The aim of this research was the genetic characterization of 218 accessions of Cucurbita moschata Duchesne, a squash, and its relationship with morphological characteristics of agronomic interest, which are part of the international collection conserved at Tropical Agricultural Research and Higher Education Center (CATIE), Costa Rica. The majority of the accessions came from Mexico and Central America; single genotypes from Curaao, Colombia, Peru and the Russian Federation were also included. The polymerase chain reaction (PCR) and single strand conformation polymorphism analysis (SSCP) were used for the analysis of the regions amplified with ITS1-ITS2 nuclear primers and tRNL-F chloroplast primers. Haplotypes were constructed according to band patterns in SSCP gels. Twenty-five haplotypes were found using the ITS1-ITS2 markers, and 24 haplotypes were found with the tRNL-F markers. Unique haplotypes were found with both markers. Two individuals of each tRNL-F haplotype were sequenced. The results indicated a high level of genetic diversity in CATIE squash collection. Using ITS1-ITS2 primers, it was found that the number of haplotypes was independent of the geographical source of the accession, and haplotypes were distributed randomly throughout the study area. Mexico had the highest values of total heterozygosity (HE), genetic diversity (H) and Shannon index (I) while Panama showed the lowest values. Sequences obtained from tRNL-F intergenic marker showed the highest diversity index values were present in the group of additional sequences and Mexico, and lower values were observed for Nicaragua, Guatemala and Panama. PCoA based on morphological data showed three groups and by ANOSIM (R) all group differences were significant. Results obtained in this study suggest that high diversity is a characteristic of C. moschata from Mesoamerica.

Tomato chlorosis virus (ToCV) is an emerging whitefly-transmitted crinivirus (2). In Costa Rica in 2007, ToCV was detected in field-grown and greenhouse tomato (Solanum lycopersicum L.) plants causing symptoms of severe yellowing and foliar chlorosis (1). To identify alternative hosts that may serve as virus reservoirs, 78 samples were collected from multiple species of common weeds growing adjacent to tomato nurseries in the Cartago Province, where ToCV was previously identified, during the autumn of 2008 and summer of 2009. The weeds were collected on the basis of the presence of whiteflies and/or symptoms of interveinal chlorosis, but not all samples were symptomatic for infection by ToCV. Total RNA was extracted from leaf tissue with TRI Reagent (Molecular Research Inc., Cincinnati, OH). Reverse transcription (RT)-PCR reactions were performed with the Qtaq One-Step qRT-PCR SYBR Kit (Clontech Laboratories, Mountain View, CA) and primers specific for the ToCV HSP70h gene (3). A 123-bp DNA fragment was amplified in five weeds, which were identified taxonomically as Ruta chalepensis (Rutaceae), Phytolacca icosandra (Phytolacaceae), Plantago major (Plantaginaceae), a Brassica sp. (Brassicaceae) (two samples), and a single plant of Cucurbita moschata (Cucurbitaceae) growing next to those weeds. The amplified DNA fragments were sequenced and BLAST analysis showed 100% nucleotide sequence identity with the HSP70h gene of the Florida ToCV isolate (GenBank Accession No. AY903448). To confirm the presence of ToCV in these six weed samples, conventional RT-PCR reactions were performed using primers specific for the ToCV CPm and p22 genes as described previously (1). Nucleotide sequence analysis of the amplified DNA fragments verified their identity as ToCV, with 100% sequence identity to the CPm of the ToCV isolate of Florida (Accession No. AY903448) and the p22 gene of the Cartago, Costa Rican isolate (Accession No. FJ809714). Although the number of samples analyzed is not sufficient to allow a determination of the role of weed reservoirs in ToCV epidemics in Costa Rican tomato crops, this report on the wider natural host range of ToCV in Costa Rica may lead to a better understanding of the epidemiology of this virus and be useful in the development of disease management strategies. To our knowledge this is the first report of these weeds as natural hosts of ToCV. References: (1) R. M. Castro et al. Plant Dis. 93:970, 2009. (2) M. I. Font et al. Plant Dis. 88:82, 2004. (3) W. M. Wintermantel et al. Phytopathology 98:1340, 2008.

Web blight is one of the main diseases that affects bean (Phaseolus vulgaris) cultivation. It infects diverse organs at any growth stage of the plant and can be present at different altitudes in a humid tropical climate. The causal agent of this disease is Thanatephorus cucumeris in its sexual stage and Rhizoctonia solani in the anamorph. The objective of this investigation was to characterize molecular isolates of R. solani obtained from bean plants from diverse production regions in Costa Rica and determine their virulence. Fifty-one samples of symptomatic bean plants were collected using a global positioning system. Virulence was evaluated using the detached leaf technique. Isolates were identified using AG 1-IA, AG 1-IB, AG 1-IC, AG 1-ID, AG 2-2, AG 2-2IIIB, AG 2-2IV and AG 4 molecular markers. ITS sequences were obtained and analyzed with BLAST, aligned, and a phylogenetic tree was constructed. A high degree of virulence and genetic variability between isolates was identified and the anastomosis subgroups of isolates were independent of their geographical origin.

Squash (Cucurbita moschata) is one of the most important crops in tropical countries. Geminiviruses are an important group of plant pathogens. In 2002 a new begomovirus was reported to naturally infect squash and some other crops in Costa Rica. Our objective was to compare, using molecular techniques, the extraction and further purification of DNA from squash by different extraction protocols and storage methods. A single infected sample was collected, half of the material was stored frozen at )70°C, and the remainder was stored dehydrated in silica gel (SG). Total nucleic acids (TNAs) were extracted by three different protocols and were quantified by fluorometry, and the quality was analysed by electrophoresis in agarose gels, polymerase chain reaction (PCR) of the virus genome, dot blot and Southern blot hybridization. Even though the tissue stored in SG yielded a higher amount of TNAs, the genetic material exhibited lower integrity and this made it useful exclusively for the detection of geminiviral DNA by PCR amplification of short viral sequences and by hybridization with short viral probes. The Dellaporta method proved to be the most effective for the detection of geminiviral DNA in infected squash tissue. Although the cetyltrimethylammonium bromide method showed similar results, the procedure is more time-consuming. Surprisingly, the citrate method showed either similar or worse results than the other methods.

The present investigation was undertaken to study the effect of gamma irradiation (dose from 10 to 100 Gy) and in vitro selection with fungus filtrate as selecting agent (concentration from 20% to 100%) on the susceptibility of the common bean to Rhizoctonia solani. The best results were found with a dose of 20 Gy or a concentration of 20% of fungus filtrate applied separately. These conditions were used to evaluate the combined effect of both approaches in a second experiment. The combined effect of irradiation and then selection adversely affected growth (height and roots) and survival of the in vitro plants. It may not be necessary to combine the variation generated by irradiation with the selection technique. For future assays we propose the application of: 1) gamma radiation, thereby inducing not only mutants with pathogen resistance, but also with other agronomic traits of interest. Later in the subculture MV4 potential fungus-resistant mutants will be evaluated in the field; or 2) selection pressure using fungus filtrate during three subcultures, which may be sufficient to induce the variation necessary to obtain in vitro plants resistant to fungus.