In situ hybridization for the localization of two pepino mosaic virus isolates in mixed infections.

In situ hybridization (ISH) is an informative and relatively accessible technique for the localization of viral genomes in plant tissue and cells. However, simultaneous visualization of related plant viruses in mixed infections may be limited by the nucleotide similarity in the genomes and the single chromogenic detection over the same sample preparation. To address this issue, we used two Pepino mosaic virus isolates and performed ISH over consecutive serial cross-sections of paraffin-embedded leaf samples of single and mixed infected Nicotiana benthamiana plants. Moreover, the probe design was optimized to reduce cross-hybridisation, and co-localization was based on the overlapping of consecutive cross-sections from mixed infected leaves; thus, our results showed that both Pepino mosaic virus isolates co-localized in the same leaf tissue. In turn, both isolates were localized in the cytoplasm of the same cells. These results provide valuable information for studying mixed infections in plants by using a simple ISH procedure that is accessible to any pathology laboratory.

Accuracy in Rietveld quantitative phase analysis: a comparative study of strictly monochromatic Mo and Cu radiations.

This study reports 78 Rietveld quantitative phase analyses using Cu Kα1, Mo Kα1 and synchrotron radiations. Synchrotron powder diffraction has been used to validate the most challenging analyses. From the results for three series with increasing contents of an analyte (an inorganic crystalline phase, an organic crystalline phase and a glass), it is inferred that Rietveld analyses from high-energy Mo Kα1 radiation have slightly better accuracies than those obtained from Cu Kα1 radiation. This behaviour has been established from the results of the calibration graphics obtained through the spiking method and also from Kullback-Leibler distance statistic studies. This outcome is explained, in spite of the lower diffraction power for Mo radiation when compared to Cu radiation, as arising because of the larger volume tested with Mo and also because higher energy allows one to record patterns with fewer systematic errors. The limit of detection (LoD) and limit of quantification (LoQ) have also been established for the studied series. For similar recording times, the LoDs in Cu patterns, ∼0.2 wt%, are slightly lower than those derived from Mo patterns, ∼0.3 wt%. The LoQ for a well crystallized inorganic phase using laboratory powder diffraction was established to be close to 0.10 wt% in stable fits with good precision. However, the accuracy of these analyses was poor with relative errors near to 100%. Only contents higher than 1.0 wt% yielded analyses with relative errors lower than 20%.

First Detection of Tomato leaf curl New Delhi virus Infecting Zucchini in Spain.

In September 2012, a novel disease syndrome was observed in zucchini (Cucurbita pepo L.) crops in Murcia Province (southeastern Spain). Symptoms included curling, vein swelling, and severe mosaic in young leaves, short internodes, and fruit skin roughness, resembling begomovirus infection. Similar symptoms were observed in May 2013 in Almería Province (southern Spain). DNA was isolated from 8 and 7 symptomatic leaf samples collected in Murcia and Almería, respectively, and analyzed by PCR with primers GemCP-V-5' and GemCP-C-3' designed to detect begomoviruses by amplifying the core of coat protein gene (CP) (3). DNA fragments of the expected size (~600 bp) were amplified supporting a begomovirus infection. The DNA sequences obtained from four samples were identical. BLAST analysis showed the highest nucleotide identity (98%) with partial CP gene sequences from isolates of Tomato leaf curl New Delhi virus (ToLCNDV) infecting cucumber in India (GenBank Accession No. KC846817). ToLCNDV, a bipartite begomovirus first reported from tomato, also infects other solanaceous and cucurbitaceous crops in India and neighboring countries (1). DNA from two samples from Murcia and three from Almería was used for rolling-circle amplification using ϕ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK) and digested with a set of restriction endonucleases. All five samples yielded amplification products with identical restriction patterns. Two samples from Murcia (MU-8.1 and MU-11.1) and one from Almería (AL-661) were selected to clone the putative DNA-A and DNA-B begomovirus genome components by using single BamHI or NcoI sites. Inserts of two clones from each sample, one corresponding to DNA-A and one to DNA-B, were completely sequenced. The cloned genomes exhibited the typical organization of Old World bipartite begomoviruses (1). Sequences were aligned with begomovirus sequences available in databases using MUSCLE and pairwise identity scores were calculated with SDT (species demarcation tool [4]). DNA-A sequences obtained from Murcia (2,738 nt, KF749224 and KF749225) and Almería (2,738 nt, KF749223) shared >99% nucleotide identity, with the highest nucleotide identity (91.3 to 91.5%) with that of an Indian ToLCNDV isolate from chilli (HM007120). DNA-B sequences (2,684 nt, KF749226, KF749227, and KF749228) shared >99% nucleotide identity, and showed the highest nucleotide identity (83.1 to 83.3%) with that of a Pakistani ToLCNDV isolate from Solanum nigrum (AJ620188). Nucleotide sequence identity of DNA-A with the most closely related begomoviruses was above the 91% threshold for species demarcation (2), thus confirming that the begomoviruses found infecting zucchini in Spain are isolates of ToLCNDV. In fall 2013, the disease was widespread in zucchini both in Murcia and Almería, and ToLCNDV has also been found infecting melon and cucumber crops. To our knowledge, this is the first report of a bipartite begomovirus in Spain and Europe. References: (1) J. K. Brown et al. Page 351 in: Virus Taxonomy. Ninth Report of the ICTV. A. M. Q. King et al., eds. Elsevier/Academic Press, London, 2012. (2) ICTV Geminiviridae Study Group. New species and revised taxonomy proposal for the genus Begomovirus (Geminiviridae). ICTV. Retrieved from http://talk.ictvonline.org/files/proposals/ taxonomy_proposals_plant1/m/plant04/4720.aspx , 10 October 2013. (3) H. Lecoq and C. Desbiez. Adv. Virus Res. 84:67, 2012. (4) B. Muhire et al. Arch. Virol. 158:1411, 2013.

