A membrane localized RTX-like protein mediates physiochemical properties of the Pantoea stewartii subsp. stewartii cell envelope that impact surface adhesion, cell surface hydrophobicity and plant colonization.

Pantoea stewartii subsp. stewartii (Pnss), is the bacterial causal agent of Stewart's wilt of sweet corn. Disease symptoms include systemic wilting and foliar, water-soaked lesions. A Repeat-in-toxin (RTX)-like protein, RTX2, causes cell leakage and collapse in the leaf apoplast of susceptible corn varieties and is a primary mediator of water-soaked lesion formation in the P. stewartii-sweet corn pathosystem. RTX toxins comprise a large family of proteins, which are widely distributed among Gram-negative bacteria. These proteins are generally categorized as cellulolysins, but the Biofilm-Associated Proteins (Bap) subfamily of RTX toxins are implicated in surface adhesion and other biofilm behaviors. The Pnss RTX2 is most phylogenetically related to other Bap proteins suggesting that Pnss RTX2 plays a dual role in adhesion to host surfaces in addition to mediating the host cell lysis that leads to water-soaked lesion formation. Here we demonstrated that RTX2 localizes to the bacterial cell envelope and influences physiochemical properties of the bacterial cell envelope that impact bacterial cell length, cell envelope integrity and overall cellular hydrophobicity. Interestingly, the role of RTX2 as an adhesin was only evident in absence of exopolysaccharide (EPS) production suggesting that RTX2 plays a role as an adhesin early in biofilm development before EPS production is fully induced. However, deletion of rtx2 severely impacted Pnss' colonization of the xylem suggesting that the dual role of RTX2 as a cytolysin and adhesin is a mechanism that links the apoplastic water-soaked lesion phase of infection to the wilting phase of the infection in the xylem.

Phylogenomic analyses and comparative genomics of Pseudomonas syringae associated with almond (Prunus dulcis) in California.

We sequenced and comprehensively analysed the genomic architecture of 98 fluorescent pseudomonads isolated from different symptomatic and asymptomatic tissues of almond and a few other Prunus spp. Phylogenomic analyses, genome mining, field pathogenicity tests, and in vitro ice nucleation and antibiotic sensitivity tests were integrated to improve knowledge of the biology and management of bacterial blast and bacterial canker of almond. We identified Pseudomonas syringae pv. syringae, P. cerasi, and P. viridiflava as almond canker pathogens. P. syringae pv. syringae caused both canker and foliar (blast) symptoms. In contrast, P. cerasi and P. viridiflava only caused cankers, and P. viridiflava appeared to be a weak pathogen of almond. Isolates belonging to P. syringae pv. syringae were the most frequently isolated among the pathogenic species/pathovars, composing 75% of all pathogenic isolates. P. cerasi and P. viridiflava isolates composed 8.3 and 16.7% of the pathogenic isolates, respectively. Laboratory leaf infiltration bioassays produced results distinct from experiments in the field with both P. cerasi and P. syringae pv. syringae, causing significant necrosis and browning of detached leaves, whereas P. viridiflava conferred moderate effects. Genome mining revealed the absence of key epiphytic fitness-related genes in P. cerasi and P. viridiflava genomic sequences, which could explain the contrasting field and laboratory bioassay results. P. syringae pv. syringae and P. cerasi isolates harboured the ice nucleation protein, which correlated with the ice nucleation phenotype. Results of sensitivity tests to copper and kasugamycin showed a strong linkage to putative resistance genes. Isolates harbouring the ctpV gene showed resistance to copper up to 600 µg/ml. In contrast, isolates without the ctpV gene could not grow on nutrient agar amended with 200 µg/ml copper, suggesting ctpV can be used to phenotype copper resistance. All isolates were sensitive to kasugamycin at the label-recommended rate of 100µg/ml.

Natural Recombination among Type I Restriction-Modification Systems Creates Diverse Genomic Methylation Patterns among Xylella fastidiosa Strains.

