Article impact statement: Human activities reduce the presence of large herbivores and predators, affecting ecosystem function, even in well-conserved forests.

Dry common bean plants (Phaseolus vulgaris) from the main production regions of Costa Rica have been affected by a disease, locally called 'amachamiento'. Main symptoms are a severe loss of bean pods due to flowering reduction or abortion, interveinal chlorosis, deformed leaves with corrugated midrib; with diseased plants remaining green at harvest. Morales et al. (1999) associated 'amachamiento' to Cowpea chlorotic mottle virus (CCMV), but CCMV was shown not to induce such symptoms in bean (Gá mez, 1976). A total of 104 plants with symptoms were collected and analyzed by DAS-ELISA for CCMV, and also three comoviruses and five potyviruses commonly infecting P. vulgaris. Negative DAS-ELISA results were shown by 57% of plants for all viruses studied. Nested PCR with universal 16S rRNA phytoplasma primers (P1 ⁄ P7 and R16R2-R16F2n) was used to search for phytoplasma in those symptom-bearing plants negative for DAS-ELISA, and three symptomless plants. R16R2-R16F2n PCR products of 1AE2-kb were amplified from more than 60 plants with symptoms, but not from the symptomless plants. RFLP patterns with RsaI, HhaI, KpnI, BfaI, HaeIII, HpaII, AluI, and MseI characterized the

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.

The giant coral tree (Erythrina poeppigiana, Fabaceae) is a common shade tree in coffee plantations in Costa Rica. Coral trees are pruned to decrease fungal infections and increase nitrogen fixation. Recently, severe shoot proliferation, internodes shortening, and leaf reduction were observed in pruned shade trees in the south of San José Province, Costa Rica. Leaf samples from 10 symptomatic E. poeppigiana trees were collected. Also, two samples from symptomless coral trees were collected from areas free of witches'-broom. Total DNA was extracted from 0.5 g of petiole tissue from all samples with the plant extraction mini kit (Qiagen GmbH, Hilden, Germany) with a modified protocol (2) and assayed by nested PCR with phytoplasma universal rDNA primers (P1/P7) (1) and R16F2n/R16R2 (3). All symptomatic trees tested positive for phytoplasmas by PCR, yielding the expected 1.2-kb band. DNA from the symptomless trees was not amplified by PCR. The restriction fragment length polymorphism analyses (HaeIII, AluI, RsaI, BfaI, HpaII, KpnI, HhaI, and MseI) and the sequence of the 1.2-kb PCR fragment (GenBank Accession No. DQ485305) revealed that the phytoplasma associated with coral tree witches'-broom belongs to the aster yellows phytoplasma group (16SrI) (4). To our knowledge, this is the first report of a phytoplasma belonging to the aster yellow group causing witches'-broom in the Erythrina genus. References: (1) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) M. J. Green et al. Plant Dis. 83:482, 1999. (3) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (4) I. M Lee et al. Int. J. Syst. Bacteriol.48:1153, 1998.

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.