First report of Tomato blistering mosaic virus infecting tomato in Argentina

Tomato (Solanum lycopersicum) ranks as the most important crop among greenhouse-planted crops in terms of cultivated areas (17,800 ha) and production in Argentina. In September 2014, farmers reported that tomato plants of cv. Elpida (Enza Zaden) showed mosaic and blistering on leaves in La Plata (Buenos Aires green-belt). The diseased plants occurred together in the same line, showing a typical pattern of mechanically transmitted pathogens. Although incidence did not exceed 1.5%, it was distributed in several distant locations within the district, posting a serious threat to the tomato production. Five samples were collected from symptomatic plants and tested for Tospovirus, Begomovirus, and Cucumber mosaic virus by reverse transcription polymerase chain reaction (RT-PCR) (Dewey et al. 1996), PCR (Rojas et al. 1993), and enzyme-linked immunosorbent assay (ELISA), respectively. All results were negative. The samples were then examined under transmission electron microscopy. Isometric virus-like particles of 25 to 30 nm in diameter were seen in leaf-dip preparations, and isometric virus-like particles aggregated in the cytoplasm of mesophyll cells and alterations of chloroplast membrane in association to virus-like particles were observed in ultrathin sections suggesting a Tymovirus. To identify this virus, leaf and stem samples were dot blotted on a nitrocellulose membrane and incubated with Tomato blistering mosaic virus (ToBMV) antiserum, kindly provided by Dr. Alice K. Inoue-Nagata (Embrapa-Horticulture, Brazil). The samples reacted to the ToBMV antiserum, indicating the presence of ToBMV or a ToBMV-related virus. Sap of the diseased samples was used to inoculate tomato cv. Elpida and several indicator plants (Datura stramonium, Nicotiana benthamiana, Nicotiana tabacum cv. TNN, Capsicum annuum cv. Almuden, and Chenopodium quinoa). The inoculated tomato plants showed foliar chlorosis, mosaic, necrosis, and blistering similar to those observed in the greenhouses, and the indicator plants reproduced the symptoms reported by de Oliveira et al. (2013). Furthermore, two samples were subjected to RT-PCR using primers specifically designed to amplify the complete capsid protein (CP) from ToBMV: TymoF3RaceFor, 5′-TCCAATCCATGCCTTTTGAT-3′; and ToBMV3EndNdeIRev, 5′-TTCATATGGTTTCCGATACCCTCGG-3′. DNA fragments of expected size (1000 bp) were amplified, cloned in pGEM-T easy (Promega), and sequenced. Sequence analysis showed the nucleotide sequences of the two isolates were identical, suggesting that they are the same variant (Accession No. KT235894). This isolate shares 92% identity with tomato isolate SC50 (Nicolini et al. 2015) and tobacco isolate BR001 (Melo et al. 2014), both from Brazil. These results indicate that the tomato disease in Buenos Aires green-belt is caused by ToBMV. To our knowledge, this is the first report of ToBMV infecting tomato in Argentina. This finding has an important epidemiological impact because ToBMV represents a new problem to tomato crops in this country.Centro de Investigaciones en FitopatologíaInstituto de Biotecnologia y Biologia Molecula

