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.

Leaf samples of Solanum lycopersicum, Capsicum annuum, Cucurbita moschata, Cucurbita pepo, Sechium edule and Erythrina spp. were collected. All samples were positive for begomoviruses using polymerase chain reaction and degenerate primers. A sequence of ∼1,100 bp was obtained from the genomic component DNA-A of 14 samples. In addition, one sequence of ∼580 bp corresponding to the coat protein (AV1) was obtained from a chayote (S. edule) leaf sample. The presence of Squash yellow mild mottle virus (SYMMoV) and Pepper golden mosaic virus (PepGMV) were confirmed. The host range reported for SYMMoV includes species of the Cucurbitaceae, Caricaceae and Fabaceae families. This report extends the host range of SYMMoV to include the Solanaceae family, and extends the host range of PepGMV to include C. moschata, C. pepo and the Fabaceae Erythrina spp. This is the first report of a begomovirus (PepGMV) infecting chayote in the Western Hemisphere.

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 degrees 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 purpose of this study was to determine the expression of periodontal disease within an extended costarican family. A periodontal assessment was conducted by measuring pocket probing depths on 6 sites of all standing teeth, using a UNC-15 probe (Hu-Friedy, Chicago, USA). General medical and dental histories were also obtained and peripheral venous blood collected in order to determine serum cytokine levels and genetic polymorphisms. Ethical committee approval was obtained and all participants gave written informed consent. All members of the family presented some degree of periodontal disease and those with more severe disease had alterations in IL-1α and the same IL-6 polymorphism. It is hypothesized that this may be related to the inflammatory status and levels of alveolar bone loss.

Maize rayado fino virus (MRFV) infects maize and appears to be restricted to, yet widespread in, the Americas. MRFV was previously unreported from Ecuador. Maize plants exhibiting symptoms of MRFV infection were collected at the Santa Catalina experiment station in Quito, Ecuador. RT-PCR reactions were performed on total RNA extracted from the symptomatic leaves using primers specific for the capsid protein (CP) gene and 3' non-translated region of MRFV and first strand cDNA as a template. Nucleotide sequence comparisons to previously sequenced MRFV isolates from other geographic regions revealed 88-91% sequence identity. Phylogenetic trees constructed using Maximum Likelihood, UPGMA, Minimal Evolution, Neighbor Joining, and Maximum Parsimony methods separated the MRFV isolates into four groups. These groups may represent geographic isolation generated by the mountainous chains of the American continent. Analysis of the sequences and the genetic distances among the different isolates suggests that MRFV may have originated in Mexico and/or Guatemala and from there it dispersed to the rest of the Americas.

A system for the genetic transformation of maize was developed for two Costa Rican varieties: CR-7 and Diamantes 8843, that can allow the subsequent transfer of viral-derived genes in order to confer resistance to the disease caused by maize rayado fino virus (MRFV). The method is based on particle bombardment of organogenic calli derived from shoot tips. On the other hand, the molecular construction pRFcp-bar, containing the coat protein gene of MRFV and the marker gene bar, was elaborated. For the visual selection of the transformed material was used also the plasmid pDM803 that contains the reporter gene uidA (GUS). The results indicate that devices evaluated: the PIG ("Particle Inflow Gun") and the Bio-Rad are both enough efficient to transfer foreign genes to the genome of the maize.

Contributions to whitefly-geminivirus management in Costa Rica. The diseases associated with the whitefly-geminivirus complex in different crops are one of the most important problems facing agriculture worldwide. This complex is distributed among tropical and subtropical regions. The control of the disease’s dissemination is complicated due to several intervening factors. Since 1994, the University of Costa Rica research program for the molecular control of geminivirus (PROGEMINI-UCR) has been developing a molecular strategy for the diagnosis and characterization of new geminiviruses, contributing to the management of the whitefly-geminivirus complex. The project’s development, associated with the Regional Action Plan for the management of whitefly and geminivirus, included: identification of new geminiviruses infecting beans, cucurbits, soy, tomatoes, pepper, papaya and wild plants; determination of the inoculum’s sources; translocation and quantification studies associated to crop, severity and yield reduction; and development of management methodologies for the study of the complex. Viral sequences discovered within the Program constitute a gene bank for the development of transgenic plants resistant to these pathogens. These sequences could also be used as specific diagnostic probes. PROGEMINI-UCR has supported the training of new scientists and technicians in Latin America. Interinstitutional cooperation links allow the development of pertinent research projects and the successful transference of technology, helping to increase the competitiveness of Costa Rican agriculture facing the challenge of a globalized economy.