Xylella fastidiosa is an important bacterial plant pathogen causing high-consequence diseases in agricultural crops around the world. Although as a species X. fastidiosa can infect many host plants, there is significant variability between strains regarding virulence on specific host plant species and other traits. Natural competence and horizontal gene transfer are believed to occur frequently in X. fastidiosa and likely influence the evolution of this pathogen. However, some X. fastidiosa strains are difficult to manipulate genetically using standard transformation techniques. Several type I restriction-modification (R-M) systems are encoded in the X. fastidiosa genome, which may influence horizontal gene transfer and recombination. Type I R-M systems themselves may undergo recombination, exchanging target recognition domains (TRDs) between specificity subunits (hsdS) to generate novel alleles with new target specificities. In this study, several conserved type I R-M systems were compared across 129 X. fastidiosa genome assemblies representing all known subspecies and 32 sequence types. Forty-four unique TRDs were identified among 50 hsdS alleles, which are arrayed in 31 allele profiles that are generally conserved within a monophyletic cluster of strains. Inactivating mutations were identified in type I R-M systems of specific strains, showing heterogeneity in the complements of functional type I R-M systems across X. fastidiosa. Genomic DNA methylation patterns were characterized in 20 X. fastidiosa strains and associated with type I R-M system allele profiles. Overall, these data suggest hsdS genes recombine among Xylella strains and/or unknown donors, and the resulting TRD reassortment establishes differential epigenetic modifications across Xylella lineages. IMPORTANCE Economic impacts on agricultural production due to X. fastidiosa have been severe in the Americas, Europe, and parts of Asia. Despite a long history of research on this pathogen, certain fundamental questions regarding the biology, pathogenicity, and evolution of X. fastidiosa have still not been answered. Wide-scale whole-genome sequencing has begun to provide more insight into X. fastidiosa genetic diversity and horizontal gene transfer, but the mechanics of genomic recombination in natural settings and the extent to which this directly influences bacterial phenotypes such as plant host range are not well understood. Genome methylation is an important factor in horizontal gene transfer and bacterial recombination that has not been comprehensively studied in X. fastidiosa. This study characterizes methylation associated with type I restriction-modification systems across a wide range of X. fastidiosa strains and lays the groundwork for a better understanding of X. fastidiosa biology and evolution through epigenetics.

Insights Regarding Resistance of 'Nemaguard' Rootstock to the Bacterium Xylella fastidiosa.

'Nemaguard' is a commonly used rootstock for almond and stone fruits due to resistance to nematodes and enhanced scion vigor. Nemaguard also happens to be resistant to strains of Xylella fastidiosa that cause almond leaf scorch disease. Previous research showed that prior to June-budding, this rootstock can prevent infection of almond nursery stock by X. fastidiosa. Further, the rootstock also promotes recovery from infection in susceptible almond scions. Objectives of this study were to 1) compare movement and bacterial populations of X. fastidiosa in almond and Nemaguard, 2) determine whether the metabolic profile of infected versus noninfected plants of each species correspond with differences in pathogen distribution, and 3) evaluate the impact of feeding on Nemaguard on transmission efficiency and pathogen populations in insects. Results showed limited or no movement of X. fastidiosa beyond the point of mechanical inoculation in Nemaguard, whereas X. fastidiosa was detected in susceptible almond and isolated from plant samples distal to the point of inoculation. Large differences in the concentration of phenolic compounds between Nemaguard and almond were also found, although this was not impacted by infection status. After acquiring X. fastidiosa from infected plants, vector access periods of up to 14 days on Nemaguard neither reduced pathogen populations in vectors nor reduced transmission efficiency of X. fastidiosa to susceptible plants when compared with similar vector-access periods on susceptible grapevines. Results suggest Nemaguard, in spite of having high phenolic concentrations in its xylem, does not directly impact X. fastidiosa survival and that future research should focus on identification of potential physical traits that prevent bacterial attachment, multiplication, or movement within the plant.

Microbe Profile: Xylella fastidiosa - a devastating agricultural pathogen with an endophytic lifestyle.

Xylella fastidiosa is a vector-borne plant vascular pathogen that has caused devastating disease outbreaks in diverse agricultural crops worldwide. A major global quarantine pathogen, X. fastidiosa can infect hundreds of plant species and can be transmitted by many different xylem sap-feeding insects. Several decades of research have revealed a complex lifestyle dependent on adaptation to the xylem and insect environments and interactions with host plant tissues.

Grapevine phenolic compounds influence cell surface adhesion of Xylella fastidiosa and bind to lipopolysaccharide.

Bacterial phytopathogen Xylella fastidiosa specifically colonizes the plant vascular tissue through a complex process of cell adhesion, biofilm formation, and dispersive movement. Adaptation to the chemical environment of the xylem is essential for bacterial growth and progression of infection. Grapevine xylem sap contains a range of plant secondary metabolites such as phenolics, which fluctuate in response to pathogen infection and plant physiological state. Phenolic compounds are often involved in host-pathogen interactions and influence infection dynamics through signaling activity, antimicrobial properties, and alteration of bacterial phenotypes. The effect of biologically relevant concentrations of phenolic compounds coumaric acid, gallic acid, epicatechin, and resveratrol on growth of X. fastidiosa was assessed in vitro. None of these compounds inhibited bacterial growth, but epicatechin and gallic acid reduced cell-surface adhesion. Cell-cell aggregation decreased with resveratrol treatment, but the other phenolic compounds tested had minimal effect on aggregation. Expression of attachment (xadA) and aggregation (fimA) related genes were altered by presence of the phenolic compounds, consistent with observed phenotypes. All four of the phenolic compounds bound to purified X. fastidiosa lipopolysaccharide (LPS), a major cell-surface component. Information regarding the impact of chemical environment on pathogen colonization in plants is important for understanding the infection process and factors associated with host susceptibility.