Mixed infections of Pepino mosaic virus strains modulate the evolutionary dynamics of this emergent virus.

Pepino mosaic virus (PepMV) is an emerging pathogen that causes severe economic losses in tomato crops (Solanum lycopersicum L.) in the Northern hemisphere, despite persistent attempts of control. In fact, it is considered one of the most significant viral diseases for tomato production worldwide, and it may constitute a good model for the analysis of virus emergence in crops. We have combined a population genetics approach with an analysis of in planta properties of virus strains to explain an observed epidemiological pattern. Hybridization analysis showed that PepMV populations are composed of isolates of two types (PepMV-CH2 and PepMV-EU) that cocirculate. The CH2 type isolates are predominant; however, EU isolates have not been displaced but persist mainly in mixed infections. Two molecularly cloned isolates belonging to each type have been used to examine the dynamics of in planta single infections and coinfection, revealing that the CH2 type has a higher fitness than the EU type. Coinfections expand the range of susceptible hosts, and coinfected plants remain symptomless several weeks after infection, so a potentially important problem for disease prevention and management. These results provide an explanation of the observed epidemiological pattern in terms of genetic and ecological interactions among the different viral strains. Thus, mixed infections appear to be contributing to shaping the genetic structure and dynamics of PepMV populations.

Recessive resistance to plant viruses.

About half of the approximately 200 known virus resistance genes in plants are recessively inherited, suggesting that this form of resistance is more common for viruses than for other plant pathogens. The use of such genes is therefore a very important tool in breeding programs to control plant diseases caused by pathogenic viruses. Over the last few years, the detailed analysis of many host/virus combinations has substantially advanced basic research on recessive resistance mechanisms in crop species. This type of resistance is preferentially expressed in protoplasts and inoculated leaves, influencing virus multiplication at the single-cell level as well as cell-to-cell movement. Importantly, a growing number of recessive resistance genes have been cloned from crop species, and further analysis has shown them all to encode translation initiation factors of the 4E (eIF4E) and 4G (eIF4G) families. However, not all of the loss-of-susceptibility mutants identified in collections of mutagenized hosts correspond to mutations in eIF4E and eIF4G. This, together with other supporting data, suggests that more extensive characterization of the natural variability of resistance genes may identify new host factors conferring recessive resistance. In this chapter, we discuss the recent work carried out to characterize loss-of-susceptibility and recessive resistance genes in crop and model species. We review actual and probable recessive resistance mechanisms, and bring the chapter to a close by summarizing the current state-of-the-art and offering perspectives on potential future developments.