Gene silencing: the counterattack of plants to infections by pathogenic viruses. Gene silencing is a defense system by which transgenic and non-transgenic plants marshal,in response to localized infections of pathogenic viruses, a sophisticated mechanism of viral RNA degradation. During their replication process, RNA viruses generate intermediary molecules of double-stranded RNA (dsRNA).Plant cells do not normally possess RNAdc. So these molecules are detected by the surveillance mechanism of the plant and subsequently degraded to small fragments of dsRNA which are finally denatured and guide the specific degradation of homologous transcripts. DNA viruses, such as pararetrovirus and geminivirus, can also be silenced by an analogous mechanism. Such a silencing response is adaptive and can be systemically spread throughout the host plant,thus protecting it against subsequent attacks by the same virus or others that have similar sequences of nucleotides. However, plant viruses have counter-defended and have evolved different molecular strategies to neutralize the silencing mechanism of host plants, thus establishing a clear challenge to our understanding of plant-pathogen interactions. Research in this field present an opportunity to design improved practices for the management of viral diseases in plants.

Geminivirus, pathogens of worldwide importance. At present, Latin America has been the region most affected by the whitefly-geminivirus complex,both by number of crops affected and by yield losses, and the agricultural area devastated. Infections by geminivirus within agrosystems are dynamic, because they are complex interactions that involve various factors that can change, such as: the geminivirus, the systems of production,the environment and the biotypes of the vector. For this reason,identification of the virus must be a permanent process, given that new pathogens will require continuous change in management strategies. In this review the organization of the genome in Begomovirus, general multiplication and viral transcription strategies as well as phylogenetic diversity and the most recent hypotheses that try to explain the molecular diversity of these pathogens, is analyzed. Also transmission by the insect vector and the use of molecular techniques as tools for identification and characterization of geminivirus, are reviewed. Furthermore, genetic engineering strategies for the management of the geminivirus-whitefly complex are included..

The complete nucleotide sequence of the single-stranded RNA genome of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus, is 6305 nucleotides (nts) in length and contains two putative open reading frames (ORFs). The largest ORF (nt 97–6180) encodes a polyprotein of 224 kDa with sequence similarities at its N-terminus to the replication-associated proteins of other viruses with positive-strand RNA genomes and to the papainlike protease domain found in tymoviruses. The C-terminus of the 224-kDa ORF also encodes the MRFV capsid protein. A smaller, overlapping ORF (nt 302-1561) encodes a putative protein of 43 kDa with unknown function but with limited sequence similarities to putative movement proteins of tymoviruses. The nucleotide sequence and proposed genome expression strategy of MRFV is most closely related to that of oat blue dwarf virus (OBDV). Unlike OBDV, MRFV RNA does not appear to contain a poly(A) tail, and it encodes a putative second overlapping open reading frame.

Se estandarizó un sistema de electroforesis de isoenzimas con tejido de hojas que permitió la identificación del polimorfismo fenético entre cuatro razas y un lubrido de pejibaye Bactris gasipaes provenientes de Brasil, Perú, Bolivia, Panamá y Costa Rica. Las isoenzimas ensayadas fueron: PRX,E ST,A CP,ME , DlA,M DH,G 6PDH,P GI,S OD, PGM, ADH, GOT, de estas solamente las dos o:ltimas no mostraron actividad. El resto de isoenzimas mostraron polimorfismo fenético en diferentes grados, por esto se consideran marcadores polimórficos potenciales para estudios de variabilidad genética en pejibaye. Se encontró un locus único en el zimograma de la enzima PRX en las muestras de Utilis-Guápiles (CR), el cual podría usarse como un marcador discriminatorio para esta raza. Se establecieron relaciones de similitud isoenzimática entre las razas Utilis-Guápiles (CR) y Tuira-Darién (Pa); Tembé-Chapare (Bo) y ParáBelem (Bra) respectivamente, mientras que el híbrido Yurimaguas (Pe) se ubicó aparte y ligeramente más cercano a Utilis y Tuira, por esto. se considera que posiblemente desciende de padres aún no identificados.

Maize rayado fino virus (MRFV) causes severe yield losses in maize (Zea mays) in Latin America with reductions of 40–50% on indigenous genotypes and 100% in some new cultivars. A collection of 20 different maize genotypes were obtained from the Center for the Improvement of Maize and Wheat (CIMMYT, México), Estación Experimental Fabio Baudrit (EEFB-Alajuela), and the Consejo Nacional de la Producción (CNP-Costa Rica). Enzyme-linked immunosorbent assay (ELISA) and symptom severity were used to evaluate the responses of the maize genotypes to MRFV infection under screenhouse conditions. Two genotypes from the CIMMYT germ plasm collection (accession nos. 2980-93 and 3974) showed mild and delayed symptoms and low MRFV concentrations as determined by ELISA.

We have examined the molecular epidemiology of the leafhopper-borne maize rayado fino virus (MRFV) in Latin America. The coat protein gene and 3' non-translated region of 14 isolates of MRFV collected from Latin America and the United States were sequenced and phylogenetic relationships examined. The nucleotide sequence revealed remarkable conservation, with a sequence similarity of 88-99%. Phylogenetic analysis of sequence data obtained from a 633 bp fragment showed that MRFV has diverged into three main clusters, i.e. the geographically distinct northern and southern isolates and the Colombian isolates. Significant differences between the isolates collected from Colombia, previously named maize rayado colombiana virus, based upon differences in symptomatology and serological relationships to MRFV, and the other MRFV isolates, provides additional evidence supporting its designation as a unique strain of MRFV.