Causal Role of Xylella fastidiosa in Oleander Leaf Scorch Disease

ABSTRACT A lethal leaf scorch disease of oleander (Nerium oleander) appeared in southern California in 1993. A bacterium, Xylella fastidiosa, was detected by culturing, enzyme-linked immunoassay, and polymerase chain reaction in most symptomatic plants but not in symptomless plants or negative controls. Inoculating oleanders mechanically with X. fastidiosa cultures from diseased oleanders caused oleander leaf scorch (OLS) disease. The bacterium was reisolated from inoculated plants that became diseased. Three species of xylem sap-feeding leafhoppers transmitted the bacterium from oleander to oleander. The bacterium multiplied, moved systemically, and caused wilting in Madagascar periwinkle (Catharanthus rosea) and leaf scorch in periwinkle (Vinca major) in a greenhouse after inoculation with needle puncture. No bacterium was reisolated from grapevine (Vitis vinifera), peach (Prunus persica), olive (Olea europaea), California blackberry (Rubus ursinus), or valley oak (Quercus lobata) mechanically inoculated with OLS strains of X. fastidiosa. A 500-bp sequence of the 16S-23S ribosomal intergenic region of oleander strains showed 99.2% identity with Pierce's disease strains, 98.4% identity with oak leaf scorch strains, and 98.6% identity with phony peach, plum leaf scald, and almond leaf scorch strains.     

Phylogenetic Relationships of Xylella fastidiosa Strains Isolated from Landscape Ornamentals in Southern California

ABSTRACT Xylella fastidiosa is an insect-borne, xylem-limited pathogenic bacterium that has been associated with a rise in incidence of diseased landscape ornamentals in southern California. The objective of this study was to genetically characterize strains isolated from ornamental hosts to understand their distribution and identity. Strains of X. fastidiosa isolated from ornamentals were characterized using a multiprimer polymerase chain reaction (PCR) system, random amplified polymorphic DNA (RAPD)-PCR, and sequence analysis of the 16S-23S rDNA intergenic spacer region (ISR). Based on RAPD-PCR and 16S-23S rDNA ISR, strains isolated from daylily, jacaranda, and magnolia clustered with members of X. fastidiosa subsp. sandyi and caused oleander leaf scorch but not Pierce's disease symptoms in glasshouse assays on oleander and grape, respectively. This demonstrated both that our groupings based on genetic characterization were valid and that strains of X. fastidiosa subsp. sandyi are present in hosts other than oleander. Strains isolated from Spanish broom, cherry, and one strain isolated from western redbud clustered with X. fastidiosa subsp. fastidiosa members. Strains isolated from purple-leafed plum, olive, peach, plum, sweetgum, maidenhair tree, crape myrtle, and another western redbud strain clustered with members of X. fastidiosa subsp. multiplex. All strains isolated from mulberry and one from heavenly bamboo formed a separate cluster that has not yet been defined as a subspecies.     

Evaluation of the Genetic Diversity of Xylella fastidiosa Strains from Citrus and Coffee Hosts by Single-Nucleotide Polymorphism Markers

ABSTRACT The aim of this study was to obtain information about genetic diversity and make some inferences about the relationship of 27 strains of Xylella fastidiosa from different hosts and distinct geographical areas. Single-nucleotide polymorphism (SNP) molecular markers were identified in DNA sequences from 16 distinct regions of the genome of 24 strains of X. fastidiosa from coffee and citrus plants. Among the Brazilian strains, coffee-dependent strains have a greater number of SNPs (10 to 24 SNPs) than the citrus-based strains (2 to 12 SNPs); all the strains were compared with the sequenced strain 9a5c. The identified SNP markers were able to distinguish, for the first time, strains from citrus plants and coffee and showed that strains from coffee present higher genetic diversity than the others. These markers also have proven to be efficient for discriminating strains from the same host obtained from different geographic regions. X. fastidiosa, the causal agent of citrus variegated chlorosis, possesses genetic diversity, and the SNP markers were highly efficient for discriminating genetically close organisms.     

