An Evolutionary Perspective of Pierce's Disease of Grapevine,Citrus Variegated Chlorosis,and Mulberry Leaf Scorch Diseases

Xylella fastidiosa causes diseases on a growing list of economically important plants. An understanding of how xylellae diseases originated and evolved is important for disease prevention and management. In this study, we evaluated the phylogenetic relationships of X. fastidiosa strains from citrus, grapevine, and mulberry through the analyses of random amplified polymorphic DNAs (RAPDs) and conserved 16S rDNA genes. RAPD analysis emphasized the vigorous genome-wide divergence of X. fastidiosa and detected three clonal groups of strains that cause Pierce's disease (PD) of grapevine, citrus variegated chlorosis (CVC), and mulberry leaf scorch (MLS). Analysis of 16S rDNA sequences also identified the PD and CVC groups, but with a less stable evolutionary tree. MLS strains were included in the PD group by the 16S rDNA analysis. The Asiatic origins of the major commercial grape and citrus cultivars suggest the recent evolution of both PD and CVC disease in North and South America, respectively, since X. fastidiosa is a New World organism. In order to prevent the development of new diseases caused by X. fastidiosa, it is important to understand the diversity of X. fastidiosa strains, how strains of X. fastidiosa select their hosts, and their ecological roles in the native vegetation. Received: 7 February 2002 / Accepted: 7 March 2002     

Utilization of the Etest Assay for Comparative Antibiotic Susceptibility Profiles of Citrus Variegated Chlorosis and Pierce’s Disease Strains of Xylella fastidiosa

Xylella fastidiosa has a wide host range. Isolates of this bacterium that cause diseases in citrus (CVC) and grapes (PD) share 98% genome homology, and 95.7% amino acid identity. Drug resistance genes show a higher level of divergence and may be involved in the X. fastidiosa–host interaction. Antibiotic susceptibility of CVC and PD strains were compared utilizing the Etest strip method (AB Biodisk). Etest is applicable for fastidious slow-growing organisms due to its reproducibility. Results showed that the CVC strain was resistant to bacitracin, cefotaxime, and trimethoprim, and susceptible to chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, and tetracycline. The PD strain was susceptible to all tested antibiotics, except kanamycin and trimethoprim. Both isolates produced a class C β-lactamase. These data support previous antibiotic studies and gene discrepancies found in the sequencing data of PD and CVC strains. These results demonstrate the efficacy of utilizing Etest assays for X. fastidiosa strains.     

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.     

A Suitable <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> CVC Strain for Post-Genome Studies

The genome sequence of the pathogen Xylella fastidiosa Citrus Variegated Chlorosis (CVC) strain 9a5c has revealed many genes related to pathogenicity mechanisms and virulence determinants. However, strain 9a5c is resistant to genetic transformation, impairing mutant production for the analysis of pathogenicity mechanisms and virulence determinants of this fastidious phytopathogen. By screening different strains, we found out that cloned strains J1a12, B111, and S11400, all isolated from citrus trees affected by CVC, are amenable to transformation, and J1a12 has been used as a model strain in a functional genomics program supported by FAPESP (São Paulo State Research Foundation). However, we have found that strain J1a12, unlike strains 9a5c and B111, was incapable of inducing CVC symptoms when inoculated in citrus plants. We have now determined that strain B111 is an appropriate candidate for post-genome studies of the CVC strain of X. fastidiosa.     

Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis

By cloning and sequencing specific randomly amplified polymorphic DNA (RAPD) products, we have developed pairs of PCR primers that can be used to detect Xylella fastidiosa in general, and X. fastidiosa that cause citrus variegated chlorosis (CVC) specifically. We also identified a CVC-specific region of the X. fastidiosa genome that contains a 28-nucleotide insertion, and single base changes that distinguish CVC and grape X. fastidiosa strains. When using RAPD products to develop specific PCR primers, we found it most efficient to screen for size differences among RAPD products rather than presence/absence of a specific RAPD band.     

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.     

