First Ocurrence of a Rhabdovirus Infecting Maize in Argentina.

Maize (Zea mays) plants showing symptoms of shortened internodes, dwarfism, panicle sterility, and a mosaic of coarse and fine yellow stripes on leaf blades and sheaths, were found from December to March in experimental maize plantings in every crop year since 2000-01. Although the disease appeared at a very low incidence (estimates less than 1%), it was found in several locations such as Santa Isabel and Venado Tuerto, Santa Fe Province; Río Cuarto, Colonia Caroya, Río Segundo, and Sampacho, Córdoba Province; and Pedro Luro, Buenos Aires Province. Leaf tissue from eight symptomatic plants collected in Colonia Caroya in December 2011 was used to perform "leaf dips" and ultrathin sections. Electron microscopy of these preparations revealed membrane-bound bullet-shaped particles characteristic of the Rhabdoviridae family in mesophyll cytoplasm and vascular bundle parenchyma. The virus was experimentally transmitted to healthy 9-day-old corn plants, with Peregrinus maidis (order Hemiptera, family Delphacidae) raised under laboratory conditions using acquisition, latency, and inoculation vector periods of 7, 21, and 7 days, respectively. The field observed symptoms were replicated in the transmitted plants. Total RNA was extracted from symptomatic and asymptomatic plants with the RNeasy Plant Mini Kit (Qiagen, Germany), and one step RT-PCR (Access RT-PCR Kit, Promega, Madison, WI) was performed, using two sets of degenerate primers targeting conserved regions of rhabdovirus L polymerase gene, primers PVO (1) and Rhab (2). The agarose gel bands shown only in symptomatic samples were 450 bp (1) and 1,000 bp (2), as expected. The approximately 1 kb amplicon, which includes that of 450 bp, was cloned into pGEM-T Easy Vector System (Promega). Five independent clones were sequenced in both directions with M13 F/R universal primers to generate a consensus sequence (GenBank Accession No. JQ715419), which was compared to similar plant rhabdovirus sequences available on GenBank. The partial L polymerase gene sequence of the corn rhabdovirus, Maize yellow striate virus had 73% and 71% sequence identity with the members of the Cytorhabdovirus genus Barley yellow striate mosaic virus isolate Zanjan-1 (BYSMV; GenBank Accession No. FJ665628) and Northern cereal mosaic virus (NCMV; GenBank Accession No. NC002251), respectively. A phylogenetic tree from the partial nucleotide L polymerase sequence indicates that the rhabdovirus infecting maize in Argentina is closely related to the cytorhabdovirus members and is separated from the nucleorhabdovirus group. To our knowledge, this is the first mention of a Rhabdoviridae family virus infecting maize detected in Argentina. References: (1) H. Bourhy et al. J. Gen. Virol. 86:2849, 2005. (2) R. L. Lamprecht et al. Eur. J. Plant Pathol. 123:105, 2009.

Exitianus obscurinervis (Hemiptera: Cicadellidae), a new experimental vector of Spiroplasma kunkelii.

"Corn stunt" caused by the mollicute Spiroplasma kunkelii (Whitcomb) is potentially one of the most severe diseases affecting the corn (Zea mays L.) crop in the Americas, and the leafhopper Dalbulus maidis (DeLong & Wolcott) is considered its most important vector. However, other insects seen quite frequently in corn crops might well be its vectors in Argentina To identify any leafhoppers species other than D. maidis that can transmit S. kunkelii, transmission assays were conducted, using individuals of Exitianus obscurinervis (Stål) collected in field and reared under controlled conditions. S. kunkelii was transmitted to corn plants by E. obscurinervis. The pathogen was transmitted to seven of the 11 plants, which showed characteristic corn stunt symptoms, and the presence of the pathogen was confirmed by DAS-ELISA. The presence of S. kunkelii in the E. obscurinervis individuals used in transmission experiments was confirmed by polymerase chain reaction and electron microscopy. The current study shows the existence of a new experimental vector of S. kunkelii, the leafhopper E. obscurinervis, which acquired spiroplasmas from infected plants and inoculated it to healthy plants.

First Report of Pear blister canker viroid in Pear Trees in Argentina.

