Specific Detection of Xylella fastidiosa Pierce's Disease Strains

Pierce's disease (PD, Xylella fastidiosa) of grapevine is the primary pathogen limiting vinifera grape production in Florida and other regions of the southeastern United States. Quick and accurate detection of PD strains is essential for PD studies and control. A unique random amplified polymorphic DNA (PD1-1-2) was isolated from a PD strain from Florida. Fragment PD1-1-2 was cloned, sequenced, and found to be 1005 bp in length. PCR primers were designed to utilize these sequence data for PD strain detection. One primer set (XF176f–XF954r) amplified a 779-bp DNA fragment from 34 PD strains including seven pathotypes of X. fastidiosa, but not from strains of Xanthomonas campestris pv. campestris, Xan. vesicatoria or Escherichia coli. A second primer set (XF176f and XF686r) amplified a 511-bp fragment specific to 98 PD strains, but not from strains of citrus variegated chlorosis, mulberry leaf scorch, oak leaf scorch, periwinkle wilt, phony peach, or plum leaf scald. Sequence analysis indicated that RAPD fragment PD1-1-2 contains a Ser-tRNA gene. The PD-specific region includes a TaqI restriction site (TCGA) and is 150 bp downstream of the Ser-tRNA gene. Received: 1 March 1999 / Accepted: 5 April 1999     

Biological Traits of Xylella fastidiosa Strains from Grapes and Almonds

Xylella fastidiosa is a xylem-limited bacterium that causes various diseases, among them Pierce's disease of grapevine (PD) and almond leaf scorch (ALS). PD and ALS have long been considered to be caused by the same strain of this pathogen, but recent genetic studies have revealed differences among X. fastidiosa isolated from these host plants. We tested the hypothesis that ALS is caused by PD and ALS strains in the field and found that both groups of X. fastidiosa caused ALS and overwintered within almonds after mechanical inoculation. Under greenhouse conditions, all isolates caused ALS and all isolates from grapes caused PD. However, isolates belonging to almond genetic groupings did not cause PD in inoculated grapes but systemically infected grapes with lower frequency and populations than those belonging to grape strains. Isolates able to cause both PD and ALS developed 10-fold-higher concentrations of X. fastidiosa in grapes than in almonds. In the laboratory, isolates from grapes overwintered with higher efficiency in grapes than in almonds and isolates from almonds overwintered better in almonds than in grapes. We assigned strains from almonds into groups I and II on the basis of their genetic characteristics, growth on PD3 solid medium, and bacterial populations within inoculated grapevines. Our results show that genetically distinct strains from grapes and almonds differ in population behavior and pathogenicity in grapes and in the ability to grow on two different media.     

Genetic Differences between Two Strains of Xylella fastidiosa Revealed by Suppression Subtractive Hybridization

Suppression subtractive hybridization was used to rapidly identify 18 gene differences between a citrus variegated chlorosis (CVC) strain and a Pierce's disease of grape (PD) strain of Xylella fastidiosa. The results were validated as being highly representative of actual differences by comparison of the completely sequenced genome of a CVC strain with that of a PD strain.     

Differentiation of Strains of Xylella fastidiosa by a Variable Number of Tandem Repeat Analysis

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although mono- and dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Nei's measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a H value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.     

Genetic Diversity of Pierce's Disease Strains and Other Pathotypes of Xylella fastidiosa

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.     

Surface motility of Xylella fastidiosa visualized by oblique illumination

Stereomicroscopic observations using oblique illuminations revealed the presence of two types of movement trails by Xylella fastidiosa strains (A- and G-genotypes) isolated from almond-leaf scorch samples on the surface of PW and PD3 culture media. The A-genotype strains showed curved motility trails, and the G-genotype strains showed straight motility trails. Haloes were found around some G-genotype colonies due to the excretion of unknown factors and (or) compounds, which might be related to bacterial surface motility.     

