Influence of Culture Medium pH on Growth, Aggregation, and Biofilm Formation of Xylella fastidiosa

The major feature of Xylella fastidiosa growing in its hosts, as well as in culture media, is its cellular aggregation and biofilm formation, leading to partial obstruction of the xylem causing water stress in the plant. We report that growth, aggregation, and biofilm formation of X. fastidiosa are influenced by the medium pH. We have verified that X. fastidiosa cell aggregation is reversibly inhibited by decreasing the medium pH from 6.6 to 6.4. Biofilm formation on glass walls was affected as well, and a concomitant decrease in cell multiplication was observed below pH 6.4. The manipulation of culture medium pH can be used as a tool for the cloning of X. fastidiosa strains isolated from plant hosts, because different strains can inhabit the same plant. Also, X. fastidiosa mutants produced by gene manipulation can be isolated from cell aggregates containing transformed and untransformed cells.     

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).     

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.     

Growth and siderophore production of Xylella fastidiosa under iron-limited conditions

In this study, the production of siderophores by Xylella fastidiosa from the citrus bacteria isolate 31b9a5c (FAPESP – ONSA, Brazil) was investigated. The preliminary evidence supporting the existence of siderophore in X. fastidiosa was found during the evaluation of sequencing data generated in our lab using the BLAST-X tool, which indicated putative open reading frames (ORFs) associated with iron-binding proteins. In an iron-limited medium siderophores were detected in the supernatant of X. fastidiosa cultures. The endophytic bacterium Methylobacterium extorquens was also evaluated. Capillary electrophoresis was used to separate putative siderophores produced by X. fastidiosa. The bacterial culture supernatants of X. fastidiosa were identified negative for hydroxamate and catechol and positive for M. extorquens that secreted hydroxamate-type siderophores.     

A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism.     

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.     

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.     

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.     

Natural competence and recombination in the plant pathogen Xylella fastidiosa

Homologous recombination is one of many forces contributing to the diversity, adaptation, and emergence of pathogens. For naturally competent bacteria, transformation is one possible route for the acquisition of novel genetic material. This study demonstrates that Xylella fastidiosa, a generalist bacterial plant pathogen responsible for many emerging plant diseases, is naturally competent and able to homologously recombine exogenous DNA into its genome. Several factors that affect transformation and recombination efficiencies, such as nutrient availability, growth stage, and methylation of transforming DNA, were identified. Recombination was observed in at least one out of every 10(6) cells when exogenous plasmid DNA was supplied and one out of every 10(7) cells when different strains were grown together in vitro. Based on previous genomic studies and experimental data presented here, there is mounting evidence that recombination can occur at relatively high rates and could play a large role in shaping the genetic diversity of X. fastidiosa.     

Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa

Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer.     

Whole-genome analysis of transporters in the plant pathogen Xylella fastidiosa.

The transport systems of the first completely sequenced genome of a plant parasite, Xylella fastidiosa, were analyzed. In all, 209 proteins were classified here as constitutive members of transport families; thus, we have identified 69 new transporters in addition to the 140 previously annotated. The analysis lead to several hints on potential ways of controlling the disease it causes on citrus trees. An ADP:ATP translocator, previously found in intracellular parasites only, was found in X. fastidiosa. A P-type ATPase is missing-among the 24 completely sequenced eubacteria to date, only three (including X. fastidiosa) do not have a P-type ATPase, and they are all parasites transmitted by insect vectors. An incomplete phosphotransferase system (PTS) was found, without the permease subunits-we conjecture either that they are among the hypothetical proteins or that the PTS plays a solely metabolic regulatory role. We propose that the Ttg2 ABC system might be an import system eventually involved in glutamate import rather than a toluene exporter, as previously annotated. X. fastidiosa exhibits fewer proteins with > or =4 alpha-helical transmembrane spanners than any other completely sequenced prokaryote to date. X. fastidiosa has only 2.7% of all open reading frames identifiable as major transporters, which puts it as the eubacterium having the lowest percentage of open reading frames involved in transport, closer to two archaea, Methanococcus jannaschii (2.4%) and Methanobacterium thermoautotrophicum (2.4%).     

