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 Xylella fastidiosa in Avocado in Costa Rica.

Since the late 1990s, chlorotic mottling, marginal scorch, deformation of leaves, defoliation, shortening of internodes, and branch dieback have been observed in avocado trees (Persea americana Mill.) in Costa Rica. The symptoms are not uniformly distributed in the tree, so some branches are symptomatic while others are not. These symptoms are similar to several leaf scorch diseases caused by the bacterium Xylella fastidiosa Wells (2,4). This bacterium has been detected in coffee and citrus plants in Costa Rica. Of 227 avocado trees tested by double-antibody sandwich (DAS)-ELISA with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) from 2000-2004, 188 were positive. Results of ELISA tests of individual trees varied with the season and branches tested. Fifteen greenhouse-grown, ELISA-negative avocado seedlings were grafted with budwood from an ELISA-positive tree. Eight of these developed scorch symptoms and one also showed chlorotic mottling and deformation, showing that the disease is graft transmitted. All of these features are characteristic of diseases caused by X. fastidiosa (2,4). Transmission electron microscopy of leaf petioles from three field trees positive by ELISA, revealed rod-shaped bacilli approximately 1.6 to 2.0 µm long and 0.3 µm in diameter with a rippled cell wall inside xylem vessels and embedded in a matrix; morphology and measurements that are consistent with those reported for X. fastidiosa (2). DNA extraction and PCR attempts have been limited by mucilaginous sap from avocado. Positive PCR results (approximately 472-bp band) were obtained from two of the grafted seedlings and seven field trees from two distinct geographical locations (Alajuela and San José provinces) with DNA extractions from the plant sap using DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) following a modified protocol (1) and nested PCR (3). Four of the PCR products, including one from the grafted seedlings, were cloned and sequenced in duplicate. GenBank sequences EU021997 to EU022000 present 99 to 100% sequence identity to a Pierce's disease strain from California (Temecula1) and 94 to 95% to a citrus variegated chlorosis strain from Brazil (Found-5). Several attempts have been made to isolate the bacterium in 'periwinkle wilt' and buffered cysteine-yeast extract media with negative results, probably because of the rapid production of mucilaginous sap when the avocado tissues were sampled. To our knowledge, this is the first report of X. fastidiosa in avocado trees. References: (1) M. J. Green et al. Plant Dis. 83:482, 1999. (2) S. S. Hearon et al. Can. J. Bot. 58:1986, 1980. (3) M. R. Pooler and J. S. Hartung. Curr. Microbiol. 31:377, 1995. (4) A. H. Purcell et al. Phytopathology 89:53, 1999.

Occurrence of Potato Powdery Scab Caused by Spongospora subterranea f. sp. subterranea in Costa Rica.

Powdery scab of potatoes, caused by Spongospora subterranea (Wallr.) Lagerheim f. sp. subterranea Tomlinson, is important worldwide due to its effect on tuber quality and transmission of Potato mop-top virus. Although powdery scab-like lesions have been observed on potato in Costa Rica (1), the presence of the pathogen has not been confirmed. During a survey in 2001, powdery scab-on was observed from a field and a greenhouse in the high elevation zone of the main potato-producing area of Costa Rica. Commercial potatoes with scab-like lesions were also obtained at a farmers' market. Scraping the lesions, and observing spore balls or cystosori with a honey-comb-like structure under light microscopy confirmed the identity of S. subterranea. The identity of the pathogen was also confirmed by enzyme-linked immunosorbent assay using monoclonal antibodies specific for S. subterranea (BioReba Ag, Reinach, Switzerland). Pathogenicity of S. subterranea was confirmed by a bioassay on tomato plants grown in nutrient solution culture (2). Tomato cv. Supermarmande plants were grown from seed in pots filled with quartz and watered with nutrient solution. Three weeks after planting, the roots were trimmed to 60 mm, and the plants were transferred to the nutrient solution for additional growth. After growing for 1 week in the nutrient solution, tomato seedlings were inoculated by replacing the nutrient solution with nutrient solution containing cystosori (20 mg/liter, wt/vol) that were scraped from the scab lesions. Zoosporangia of S. subterranea were observed in root hairs and epidermal cells of the seedlings 2 weeks after inoculation. To our knowledge, this is the first report that confirms the presence of S. subterranea on potato in Costa Rica. References: (1) R. Amador. Invest. Agri. Costa Rica. 1(1):16, 1987. (2) U. Merz. Bull. OEPP 19:585, 1989.