Carmen Rivera

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.

First Report of Xylella fastidiosa Infecting Citrus in Costa Rica

Citrus variegated chlorosis (CVC) is an important disease mainly of sweet orange (Citrus sinensis (L.) Osbeck) cultivars. It was first described in Brazil in the state of Sā Paulo in 1987 (4). The disease has spread to all Brazilian states that grow citrus and is affecting more than one-third of the orange trees grown in Brazil. CVC is caused by Xylella fastidiousa, a xylem-limited, gram-negative bacterium. During the last 4 years, symptoms including leaf interveinal chlorosis, stunting, canopy dieback, and hard and undersized fruits, similar to those caused by CVC (3), appeared in sweet orange trees used as shade plants for coffee plantations and as fence posts in Costa Rica. Necrotic lesions on the abaxial side of the leaves as reported in Brazil were rarely observed. Leaf petiole samples from 25 symptomatic sweet orange trees reacted positively with a X fastidiosa-specific antiserum (AGDIA Inc., Elkart, IN) in a double-sandwich antibody enzyme-linked immunosorbent assay (DAS-ELISA). A fastidious, gram-negative bacterium identified as X. fastidiosa using DAS-ELISA was isolated on perwinkle wilt (PW) medium plates (1) from citrus stems showing CVC symptoms, but not from asymptomatic trees. The isolated colonies were circular and opalescent with diameters of 2 to 3 mm and were clearly visible within 6 to 7 days after streaking. Petiole sections from symptomatic plants observed with scanning electron microscopy showed rod-shaped bacteria with rippled cell walls tightly packed in xylem vessels, as described for X. fastidiosa previously (2), and with transmission electron microscopy, the bacteria were morphologically similar to those reported previously for CVC (2). To our knowledge, this is the first report of X. fastidiosa associated with citrus in Costa Rica. References: (1) M. J. Davis et al. Curr. Microbiol. 6:309, 1981. (2) J. S. Hartung et al. Phytopathology 84:591, 1994. (3) R. F. Lee et al. Summa Phytopathol. 19:123, 1993. (4) V. Rossetti et al. 1990, C.R. Acad. Sci. (Paris) 310:345-349.

Genetic diversity of Xylella fastidiosa strains from costa rica, sao paulo, brazil, and United States

ABSTRACT The diversity of 42 Xylella fastidiosa strains from Costa Rica, São Paulo, Brazil, and the United States were analyzed using the sequence of the 16S rRNA gene by variable number of tandem repeat (VNTR) fragment analysis and by restriction fragment length polymorphisms (RFLP) of a specific polymerase chain reaction (PCR)-amplification product using enzyme CfoI. Limited variability in the sequence of the 16S rRNA gene was observed and, although the separation was not absolute, most strains from Costa Rica clustered with strains from the United States and not with strains from São Paulo. The PCR-RFLP produced different patterns of DNA bands. The same pattern was shared by strains from Costa Rica, the United States, and two coffee strains from São Paulo, but a different pattern was observed in six coffee and orange strains from Brazil. In all, 32 amplification products were scored in the VNTR fragment analysis. The total variation observed among the X. fastidiosa strains had significant (P < 0.001) contributions from both geography and host origin as inferred by Neis values of genetic diversity and WINAMOVA statistics. The strains from Costa Rica were isolated from diseased grapevines, coffee, and sweet orange and these strains grouped together and could be distinguished from strains from grapevine from the United States or from either coffee or sweet orange from São Paulo. The strains tested from Costa Rica are most likely of local origin, although the possibility that they have been introduced along with horticultural crops cannot be excluded. In either case, they are examples of independent selection of strains of X. fastidiosa affecting coffee and sweet orange. Greater genetic similarity was observed between strains from Costa Rica and the United States than with those from São Paulo.

Incidence, Distribution, and Association of Spongospora subterranea and Potato mop-top virus in Costa Rica

A survey was conducted in 39 potato (Solanum tuberosum) fields in Costa Rica to determine incidence and association of Spongospora subterranea f. sp. subterranea and Potato mop-top pomovirus (PMTV). The fields were located in Costa Ricas two major potato-production regions and were further characterized by their altitude. In all, 633 paired samples of leaf tissue and corresponding tubers were collected, assessed visually for disease, and subsequently assayed by enzyme-linked immunosorbent assay (ELISA). S. subterranea presence in tuber tissue was tested by double-antibody sandwich (DAS)-ELISA and PMTV presence in leaf and tuber tissues was tested by triple-antibody sandwich (TAS)-ELISA. Moreover, soil samples were collected from 10 fields surveyed and were evaluated for both pathogens via ELISA and bioassay. The incidence of both diseases ranged from 0 to 100% within individual fields, with incidences lower than 40% occurring in more than 70% of the fields. Higher incidences were found in fields located at higher altitudes. Of the 633 paired samples, 179 and 146 were positive for PMTV and S. subterranea, respectively, according to ELISA in either the foliage or tubers. A low correlation was found for PMTV visual symptoms and ELISA test results. Only 14 of the 81 foliar samples testing positive for PMTV had visual symptoms; the remaining 67 samples were asymptomatic. Conversely, comparison of visual evaluation with detection of S. subterranea by ELISA on tubers showed that 70% of the results were coincident. S. subterranea was detected in 4 of 10 soil samples tested by ELISA. Soilborne PMTV was detected by ELISA in roots of bait plants sown in these soil samples. Co-occurrence of both pathogens was detected in 64 samples. A significant but low degree of association for vector and virus was determined, and data suggests that S. subterranea is participating in the transmission of PMTV in Costa Rica in low frequency.