First report of Tomato blistering mosaic virus infecting tomato in Argentina

Tomato (Solanum lycopersicum) ranks as the most important crop among greenhouse-planted crops in terms of cultivated areas (17,800 ha) and production in Argentina. In September 2014, farmers reported that tomato plants of cv. Elpida (Enza Zaden) showed mosaic and blistering on leaves in La Plata (Buenos Aires green-belt). The diseased plants occurred together in the same line, showing a typical pattern of mechanically transmitted pathogens. Although incidence did not exceed 1.5%, it was distributed in several distant locations within the district, posting a serious threat to the tomato production. Five samples were collected from symptomatic plants and tested for Tospovirus, Begomovirus, and Cucumber mosaic virus by reverse transcription polymerase chain reaction (RT-PCR) (Dewey et al. 1996), PCR (Rojas et al. 1993), and enzyme-linked immunosorbent assay (ELISA), respectively. All results were negative. The samples were then examined under transmission electron microscopy. Isometric virus-like particles of 25 to 30 nm in diameter were seen in leaf-dip preparations, and isometric virus-like particles aggregated in the cytoplasm of mesophyll cells and alterations of chloroplast membrane in association to virus-like particles were observed in ultrathin sections suggesting a Tymovirus. To identify this virus, leaf and stem samples were dot blotted on a nitrocellulose membrane and incubated with Tomato blistering mosaic virus (ToBMV) antiserum, kindly provided by Dr. Alice K. Inoue-Nagata (Embrapa-Horticulture, Brazil). The samples reacted to the ToBMV antiserum, indicating the presence of ToBMV or a ToBMV-related virus. Sap of the diseased samples was used to inoculate tomato cv. Elpida and several indicator plants (Datura stramonium, Nicotiana benthamiana, Nicotiana tabacum cv. TNN, Capsicum annuum cv. Almuden, and Chenopodium quinoa). The inoculated tomato plants showed foliar chlorosis, mosaic, necrosis, and blistering similar to those observed in the greenhouses, and the indicator plants reproduced the symptoms reported by de Oliveira et al. (2013). Furthermore, two samples were subjected to RT-PCR using primers specifically designed to amplify the complete capsid protein (CP) from ToBMV: TymoF3RaceFor, 5′-TCCAATCCATGCCTTTTGAT-3′; and ToBMV3EndNdeIRev, 5′-TTCATATGGTTTCCGATACCCTCGG-3′. DNA fragments of expected size (1000 bp) were amplified, cloned in pGEM-T easy (Promega), and sequenced. Sequence analysis showed the nucleotide sequences of the two isolates were identical, suggesting that they are the same variant (Accession No. KT235894). This isolate shares 92% identity with tomato isolate SC50 (Nicolini et al. 2015) and tobacco isolate BR001 (Melo et al. 2014), both from Brazil. These results indicate that the tomato disease in Buenos Aires green-belt is caused by ToBMV. To our knowledge, this is the first report of ToBMV infecting tomato in Argentina. This finding has an important epidemiological impact because ToBMV represents a new problem to tomato crops in this country.Centro de Investigaciones en FitopatologíaInstituto de Biotecnologia y Biologia Molecula

Interacción de <i>Pseudomonas corrugata</i> y <i>Pseudomonas viridiflava</i> y diferentes genotipos de tomate