Whole Genome Sequences of Two Xylella fastidiosa Strains (M12 and M23) Causing Almond Leaf Scorch Disease in California

Xylella fastidiosa is a Gram-negative plant-pathogenic bacterium causing many economically important diseases, including almond leaf scorch disease (ALSD) in California. Genome information greatly facilitates research on this nutritionally fastidious organism. Here we report the complete genome sequences of two ALSD strains of this bacterium, M12 and M23.Xylella fastidiosa is a Gram-negative and nutritionally fastidious plant-pathogenic bacterium that causes almond leaf scorch disease (ALSD) and Pierce''s disease (PD) of grapevine. In 2003, we isolated two ALSD strains of X. fastidiosa from almond trees in Kern County in the San Joaquin Valley of California. Strain M12 caused only ALSD, and strain M23 caused both ALSD and PD. 16S rRNA gene sequences were analyzed; strain M12 was regarded as A genotype and strain M23 as G genotype (1), corresponding to X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa (4), respectively.Genomic DNAs of X. fastidiosa strains M12 and M23 were extracted from pure culture in PW medium (1). The random shotgun method was used for genome sequencing. Large-insert (40-kb), medium-insert (8-kb), and small-insert (3-kb) random libraries were partially sequenced, and sequences were assembled with parallel Phrap (High Performance Software, LLC). Possible misassemblies were corrected with the Dupfinisher software program (2). Gaps between contigs were closed by custom primer walking through PCR amplification. Annotation of the assembled genome sequence was carried out with the genome annotation system Oak Ridge Genome Annotation and Analysis (ORGAA) Pipelines and JGI Integrated Microbial Genomes (IMG) server (3). A combined gene prediction strategy was applied by means of the GLIMMER 2.0 system and the CRITICA program suite, along with postprocessing by the RBSfinder tool. tRNA genes were identified using the tRNAscan-SE server. The deduced proteins were functionally characterized by automated searches in public databases, including SWISS-PROT and TrEMBL, Pfam, TIGRFAM, InterPro, and KEGG. Additionally, the SignalP, helix-turn-helix, and TMHMM software programs were applied. Finally, each gene was functionally classified by assigning a clusters of orthologous groups (COG) number and corresponding COG category and gene ontology numbers. Detailed information about the genome properties, genome annotation, and its related references can be obtained from the JGI Integrated Microbial Genomes website at http://img.jgi.doe.gov/pub/.The genome of X. fastidiosa M12 consists of a single, circular, 2,475,130-nucleotide (nt) chromosome that has a GC content of 51.9%. A total of 2,368 protein-encoding genes are predicted, 2,104 of which have been assigned a tentative function. The genome of X. fastidiosa M23 consists of a single, circular, 2,535,690-nt chromosome that has a GC content of 51.7%. A total of 2,280 protein-encoding genes are predicted, 2,161 of which have been assigned a tentative function. In addition, a circular plasmid of 38,297 nt, pXFAS01, with a GC content of 49%, was also identified in strain M23 but was absent in strain M12. Both strains had two identical rRNA operons in their chromosomes.     

Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain ofXylella fastidiosa

A xylem-limited bacterium resemblingXylella fastidiosa has been shown previously by electron mmcroscopy to be associated with citrus variegated chlorosis (CVC), a new disease of sweet organe tress in Brazil. A bacterium was consistently cultured from plant tissues from CVC twigs of sweet orange trees but not from tissues of healthy trees on several cell-free media known to support the growth ofXylella fastidiosa. Bacterial colonies typical ofX. fastidiosa became visible on PW, CS20, and PD2 agar media after 5 and 7–10 days of incubation, respectively. The cells of the CVC bacterium were rod-shaped, 1.4–3 m in length, and 0.2–0.4 m in diameter, with rippled walls. An antiserum against an isolate (8.1.b) of the bacterium gave strong positive reactions to double-antibody-sandwich (DAS), enzyme-linked immunosorbent assay (ELISA) with other cultured isolates from CVC citrus, as well as with several type strains ofX. fastidiosa. This result indicates that the CVC bacterium is a strain ofX. fastidiosa. ELISA was also highly positive with all leaves tested from CVC-affected shoots. Leaves from symptomless tress reacted negatively. Sweet organe seedlings inoculated with a pure culture of the CVC bacterium supported multiplication of the bacterium, which became systemic with 6 months after inoculation and could be reisolated from the inoculated seedlings. Symptoms characteristic of CVC developed 9 months post inoculation.     

Strains of Xylella fastidiosa Rapidly Distinguished by Arbitrarily Primed-PCR

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0.872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors. Received: 12 October 1999 / Accepted: 16 November 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.     

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.     