Site-Directed Disruption of the fimA and fimF Fimbrial Genes of Xylella fastidiosa

ABSTRACT Xylella fastidiosa causes Pierce's disease, a serious disease of grape, citrus variegated chlorosis, almond and oleander leaf scorches, and many other similar diseases. Although the complete genome sequences of several strains of this organism are now available, the function of most genes in this organism, especially those conferring virulence, is lacking. Attachment of X. fastidiosa to xylem vessels and insect vectors may be required for virulence and transmission; therefore, we disrupted fimA and fimF, genes encoding the major fimbrial protein FimA and a homolog of the fimbrial adhesin MrkD, to determine their role in the attachment process. Disruption of the fimA and fimF genes in Temecula1 and STL grape strains of X. fastidiosa was obtained by homologous recombination using plasmids pFAK and pFFK, respectively. These vectors contained a kanamycin resistance gene cloned into either the fimA or fimF genes of X. fastidiosa grape strains Temecula1 or STL. Efficiency of transformation was sufficiently high ( approximately 600 transformants per mug of pFFK DNA) to enable selection of rare recombination events. Polymerase chain reaction and Southern blot analyses of the mutants indicated that a double crossover event had occurred exclusively within the fimA and fimF genes, replacing the chromosomal gene with the disrupted gene and abolishing production of the corresponding proteins, FimA or FimF. Scanning electron microscopy revealed that fimbriae size and number, cell aggregation, and cell size were reduced for the FimA or FimF mutants of X. fastidiosa when compared with the parental strain. FimA or FimF mutants of X. fastidiosa remained pathogenic to grapevines, with bacterial populations slightly reduced compared with those of the wild-type X. fastidiosa cells. These mutants maintained their resistance to kanamycin in planta for at least 6 months in the greenhouse.     

Genetic Diversity of Xylella fastidiosa Strains from Costa Rica, São Paulo, Brazil, and United States

ABSTRACT The diversity of 42 Xylella fastidiosa strains from Costa Rica, S?o 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 S?o 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 S?o 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 Nei's 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 S?o 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 S?o Paulo.     

Vitis Resistance to Pierce's Disease Is Characterized by Differential Xylella fastidiosa Populations in Stems and Leaves

ABSTRACT The pattern of Xylella fastidiosa infection in resistant and susceptible grapevines representing a diverse selection of Vitis spp. was characterized through measurements of X. fastidiosa bacterial movement and accumulation in artificially inoculated greenhouse-grown grapevines. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was optimized for quantification of X. fastidiosa populations and tested on known amounts of X. fastidiosa added to grape tissue extracts. Predicted versus known X. fastidiosa concentrations proved to be highly correlated (R(2) = 0.99). Populations of X. fastidiosa in stem internode, stem node, petiole, and leaf blade samples from the genotypes in this study were measured at 12 weeks postinoculation using the optimized ELISA procedure. Samples from each plant part were taken at eight positions along the inoculated shoots. Systemic infection was detected in both susceptible and resistant genotypes. Resistant genotypes were characterized by significant differences in X. fastidiosa populations between stem internodes and leaves (1.0 x 10(6) and 1.1 x 10(7) cells/g of sample, respectively). In contrast, the susceptible genotypes were characterized by high mean X. fastidiosa populations in both stems and leaves (5.6 x 10(7) and 4.8 x 10(7) cells/g, respectively) the latter of which were not significantly different from the resistant genotypes. A high correlation (R(2) = 0.97) between stem X. fastidiosa numbers to previously characterized field Pierce's disease (PD) performance indicates that the quantitative ELISA measurements of X. fastidiosa in greenhouse-grown grapevines should be a useful tool for predicting PD resistance under field conditions.     

An Evaluation of the Genetic Diversity of Xylella fastidiosa Isolated from Diseased Citrus and Coffee in São Paulo, Brazil

ABSTRACT Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Café, respectively, were indistinguishable based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of S?o Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.     