En este trabajo, se evalu&oacute; el efecto de la cepa bacteriana, el cultivar del hospedante y la interacci&oacute;n de ambos factores sobre la expresi&oacute;n de los s&iacute;ntomas de la enfermedad conocida como necrosis medular del tomate, ocasionada por distintas especies del g&eacute;nero Pseudomonas. Se trabaj&oacute; con 9 cepas de Pseudomonas corrugata provenientes de Argentina, 7 cepas de referencia de P. corrugata y 3 de Pseudomonas mediterranea de distintas colecciones internacionales y 4 cepas de Pseudomonas viridiflava provenientes de Argentina. Las 21 cepas fueron inoculadas artificialmente sobre plantas de tomate de los cultivares FA 144 , 110 , Presto , Iv&aacute;n, Ringo y Platense L&iacute;nea 7 . Cuarenta y cinco d&iacute;as despu&eacute;s de la inoculaci&oacute;n se diagnosticaron los s&iacute;ntomas y se cuantific&oacute; la longitud de la necrosis medular. Tanto la cepa bacteriana como el cultivar de tomate y la interacci&oacute;n entre ambos factores tuvieron efecto significativo sobre la severidad de la enfermedad. Las cepas europeas de P. corrugata determinaron sobre Ringo y Platense L&iacute;nea 7 lesiones significativamente m&aacute;s severas que todas las cepas argentinas de P. corrugata y P. viridiflava. Dentro de los cepas argentinas, a su vez, destacan por su virulencia Pc4, Pc5 y Pc16 que resultaron los m&aacute;s patog&eacute;nicos para FA 144 , 110 y L&iacute;nea 7 . Los resultados obtenidos sugieren la existencia de una alta variabilidad intra-especie tanto en las poblaciones de P. corrugata como en las de P. viridiflava que resulta necesario conocer a fin de que la elecci&oacute;n del cultivar sea una herramienta &uacute;til a los fines de reducir el impacto econ&oacute;mico de esta enfermedad.The severity of symptoms of tomato pith necrosis caused by different species of Pseudomonas was evaluated taking into account the effect of bacterial strains, tomato cultivars and the interaction between strains and cultivars. Nine Pseudomonas corrugata strains from Argentina; 7 reference strains of Pseudomonas corrugata and 3 reference strains of Pseudomonas mediterranea from International Collections; and four Pseudomonas viridiflava strains from Argentina were artificially inoculated on tomato cultivars FA 144 , 110 , Presto , Iv&aacute;n, Ringo y Platense L&iacute;nea 7 . Forty-five days after inoculation, symptoms were diagnosed and stem pith necrosis was quantified. Bacterial strain, tomato cultivar and the interaction among them had significant effects on disease severity. European strains of P. corrugata induced on Ringo y Platense L&iacute;nea 7 lesions significantly more severe than Argentinean strains of both P. corrugata and P. viridiflava. Among Argentinean strains, Pc4, Pc5, and Pc16 were the most virulent for FA 144 , 110 y L&iacute;nea 7 . Results reported here suggest the existence of high intra-species variability in both P. corrugata and P. viridiflava populations that is necessary take into account before the selection of a tomato cultivar as a tool to reduce the economical impact of the disease

Los virus en especies forestales

p.93-9

First report of a resistance-breaking isolate of Tomato spotted wilt virus infecting sweet pepper harboring the Tsw gene in Argentina

The disease caused by Tomato spotted wilt virus (TSWV) is endemic on vegetable crops in the Buenos Aires green belt, the most important vegetable-production area in Argentina (18,000 ha). Approximately half of the greenhouse surface planted with sweet pepper (Capsicum annuum L.) in the country is located in La Plata (Buenos Aires). In this area, TSWV had limited sweet pepper production until the introduction of resistant genotypes harboring the Tsw resistance gene that replaced 100% of the susceptible cultivars. However, in November 2013, resistant peppers showing typical Tospovirus symptoms were observed in La Plata. Symptoms appeared 20 days after transplanting in early spring, and by midsummer 100% of the plants were affected in many greenhouses, causing important economic losses in this season. Samples from symptomatic plants were analyzed by ELISA with antisera against the viruses: TSWV, Groundnut ringspot virus (GRSV), Tomato chlorotic spot virus (TCSV), Potato virus Y (PVY), Cucumber mosaic virus (CMV), and Tobacco mosaic virus (TMV). All samples were positive only for TSWV. Presence of TSWV was confirmed by RT-PCR with primers designed from a conserved sequence of the N gene that amplified a fragment of about 450 bp. Further, RFLP using BstNI and HinCII enzymes that cut the 450-bp fragment specifically (Dewey et al. 1996) showed the typical restriction pattern of TSWV. To test the ability to overcome the resistance, this greenhouse-isolate, named TSWV-A2, was mechanically transmitted to two commercial pepper cultivars carrying the Tsw gene, cvs. Almuden and Platero (10 plants each). After two weeks, all plants were systemically infected, showing the typical symptoms of TSWV infection. RT-PCR (as previously described) from total RNA extracted from symptomatic leaves of these plants confirmed the presence of TSWV-A2. Therefore, we demonstrated that TSWV-A2 is able to overcome Tsw gene resistance. Furthermore, to amplify the complete N gene of TSWV-A2 isolate, a new RT-PCR was carried out (Lovato et al. 2008). A specific 800-bp product was cloned and sequenced (GenBank Accession No. KP719131). BLAST analysis showed that the sequence was 99% homologous at the nucleotide as well as the amino acid sequence to the N gene of isolates EF195230 and EF195224 from South Korea (Kim et al. 2004). In addition, TSWV-A2 shared common characteristics with the resistant-breaking isolates infecting sweet pepper cultivars carrying the Tsw gene, previously reported in Italy (Roggero et al. 2002) and Spain (Margaria et al. 2004). Overall, based on biological, serological, and molecular features, this is the first report of a local isolate of TSWV breaking the Tsw-resistance in sweet pepper in Argentina.Facultad de Ciencias Agrarias y Forestale

