Mauricio Montero-Astúa

Population Genomic Analysis of a Bacterial Plant Pathogen: Novel Insight into the Origin of Pierce's Disease of Grapevine in the U.S.

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 Pierces 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.

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.

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 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.

Potato powdery scab, caused bySpongospora subterranea f. sp.subterranea, in Costa Rica

SummarySeven samples of potato powdery scab like-lesions were used to determine and confirm the occurrence ofSpongospora subterranea f.sp.subterranea in Costa Rica. Light microscope observations of spore balls and zoosporangia in tomato bait plants identified the organism asS. subterranea. Positive DAS-ELISA test results from all seven samples and PCR specific amplification products from five samples confirmed its identity. The geographical location of the samples source plantations suggests wide distribution of the plant pathogen across Costa Rica main potato growing areas. Different lesion types may indicate host-pathogen or environment-pathogen interactions. The symptoms differences and PCR amplification results among specific primer pairs suggest that some degree of genetic variation among theS. subterranea samples may exist; however, further testing is required.

First report of Iris yellow spot virus in Costa Rica

Iris yellow spot virus (IYSV), an emerging disease of onion crops, was identified by transmission electron microscopy, enzyme linked immunosorbent assay, and reverse transcription polymerase chain reaction in Costa Rica. Onion plants had straw-colored, elongated lesions and tip dieback. Costa Rican IYSV nucleocapsid partial sequences (15 isolates) grouped with isolates from North and Central America and from New Zealand.

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.

Detection of Plantago asiatica mosaic virus in lily hybrid plants (Lilium spp.) in Costa Rica grown from imported bulbs

Plantago asiatica mosaic virus (PlAMV) was detected in Costa Rica infecting lily plants showing foliar chlorotic and necrotic streaking. Virus identity was established by TEM, sequencing and ELISA. Pathogenicity was confirmed on Nicotiana benthamiana by mechanical inoculation, and PlAMV was detected by ELISA and RT-PCR.