Quantification of Xylella fastidiosa from Citrus Trees by Real-Time Polymerase Chain Reaction Assay

ABSTRACT Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease of sweet orange cultivars in Brazil. Polymerase chain reaction (PCR)-based assays constitute the principal diagnostic method for detection of these bacteria. In this work, we established a real-time quantitative PCR (QPCR) assay to quantify X. fastidiosa in naturally and artificially infected citrus. The X. fastidiosa cell number detected in the leaves increased according to the age of the leaf, and bacteria were not detected in the upper midrib section in young leaves, indicating temporal and spatial distribution patterns of bacteria, respectively. In addition, the X. fastidiosa cell number quantified in leaves of 'Pera' orange and 'Murcott' tangor reflected the susceptible and resistant status of these citrus cultivars. None of the 12 endophytic citrus bacteria or the four strains of X. fastidiosa nonpathogenic to citrus that were tested showed an increase in the fluorescence signal during QPCR. In contrast, all 10 CVC-causing strains exhibited an increase in fluorescence signal, thus indicating the specificity of this QPCR assay. Our QPCR provides a powerful tool for studies of different aspects of the Xylella-citrus interactions, and can be incorporated into breeding programs in order to select CVC-resistant plants more quickly.     

Isolation of Peirce's disease bacteria from grapevines in Europe

Xylella fastidiosa, the causal agent of Pierce's disease (PD) of grape, was isolated from diseased grapevines grown in Kosova, Yugoslavia. The Kosova isolate was a rod-shaped bacterium which showed a typically rippled cell wall under electron microscopy. ELISA comparisons indicated that the Kosova isolate was closely related to the U.S. PD strains and to several other strains of X. fastidiosa. When DNA extracted from diseased grapevines collected from Kosova was used as template in PCR with primer sets specific for X. fastidiosa, a band of about 730 bp diagnostic for PD bacteria was detected. DNA from the isolated Kosova bacteria and the type strain of PD yielded the same length of DNA fragment in PCR assay. The Kosova isolate was inoculated into young healthy grapevines through the roots with negative pressure applied to the shoots. Typical scald and scorch symptoms appeared on the leaves of the inoculated grapevines 40–80 days after inoculation. The same bacteria were reisolated from these inoculated diseased plants and used to reinoculate young grapevines. The reinoculated grapevines produced the same symptoms, thereby fulfilling Koch's postulates. This is the first confirmation that PD of grapes occurs in Europe.     

Grapevine phenolic compounds in xylem sap and tissues are significantly altered during infection by Xylella fastidiosa

Pierce's disease of grapevine (PD), caused by the bacterial pathogen Xylella fastidiosa, remains a serious problem for grape production in California and elsewhere. This research examined induction of phenolic compounds in grapevines ('Thompson Seedless') infected with X. fastidiosa over a 6-month period. Two months postinoculation with X. fastidiosa, catechin, digalloylquinic acid, and astringin were found at greater levels in xylem sap; multiple catechins, procyanidins, and stilbenoids were found at greater levels in xylem tissues; and precursors to lignin and condensed tannins were found at greater levels in xylem cell walls. However, such large-scale inductions of phenolic compounds were not observed 4 months after inoculation. Six months after inoculation, infected plants had significantly reduced phenolic levels in xylem sap and tissues when compared with control plants, including lowered levels of lignin and condensed tannins. At 6 months, PD symptoms were severe in infected plants and most photosynthetic tissue was abscised. These results suggest that, even though grapevine hosts may initially respond to X. fastidiosa infections with increased production of phenolic compounds, ultimately, PD causes grapevines to enter a state of decline whereby diseased hosts no longer have the resources to support secondary metabolite production, including defense-associated phenolic compounds.     

Presence of Xylella fastidiosa in Sweet Orange Fruit and Seeds and Its Transmission to Seedlings

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.     

