W. Villalobos

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.

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 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.

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.

First report of the cymbidium mosaic potexvirus (CymMV) infecting the terrestrial orchid Phaius tankervillieae in Costa Rica.

In 1996, plants of the terrestrial orchid Phaius tankervilliae from a nursery in the Central Valley of Costa Rica were observed with mild to severe foliar symptoms of chlorotic streak. No differences were observed in growth, bulb production, flowers, or flowering time between symptomatic and asymptomatic plants, except the symptomatic plants had earlier senescence. Occasionally, the flowers displayed symptoms of chlorosis and white rings in the sepals. Extracts from symptomatic leaves were concentrated by differential centrifugation and analyzed after sucrose gradients. Negative staining of fractions from gradients from symptomatic plants showed the presence of filamentous viral particles 500 by 17 nm. Purified particles contained a single major protein of about 28 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single RNA of about 7 kb, which is greater than the 6.2 kb reported (GenBank). These data suggest the presence of a potexvirus in symptomatic plants (1,2). In enzyme-linked immunosorbent assays, symptomatic plants reacted strongly with antiserum specific for Cymbidium mosaic potexvirus (CymMV). This is the first report of CymMV in P. tankervilliae in Costa Rica. References: (1) J. A. Frowd and J. H. Tremaine. Phytopathology 67:43, 1977. (2) H. T. Hsu et al. Phytopathology 82:491, 1992.

Genipa americana and Ageratina anisochroma, two new hosts of Candidatus Phytoplasma asteris in Costa Rica

We report two new plant species hosts for Candidatus Phytoplasma asteris: Genipa americana (Rubiaceae) and Ageratina anisochroma (Asteraceae). Phytoplasma infections were detected by real-time loop-mediated isothermal amplification and nested PCR. Consensus sequences from both hosts share 99% identity to the 16SrI-B subgroup using BLAST; however, potential new subgroups are suggested due to unique RFLP patterns of the 16S rDNA F2nR2 fragment.

First Report of Citrus Blight in Costa Rica

Citrus blight (CB), causing a chronic decline of citrus, has been an important disease in Florida for over 100 years. CB was first reported in Brazil in the 1980s and is now responsible for the removal of nearly 10% of the trees from production annually. No causal agent has been identified, but CB has been root-graft transmitted to healthy trees, suggesting that the causal agent is infectious (3). Since 1997, CB symptoms were observed in several groves in northern Costa Rica, the most important citrus area of approximately 25,000 ha. Symptoms observed include a general decline and wilt of the tree canopy, off-color leaves, leaf drop, twig dieback, small fruit, delayed blossom, poor growth, and death. A survey near Guanacaste revealed CB symptoms in 7-yr-old Valencia and Pineapple orange trees (Citrus sinensis (L.) Osbeck) grafted on Carrizo citrange (C. sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.) rootstock. Since 1997, 6% of the trees in this area have been replanted annually because of CB symptoms. Similar situations were observed in other groves in the northern citrus area. Dot immunobinding assays (DIBA) (1) were used to detect the P12 protein associated with CB with 20 of 22 trees showing CB-like symptoms giving a positive test. Zinc (Zn) accumulation in trunk wood and water uptake tests were done according to Roistacher (2) in 8 healthy and 20 symptomatic trees which were positive for CB using DIBA. The average Zn concentration of 16 declining trees was 4.6 ± 1.9, whereas the average concentration for 8 healthy trees was 2.0 ± 0.9. The average water uptake in 1 min was 14 ml for healthy trees, and virtually zero for the 20 symptomatic trees. These diagnostic tests confirm the presence of CB in the northern citrus area of Costa Rica, and the surveys indicate the disease is beginning to spread and become economically important. To our knowledge, this is the first report of CB in commercial citrus in Costa Rica. References: (1) K. S. Derrick et al. Plant Dis. 74:168, 1990. (2) C. N. Roistacher. Pages 57-66 in: Graft-Transmissible Diseases of Citrus. Handbook for Detection and Diagnosis. C.N. Roistacher, ed. Food and Agriculture Organization, Rome, 1991. (3) D. P. H. Tucker et al. Plant Dis. 68:979, 1984.