Nexo causal de neumotórax asociado con acupuntura. A propósito de un caso / Pneumothorax related to acupuncture therapy. A case report of pneumothorax

Se describe el caso de un paciente sometido a tratamiento con acupuntura y electroestimulación que sufrió un cuadro de neumotórax al terminar una de las sesiones. Se trata de un varón de 26 años, diagnosticado de fractura tercio distal del radio derecho tratado quirúrgicamente. En su evolución presentó algodistrofia de la muñeca y la mano derechas, fue sometido a tratamiento con acupuntura y electroestimulación y presentó, al terminar una de las sesiones, un neumotórax derecho. El interés que presenta este caso es la valoración pericial del nexo causal entre el neumotórax y el tratamiento con acupuntura, mediante el análisis de los criterios de causalidad médico-legal aplicables al caso. Aunque los efectos adversos graves debidos a la acupuntura son raros y la literatura médica no proporciona adecuados datos relacionados con la incidencia de tales complicaciones, la mayoría de los autores atribuyen el neumotórax, en general, a un error del terapeuta y no a los riesgos específicos de la acupuntura

A case-report of pneumothorax related to acupuncture therapy and electrostimulation is described. A 26-year-old male had an accident and as a result, a right distal radius fracture. After surgery, he developed algodystropy of the right wrist and hand and underwent treatment with acupuncture and electrostimulation. At the end of one of the sessions, he suffered an immediate right pneumothorax. The interest of this case is the expert evaluation of the causal connection between the pneumothorax and treatment with acupuncture by means of the analysis of the medical-legal causality criteria applicable to the case. Although serious adverse events due to acupuncture therapy are rare, the medical literature does not provide data about the incidence of such complications. The majority of the authors generally attribute the pneumothorax to an error by the therapist and not to the specific risks of the acupuncture

Tomato torrado virus is Transmitted by Bemisia tabaci and Infects Pepper and Eggplant in Addition to Tomato.

Torrao or torrado is an emerging disease that is causing serious economic losses in tomato crops of southeastern Spain. The causal agent has been shown to be a new picorna-like plant virus, tentatively named Tomato torrado virus (ToTV) (4). By using trap tomato plants in a greenhouse affected by torrado located in the Murcia Region of Spain, we obtained a ToTV isolate (ToTV-CE) that we have biologically and molecularly characterized. Subtracted cDNA libraries (1) and expressed sequence tags sequencing were used to determine the partial nucleotide sequence of ToTV-CE. We covered ≈53% of the virus genome (GenBank Accession Nos. EU476181 and EU476182) and found that ToTV-CE RNAs 1 and 2 had a high nucleotide similarity (98 and 99%, respectively) with the ToTV published sequences (2,4). ToTV-CE sequences also showed a 70% nt similarity with those of Tomato apex necrosis virus, a newly identified virus in tomato crops of the Culiacan area (Sinaloa, Mexico) (3). To characterize the host range of ToTV-CE, 6 to 10 plants belonging to 14 species were mechanically inoculated with extracts from ToTV-CE-infected Nicotiana benthamiana plants. The presence of ToTV in these plants was analyzed at 3 and 6 weeks postinoculation (PI) by molecular hybridization in dot-blots. The determined host range was in agreement with that described earlier (2,4), but additional hosts and nonhosts were identified. Thus, the virus did not infect melon (Cucumis melo var. cantaloupe), cucumber (C. sativus cv. Marketmore), squash (Cucurbita pepo cv. Negro Belleza), Chenopodium album ssp. Amaranticolor, or Chenopodium quinoa. The virus infected systemically N. benthamiana, N. glutinosa, N. rustica, tobacco (N. tabacum cvs. Xanthi nc and Samsun), Physalis floridana, pepper (Capsicum annuum cv. Italian Long Sweet), tomato (Solanum lycopersicum cv. Boludo), and eggplant (S. melongena cv. Black Beauty). Pepper plants displayed severe symptoms of infection consisting of marked mosaics and stunting (but no necrosis), but eggplant remained asymptomatic for up to 6 weeks PI. A simple assay was devised to analyze whether ToTV can be transmitted by whiteflies. ToTV-CE-infected tomato plants were placed together with three to eight healthy tomato seedlings inside insect-proof glass boxes. Adult Bemisia tabaci (100 to 800 individuals in three replicates) or Trialeurodes vaporariorum (100 individuals in one replicate) were released into each box. For both treatments, symptoms typically induced by ToTV appeared in one to seven tomato seedlings by 1 week after the release of the whiteflies. ToTV infection was confirmed by molecular hybridization in tissue prints of petiole cross sections at 10 days PI. These data are in agreement with those reported by Pospieszny et al. (2) and strongly suggest that both B. tabaci and T. vaporariorum can transmit ToTV. References: (1) L. Diachenko et al. Proc. Natl. Acad. Sci. USA 93:6025, 1996. (2) H. Pospieszny et al. Plant Dis. 91:1364, 2007 (3) M. Turina et al. Plant Dis. 91:932, 2007. (4) M. Verbeek et al. Arch. Virol. 152:881, 2007.