During December 2009 and January 2010, pear trees (Pyrus communis L.) from five orchards located in Allen (Alto Valle) and one in Rio Colorado, both in Rio Negro Province in Argentina, were randomly sampled for Pear blister canker viroid (PBCVd). Ninety-six trees were tested, 20 of cv. Williams, 4 of Abate Fetel, 30 of D'Anjou, and 38 of Packhamn, that showed no symptoms of PBCVd, plus four trees of cv. Red Bartlett that exhibited symptoms of bark pustules and rounded, scaly cankers varying from 2 to 6 cm in diameter on the stems. Purified dsRNA from leaves of 96 trees were analyzed by dot-blot hybridization with a specific probe for PBCVd (2). Of the plants tested, 18 were positive for PBCVd. Three of the positive dsRNAs, with a higher dot-blot signal, were analyzed by electrophoresis in 5% nondenaturing polyacrylamide and a second denaturing polyacrylamide gel. A band, in the portion of viroids migration, was detected with ethidium bromide. The segment corresponding to the three bands was excised and electroeluted. Two-step reverse transcription (RT)-PCR was performed using Moloney-murine leukemia virus (M-MLV) reverse-transcriptase (Promega Corporation, Madison, WI), retrotranscriptase, and PBCVd primers F-5'-GCGGGACAGAAGACGAGGCTCAGGCAGGAAGCAAC-3' and R-5'-TATAAAAGAAAAAAGCGCTTCGGCGGTGCTCGGG-3' (3). The product was legated into pUC 19 vector (Fermentas Inc., Glen Burnie, MD) and cloned following the manufacturer's instructions. Four clones were sequenced by the Unidad de Genómica, Instituto de Biotecnología (Argentina) and the sequences were analyzed with the Lasergene Biocomputing Software for Windows (version 8.0.2; DNASTAR, Madison, WI). The four partial sequences of 296 nucleotides from the local sequences were identical to each other and had the highest nucleotide identity (99.7%) with the Spanish PBCVd (GenBank Accession No. D12823). The local sequence was submitted to GenBank (Accession No. HQ606079). PBCVd is a member of the genus Apscaviroid within the family Pospiviroidae. PBCVd is a 316-nucleotide viroid responsible for pear blister canker disease. It causes pustules, cankers, and/or bark symptoms on the pear indicators A 20 and Fieud 37, whereas infections on most commercial pear cultivars remain symptomless (1). These lesions are entry points for other pathogens to infect the plant. This research indicates the need to test pear propagation material in Argentina, since this is the primary way of spreading this pathogen. To our knowledge, this is the first report of PBCVd in Argentina. References: (1) J. Desvignes et al. Plant Dis. 83:419, 1999. (2) R. Flores et al. J. Gen. Virol. 72:1199, 1991. (3) C. Hernandez et al. J. Gen. Virol. 73:2503, 1992.

First Report of a Rhabdovirus Infecting Alfalfa in Argentina.

Alfalfa (Medicago sativa L.) is a major forage crop in Argentina with an estimated cultivated area of 4 million ha in the 2009-2010 season, which constitutes a primary component for the animal production chain. In early summer of 2010, alfalfa plants showing virus-like symptoms were identified in 20 commercial fields in La Pampa Province with 95% disease prevalence. Diseased plants had shortened internodes, a bushy appearance, deformations, puckering, epinasty of leaflet blades, vein enations, and varying sized papillae on the adaxial leaflet surfaces. Similar symptoms were observed in alfalfa crops in Buenos Aires, Cordoba, Santa Fe, and Santiago del Estero provinces. Electron microscopy (EM) and molecular assays were performed on leaf tissue from one asymptomatic and two symptomatic plants. EM of ultrathin sections revealed membrane-bound bullet-shaped particles associated with the endoplasmic reticulum of phloem cells from symptomatic plants only. Total RNA was extracted from symptomatic and asymptomatic plants with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) for a template in one-step reverse transcription (RT)-PCR assays with the Access RT-PCR Kit (Promega, Madison, WI). RT-PCR assays employed degenerate primers targeting conserved regions of plant rhabdovirus polymerase (L) genes (2). An amplicon of approximately 1 kilobase pairs (detected only from symptomatic plants) was gel purified with the Wizard SV Gel and PCR Clean-Up System (Promega), cloned into pGEM-T Easy Vector System (Promega), and sequenced. Three independents clones were sequenced in both directions at Macrogen Inc. (Korea Republic) to generate a consensus sequence (GenBank Accession No. HQ380230) and compared to sequences of other plant rhabdoviruses available on GenBank. The partial L gene sequence of the alfalfa-infecting rhabdovirus shared highest nucleotide (68.0%) and amino acid (76.5%) sequence identity with the cytorhabdovirus Strawberry crinkle virus (Accession No. AY331390). A phylogenetic tree based on partial amino acid sequences of the polymerase gene indicated the alfalfa-infecting virus was more closely related to cytorhabdoviruses than to nucleorhabdoviruses. Symptoms observed resembled those reported for alfalfa plants infected with a plant rhabdovirus named Alfalfa enation virus (1), which has never been fully characterized. Collectively, the data implicate the observed rhabdovirus as the etiological agent. To our knowledge, this is the first report in Argentina (and South America) of a rhabdovirus infecting alfalfa. Additional field surveys and monitoring of vector/s and yield losses need to be conducted. References: (1) B. Alliot and P. A. Signoret. Phytopathol. Z. 74:69, 1972. (2) R. L. Lamprecht et al. Eur. J. Plant Pathol. 123:105, 2009.

