Pilar Ramírez

Evidence that the proliferation stage of micropropagation procedure is determinant in the expression of Banana streak virus integrated into the genome of the FHIA 21 hybrid (Musa AAAB)

Summary. Banana streak virus (BSV) is causing increasing concern in almost every producing area of banana and plantain (Musa spp.) worldwide. This situation appeared partially linked to some breeding lines and micropropagated hybrids. A complete BSV sequence integrated into the genome of a triploid plantain has been recently characterised and it has been hypothesised that it could give rise to infectious virus via recombination. In this study, we evaluated the effect of a routine micropropagation procedure on the expression of BSV in the FHIA 21 tetraploid hybrid. The widespread presence of integrated sequences and the absence of episomal BSV in thirty FHIA 21 “mother plants” selected for micropropagation were first confirmed by specific PCR and IC-PCR tests. The proliferation stage of the procedure, characterised by an intensive production of neoformed buds, appeared determinant in BSV expression whereas the rooting and acclimatisation stages had little or no effect. The duration in culture and the way of subdividing the clumps of proliferation influenced greatly the percentage of episomal BSV infections, reaching 58% of infected micropropagated lines after six in vitro subcultures. These data suggest that the expression of episomal BSV observed during the in vitro procedure is correlated with the presence of an integrated form.

Newly Discovered Natural Hosts of Tomato chlorosis virus in Costa Rica

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.

Begomoviruses Associated with Bean Golden Mosaic Disease in Nicaragua

Begomovirus spp. cause substantial losses in bean crops in tropical and subtropical regions of the Americas. The predominant Begomovirus sp. in Central America associated with golden mosaic symptoms in bean is Bean golden yellow mosaic virus (BGYMV). However, Calopogonium golden mosaic virus was previously found to infect bean crops in the northern region of Costa Rica. The objective of this research was to identify Begomovirus spp. that infect bean plants in different geographical regions of Nicaragua. In all, 126 samples of young bean leaves with symptoms of golden mosaic were collected from eight different regions of Nicaragua. Using DNA hybridization with specific probes, 120 samples tested positive for BGYMV, 14 samples tested positive for Squash yellow mild mottle virus, and 7 samples tested positive for Calopogonium golden mosaic virus. Sequence analysis of polymerase chain reaction-amplified products from three samples (MA-9 Managua, BE-8 Rivas, and SO-9 Granada) also indicated that the symptoms of golden mosaic in bean are associated with viral sequences from three different Begomovirus spp. Management of bean golden mosaic disease must take into account that BGYMV is the predominant virus (95% of the samples) and that 12% of the samples exhibited possible mixed infections or recombination events in the south and central geographical regions of Nicaragua.

Evaluation of Maize germ plasm for resistance to Maize rayado fino virus

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.

Calopogonium golden mosaic virus Identified in Phaseolus vulgaris from Western and Northern Regions of Costa Rica

Bean golden yellow mosaic virus (BGYMV, GenBank Accession No. M91604) is reported to be the main bean-infecting (Phaseolus vulgaris) begomovirus in Central America (1,2). Another begomovirus, Bean calico mosaic virus (BCaMV, GenBank Accession No. AF110189), is known to infect P. vulgaris in central Mexico. Bean samples with the viral symptom of foliar chlorosis were collected in different regions of Costa Rica from 1994 to 2001. Total nucleic acids were extracted and tested using polymerase chain reaction (PCR) with degenerate primer pair PBL1v2039/PCRc2 for DNA-B, which can give a different fragment size for some begomoviruses (4). PCR fragments were obtained from 119 bean samples. Fifty samples from the central valley produced 600-bp PCR fragments, which is typical for BGYMV (4), and one sample produced two fragments (500 and 600 bp). One sample from western Costa Rica had a 550-bp fragment, and 69 samples from the western and northern regions produced 500-bp fragments. One of the latter samples (No. 01-1152) was selected for PCR with degenerate primer pairs PAL1v1978/PAR1c715 for DNA-A and PBL1v2039/PBR1v800 for DNA-B (4). The amplified PCR fragments were 1.35 and 1.4 kb for DNA-A and DNA-B, respectively. These are expected sizes for a Western Hemisphere begomovirus. The DNA-A fragment was cloned and sequenced (GenBank Accession No. AF439402). Sequence comparisons indicated that this uncharacterized begomovirus (No. 01-1152) had 98, 97, and 93% nucleotide identities with the rep gene, common region, and coat protein gene promoter of Calopogonium golden mosaic virus (CalGMV, GenBank Accession Nos. AF439748 and AF439749, respectively) (3). CalGMV had been isolated from a weed, Calopogonium sp., collected in 1991 near Quepos in the west and Tilaran in the north of Costa Rica (3). Phylogenetic analysis of the viral sequence of the begomovirus associated with No. 01-1152 placed it in the Squash leaf curl virus clade (1). BCaMV is also in this phylogenetic clade, but sequence comparisons between CalGMV and BCaMV had <90% nucleotide identity, and so are distinct species. CalGMV is the begomovirus associated with No. 01-1152, and this is an example of the movement of a weed-infecting begomovirus into an agricultural crop. Since this Calopogonium sp. is widespread in Central America, it is important to determine if CalGMV is present in beans in other regions. The data also indicate that BGYMV and CalGMV infect beans in two geographically distinct regions of Costa Rica. References: (1) J. C. Faria et al. Phytopathology 84:321, 1994. (2) W. McLaughlin et al. Plant Dis. 78:1220, 1994. (3) M. R. Rojas et al. Annu. Rep. Bean Improv. Coop. 35:150, 1992. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Virulence and molecular characterization of Costa Rican isolates of Rhizoctonia solani from common bean

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.

Occurrence of Squash yellow mild mottle virus and Pepper golden mosaic virus in Potential New Hosts in Costa Rica.

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.