Effects of Date of Inoculation on the Within-Plant Movement of Xylella fastidiosa and Persistence of Pierce's Disease Within Field Grapevines

ABSTRACT The effects of date of inoculation on the development of Pierce's disease (PD) were evaluated in California grapevines during 1997 through 2000 at four locations. Some vines that had been inoculated either by using blue-green sharpshooters (Graphocephala atropunctata) as vectors or mechanically by needle puncture with the PD causal bacterium Xylella fastidiosa became infected during each month and at each location where infection was attempted. Vines inoculated on the earliest inoculation dates (April to May) developed more extensive and severe PD symptoms, and only 54% of these vines recovered from PD after the following winter, compared with vines that had been inoculated during June through August, of which 88% recovered from PD after the following winter. For the 1999 inoculations, the number of vines infected at a central California site (Parlier) was higher than the number of vines infected at a north coastal site (Hopland). For the best-fitting regression equation, percent recovery of vines infected with X. fastidiosa increased significantly with date of inoculation (r(2) = 0.737) at all sites excluding Hopland. The Hopland site had the highest percentage of vines that recovered from PD (100%). At most sites, only early infection (April and May) resulted in chronic disease unless the vines were inoculated at the bases instead of the distal tips of canes. Vines inoculated early in the growing season (April and May) have less chance to recover from Pierce's disease than vines inoculated later (July and August).     

Analysis of the Spatial Patterns of Pierce's Disease Incidence in the Lower San Joaquin Valley in California

ABSTRACT The incidence of Pierce's disease (PD), caused by Xylella fastidiosa, was monitored in 11 naturally infested commercial vineyards to determine the presence of an X. fastidiosa vector, Homalodisca coagulata (glassy-winged sharpshooter [GWSS]), to examine the spatial patterns of the disease and elucidate possible influences of surrounding environments. Disease incidence ranged from <1 to 65% among vineyards in 2001. Our efforts to trap or capture potential disease vectors have indicated that the GWSS is the most likely vector. Disease incidence doubled in most vineyards during the 2002 production season. Spatial patterns of symptomatic vines in 2001 and 2002, as determined by ordinary runs analysis, showed strong evidence for within- and across-row aggregation of infected vines. In most fields, they were no disease gradients observed relative to GWSS source (e.g., citrus). Within fields, however, disease incidence displayed strong spatial dependence and a high degree of anisotropy, indicating strongly aggregated patterns of disease with distinct directional orientation. The within-row (0 degrees ) and across-row (90 degrees ) orientations generally were the predominant directions of increased disease incidence, consistent with vine-to-vine spread of X. fastidiosa. We concluded that the distribution of PD in vineyards reflected the feeding pattern of vectors carrying X. fastidiosa. Based on these results, effective PD management is likely to be based on practices that reduce significant insect vector populations and remove infected vines as soon as identified and on the use of resistant cultivars.     

Analysis of resistance to Xylella fastidiosa within a hybrid population of Pera sweet orange × Murcott tangor

Resistance to Xylella fastidiosa was evaluated within a population of 20 interspecific hybrids of Pera sweet orange and Murcott tangor under greenhouse conditions. Efficiency of inoculation, multiplication of bacteria within the plants, xylem vessel morphology, and symptom expression were analysed. The rate of infection ranged from 40 to 100% (average 70%) for all genotypes analysed. Xylella fastidiosa populations ranged from log 0·59 to log 2·13 cells mg⁻¹ tissue for the resistant hybrids. These values were significantly different ( P  = 0·05) from those obtained for the tolerant (no symptoms but bacteria recovered) or susceptible (symptoms and bacteria recovered) hybrids (log 3·02 to log 4·06 cells mg⁻¹). Xylella fastidiosa was recovered from all hybrids (log 2·31 to 5·03 CFU mg⁻¹ tissue) except the resistant ones. The first foliar symptoms appeared at least 90 days post-inoculation, the time varying according to genotype. No correlation between xylem vessel morphology and disease expression was observed, indicating that the resistance was the result of a genetic response of the host. According to this hypothesis, a high broad-based heritability index for resistance was obtained (0·96) at 210 days from X. fastidiosa inoculations, using bacterial quantification by real-time PCR, which indicated that the influence of the number of bacteria was the result of genetic rather than environmental variations.     