Grapevine xylem sap enhances biofilm development by Xylella fastidiosa

Xylella fastidiosa is able to form biofilms within xylem vessels of many economically important crops. Vessel blockage is believed to be a major contributor to disease development caused by this bacterium. This report shows that Vitis riparia xylem sap increases growth rate and induces a characteristic biofilm architecture as compared with biofilms formed in PD2 and PW media. In addition, stable cultures could be maintained, frozen and reestablished in xylem sap. These findings are important as xylem sap provides a natural medium that facilitates the identification of virulence determinants of Pierce's disease.     

Specific Detection and Identification of American Mulberry-Infecting and Italian Olive-Associated Strains of Xylella fastidiosa by Polymerase Chain Reaction

Xylella fastidiosa causes bacterial leaf scorch in many landscape trees including elm, oak, sycamore and mulberry, but methods for specific identification of a particular tree host species-limited strain or differentiation of tree-specific strains are lacking. It is also unknown whether a particular landscape tree-infecting X. fastidiosa strain is capable of infecting multiple landscape tree species in an urban environment. We developed two PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced to date. PCR using the primers allowed for detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or non-specific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally X. fastidiosa-infected oak, elm and sycamore trees growing in the same region where naturally infected mulberry trees were grown. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly but interestingly, the unique open reading frame conserved in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. When the primer set was tested against naturally infected olive plant samples collected in Italy, it allowed for detection of olive-associated strains of X. fastidiosa in Italy. This PCR assay, therefore, will also be useful for detection and identification of the Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution and biology of this new group of X. fastidiosa strains.     

Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa

A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics.     

Mapping analysis of the Xylella fastidiosa genome

A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.fastidiosa sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.     

Prediction of potential thermostable proteins in Xylella fastidiosa

The average protein (E+K)/(Q+H) ratio in organisms has already been demonstrated to have a strong correlation with their optimal growth temperature. Employing the Thermo-Search web tool, we used this ratio as a basis to look for thermostable proteins in a mesophile, Xylella fastidiosa. Nine proteins were chosen to have their three-dimensional structures modeled by homology, using mainly proteins from mesophiles as templates. Resulting models featured a high number of hydrophobic interactions, a property that has been previously associated with thermostability. These results demonstrate the interesting possibility of using the (E+K)/(Q+H) ratio to find individual thermostable proteins in mesophilic organisms.     

Xylella fastidiosa Does Not Occur in Lebanon

Xylella fastidiosa has been reported as responsible for a devastating disease on olive trees in Apulia region (south‐eastern Italy), characterized by a quick decline syndrome. In Lebanon, the pathogen was recently associated with leaf scorch symptoms on oleander, and reports on leaf scorch and dieback of olive trees branches by technicians and farmers have shown an increasing trend in the main agricultural areas. To assess the occurrence and distribution of the pathogen in Lebanon, samples of twigs from olive trees (82), olive seedlings (26), grapevine (30), oleander (32) and ornamentals imported from Italy (48) were analysed by isolation on four agarized media, serological techniques (ELISA and DTBIA) using Xylella fastidiosa‐specific antibodies and by PCR, using three specific sets of primers. Results unequivocally demonstrated that all the collected samples were free from the pathogen. As well, both detection protocols and attempts at isolating the pathogen on agarized media demonstrated that oleander samples gathered from American University campus in Beirut, where X. fastidiosa was previously reported, were not infected. Nevertheless, continuous monitoring and rigorous control measures of propagative materials are necessary to prevent the introduction of Xylella fastidiosa in Lebanon.     

Initial Genetic Analysis of Xylella fastidiosa in Texas

Xylella fastidiosa is the causative agent of Pierce’s Disease of grape. No published record of X. fastidiosa genetics in Texas exists despite growing financial risk to the U.S. grape industry, a Texas population of the glassy-winged sharpshooter insect vector (Homalodisca vitripennis) now spreading in California, and evidence that the bacterium is ubiquitous to southern states. Using sequences of conserved gyrB and mopB genes, we have established at least two strains in Texas, grape strain and ragweed strain, corresponding genetically with subsp. piercei and multiplex, respectively. The grape strain in Texas is found in Vitis vinifera varieties, hybrid vines, and wild Vitis near vineyards, whereas the ragweed strain in Texas is found in annuals, shrubs, and trees near vineyards or other areas. RFLP and QRT PCR techniques were used to differentiate grape and ragweed strains with greater efficiency than sequencing and are practical for screening numerous X. fastidiosa isolates for clade identity.     