First report of Tomato blistering mosaic virus infecting tomato in Argentina

Tomato (Solanum lycopersicum) ranks as the most important crop among greenhouse-planted crops in terms of cultivated areas (17,800 ha) and production in Argentina. In September 2014, farmers reported that tomato plants of cv. Elpida (Enza Zaden) showed mosaic and blistering on leaves in La Plata (Buenos Aires green-belt). The diseased plants occurred together in the same line, showing a typical pattern of mechanically transmitted pathogens. Although incidence did not exceed 1.5%, it was distributed in several distant locations within the district, posting a serious threat to the tomato production. Five samples were collected from symptomatic plants and tested for Tospovirus, Begomovirus, and Cucumber mosaic virus by reverse transcription polymerase chain reaction (RT-PCR) (Dewey et al. 1996), PCR (Rojas et al. 1993), and enzyme-linked immunosorbent assay (ELISA), respectively. All results were negative. The samples were then examined under transmission electron microscopy. Isometric virus-like particles of 25 to 30 nm in diameter were seen in leaf-dip preparations, and isometric virus-like particles aggregated in the cytoplasm of mesophyll cells and alterations of chloroplast membrane in association to virus-like particles were observed in ultrathin sections suggesting a Tymovirus. To identify this virus, leaf and stem samples were dot blotted on a nitrocellulose membrane and incubated with Tomato blistering mosaic virus (ToBMV) antiserum, kindly provided by Dr. Alice K. Inoue-Nagata (Embrapa-Horticulture, Brazil). The samples reacted to the ToBMV antiserum, indicating the presence of ToBMV or a ToBMV-related virus. Sap of the diseased samples was used to inoculate tomato cv. Elpida and several indicator plants (Datura stramonium, Nicotiana benthamiana, Nicotiana tabacum cv. TNN, Capsicum annuum cv. Almuden, and Chenopodium quinoa). The inoculated tomato plants showed foliar chlorosis, mosaic, necrosis, and blistering similar to those observed in the greenhouses, and the indicator plants reproduced the symptoms reported by de Oliveira et al. (2013). Furthermore, two samples were subjected to RT-PCR using primers specifically designed to amplify the complete capsid protein (CP) from ToBMV: TymoF3RaceFor, 5′-TCCAATCCATGCCTTTTGAT-3′; and ToBMV3EndNdeIRev, 5′-TTCATATGGTTTCCGATACCCTCGG-3′. DNA fragments of expected size (1000 bp) were amplified, cloned in pGEM-T easy (Promega), and sequenced. Sequence analysis showed the nucleotide sequences of the two isolates were identical, suggesting that they are the same variant (Accession No. KT235894). This isolate shares 92% identity with tomato isolate SC50 (Nicolini et al. 2015) and tobacco isolate BR001 (Melo et al. 2014), both from Brazil. These results indicate that the tomato disease in Buenos Aires green-belt is caused by ToBMV. To our knowledge, this is the first report of ToBMV infecting tomato in Argentina. This finding has an important epidemiological impact because ToBMV represents a new problem to tomato crops in this country.Centro de Investigaciones en FitopatologíaInstituto de Biotecnologia y Biologia Molecula

Pseudomonas populations causing pith necrosis of tomato and pepper in Argentina are highly diverse

