genotype. This is the first report describing a necrotic type of PepMV of the Ch2 genotype.     

Coat Protein Gene of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island

Nucleotide and amino acid sequences of the coat protein (CP) of 12 isolates of Cymbidium mosaic virus from Vanilla fragrans in Reunion Island (CyMV‐R) were compared with each other and with those of previously described Asian strains. Alignment revealed that CyMV‐R isolates were highly homologous, suggesting that one strain is prevalent in Reunion. This strain also showed high homology with the Korean CyMV‐K2 and Singapore CyMV‐S2 strains, but nucleotide additions resulted in the carboxy‐terminal ends of the CP sequences differing from those of the Korean CyMV‐K1 and Singapore CyMV‐K1 strains.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Serological detection of Cucumber mosaic virus on some important host crops in the north region of Iran

Plant virus diseases cause major losses in agricultural and horticultural products, especially in tropical and subtropical regions. The first step to manage these diseases is detecting, identifying and determining the pathogen characteristics. Cucumber mosaic disease is one of the most prevalent and damaging plant diseases in the world which is caused by Cucumber mosaic virus (CMV). Each year, this virus causes yield decreasing and substantial economic damages in its host plants worldwide including the north of Iran. In order to study and identify CMV, 935 leaf samples were collected based on typical symptoms from 10 crops (tomato, pea, tobacco, soybean, watermelon, broad bean, squash, cucumber, eggplant and lettuce) in different regions of Golestan and Mazandaran provinces (north of Iran) during 2009–2010. Suspicious samples were analysed by DAS-ELISA and polyclonal antibodies. The results showed that 275 samples (29.4%) were infected by CMV. Between these hosts, the highest and the lowest CMV infection was associated to watermelon (62.44%) and lettuce (0%), respectively. Among sampling locations, Behshahr (100%) and Minoodasht (3.47%) showed the maximum and minimum infection, respectively.     

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

The potential use of a viral coat protein gene as a transgene screening marker and multiple virus resistance of pepper plants coexpressing coat proteins of cucumber mosaic virus and tomato mosaic virus

Transgenic pepper plants coexpressing coat proteins (CPs) of cucumber mosaic virus (CMV-Kor) and tomato mosaic virus (ToMV) were produced by Agrobacterium-mediated transformation. To facilitate selection for positive transformants in transgenic peppers carrying an L gene, we developed a simple and effective screening procedure using hypersensitive response upon ToMV challenge inoculation. In this procedure, positive transformants could be clearly differentiated from the nontransformed plants. Transgenic pepper plants expressing the CP genes of both viruses were tested for resistance against CMV-Kor and pepper mild mottle virus (PMMV). In most transgenic plants, viral propagation was substantially retarded when compared to the nontransgenic plants. These experiments demonstrate that our transgenic pepper plants might be a useful marker system for the transgene screening and useful for classical breeding programs of developing virus resistant hot pepper plants.     

Occurrence of fungally transmitted wheat mosaic viruses in China

A soil-borne mosaic disease of winter wheat in Sichuan, Shaanxi, Hubei and Henan provinces was associated with infection by a virus with filamentous particles and that in Shandong, Anhui, Jiangsu and Zhejiang provinces by co-infection with this virus and soil-borne wheat mosaic virus. The virus with filamentous particles was identified serologically, by particle sizes, cytopathology and the molecular weights of the coat protein and the two RNA species to be either wheat spindle streak mosaic virus (WSSMV) or wheat yellow mosaic virus. These viruses are probably isolates of the same virus and the name WSSMV is preferred. In baiting tests using infested soil, the dilution endpoints for detecting WSSMV were 1/625-1/15625, and for the fungus vector, Polymyxa graminis, 1/3125-1/15625.     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

Completion of the Nucleotide Sequence of a Brazilian Isolate of Southern bean mosaic virus

The sequence of the central part (ORF2) of a Brazilian isolate of Southern bean mosaic virus (SBMV^SP) is described. This ORF is 2888 nt long and together with the previously-sequenced 5' and 3' ends provides the complete nucleotide sequence of this virus isolate. The SBMV^SP RNA encodes four overlapping open reading frames (ORF1, ORF2a, ORF2b, ORF4) and has a genome organization similar to that of the Cocksfoot mottle sobemovirus .     

Homology between the proteins encoded by tobacco mosaic virus and two tricornaviruses

Summary A comparison was made of the amino acid sequences of the proteins encoded by RNAs 1 and 2 of alfalfa mosaic virus (A1MV) and brome mosaic virus (BMV), and the 126K and 183K proteins encoded by tobacco mosaic virus (TMV). Three blocks of extensive homology of about 200 to 350 amino acids each were observed. Two of these blocks are located in the A1MV and BMV RNA 1 encoded proteins and the TMV encoded 126K protein; they are situated at the N-terminus and C-terminus, respectively. The third block is located in the A1MV and BMV RNA 2 encoded proteins and the C-terminal part of the TMV encoded 183K protein. These homologies are discussed with respect to the functional equivalence of these putative replicase proteins and a possible evolutionary connection between A1MV, BMV and TMV.