Low temperature crystal structures of apatite oxygen-conductors containing interstitial oxygen.

Oxygen-stoichiometric La(9.33) square(0.67)(Si(6)O(24))O2 and oxygen-excess La(8.65)Sr(1.35)(Ge(6)O(24))O(2.32) and La(8.65)Sr(1.35)(Si(6)O(24))O(2.32) oxy-apatites have been structurally characterized at low temperatures by the Rietveld method. Oxygen-interstitial distribution has been studied at 15 K for La(9.33) square(0.67)(Si(6)O(24))O2 and La(8.65)Sr(1.35)(Ge(6)O(24))O(2.32) by time-of-flight neutron powder diffraction and at 4 K for La(8.65)Sr(1.35)(Si(6)O(24))O(2.32) by constant-wavelength neutron powder diffraction. The low temperature structural study was undertaken in order to distinguish between the effects of static disorder, originated mainly from the presence of interstitial oxygens, and the anisotropic thermal vibrations. At such low temperatures, the influence of the anisotropic thermal vibrations is minimised. This structural study has firmly established the existence of interstitial oxygens in these materials, which may be useful as electrolytes for solid oxide fuel cells.

Cicatrización epidural en presencia de dos sellantes durales: Duraseal® y Tissucol®. Estudio experimental / Epidural scar formation in presence of two dural sealants: Duraseal® and Tissucol®. An experimental study

Objetivo: Comparar la cicatrización epidural inducida por la presencia de Duraseal®, un material biológico generalmente utilizado como sellante dural, con respecto a la cicatriz epidural en animales control y en presencia de otro sellante dural (Tissucol®). Métodos: Se utilizaron 15 ratas Wistar, sometidas a laminectomía D6-D8. En los grupos de Duraseal® y Tissucol ®, se colocaron estos materiales en espacio epidural. Tres meses después se realizó un estudio histológico de la cicatriz epidural, comparándose el grosor de la cicatriz en los diferentes grupos experimentales. Resultados: No existen diferencias significativas en el grosor de la cicatriz epidural a nivel raquídeo inducida por Duraseal ® respecto al grupo control o al grupo de Tissucol®. Conclusión: Los dos sellantes biológicos utilizados en este estudio pueden ser utilizados en espacio epidural raquídeo, sin que aumenten la fibrosis epidural postquirúrgica

Objective: To study epidural scar induced by the presence of Duraseal® and Tissucol® after laminectomy. Methods: 15 Wistar rats were used to perform a D6-D8 laminectomy with dural exposure. In control groups no sealants were used after laminectomy and in the other two groups Duraseal® and Tissucol® were used, respectivetly. Three months after laminectomy, the width of the scar was measured in each goup, and compared. Results: No statistical differences were found in the width of the epidural scar produced by Duraseal® when compared to controls or Tissucol®. Conclusions: Duraseal® and Tissucol® can be used in space epidural space after a laminectomy, without the epidural scarring increases

Cucurbit aphid-borne yellows virus Is Prevalent in Field-Grown Cucurbit Crops of Southeastern Spain.