Pseudomonas species causing pith necrosis symptoms on tomato and pepper collected in different areas of Argentina were identified as Pseudomonas corrugata, P. viridiflava and Pseudomonas spp. Their diversity was analysed and compared with reference strains on the basis of their phenotypic characteristics, copper and antibiotic sensitivity tests, serology, pathogenicity, DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis of a 16S rRNA gene fragment. All P. corrugata strains tested were copper-resistant while P. viridiflava strains were more variable. Numerical analysis of phenotypic data showed that all P. corrugata strains formed a single phenon that clustered at a level of about 93%, while all the P. viridiflava strains clustered in a separated phenon at a level of 94%. Genomic analysis by repetitive (rep)-PCR and 16S rRNA-RFLP fingerprinting and serological analysis showed that the two species contained considerable genetic diversity. Inoculations of tomato and pepper plants with strains from both hosts caused similar pith necrosis symptoms. Strains of both P. corrugata and P. viridiflava were grouped according to their geographical origin and not according to the original host. This is the first report of Pseudomonas viridiflava causing pith necrosis on pepper.Facultad de Ciencias Agrarias y ForestalesInstituto de Biotecnologia y Biologia Molecula

Pseudomonas populations causing pith necrosis of tomato and pepper in Argentina are highly diverse

Pseudomonas species causing pith necrosis symptoms on tomato and pepper collected in different areas of Argentina were identified as Pseudomonas corrugata, P. viridiflava and Pseudomonas spp. Their diversity was analysed and compared with reference strains on the basis of their phenotypic characteristics, copper and antibiotic sensitivity tests, serology, pathogenicity, DNA fingerprinting and restriction fragment length polymorphism (RFLP) analysis of a 16S rRNA gene fragment. All P. corrugata strains tested were copper-resistant while P. viridiflava strains were more variable. Numerical analysis of phenotypic data showed that all P. corrugata strains formed a single phenon that clustered at a level of about 93%, while all the P. viridiflava strains clustered in a separated phenon at a level of 94%. Genomic analysis by repetitive (rep)-PCR and 16S rRNA-RFLP fingerprinting and serological analysis showed that the two species contained considerable genetic diversity. Inoculations of tomato and pepper plants with strains from both hosts caused similar pith necrosis symptoms. Strains of both P. corrugata and P. viridiflava were grouped according to their geographical origin and not according to the original host. This is the first report of Pseudomonas viridiflava causing pith necrosis on pepper.Facultad de Ciencias Agrarias y ForestalesInstituto de Biotecnologia y Biologia Molecula

Identification of Mirafiori lettuce big-vein virus and Lettuce big-vein associated virus infecting Lactuca sativa with symptoms of lettuce big-vein disease in Argentina

Lettuce big-vein disease (BVD) affects all major lettuce-producing areas of the world. The causal agent is Mirafiori lettuce big-vein virus (MLBVV), an ophiovirus transmitted by the soil-borne fungus Olpidium brassicae (Lot et al., 2002). MLBVV has been detected in many different areas of the world but never in Argentina. La Plata has about 700 ha of lettuce with a production of about 13 000 tonnes, and with about 70% of the total production from Buenos Aires Province. BVD has been detected in different areas in the north and west of the La Plata horticultural green belt. Many of the plants with BVD symptoms had leaf distortions of moderate severity, which affected their commercial value.Facultad de Ciencias ExactasFacultad de Ciencias Agrarias y Forestale

Identification of Mirafiori lettuce big-vein virus and Lettuce big-vein associated virus infecting Lactuca sativa with symptoms of lettuce big-vein disease in Argentina

Lettuce big-vein disease (BVD) affects all major lettuce-producing areas of the world. The causal agent is Mirafiori lettuce big-vein virus (MLBVV), an ophiovirus transmitted by the soil-borne fungus Olpidium brassicae (Lot et al., 2002). MLBVV has been detected in many different areas of the world but never in Argentina. La Plata has about 700 ha of lettuce with a production of about 13 000 tonnes, and with about 70% of the total production from Buenos Aires Province. BVD has been detected in different areas in the north and west of the La Plata horticultural green belt. Many of the plants with BVD symptoms had leaf distortions of moderate severity, which affected their commercial value.Facultad de Ciencias ExactasFacultad de Ciencias Agrarias y Forestale