For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomat... more 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.
Since the summer of 1993, transgenic tomato plants expressing the coat protein (CP) genes of cucu... more Since the summer of 1993, transgenic tomato plants expressing the coat protein (CP) genes of cucumber mosaic cucumovirus have been tested under field conditions to assess the level of resistance and agronomic performance. Trials were performed in different areas in Italy and the target virus in the majority of tests was spread naturally by the indigenous aphid populations. Twenty-three homozygous lines of variety UC82B, transformed to contain four different CP genes of CMV, were evaluated. The lines were preselected for CP expression, single gene copy, and virus resistance in growth chamber experiments. In general, CMV resistance was confirmed under field conditions though resistance in the field was less effective than what was observed in growth chamber experiments. The resistance observed in multi-year and multi-location experiments is of commercial value for several of the most resistant lines. Engineered resistance upon transfer to Italian varieties by breeding or direct transformation will be used in tomato production in Italy or elsewhere.
Biomolecular and serological methods were used to classify 52 Italian isolates of cucumber mosaic... more Biomolecular and serological methods were used to classify 52 Italian isolates of cucumber mosaic cucumovirus (CMV) into S and WT subgroups. Two synthetic oligonucleotides, specific for the coat-protein genes of the S and WT subgroups, were designed. These probes, used in Northern blot assays, were able to identify viral RNAs present in total nucleic-acid extracts from fruits and leaves of infected field tomatoes. CMV-specific primers, flanking the coat-protein gene, were used to amplify, by RT-PCR assay, a fragment of approximately 870 bp from either dsRNA replicative intermediates or purified genomic ssRNA. The restriction pattern analysis of the PCR product distinguished between the two subgroups. Monoclonal antibodies specific for CMV isolates belonging to the S and WT subgroups were produced. Five of these were able to detect and distinguish the strains in indirect ELISA using crude extracts from field samples. The three strategies gave concordant results and were useful in detecting and typing CMV subgroups on tomato.
Since 1995, a severe necrosis disease has been observed in winter melon (Cucumis melo var. inodor... more Since 1995, a severe necrosis disease has been observed in winter melon (Cucumis melo var. inodorus) grown in the open in Sardegna region. At the stage of fruit ripening, infected plants show decline and premature death, a syndrome known as‘collapse’. Fungal pathogens have never been detected. In 1998, melon necrotic spot carmovirus (MNSV) was isolated from diseased plants showing necrotic symptoms on leaves and branches. The virus was identified by biological and serological assays. MNSV may represent a limiting factor to winter melon production in Sardegna where the‘inodorus’group is widely cultivated and appreciated for local consumption. This is the first occurrence of MNSV in Italy.
... In different areas of northern Italy, samples were collected from 48 stocks of cv. Hermes, th... more ... In different areas of northern Italy, samples were collected from 48 stocks of cv. Hermes, the most important processing cultivar in Italy, and from 12 stocks of other potato cultivars (Ernterstolz, Liseta, Monalisa, Primura, Kennebec). ...
Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin-point yell... more Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin-point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme-linked immunosorbent assay to the most important viruses-infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV-infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.
In this manuscript we provide evidence for the seed transmission of Pepino mosaic virus (PepMV) i... more In this manuscript we provide evidence for the seed transmission of Pepino mosaic virus (PepMV) in tomato. Fruit was harvested from a tomato crop artificially infected with both European and CH2 genotypes of PepMV and more than 100,000 seeds were extracted and cleaned using an enzymatic treatment without disinfection. Infection assays using indicator plants confirmed the presence of viable virus on the seeds. Seeds were distributed to ten different laboratories in three separate batches, where they were germinated and the young plants tested by ELISA. In total over 87,000 plants were tested and 23 positives detected, indicating an overall transmission rate of 0.026%. However, the observed seed transmission rates varied from 0.005% to 0.057%, depending on the seed batch used. Results clearly showed that PepMV can be transmitted from seeds contaminated with virus to seedlings, highlighting the risk of using seeds from PepMV-infected plants and the potential for seed transmission to contribute to the further spread of PepMV.