Despite the importance of field-grown cucurbits in Spain, only limited information is available about the impact of disease on their production. During the 2003 and 2004 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo) and squash (Cucurbita pepo) crops of Murcia Province (Spain). The fields were chosen with no previous information regarding their sanitation status, and samples were taken from plants showing viruslike symptoms. Samples were analyzed using molecular hybridization to detect Beet pseudo-yellows virus (BPYV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus (ZYMV). We collected 924 samples from 48 field plots. Out of these, almost 90% were infected by at least one of the viruses considered, usually CABYV, which was present in 83 and 66% of the melon and squash samples, respectively. In the case of melon, CYSDV, BPYV, and WMV followed CABYV in relative importance, with frequencies of around 20 to 30%, while in squash, CVYV and BPYY showed frequencies between 28 and 21%. The number of multiple infections was very high, 66 and 56% of the infected samples of melon and squash, respectively, being afflicted. CABYV was present in all multiple infections. The high incidence of CABYV in single and multiple infections suggests that this virus may well become an important threat for cucurbit crops in the region. Restriction fragment length polymorphism (RFLP) analysis revealed that CABYV isolates can be grouped into two genetic types, both of which seemed to be present during the 2003 epidemic episode, but only one of the types was found in 2004.

Genetic diversity of a natural population of Cucurbit yellow stunting disorder virus.

An analysis of nucleotide sequences in five coding and one non-coding genomic regions of 35 Cucurbit yellow stunting disorder virus (CYSDV) isolates collected on a local scale over an 8 year period is reported here. In total, 2277 nt were sequenced for each isolate, representing about 13 % of the complete virus genome. Mean nucleotide diversity for the whole population in synonymous positions in the coding regions was 0.00068, whilst in the 5' untranslated region (5' UTR) of genomic RNA2, it was 0.00074; both of these values are very small, compared with estimates of nucleotide diversity for populations of other plant viruses. Nucleotide diversity was also determined independently for each of the ORFs and for the 5' UTR of RNA2; the data showed that variability is not distributed evenly among the different regions of the viral genome, with the coat protein gene showing more diversity than the other four coding regions that were analysed. However, the low variability found precluded any inference of selection differences among gene regions. On the other hand, no evidence of selection associated with host adaptation was found. In contrast, at least a single amino acid change in the coat protein appears to have been selected with time.

First Report of Cucurbit aphid-borne yellows virus in Spain.

In late spring 2003, field-grown melon plants (Cucumis melo L.) showing bright yellowing of older leaves were observed near Valladolises in Campo de Cartagena, Murcia, Spain. Symptoms resembled those caused by viruses of the genus Crinivirus (family Closteroviridae), but absence or very low populations of whiteflies were observed. However, diseased foci showed clear indications of heavy aphid infestations. Later, during the fall of 2003, squash plants (Cucurbita pepo L.) grown in open fields in the same area showed similar symptoms. Tissue print hybridizations to detect Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo yellows virus (BPYV) in symptomatic samples were negative. CYSDV and BPYV are two yellowing-inducing criniviruses previously described in Spain. In contrast, standard double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with antiserum against Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) that was kindly provided by H. Lecoq (INRA-Montfavet Cedex, France) were consistently positive. Definitive confirmation of CABYV associated with symptomatic samples was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for the CABYV coat protein gene. Total RNA extracts (TRI reagent; Sigma Chemical, St. Louis, MO) were obtained from symptomatic and asymptomatic leaf samples and RT-PCR reactions were carried out using the primers 5'-GAATACGGTCGCGGCTAGAAATC-3' (CE9) and 5'-CTATTTCGGGTTCTGGACCTGGC-3' (CE10) based on the CABYV sequence published by Guilley et al. (2). A single DNA product of approximately 600 bp was obtained only from symptomatic samples. Amplified DNA fragments from two independent samples (samples 36-2 and 37-5) were cloned in E. coli and sequenced (GenBank Accession Nos. AY529653 and AY529654). Sequence comparisons showed a 95% nucleotide sequence identity between the two sequences. A 97% and 94% nucleotide sequence identity was found among 36-2 and 37-5, respectively and the CABYV sequence published by Guilley et al. (2). CABYV seems to be widespread throughout the Mediterranean Basin (1,3) but to our knowledge, it has not previously been described in Spain. Additionally, our data suggest that significant genetic variability might be present in the Spanish CABYV populations. References: (1) Y. Abou-Jawdah et al. Crop Prot. 19:217, 2000. (2) H. Guilley et al. Virology 202:1012, 1994. (3) H. Lecoq et al. Plant Pathol. 41:749, 1992.

Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot virus.