Xylella fastidiosa and Glassy-Winged Sharpshooter Population Dynamics in the Southern San Joaquin Valley of California.

Xylella fastidiosa is a vector-transmitted bacterial plant pathogen that affects a wide array of perennial crops, including grapevines (Pierce's disease). In the southern San Joaquin Valley of California, epidemics of Pierce's disease of grapevine were associated with the glassy-winged sharpshooter, Homalodisca vitripennis. During the growing season, rates of X. fastidiosa spread in vineyards are affected by changes in pathogen distribution within chronically infected grapevines and by vector population dynamics. Grapevines chronically infected with X. fastidiosa rarely tested positive for the pathogen prior to July, suggesting vector acquisition of X. fastidiosa from grapevines increases as the season progresses. This hypothesis was supported by an increase in number of X. fastidiosa-positive glassy-winged sharpshooters collected from vineyards during July through September. Analysis of insecticide records indicated that vineyards in the study area were typically treated with a systemic neonicotinoid in spring of each year. As a result, abundance of glassy-winged sharpshooters was typically low in late spring and early summer, with abundance of glassy-winged sharpshooter adults increasing in late June and early July of each year. Collectively, the results suggest that late summer is a crucial time for X. fastidiosa secondary spread in vineyards in the southern San Joaquin Valley, because glassy-winged sharpshooter abundance, number of glassy-winged sharpshooters testing positive for X. fastidiosa, and grapevines with detectable pathogen populations were all greatest during this period.

Complete Genome Sequence Data of Three Xylella fastidiosa subsp. multiplex Strains Isolated from Olive Trees in California, U.S.A.

Xylella fastidiosa is a xylem-limited bacterial plant pathogen that causes disease on numerous hosts. Additionally, X. fastidiosa asymptomatically colonizes a wide range of plant species. X. fastidiosa subsp. multiplex has been detected in olive (Olea europaea) trees grown in California, U.S.A., as well as in Europe. Strains of X. fastidiosa subsp. multiplex isolated from California olive trees are not known to cause disease on olive, although some can induce leaf-scorch symptoms on almond (Prunus dulcis). No genome assemblies currently exist for olive-associated X. fastidiosa subsp. multiplex strains; therefore, a hybrid assembly method was used to generate complete genome sequences for three X. fastidiosa subsp. multiplex strains (Fillmore, LM10, and RH1) isolated from olive trees grown in Ventura and Los Angeles counties of California.

Lost and found: Rediscovery and genomic characterization of sowthistle yellow vein virus after a 30+ year hiatus.

Beginning in the 1960's, sowthistle yellow vein virus (SYVV) was the subject of pioneering research that demonstrated propagation of a plant virus in its insect vector. Since the 1980's there has been a paucity of research on SYVV, with historic isolates no longer maintained and no genomic sequence available. Once commonly observed infecting sowthistle (Sonchus oleraceous L.) in California, SYVV incidence declined ca. 1990, likely due to displacement of the black currant aphid (Hyperomyzus lactucae L.) by an invasive non-vector aphid. In 2018, SYVV was fortuitously rediscovered infecting sowthistle in an organic citrus grove in Kern County, CA. The SYVV genome sequence (13,719 nts) obtained from the 2018 sample (designated HWY65) encoded all six expected genes: N, P, MP, M, G, and L. Nucleotide sequence (representing ∼86 % of the genome) of the SYVV Berkeley lab isolate, used by E. S. Sylvester and colleagues for the paradigm-shifting research mentioned above, was determined from an archived library of cDNA clones constructed in 1986. The two nucleotide sequences share 98.5 % identity, confirming both represent the same virus, thereby linking biology of the historic isolate with extant SYVV rediscovered in 2018. Phylogenetic analysis of the L protein indicated SYVV is positioned within a clade containing a subset of viruses currently assigned to the genus Nucleorhabdovirus. As Nucleorhabdovirus is paraphyletic, the International Committee on the Taxonomy of Viruses has proposed abolishment of the genus and establishment of three new genera. In this revised taxonomy, the clade containing SYVV constitutes a new genus designated Betanucleorhabdovirus.