For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomat... more 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.
Since the summer of 1993, transgenic tomato plants expressing the coat protein (CP) genes of cucu... more Since the summer of 1993, transgenic tomato plants expressing the coat protein (CP) genes of cucumber mosaic cucumovirus have been tested under field conditions to assess the level of resistance and agronomic performance. Trials were performed in different areas in Italy and the target virus in the majority of tests was spread naturally by the indigenous aphid populations. Twenty-three homozygous lines of variety UC82B, transformed to contain four different CP genes of CMV, were evaluated. The lines were preselected for CP expression, single gene copy, and virus resistance in growth chamber experiments. In general, CMV resistance was confirmed under field conditions though resistance in the field was less effective than what was observed in growth chamber experiments. The resistance observed in multi-year and multi-location experiments is of commercial value for several of the most resistant lines. Engineered resistance upon transfer to Italian varieties by breeding or direct transformation will be used in tomato production in Italy or elsewhere.
Biomolecular and serological methods were used to classify 52 Italian isolates of cucumber mosaic... more Biomolecular and serological methods were used to classify 52 Italian isolates of cucumber mosaic cucumovirus (CMV) into S and WT subgroups. Two synthetic oligonucleotides, specific for the coat-protein genes of the S and WT subgroups, were designed. These probes, used in Northern blot assays, were able to identify viral RNAs present in total nucleic-acid extracts from fruits and leaves of infected field tomatoes. CMV-specific primers, flanking the coat-protein gene, were used to amplify, by RT-PCR assay, a fragment of approximately 870 bp from either dsRNA replicative intermediates or purified genomic ssRNA. The restriction pattern analysis of the PCR product distinguished between the two subgroups. Monoclonal antibodies specific for CMV isolates belonging to the S and WT subgroups were produced. Five of these were able to detect and distinguish the strains in indirect ELISA using crude extracts from field samples. The three strategies gave concordant results and were useful in detecting and typing CMV subgroups on tomato.
Since 1995, a severe necrosis disease has been observed in winter melon (Cucumis melo var. inodor... more Since 1995, a severe necrosis disease has been observed in winter melon (Cucumis melo var. inodorus) grown in the open in Sardegna region. At the stage of fruit ripening, infected plants show decline and premature death, a syndrome known as‘collapse’. Fungal pathogens have never been detected. In 1998, melon necrotic spot carmovirus (MNSV) was isolated from diseased plants showing necrotic symptoms on leaves and branches. The virus was identified by biological and serological assays. MNSV may represent a limiting factor to winter melon production in Sardegna where the‘inodorus’group is widely cultivated and appreciated for local consumption. This is the first occurrence of MNSV in Italy.
... In different areas of northern Italy, samples were collected from 48 stocks of cv. Hermes, th... more ... In different areas of northern Italy, samples were collected from 48 stocks of cv. Hermes, the most important processing cultivar in Italy, and from 12 stocks of other potato cultivars (Ernterstolz, Liseta, Monalisa, Primura, Kennebec). ...
Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin-point yell... more Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin-point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme-linked immunosorbent assay to the most important viruses-infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV-infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.
In this manuscript we provide evidence for the seed transmission of Pepino mosaic virus (PepMV) i... more In this manuscript we provide evidence for the seed transmission of Pepino mosaic virus (PepMV) in tomato. Fruit was harvested from a tomato crop artificially infected with both European and CH2 genotypes of PepMV and more than 100,000 seeds were extracted and cleaned using an enzymatic treatment without disinfection. Infection assays using indicator plants confirmed the presence of viable virus on the seeds. Seeds were distributed to ten different laboratories in three separate batches, where they were germinated and the young plants tested by ELISA. In total over 87,000 plants were tested and 23 positives detected, indicating an overall transmission rate of 0.026%. However, the observed seed transmission rates varied from 0.005% to 0.057%, depending on the seed batch used. Results clearly showed that PepMV can be transmitted from seeds contaminated with virus to seedlings, highlighting the risk of using seeds from PepMV-infected plants and the potential for seed transmission to contribute to the further spread of PepMV.
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