We have studied the biological and molecular characteristics of a MNSV isolate collected in Spain (MNSV-Malpha5) and generated a full-length cDNA clone from which infectious RNA transcripts can be produced. The host range of MNSV-Malpha5 appeared to be limited to cucurbits and did not differ from that of MNSV-Dutch [4, 21]. However, differences were observed in the type of symptoms that both isolates could induce. A full-length cDNA of MNSV-Malpha5 was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) using a 5'-end primer anchoring a T7 RNA promoter sequence and a 3'-end primer, and cloned. Uncapped RNAs transcribed from this cDNA clone were infectious and caused symptoms indistinguishable from those caused by viral RNA when mechanically inoculated onto melon, cucumber or watermelon plants. The complete genome sequence of MNSV-Malpha5 was deduced from the full length cDNA clone. It is 4271 nt long and, similarly to MNSV-Dutch, consists of 5' and 3' untranslated regions (UTRs) and five open reading frames (ORFs) coding for 29, 89, 42 and two small 7 kDa proteins. One notable difference between MNSV-Malpha5 and other sequenced MNSV isolates was found, as for MNSV-Malpha5 the first of the two small ORFs, which are contiguous in the genome, terminates with a genuine stop codon, whereas for MNSV-Dutch and other sequenced MNSV isolates it terminates with an amber codon. This suggested that the putative p14 readthrough protein that could be expressed from the MNSV-Dutch and other MNSV genomes could not be expressed from the MNSV-Malpha5 genome. Also, the nucleotide and amino acid sequences comparisons showed a distant relationship of MNSV-Malpha5 with other known MNSV isolates.

Complete sequence of the Pepino mosaic virus RNA genome.

We have determined the complete nucleotide sequence (Accession No. AF484251) of the Pepino mosaic virus (PepMV) RNA genome. PepMV is the etiological agent of a new disease which affects tomato crops in Europe and North America. The PepMV genome consists of one single stranded positive sense RNA 6410 nt long that contains five open reading frames (ORFs). ORF 1 is the putative RNA dependent RNA polymerase (RdRp), as it has the characteristic methyltransferase, NTP-binding and polymerase motifs. ORF 2 to 4 form the PepMV triple gene block. ORF 5 codes for the capsid protein. Two short untranslated regions flank the coding regions and there is a poly(A) tail at the 3'end of the genomic RNA. Thus, the genome organization of PepMV is that of a typical member of the genus Potexvirus. The nucleotide sequence obtained shares an overall 99% identity with the genomic RNA of a PepMV isolate from UK which has been partially sequenced. Protein coded by ORF4 is the least conserved between both isolates (95% amino acid identity), whereas proteins coded by ORF3 and ORF5 are identical.

High Genetic Stability of the Begomovirus Tomato yellow leaf curl Sardinia virus in Southern Spain Over an 8-Year Period.

ABSTRACT The evolution of the plant single-stranded DNA virus Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae) has been monitored for 8 years after its appearance in southern Spain. Variation within three genomic regions of 166 TYLCSV isolates collected from three locations was assessed by single-strand conformation polymorphism (SSCP) analysis. According to SSCP, the intergenic region (IR) was the most variable. Low genetic diversity was found within the population and geographical or temporal differences were not evident. Nucleotide sequences of specific genomic regions of haplotypes identified by SSCP indicated close relationships among them. Therefore, the Spanish TYLCSV population appears to represent a single, undifferentiated population. The analysis of IR sequences for a subsample of 76 randomly chosen isolates confirmed the limited genetic diversity revealed by the SSCP analysis. A tendency to a lineal increase in diversity over time was observed in Málaga and Almería subpopulations; however, no accumulation of mutations in single isolates was evident. Negative selection to variation seems to operate to conserve certain regions of the genome. Thus, the low genetic diversity found in the studied TYLCSV population might be the result of a founder effect with subsequent selection against less fit variants arising by mutation.

Spanish Melon necrotic spot virus Isolate Overcomes the Resistance Conferred by the Recessive nsv Gene of Melon.