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     

New Coffee Plant-Infecting Xylella fastidiosa Variants Derived via Homologous Recombination

Xylella fastidiosa is a xylem-limited phytopathogenic bacterium endemic to the Americas that has recently emerged in Asia and Europe. Although this bacterium is classified as a quarantine organism in the European Union, importation of plant material from contaminated areas and latent infection in asymptomatic plants have engendered its inevitable introduction. In 2012, four coffee plants (Coffea arabica and Coffea canephora) with leaf scorch symptoms growing in a confined greenhouse were detected and intercepted in France. After identification of the causal agent, this outbreak was eradicated. Three X. fastidiosa strains were isolated from these plants, confirming a preliminary identification based on immunology. The strains were characterized by multiplex PCR and by multilocus sequence analysis/typing (MLSA-MLST) based on seven housekeeping genes. One strain, CFBP 8073, isolated from C. canephora imported from Mexico, was assigned to X. fastidiosa subsp. fastidiosa/X. fastidiosa subsp. sandyi. This strain harbors a novel sequence type (ST) with novel alleles at two loci. The two other strains, CFBP 8072 and CFBP 8074, isolated from Coffea arabica imported from Ecuador, were allocated to X. fastidiosa subsp. pauca. These two strains shared a novel ST with novel alleles at two loci. These MLST profiles showed evidence of recombination events. We provide genome sequences for CFBP 8072 and CFBP 8073 strains. Comparative genomic analyses of these two genome sequences with publicly available X. fastidiosa genomes, including the Italian strain CoDiRO, confirmed these phylogenetic positions and provided candidate alleles for coffee plant adaptation. This study demonstrates the global diversity of X. fastidiosa and highlights the diversity of strains isolated from coffee plants.     

Enhanced Reliability and Accuracy for Field Deployable Bioforensic Detection and Discrimination of Xylella fastidiosa subsp. pauca,Causal Agent of Citrus Variegated Chlorosis Using Razor Ex Technology and TaqMan Quantitative PCR

A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.     

TolC is required for pathogenicity of Xylella fastidiosa in Vitis vinifera grapevines

Xylella fastidiosa infects a wide range of hosts and causes serious diseases on some of them. The complete genomic sequences of both a citrus variegated chlorosis (CVC) and a Pierce's disease (PD) strain revealed two type I protein secretion plus two multidrug resistance efflux systems, and all evidently were dependent on a single tolC homolog. Marker exchange mutagenesis of the single tolC gene in PD strain Temecula resulted in a total loss of pathogenicity on grape. Importantly, the tolC- mutant strains were not recovered after inoculation into grape xylem, strongly indicating that multidrug efflux is critical to survival of this fastidious pathogen. Both survival and pathogenicity were restored by complementation using tolC cloned in shuttle vector pBBR1MCS-5, which was shown to replicate autonomously, without selection, for 60 days in Temecula growing in planta. These results also demonstrate the ability to complement mutations in X. fastidiosa.     

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.     

Characterization of the Xylella fastidiosa PD1311 gene mutant and its suppression of Pierce's disease on grapevines

Xylella fastidiosa causes Pierce's disease (PD) on grapevines, leading to significant economic losses in grape and wine production. To further our understanding of X. fastidiosa virulence on grapevines, we examined the PD1311 gene, which encodes a putative acyl‐coenzyme A (acyl‐CoA) synthetase, and is highly conserved across Xylella species. It was determined that PD1311 is required for virulence, as the deletion mutant, ΔPD1311, was unable to cause disease on grapevines. The ΔPD1311 strain was impaired in behaviours known to be associated with PD development, including motility, aggregation and biofilm formation. ΔPD1311 also expressed enhanced sensitivity to H₂O₂ and polymyxin B, and showed reduced survival in grapevine sap, when compared with wild‐type X. fastidiosa Temecula 1 (TM1). Following inoculation, ΔPD1311 could not be detected in grape shoots, which may be related to its altered growth and sensitivity phenotypes. Inoculation with ΔPD1311 2 weeks prior to TM1 prevented the development of PD in a significant fraction of vines and eliminated detectable levels of TM1. In contrast, vines inoculated simultaneously with TM1 and ΔPD1311 developed disease at the same level as TM1 alone. In these vines, TM1 populations were distributed similarly to populations in TM1‐only inoculated plants. These findings suggest that, through an indirect mechanism, pretreatment of vines with ΔPD1311 suppresses pathogen population and disease.     

Expression of Xylella fastidiosa Fimbrial and Afimbrial Proteins during Biofilm Formation

Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.Aggregative growth is a common feature in the microbial world, and its discovery radically changed our concept of microbial growth dynamics. A cellular aggregate adhering to a surface is known as a biofilm. It has important characteristics, such as greater resistance to antimicrobial compounds (34, 54), increased capacity of the cells to take up nutrients from the environment (59), and higher detoxification efficiency resulting from an increase in expression of genes encoding efflux pumps (43). These characteristics give the biofilm cells a great adaptive advantage.Biofilm growth also confers advantages to plant pathogens by promoting virulence and protection against plant defense responses. Bacteria can colonize different niches in the plant, from aerial surfaces to roots and the vascular system, and biofilm formation can play a role at all of these sites of colonization. In the vessels, biofilms are very important since the cells need to survive in a competitive habitat where plant defense compounds are produced in response to infection (7).Biofilm development is divided into at least the following five phases: (i) reversible attachment, (ii) irreversible attachment, (iii) beginning of maturation, (iv) mature biofilm, and (v) dispersion (13, 50). In Xylella fastidiosa strain 9a5c, the maturation phase occurs between days 15 and 20 in vitro, while dispersion occurs between days 25 and 30, as observed by our analysis of biofilm formation using different methods, including scanning electron microscopy and quantification of exopolysaccharides, biomass, and the total protein (unpublished data). The establishment and development of biofilms of plant-colonizing bacteria share several features with the establishment and development of biofilms of human bacterial pathogens, such as regulation by quorum sensing, nutrient starvation regulation, and phase variation. Motility is also an important factor not only for the initiation and development of the biofilm but also for dispersion (50). Attachment is mediated by surface-associated structures, which include both polysaccharides and proteins classified as fimbrial and afimbrial adhesins, depending on the structure to which they contribute. Fimbrial adhesins form filamentous structures, while afimbrial adhesins produce projections on the outer membrane (23).X. fastidiosa, a Gram-negative phytopathogen that grows as a biofilms in both plant xylem vessels and the cybarium of insect vectors, is a major threat to plant production around the world. In Brazil, it has a major economic impact on citriculture since it causes citrus variegated chlorosis disease (CVC) (35, 39, 42). The biofilm formed by X. fastidiosa blocks the xylem vessels of susceptible citrus plants, impairing water flow. This blockage leads to a drastic reduction in fruit size (32) and, consequently, severe economic losses resulting from reduced plant productivity (4).Due to the economic damage caused by CVC, there has been a major effort to generate more information about its biology. This led to sequencing of the genome of the pathogen. The X. fastidiosa genome harbors a wide variety of genes encoding adhesins (53). Bacterial cell surface adhesins are important in the initial phases of adherence to surfaces, as well as in bacterium-bacterium interactions and microcolony development (15). Insight into X. fastidiosa has also come from genome analysis of a strain of X. fastidiosa which causes Pierce''s disease of grape (58). Studies of this strain showed that the cellular aggregation process involves type I and type IV fimbrial adhesins. The two types of fimbriae present different adhesion forces that help bacteria adhere to a substrate (10, 30). Adhesion proteins have also been demonstrated to mediate adherence to carbohydrates of leafhopper foregut surfaces (27). In addition, both fimbrial and afimbrial adhesins are important for plant pathogenicity (38, 41). However, the expression of these proteins during X. fastidiosa biofilm formation either in vitro or in planta is still poorly understood. For X. fastidiosa strains causing CVC, nothing is known about the role of these proteins in pathogenicity or biofilm formation, although some adhesion-encoding genes, such as pilA2, pilC, xadA1, and xadA2, were found to be upregulated either in virulent strains of X. fastidiosa or during biofilm formation (12, 14). These results suggest that the biofilm mode of growth is important for successful colonization of the citrus host by X. fastidiosa strains that cause CVC. In this work we focused on the temporal expression of the PilA2 and PilC fimbrial proteins and XadA1 and XadA2, which are afimbrial adhesins, during in vitro development of X. fastidiosa CVC biofilms. We demonstrated that the temporal and spatial patterns of expression of the fimbrial and afimbrial adhesins are very different during biofilm development in vitro. Moreover, we also verified that these adhesins are present in X. fastidiosa cells in symptomatic plants of three different hosts (citrus, periwinkle, and hibiscus).     

Detection and Visualization of an Exopolysaccharide Produced by Xylella fastidiosa In Vitro and In Planta

Many phytopathogenic bacteria, such as Ralstonia solanacearum, Pantoea stewartii, and Xanthomonas campestris, produce exopolysaccharides (EPSs) that aid in virulence, colonization, and survival. EPS can also contribute to host xylem vessel blockage. The genome of Xylella fastidiosa, the causal agent of Pierce's disease (PD) of grapevine, contains an operon that is strikingly similar to the X. campestris gum operon, which is responsible for the production of xanthan gum. Based on this information, it has been hypothesized that X. fastidiosa is capable of producing an EPS similar in structure and composition to xanthan gum but lacking the terminal mannose residue. In this study, we raised polyclonal antibodies against a modified xanthan gum polymer similar to the predicted X. fastidiosa EPS polymer. We used enzyme-linked immunosorbent assay to quantify production of EPS from X. fastidiosa cells grown in vitro and immunolocalization microscopy to examine the distribution of X. fastidiosa EPS in biofilms formed in vitro and in planta and assessed the contribution of X. fastidiosa EPS to the vascular occlusions seen in PD-infected grapevines.