Multiplication of Maize Rayado Fino Virus in the Leafhopper Vector Dalbulus maidis

The enzyme-linked immunosorbent assay (ELISA) was used to demonstrate the increase in titer of maize rayado fino virus (MRFV) in its leafhopper vector, Dalbulus maidis. Viral antigen concentration attained a maximum in the body of the insect 25 days after virus acquisition and decreased thereafter. Substantial differences in concentration were observed among viruliferous leafhoppers. MRFV was serially passed through 5 successive leafhopper populations. The results provide further evidence of multiplication of MRFV in D. maidis.

Isolation and molecular characterization of Xylella fastidiosa from coffee plants in Costa Rica

Coffee plants exhibiting a range of symptoms including mild to severe curling of leaf margins, chlorosis and deformation of leaves, stunting of plants, shortening of internodes, and dieback of branches have been reported since 1995 in several regions of Costa Rica’s Central Valley. The symptoms are referred to by coffee producers in Costa Rica as “crespera” disease and have been associated with the presence of the bacterium Xylella fastidiosa. Coffee plants determined to be infected by the bacterium by enzyme linked immunosorbent assay (ELISA), were used for both transmission electron microscopy (TEM) and for isolation of the bacterium in PW broth or agar. Petioles examined by TEM contained rod-shaped bacteria inside the xylem vessels. The bacteria measured 0.3 to 0.5 μm in width and 1.5 to 3.0 μm in length, and had rippled cell walls 10 to 40 nm in thickness, typical of X. fastidiosa. Small, circular, dome-shaped colonies were observed 7 to 26 days after plating of plant extracts on PW agar. The colonies were comprised of Gram-negative rods of variable length and a characteristic slight longitudinal bending. TEM of the isolated bacteria showed characteristic rippled cell walls, similar to those observed in plant tissue. ELISA and PCR with specific primer pairs 272-l-int/272-2-int and RST31/RST33 confirmed the identity of the isolated bacteria as X. fastidiosa. RFLP analysis of the amplification products revealed diversity within X. fastidiosa strains from Costa Rica and suggest closer genetic proximity to strains from the United States of America than to other coffee or citrus strains from Brazil.

First Report of Orchid fleck virus in Costa Rica

Orchid fleck virus (OFV), a tentative member of the family Rhabdoviridae, infects orchids in several countries. The virus is vectored worldwide by the mite Brevipalpus californicus (Banks) (Acari: Tenuipalpidae). Eleven plants of Oncidium spp. and one plant each of the genera Cymbidium and Maxillaria exhibiting numerous yellow flecks and necrotic ringspot lesions on leaves were collected in two private orchid collections in Costa Rica. Presence of OFV was assessed by plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) using an antiserum developed against an OFV isolate in Japan (2), analyses of ultrathin sections of the host cell with transmission electron microscopy (TEM), and reverse transcription-polymerase chain reaction (RT-PCR) amplification using specific primers for the viral nucleocapsid gene (1). Eight of eleven Oncidium samples, and both Cymbidium and Maxillaria samples tested positive for OFV with PTA-ELISA having A405 values ranging from 3.9 to 14.6 times higher than negative controls. Thin sections from individual samples of Cymbidium, Oncidium, and Maxillaria revealed electron-lucent intranuclear viroplasm and short, rodlike particles (40 to 50 × 100 nm) in the nucleus or cytoplasm typical of OFV-infected cells. RT-PCR amplifications from one sample of each genera resulted in PCR-product bands of approximately 800 bp. The Cymbidium RT-PCR product was cloned into a pGEM-T-Easy expression vector and sequenced using an ABI 3700 sequencer. The 619-bp nucleocapsid gene consensus sequence had 98% homology with the OFV isolate 0023 identified in Germany (GenBank Accession No. AF343870) (1). However, it had only approximately 85% nucleocapsid gene homology with other OFV isolates available through GenBank, including those from countries geographically closer to Costa Rica, such as Brazil (1). To our knowledge, this is the first report of OFV infecting orchids in Costa Rica. References: (1) A. L. Blanchfield et al. J. Phytopathol. 149:713, 2001. (2) H. Kondo et al. Bull. Res. Inst. Bioresour. Okayama Univ. 4:149, 1996.

First report of new phytoplasma diseases associated with soybean, sweet pepper, and passion fruit in Costa Rica.