Melon necrotic spot virus (MNSV, genus Carmovirus, family Tombusviridae) is a worldwide natural pathogen that can cause significant economic losses in cucurbit crops (2). Use of commercial cultivars that carry the recessive nsv gene, the only resistance to MNSV described until now (1), is an effective means of controlling MNSV on melon crops. We report here the appearance of MNSV isolates able to overcome nsv resistance. Typical MNSV symptoms were observed in plants with an nsv/nsv genotype during a survey of melon in Almería (southeastern Spain) in 1999. The presence of MNSV in symptomatic plants was confirmed by standard double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using commercial antisera (Loewe Biochemica GmbH, Sauerlach, Germany). MNSV was isolated from these samples by three serial single-lesion inoculation passages in melon (cv. Bola de Oro). The isolate obtained (MNSV-264) was characterized by sequencing a fragment of its RNA genome corresponding to most of the capsid protein (CP) gene. Briefly, total RNA extracts (TRI reagent, Sigma Chemical, St. Louis, MO) of MNSV-264-infected plants were used in a reverse transcription polymerase chain reaction (RT-PCR) in which the oligonucleotide for the reverse transcription was 5'-TTAGGCGAGGTAAGCAGTTTC-3' (MA34), and the oligonucleotides for the PCR were MA34 and 5'-ATGGCGATGGTTAAACGC-3' (MA32). MA32 and MA34 were designed based on an alignment of nucleotide sequences of CP genes of diverse MNSV isolates. A DNA product of approximately 1.1 kbp was obtained, purified, and sequenced (GenBank Accession No. AF488692). Sequence comparisons (GCG Software Package, Madison, WI) of MNSV-264 and MNSV-Dutch (2) showed a 93% nucleotide sequence identity and a 96% similarity of the deduced amino acid sequence. Therefore, both isolates appear to be closely related. MNSV-264 was inoculated on melon accessions known to be susceptible (cvs. Panal and Bola de Oro) or resistant (cvs. Primal F1, Planters Jumbo, and PI161375) to common MNSV strains. Inoculations with MNSV-Dutch were used as a control for this experiment. The results showed that MNSV-264 was able to infect plants of all genotypes, whereas MNSV-Dutch infected only plants of the susceptible genotypes. Therefore, MNSV-264 is an isolate able to overcome the resistance conferred by nsv. The genetic determinant of the MNSV-264 virulence on resistant genotypes is currently under investigation. From 55 MNSV isolates obtained from fields in southeastern Spain during the last 3 years, only one could overcome nsv, like MNSV-264. Therefore, resistance-breaking isolates are not frequent in the MNSV populations in Spain. It is plausible that the situation may change as a consequence of the widespread use of commercial melon cultivars with the nsv genotype. A search for new sources of genetic resistance to MNSV isolates like MNSV-264 is in progress in our laboratory. References: (1) D. L. Coudriet et al. J. Am. Soc. Hortic. Sci. 106:789, 1981. (2) C. J. Riviere et al. J. Gen. Virol. 70:3033, 1989.

Synthesis of silicon-based infrared semiconductors in the Ge-Sn system using molecular chemistry methods.

Growth reactions based on a newly developed deuterium-stabilized Sn hydride [(Ph)SnD(3)] with Ge(2)H(6) produce a new family of Ge-Sn semiconductors with tunable band gaps and potential applications in high-speed, high-efficiency infrared optoelectronics. Metastable diamond-cubic films of Ge(1-x)Sn(x) alloys are created by chemical vapor deposition at 350 degrees C on Si(100). These exhibit unprecedented thermal stability and superior crystallinity despite the 17% lattice mismatch between the constituent materials. The composition, crystal structure, electronic structure, and optical properties of these materials are characterized by Rutherford backscattering, high-resolution electron microscopy, and X-ray diffraction, as well as Raman, IR, and spectroscopic ellipsometry. Electron diffraction reveals monocrystalline and perfectly epitaxial layers with lattice constants intermediate between those of Ge and alpha-Sn. X-ray diffraction in the theta-2theta mode shows well-defined peaks corresponding to random alloys, and in-plane rocking scans of the (004) reflection confirm a tightly aligned spread of the crystal mosaics. RBS ion-channeling including angular scans confirm that Sn occupies substitutional lattice sites and also provide evidence of local ordering of the elements with increasing Sn concentration. The Raman spectra show bands corresponding to Ge-Ge and Sn-Ge vibrations with frequencies consistent with random tetrahedral alloys. Resonance Raman and ellipsometry spectra indicate a band-gap reduction relative to Ge. The IR transmission spectra suggest that the band gap decreases monotonically with increasing Sn fraction. The synthesis, characterization, and gas-phase electron diffraction structure of (Ph)SnD(3) are also reported.