Pierce's Disease of Grapevines: Identification of the Primary Vectors in North Carolina

ABSTRACT In the past 10 years, the winegrape industry in the southeastern United States has experienced rapid growth; however, further expansion may be inhibited by Pierce's disease (PD). Epidemiological studies were conducted to identify the primary vectors of Xylella fastidiosa, the cause of PD of grape, by surveying sharpshooter population dynamics in the eastern Piedmont and Coastal Plain regions of North Carolina. Sharpshooter species were assessed for the presence of X. fastidiosa in the field. Leafhoppers were trapped in three vineyards in the eastern Piedmont and one vineyard in the northeastern Coastal Plain in 2004 and 2005. Four insects were identified as most abundant: Oncometopia orbona, Graphocephala versuta, Paraphlepsius irroratus, and Agalliota constricta. Adult specimens of O. orbona, G. versuta, and P. irroratus were tested for the presence of X. fastidiosa by nested polymerase chain reaction. In all, 27% of O. orbona, 28% of G. versuta, and 33% of P. irroratus trapped were positive for X. fastidiosa over the two seasons. Transmission experiments demonstrated that both O. orbona and G. versuta have the ability to transmit X. fastidiosa to grape. These vectors are likely to be important in all winegrowing regions of the Southeast, because their presence has been documented throughout the southern states. In DNA analyses, X. fastidiosa strains from insects trapped in North Carolina were genetically similar to one another and to the known "PD strain" from California. This is the first report of these two leafhopper species transmitting X. fastidiosa to grapevines in the Southeast.     

Efficacy of olive mill waste water and its derivatives in the suppression of crown gall disease of bitter almond

Olive mill waste water (OMW) and some of its indigenous bacterial strains were tested in vitro and in planta for their efficacy against crown gall disease caused by Agrobacterium tumefaciens. OMW and polyphenols displayed a high level of antibacterial activity, however the volatile fraction was less efficient as only a bacteriostatic effect was observed. In pot experiments, the percentage of bitter almond rootstock showing symptoms of crown gall was significantly reduced with the dosage rate of OMW 1% as compared to the control (highly natural infected soil treated with water). Five indigenous bacterial strains isolated from OMW exhibited an antagonistic effect against the bacterium. Based on the gene 16S rRNA sequence analysis, one isolate showed 99.2% similarity to known sequences of Bacillus subtilis, one isolate demonstrated high percentage similarities (99.3%) to the genera Bacillus pumilis, and two isolates were associated with Stenotrophomonas maltophilia and Pseudomonas putida 100% and 99.6% similarities respectively. Among these bacteria, the strain B1 proved efficient against the soil borne pathogen in vitro and pot experiments. Our study in controlled conditions suggested that the addition of OMW to soil exerts significant disease suppressiveness against A. tumefaciens. Thabet Yangui and Ali Rhouma contributed equally to this work and are regarded as joint first authors.     

Spatiotemporal colonization of Xylella fastidiosa in its vector supports the role of egestion in the inoculation mechanism of foregut-borne plant pathogens

The pathogen that causes Pierce's disease of grapevine, Xylella fastidiosa, is the only known bacterial, arthropod-transmitted plant pathogen that does not circulate in the vector's hemolymph. Instead, bacteria are foregut-borne, persistent in adult vectors but semipersistent in immatures (i.e., bacteria colonize cuticular surfaces of the anterior foregut, are retained for hours to days, but are lost during molting). Yet, exactly how a sharpshooter vector inoculates bacteria from foregut acquisition sites is unknown. The present study used confocal laser-scanning microscopy to identify locations in undissected, anterior foreguts of the glassy-winged sharpshooter colonized by green fluorescent protein-expressing X. fastidiosa. Spatial and temporal distributions of colonizing X. fastidiosa were examined daily over acquisition access periods of 1 to 6 days for both contaminated field-collected and clean laboratory-reared Homalodisca vitripennis. Results provide the first direct, empirical evidence that established populations of X. fastidiosa can disappear from vector foreguts over time. When combined with existing knowledge on behavior, physiology, and functional anatomy of sharpshooter feeding, present results support the idea that the disappearance is caused by outward fluid flow (egestion) not inward flow (ingestion) (i.e., swallowing). Thus, results support the hypothesis that egestion is a critical part of the X. fastidiosa inoculation mechanism. Furthermore, results suggest a cyclical, spatiotemporal pattern of microbial colonization, disappearance, and recolonization in the precibarium. Colonization patterns also support two types of egestion, termed rinsing and discharging egestion herein. Finally, comparison of acquisition results for field-collected versus laboratory-reared sharpshooters suggest that there may be competitive binding for optimum acquisition sites in the foregut. Therefore, successful inoculation of X. fastidiosa may depend, in large part, on vector load in the precibarium.     