A new soybean disease outbreak occurred in 2002 in a soybean (Glycine max) plantation in Alajuela Province, Costa Rica. Symptoms on the affected plants included general stunting, small leaves, formation of excessive buds, and aborted seed pods. In the same region, two other diseases, one in sweet pepper (Capsicum annuum) fields and another affecting passion fruit (Passiflora edulis) vines, were also found. Symptoms on sweet pepper plants included unusually dark green leaves, some of which exhibited a rugose symptom with a zigzag pattern to the midvein, and purple vein discoloration. Passion fruit vines exhibited bud proliferation. Collectively, symptoms resembled those commonly attributed to phytoplasmal infections. Total nucleic acid was extracted from veinal tissues of leaves or buds (soybean). A nested PCR assay using primer pair P1/P7 followed by R16F2n/R16R2 (1) was employed for the detection of putative phytoplasmas that might be associated within symptomatic plants. All seven symptomatic plants (three soybean, three sweet pepper, and one passion fruit) tested, but not healthy controls, yielded positive results. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products using restriction enzymes AluI, BfaI, HhaI, MseI, and RsaI indicated that the three diseases were associated with a very similar or identical phytoplasma. RFLP patterns and sequence analysis of cloned 16S rDNAs (GenBank Accession Nos. FJ226068-FJ226073) revealed that the phytoplasma shared less than 97.5% sequence homology with all previously classified phytoplasmas, and, as such, represents a new taxon most closely related to 16SrXII group (1) strains. To our knowledge, this is the first report of a new phytoplasma associated with diseases of soybean, sweet pepper, and passion fruit in Costa Rica. Reference: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

Chayote Mosaic, a New Disease in Sechium edule Caused by a Tymovirus

A sap-transmissible virus was isolated from chayote (Sechium edule) in Costa Rica. Infected plants showed chlorotic spots and rings, and blotchy mosaics, which often coalesced to give a complete mosaic and leaf deformation. By electron microscopy, spherical virus-like particles of approximately 29 nm in diameter were visible, and cytological changes associated with the chloroplasts were observed. The virus particles sedimented in sucrose density gradients as two components, a top component of empty protein shells and a bottom component of electron-dense particles. Electrophoretic analysis showed a single-stranded RNA of approximately 5.7 kb and capsid protein (CP) subunits of ∼22 kDa. The virus was identified as a member of the tymovirus group on the basis of particle morphology, size, sedimentation in sucrose gradients, cytopathological effects, and capsid protein and genome properties, and it was tentatively named chayote mosaic virus (ChMV).

First report of Xylella fastidiosa in Nerium oleander in Costa Rica.

Oleander (Nerium oleander L.) shrubs presenting mottling, leaf tip and margin scorch, short internodes, defoliation, and branch dieback were observed at different localities in the Central Valley in Costa Rica. Severity of the symptoms ranged widely, and most plants showed both diseased and healthy branches. In severe cases, entire sections of the plant were defoliated. Symptoms resembled those described for oleander leaf scorch (OLS) caused by the bacterium Xylella fastidiosa in the United States (3). This bacterium has been reported in coffee and citrus plants in Costa Rica. Sixty plants from five different places were sampled and tested using ELISA (Agdia Inc., Elkhart, IN) against X. fastidiosa. Thirty-five plants showed absorbance mean value of duplicate wells greater than the mean of control wells plus three times the standard deviation, and therefore were considered positive. Thirty-three of the sixty samples were processed for an immunofluorescence assay modified from Carbajal et al. (1) with antibody to X. fastidiosa (Agdia Inc.). Thirteen samples showed fluorescent rod-shaped bacilli with morphology similar to those observed from a pure culture of X. fastidiosa obtained from coffee. Ten of these thirteen samples were positive by ELISA. DNA extracts (2) from three of the oleander plants with high ELISA absorbance values were tested by nested PCR with primer pair 272-1/272-2 followed by the pair 272-1 int/272-2 int (4). Two of the samples were positive for the bacterium and one of the PCR products was cloned and sequenced in both directions (GenBank Accession No. EU009615). The negative (PCR mix) and positive (pure culture of X. fastidiosa isolated from grapevine) controls for nested-PCR were indeed negative and positive, respectively. The BLAST program was used to compare the sequence to the nucleotide collection (nr/nt) and Microbe Assembled Genomes databases in GenBank. All matches corresponded to X. fastidiosa sequences. The sequence showed 97% similarity with strains Found-4 (coffee strain from Brazil) and Found-5 (citrus strain from Brazil) and 96% similarity with strain Ann-1 from oleander in California. On the basis of serological, microscopic, and molecular detection of X. fastidiosa from oleander exhibiting symptoms of OLS similar to those reported in the literature, this pathogen likely is causing the symptoms we observed in Costa Rica. References: (1) D. Carbajal et al. Curr. Microbiol. 49:372, 2004. (2) M. J. Green et al. Plant Dis. 83:482, 1999. (3) Q. Huang et al. Plant Dis. 88:1049, 2004. (4) M. R. Pooler and J. S. Hartung. Curr. Microbiol. 31:377, 1995.