Antibacterial Activity of Phenolic Compounds Against the Phytopathogen Xylella fastidiosa

Xylella fastidiosa is a pathogenic bacterium that causes diseases in many crop species, which leads to considerable economic loss. Phenolic compounds (a group of secondary metabolites) are widely distributed in plants and have shown to possess antimicrobial properties. The anti-Xylella activity of 12 phenolic compounds, representing phenolic acid, coumarin, stilbene and flavonoid, was evaluated using an in vitro agar dilution assay. Overall, these phenolic compounds were effective in inhibiting X. fastidiosa growth, as indicated by low minimum inhibitory concentrations (MICs). In addition, phenolic compounds with different structural features exhibited different anti-Xylella capacities. Particularly, catechol, caffeic acid and resveratrol showed strong anti-Xylella activities. Differential response to phenolic compounds was observed among X. fastidiosa strains isolated from grape and almond. Elucidation of secondary metabolite-based host resistance to X. fastidiosa will have broad implication in combating X. fastidiosa-caused plant diseases. It will facilitate future production of plants with improved disease resistance properties through genetic engineering or traditional breeding approaches and will significantly improve crop yield.     

A genomic approach to the understanding of Xylella fastidiosa pathogenicity

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes several economically important plant diseases, including citrus variegated chlorosis (CVC). X. fastidiosa is the first plant pathogen to have its genome completely sequenced. In addition, it is probably the least previously studied of any organism for which the complete genome sequence is available. Several pathogenicity-related genes have been identified in the X. fastidiosa genome by similarity with other bacterial genes involved in pathogenesis in plants, as well as in animals. The X. fastidiosa genome encodes different classes of proteins directly or indirectly involved in cell-cell interactions, degradation of plant cell walls, iron homeostasis, anti-oxidant responses, synthesis of toxins, and regulation of pathogenicity. Neither genes encoding members of the type III protein secretion system nor avirulence-like genes have been identified in X. fastidiosa.     

A putative twin-arginine translocation system in the phytopathogenic bacterium Xylella fastidiosa

The twin-arginine translocation (Tat) pathway of the xylem-limited phytopathogenic bacterium Xylella fastidiosa strain 9a5c, responsible for citrus variegated chlorosis, was explored. The presence of tatA, tatB, and tatC in the X. fastidiosa genome together with a list of proteins harboring 2 consecutive arginines in their signal peptides suggested the presence of a Tat pathway. The functional Tat dependence of X. fastidiosa OpgD was examined. Native or mutated signal peptides were fused to the β-lactamase. Expression of fusion with intact signal peptides mediated high resistance to ampicillin in Escherichia coli tat+ but not in the E. coli tat null mutant. The replacement of the 2 arginines by 2 lysines prevented the export of β-lactamase in E. coli tat+, demonstrating that X. fastidiosa OpgD carries a signal peptide capable of engaging the E. coli Tat machinery. RT-PCR analysis revealed that the tat genes are transcribed as a single operon. tatA, tatB, and tatC genes were cloned. Complementation assays in E. coli devoid of all Tat or TatC components were unsuccessful, whereas X. fastidiosa Tat components led to a functional Tat translocase in E. coli TatB-deficient strain. Additional experiments implicated that X. fastidiosa TatB component could form a functional heterologous complex with the E. coli TatC component.     

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.     

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.     

Xylella fastidiosa in Europe: From the Introduction to the Current Status

Xylella fastidiosa is xylem-limited bacterium capable of infecting a wide range of host plants, resulting in Pierce’s disease in grapevine, citrus variegated chlorosis, olive quick decline syndrome, peach phony disease, plum leaf scald, alfalfa dwarf, margin necrosis and leaf scorch affecting oleander, coffee, almond, pecan, mulberry, red maple, oak, and other types of cultivated and ornamental plants and forest trees. In the European Union, X. fastidiosa is listed as a quarantine organism. Since its first outbreak in the Apulia region of southern Italy in 2013 where it caused devastating disease on Olea europaea (called olive leaf scorch and quick decline), X. fastidiosa continued to spread and successfully established in some European countries (Corsica and PACA in France, Balearic Islands, Madrid and Comunitat Valenciana in Spain, and Porto in Portugal). The most recent data for Europe indicates that X. fastidiosa is present on 174 hosts, 25 of which were newly identified in 2021 (with further five hosts discovered in other parts of the world in the same year). From the six reported subspecies of X. fastidiosa worldwide, four have been recorded in European countries (fastidiosa, multiplex, pauca, and sandyi). Currently confirmed X. fastidiosa vector species are Philaenus spumarius, Neophilaenus campestris, and Philaenus italosignus, whereby only P. spumarius (which has been identified as the key vector in Apulia, Italy) is also present in Americas. X. fastidiosa control is currently based on pathogen-free propagation plant material, eradication, territory demarcation, and vector control, as well as use of resistant plant cultivars and bactericidal treatments.