Invasive diseases present an increasing problem worldwide; however, genomic techniques are now available to investigate the timing and geographical origin of such introductions. We employed genomic techniques to demonstrate that the bacterial pathogen causing Pierce's disease of grapevine (PD) is not native to the US as previously assumed, but descended from a single genotype introduced from Central America. PD has posed a serious threat to the US wine industry ever since its first outbreak in Anaheim, California in the 1880s and continues to inhibit grape cultivation in a large area of the country. It is caused by infection of xylem vessels by the bacterium Xylella fastidiosa subsp. fastidiosa, a genetically distinct subspecies at least 15,000 years old. We present five independent kinds of evidence that strongly support our invasion hypothesis: 1) a genome-wide lack of genetic variability in X. fastidiosa subsp. fastidiosa found in the US, consistent with a recent common ancestor; 2) evidence for historical allopatry of the North American subspecies X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa; 3) evidence that X. fastidiosa subsp. fastidiosa evolved in a more tropical climate than X. fastidiosa subsp. multiplex; 4) much greater genetic variability in the proposed source population in Central America, variation within which the US genotypes are phylogenetically nested; and 5) the circumstantial evidence of importation of known hosts (coffee plants) from Central America directly into southern California just prior to the first known outbreak of the disease. The lack of genetic variation in X. fastidiosa subsp. fastidiosa in the US suggests that preventing additional introductions is important since new genetic variation may undermine PD control measures, or may lead to infection of other crop plants through the creation of novel genotypes via inter-subspecific recombination. In general, geographically mixing of previously isolated subspecies should be avoided.

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Background and Aims: A hybrid grapevine biparental mapping population was evaluated for resistance to grape phylloxera (Daktulosphaira vitifoliae Fitch) in the greenhouse and in the field. The family (n = 125) was derived from a cross of two University of Minnesota breeding selections from a complex pedigree with multiple Vitis species. Methods and Results: The vines were evaluated for foliar phylloxera severity using visual rating in the field for multiple years. In the greenhouse, a replicated experimental design was used to quantify phylloxera severity using different traits including number of galls per plant, area under the disease progress curve, proportion of leaves with galls, average number of galls per leaf and visual rating. The plants used in the greenhouse experiment were also visually rated for root infestation. Conclusions: Quantitative trait loci (QTL) mapping for the foliar resistance traits (reduced severity) routinely identified the region spanning~10 to 30 cM on linkage group (LG) 14 to be associated with resistance, and inherited from the mother parent MN1264. The plants used in the greenhouse experiment were also visually rated for root infestation, and subsequent QTL mapping identified regions on LG 5 of the maternal map and LG 10 of the paternal map (MN1246) associated with root resistance.Significance of the Study: The new QTLs identified in this report are potential novel sources of resistance and the first report of QTL for foliar phylloxera resistance in a hybrid grapegrowing region of North America. Molecular markers linked to resistance can be utilised in marker-assisted breeding.

A novel phytoplasma, designated strain SoyST1c1, associated with a newly emerging disease in soybean (Glycine max), known as soybean stunt (SoyST), was found in 2002 in a soybean plantation in Alajuela Province, Costa Rica. The same phytoplasma, or a very closely related strain, also infected sweet pepper (Capsicum annuum) with purple vein syndrome (SwPPV) and passion fruit vine (Passiflora edulis) with bud proliferation disease (PasFBP) in the same region. Sequence analysis of cloned 16S rRNA gene sequences (GenBank accession nos FJ226068-FJ226073 and HQ225624-HQ225635) indicated that all three affected plants were infected by phytoplasmas that shared ,97.5 % sequence similarity with previously described phytoplasmas. The SoyST-causing phytoplasma represents a new taxon, most closely related to phytoplasma group 16SrI and 16SrXII strains. Virtual RFLP analysis indicated that the SoyST-causing phytoplasma and its closely related strains represent a novel 16Sr group, designated 16SrXXXI. Phylogenetic analysis of 16S rRNA gene sequences from the new phytoplasma strains, those previously described as 'Candidatus Phytoplasma spp.' and other distinct, as yet unnamed, phytoplasmas indicated that the SoyST-causing phytoplasma represents a distinct lineage within the aster yellows/stolbur branch on the phylogenetic tree. On the basis of its unique 16S rRNA gene sequence and biological properties, strain SoyST1c1 represents a novel taxon, for which the name 'Candidatus Phytoplasma costaricanum' is proposed with SoyST1c1 as the reference strain.

In 1995, severe symptoms were observed on ‘Caturra’ and ‘Catuaí’ coffee (Coffea arabica L.) varieties in farms in the southern part of the Central Valley in Costa Rica. Symptoms were reduced leaf size, malformation of leaves, curling of leaf edges, shortening of internodes, and severe leaf chlorotic mosaic, which sometimes became necrotic. Abortion of flowers and young beans was also observed, with a reduction in yield. Plants also showed irregular growth with an atypical curling appearance that gave rise to the Spanish name “crespera.” Ten and three healthy plants were inoculated by grafting in the greenhouse, using infected and healthy budwoods, respectively. Approximately 6 months after inoculation, 3 of 10 grafted plants with infected budwoods showed symptoms of leaf chlorosis, curling, and malformation of leaves and bunched new flushes. Samples of 39 symptomatic plants collected from the field and samples of 3 healthy plants maintained in the greenhouse were tested by enzyme-linked immunosorbent assay (ELISA). All (100%) analyzed symptomatic samples were positive for X. fastidiosa, and all healthy controls were negative. The symptoms observed in Costa Rica are different from those described for coffee leaf scorch in Brazil (1,2), but the climatological conditions and soil type present in Costa Rica are also very different from the areas where X. fastidiosa occurs in Brazil. Leafhoppers were collected randomly in one of the most affected regions. Graphocephala permagna and Erythrogonia sonora were the most frequent insect species found associated with coffee. In ELISA, 34.5% (10 of 29) and 23.8% (5 of 21) of the collected specimens belonging to G. permagna and E. sonora, respectively, tested positive for X. fastidiosa. These positive ELISAs do not necessarily mean that the insect is a vector. The results presented here extend the known geographic distribution of X. fastidiosa. To our knowledge, this is the first report of X. fastidiosa in coffee in Costa Rica. References: (1) M. J. G. Beretta et al. Plant Dis. 80:821, 1996. (2) de Lima et al. Plant Dis. 82:94, 1998.

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 microm in width and 1.5 to 3.0 microm 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.

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.