Growth optimization procedures for the phytopathogen Xylella fastidiosa

For the first time, growth curves are shown for the phytopathogen Xylella fastidiosa on traditional growth media such as PW (periwinkle wilt), BCYE (buffered charcoal yeast extract), and on new ones such as GYE (glutamate yeast extract) and PYE (phosphate yeast extract) that were developed in this work. The optimal growth conditions on solid and liquid media as well as their measurements are presented, by using total protein content and turbidity determinations. The results demonstrated that yeast extract provided sufficient nutrients for X. fastidiosa, since the cells grew well on PYE medium.     

Transposon mutagenesis of Xylella fastidiosa by electroporation of Tn5 synaptic complexes

Pierce's disease, a lethal disease of grapevine, is caused by Xylella fastidiosa, a gram-negative, xylem-limited bacterium that is transmitted from plant to plant by xylem-feeding insects. Strains of X. fastidiosa also have been associated with diseases that cause tremendous losses in many other economically important plants, including citrus. Although the complete genome sequence of X. fastidiosa has recently been determined, the inability to transform or produce transposon mutants of X. fastidiosa has been a major impediment to understanding pathogen-, plant-, and insect-vector interactions. We evaluated the ability of four different suicide vectors carrying either Tn5 or Tn10 transposons as well as a preformed Tn5 transposase-transposon synaptic complex (transposome) to transpose X. fastidiosa. The four suicide vectors failed to produce any detectable transposition events. Electroporation of transposomes, however, yielded 6 x 10(3) and 4 x 10(3) Tn5 mutants per microg of DNA in two different grapevine strains of X. fastidiosa. Molecular analysis showed that the transposition insertions were single, independent, stable events. Sequence analysis of the Tn5 insertion sites indicated that the transpositions occur randomly in the X. fastidiosa genome. Transposome-mediated mutagenesis should facilitate the identification of X. fastidiosa genes that mediate plant pathogenicity and insect transmission.     

Detection and characterization of protease secreted by the plant pathogen Xylella fastidiosa

Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a copolymerized substrate. Three major protein bands were produced by the citrus strain with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9,713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) activity gel indicated that the protease of Xylella fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30 degrees C with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and beta-1,3-glucanase activities were also detected in cultures of Xylella fastidiosa (citrus). From these results, it is suggested that proteases produced by strains of Xylella fastidiosa from citrus and grape, belong to the serine- and metallo-protease group, respectively.     

Upstream migration of Xylella fastidiosa via pilus-driven twitching motility

Xylella fastidiosa is a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plant's vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered that X. fastidiosa migrates via type IV-pilus-mediated twitching motility at speeds up to 5 mum min(-1) against a rapidly flowing medium (20,000 mum min(-1)). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 mum) and short type I pili (0.4 to 1.0 mum). We further demonstrated that two knockout mutants (pilB and pilQ mutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions in pilB or pilQ (possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion in fimA (possessing only type IV pili) is biofilm deficient.     

Molecular models for shikimate pathway enzymes of Xylella fastidiosa

The Xylella fastidiosa is a bacterium that is the cause of citrus variegated chlorosis (CVC). The shikimate pathway is of pivotal importance for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Putative structural differences in the enzymes from the shikimate pathway, between the proteins of bacterial origin and those of plants, could be used for the development of a drug for the control of CVC. However, inhibitors for shikimate pathway enzymes should have high specificity for X. fastidiosa enzymes, since they are also present in plants. In order to pave the way for structural and functional efforts towards antimicrobial agent development, here we describe the molecular modeling of seven enzymes of the shikimate pathway of X. fastidiosa. The structural models of shikimate pathway enzymes, complexed with inhibitors, strongly indicate that the previously identified inhibitors may also inhibit the X. fastidiosa enzymes.