Specific characters of 16S rRNA gene and 16S–23S rRNA internal transcribed spacer sequences of Xylella fastidiosa pear leaf scorch strains

Pear leaf scorch, the only Xylella fastidiosa-induced disease reported from Taiwan, was found in area where the variety Hengshan (Pyrus pyrifolia) was grown. Strains of pear leaf scorch Xyl. fastidiosa (XF-PLS) shared similarities to strains of other host origins in the requirement of complex medium and the exhibition of rippled cell walls, however, recent serological and molecular biology studies showed difference among them. Five strains of XF-PLS were compared with 20 other strains originally isolated from almond, oleander, pecan, plum, peach, mulberry, grapes, citrus, coffee, and sycamore by sequence analyses of the 16S rRNA gene and 16S–23S rRNA internal transcribed spacer region (ITS). When sequences of 16S rRNA gene based on fragment size of 1,537–1,540 bp were compared, the similarity index among 5 XF-PLS strains was 99.3–99.8%, whereas it was 97.8–98.6% between XF-PLS strains and strains from other hosts. When sequences of 16S–23S rRNA ITS based on fragment size of 510–540 bp were compared, the similarity index among 5 XF-PLS strains was 99.0–100%, whereas it was 80.7–82% between XF-PLS strains and strains from other hosts. Multiple sequence alignments led to the identification of 5 polymorphic nucleotides in the 16S rRNA gene among the 25 Xyl. fastidiosa strains, and there were considerable variations in the nucleotide sequences of 16S–23S rRNA ITS between XF-PLS and the other 20 Xyl. fastidiosa strains. The phylogenetic trees revealed that XF-PLS strains were separated from strains of other hosts. Strains of other hosts were divided into four subgroups: strains from (1) oleander, (2) grape, almond M23 and mulberry, (3) citrus and coffee, and (4) pecan, peach, plum, sycamore and almond M12. Results indicate that XF-PLS strains were not closely related to the above-mentioned strains from other hosts and could possibly belong to a new subspecies of Xyl. fastidiosa.     

Antagonistic Interactions Between Strains of Xanthomonas oryzae pv. oryzae

ABSTRACT The ability of some phytopathogenic bacterial strains to inhibit the growth of others in mixed infections has been well documented. Here we report that such antagonistic interactions occur between several wild-type strains of the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae. In mixed inoculations, a wild-type Philippine strain was found to inhibit the growth of a wild-type Korean strain. Furthermore, a nonpathogenic mutant of the Philippine strain maintained these antagonistic properties. Growth curve analysis indicated that both the wild-type Philippine strain and its nonpathogenic mutant inhibited the growth of the Korean strain 2 days after infection and prior to the onset of disease symptoms. When mixed with the nonpathogenic mutant, 10 out of 18 diverse wild-type X. oryzae pv. oryzae strains did not cause disease. Conversely, three of the strains that were not affected by the nonpathogenic mutant were found to inhibit the growth of both the wild-type and mutant Philippine strains, indicating that antagonism is widespread and strain specific. The observed growth inhibition occurred only in planta and did not correlate with bacteriocin activity in vitro. Antagonistic interactions also were found to affect resistance (R) gene-mediated resistance. The R gene Xa21 was capable of protecting rice plants coinoculated with nonantagonistic virulent and avirulent strains; however, when avirulent strains were coinoculated with virulent antagonistic strains, disease ensued. Taken together, these results indicate that X. oryzae pv. oryzae has evolved strategies to compete with rival strains in a fashion that allows virulent strains to evade R gene-mediated protection even when avirulent strains are present in the inoculum.