First Report of Peach latent mosaic viroid in Peach Trees in Argentina.

Peach trees (Prunus persica L.) with yellowing symptoms on leaves were found in December (summer season) of 2005 in commercial fields in Colonia Caroya, Córdoba, Argentina. This symptom resembled Peach latent mosaic viroid (PLMVd). The viroid-affected trees usually showed vein banding and yellowing symptoms on leaves, fruit suture splitting, delays in budding, flowering and fruit ripening, reduced foliage density, and a spindly appearance. PLMVd is mainly transmitted by the propagation of infected budwood and to a lesser extent by pruning tools and aphids (1). Extraction and partial purification of nucleic acids was made from symptomatic leaves (2) collected during the indicated summer season from seven peach trees from commercial plots. Preliminary identification of the viroid was made by dot-blot hybridization with a PLMVd probe (provided by R. Flores). The positive plants were tested also by reverse transcription (RT)-PCR with PLMVd primers RF-43 (5'-CTGGATCACACCCCCCTCGGAACCAACCGCT-3') and RF-44 (5'-TGTGATCCAGGTACCGCCGTAGAAACT-3'), which amplify the complete genome. All identified positive samples yielded the correct sized PCR product and four were cloned and sequenced. This local sequence (GenBank Accession No. EU429320) had 97% nucleotide sequence identity with a reported PLMVd sequence (GenBank Accession No. M83545), confirming the identity of the local pathogen as Peach latent mosaic viroid. This result suggests that the source of the pathogen may have been infected budwood introduced from Europe. To our knowledge, this is the first report of this viroid in Argentina. References: (1) R. Flores et al. Mol. Plant Pathol. 7:209, 2006. (2) V. Pallás et al. J. Gen. Virol. 68:3201, 1987.

[Hypospadias repair with Snodgrass' technique]. / Reparación de hipospadias mediante la Técnica de Snodgrass.

Retrospective study of 124 patients (average age: 3.8 years) with midpenile hypospadias: 48.3% (60 children), distal penile: 45.9% (57) and coronal 5.6% (7), of which the 25.8% (16) presented ventral curvature and the 4.8% (6) resulting from the complication of another previous technique. All of them were operated according to Snodgrass' technique, removing the catheter between the 6th and 7th day in most of them. The global rate of complications was of 12%: 9 fistulae (7.2%) and 6 meatal stenosis (4.8%). Aesthetic result was satisfactory in all cases, getting glans covered by foreskin in 57.3%.

Reparación de hipospadias mediante la Técnica de Snodgrass / Hypospadias repair with Snodgrass´Technique

Estudio retrospectivo de 124 pacientes (edad media: 3,8 años) con hipospadias peneano medio: 48,3% (60 niños), peneano distal: 45,9% (57) y coronal 5,6% (7), de los cuales el 25,8% (16) presentaban incurvación ventral y el 4,8% (6) procedían del fracaso de otra técnica. Todos ellos intervenidos según técnica de Snodgrass, retirando la sonda entre el 6 y 7º día en la mayoría. La tasa global de complicaciones fue del 12%: 9 fístulas (7,2%) y 6 estenosis de meato (4,8%). El resultado estético fue satisfactorio en todos los casos, quedando incluso el glande cubierto por prepucio el 57,3%

Retrospective study of 124 patients (average age: 3.8 years) with midpenile hypospadias: 48,3% (60 children), distal penile: 45,9% (57) and coronal 5,6% (7), of which the 25.8% (16) presented ventral curvature and the 4,8% (6) resulting from the complication of another previous technique. All of them were operated according to Snodgrass´ technique, removing the catheter between the 6th and 7th day in most of them. The global rate of complications was of 12%: 9 fistulae (7,2%) and 6 meatal stenosis (4,8%). Aesthetic result was satisfactory in all cases, getting glans covered by foreskin in 57,3%

Localization of sweet potato chlorotic stunt virus (SPCSV) in synergic infection with potyviruses in sweet potato

Among diseases reported worldwidely for sweet potato (Ipomoea batatas (L) Lam) crop, one of the most frequent is the Sweet potato virus disease (SPVD), caused by sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) co-infection. In Argentina, there exists the sweet potato chlorotic dwarf (SPCD), a sweet potato disease caused by triple co-infection with SPCSV, SPFMV and sweet potato mild speckling virus (SPMSV). Both diseases cause a synergism between the potyviruses (SPFMV and SPMSV) and the crinivirus (SPCSV). Up to date, studies carried out on the interaction among these three viruses have not described their localization in the infected tissues. In single infections, virions of the crinivirus genus are limited to the phloem while potyviral virions are found in most tissues of the infected plant. The purpose of this work was to localize the heat shock protein 70 homolog (HSP70h), a movement protein for genus crinivirus, of an Argentinean SPCSV isolate in its single infection and in its double and triple co-infection with SPFMV and SPMSV. The localization was made by in situ hybridization (ISH) for electron microscopy (EM) on ultrathin sections of sweet potato cv. Morada INTA infected tissues. The results demonstrated that viral RNA coding HSP70h is restricted to phloem cells during crinivirus single infection, while it was detected outside the phloem in infections combined with the potyviruses involved in chlorotic dwarf disease.

Localization of sweet potato chlorotic stunt virus (SPCSV) in synergic infection with potyviruses in sweet potato

Among diseases reported worldwidely for sweet potato (Ipomoea batatas (L) Lam) crop, one of the most frequent is the Sweet potato virus disease (SPVD), caused by sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) co-infection. In Argentina, there exists the sweet potato chlorotic dwarf (SPCD), a sweet potato disease caused by triple co-infection with SPCSV, SPFMV and sweet potato mild speckling virus (SPMSV). Both diseases cause a synergism between the potyviruses (SPFMV and SPMSV) and the crinivirus (SPCSV). Up to date, studies carried out on the interaction among these three viruses have not described their localization in the infected tissues. In single infections, virions of the crinivirus genus are limited to the phloem while potyviral virions are found in most tissues of the infected plant. The purpose of this work was to localize the heat shock protein 70 homolog (HSP70h), a movement protein for genus crinivirus, of an Argentinean SPCSV isolate in its single infection and in its double and triple co-infection with SPFMV and SPMSV. The localization was made by in situ hybridization (ISH) for electron microscopy (EM) on ultrathin sections of sweet potato cv. Morada INTA infected tissues. The results demonstrated that viral RNA coding HSP70h is restricted to phloem cells during crinivirus single infection, while it was detected outside the phloem in infections combined with the potyviruses involved in chlorotic dwarf disease.(AU)

Obstructive uropathy in a HIV+ infant under indinavir treatment.

The case is presented of a 10-year-old HIV+ male with renoureteral pain, who developed an obstructive uropathy with renal function impairment and required endoscopic placement of a ureteral stent. Certain aspects of the epidemiology, clinical presentation, diagnosis, treatment and prevention are discussed.

Occurrence of Maize rayado fino virus in Maize in Argentina.

Symptoms of fine chlorotic stipple-striping of the veins, chlorosis, numerous dots and stripes, formation of holes in the leaf blade, and ears reduced in size, bearing few grains, were observed in maize crops in Tafí del Valle (Tucumán Province), Orán, El Galpón (Salta Province), Tilcara and Yaví (Jujuy Province), the subtropical area of northwest Argentina where the leafhopper vector Dalbulus maidis (DeLong & Wolcott) is present. Maize rayado fino virus (MRFV) was detected in these samples by a positive reaction in double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) using an AGDIA kit. Electron microscopy revealed abundant isometric particles about 30 nm in diameter in the cytoplasm and vacuoles of phloem cells and xylem parenchyma cells. The virus was also detected by reverse transcription polymerase chain reaction (RT-PCR) using a primer pair MRFV-09/MRFV-10. Primers and PCR conditions were as previously described (1). Virus amplification was observed only in samples from symptomatic plants. In 1981 (2), the presence of MRFV in Argentina was revealed by serological assay in plants from temperate central areas. No further reports were released since then. This is the first evidence of MRFV in subtropical areas of Argentina and identification of the virus by combining DAS-ELISA, particle size, relation with plant tissues, and RTPCR. References: (1) R. W. Hammond et al. J. Gen. Virol. 78:3153, 1997. (2) S. F. Nome et al. RIA XIX:257, 1984.