Infection of Blueberry Cultivar 'Emerald' with a California Pierce's Disease Strain of Xylella fastidiosa and Acquisition by Glassy-Winged Sharpshooter.

Bacterial leaf scorch disease caused by Xylella fastidiosa occurs in southern highbush blueberry varieties in the southeastern United States. Susceptibility to X. fastidiosa varies by blueberry cultivar, and these interactions are often strain-specific. Xylella fastidiosa subsp. fastidiosa is the causal agent of Pierce's disease in grapevines, and it has been problematic in the San Joaquin Valley of California since the introduction of the glassy-winged sharpshooter (Homalodisca vitripennis). The glassy-winged sharpshooter is known to feed on blueberry, a crop that is expanding in the San Joaquin Valley. Currently, little is known about the potential for the spread of X. fastidiosa between grape and blueberry in this region. The ability of a Pierce's disease strain of X. fastidiosa from the San Joaquin Valley to cause disease in southern highbush blueberry and the potential for the glassy-winged sharpshooter to transmit X. fastidiosa between blueberry and grapevine were investigated. Experimental inoculations showed that the X. fastidiosa subsp. fastidiosa strain Bakersfield-1 can cause disease in blueberry cv. Emerald, and that the glassy-winged sharpshooter can acquire X. fastidiosa from artificially inoculated blueberry plants under laboratory conditions. Understanding the possibility for X. fastidiosa strains from the San Joaquin Valley to infect multiple crops grown in proximity is important for area-wide pest and disease management.

Novel amplification targets for rapid detection and differentiation of Xylella fastidiosa subspecies fastidiosa and multiplex in plant and insect tissues.

Xylella fastidiosa is an insect-transmitted bacterial plant pathogen which causes a variety of economically important diseases worldwide. Molecular identification of X. fastidiosa is used for quarantine screening, surveillance, and research applications; many of which require subspecies level differentiation of pathogen isolates. This study describes quantitative PCR (qPCR) and isothermal amplification assays which can rapidly identify X. fastidiosa isolates belonging to the fastidiosa and multiplex subspecies. The TaqMan qPCR primers described here are used to differentiate X. fastidiosa strains by subspecies in plant and insect tissue in a single reaction, with the inclusion of a general amplification control probe to identify potential false negative samples. This TaqMan qPCR protocol can identify between 103 and 104 cfu/ml concentrations of X. fastidiosa at the subspecies level in a variety of sample types. Additionally, loop-mediated isothermal amplification (LAMP) targets were designed for faster detection of X. fastidiosa subspecies fastidiosa and multiplex, applicable to a field setting. These assays are effective for strain differentiation in artificially and naturally inoculated plant material, and in field collected insect vectors.

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions.

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa, but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb, putatively encoding a conjugative type IV secretion system, are found in some but not all X. fastidiosa isolates, often on native plasmids. X. fastidiosa strains that carry the conjugative transfer genes can belong to different subspecies and frequently differ in host ranges. Using X. fastidiosa strain M23 (X. fastidiosa subsp. fastidiosa) or Dixon (X. fastidiosa subsp. multiplex) as the donor strain and Temecula (X. fastidiosa subsp. fastidiosa) as the recipient strain, plasmid transfer was characterized using the mobilizable broad-host-range vector pBBR5pemIK. Transfer of plasmid pBBR5pemIK was observed under in vitro conditions with both donor strains and was dependent on both tra and trb operon functions. A conjugative mechanism likely contributes to gene transfer between diverse strains of X. fastidiosa, possibly facilitating adaptation to new environments or different hosts.IMPORTANCEXylella fastidiosa is an important plant pathogen worldwide, infecting a wide range of different plant species. The emergence of new diseases caused by X. fastidiosa, or host switching of existing strains, is thought to be primarily due to the high frequency of HGT and recombination in this pathogen. Transfer of plasmids by a conjugative mechanism enables movement of larger amounts of genetic material at one time, compared with other routes of gene transfer such as natural transformation. Establishing the prevalence and functionality of this mechanism in X. fastidiosa contributes to a better understanding of HGT, adaptation, and disease emergence in this diverse pathogen.

The DinJ/RelE Toxin-Antitoxin System Suppresses Bacterial Proliferation and Virulence of Xylella fastidiosa in Grapevine.

Xylella fastidiosa, the causal agent of Pierce's disease of grapes, is a slow-growing, xylem-limited, bacterial pathogen. Disease progression is characterized by systemic spread of the bacterium through xylem vessel networks, causing leaf-scorching symptoms, senescence, and vine decline. It appears to be advantageous to this pathogen to avoid excessive blockage of xylem vessels, because living bacterial cells are generally found in plant tissue with low bacterial cell density and minimal scorching symptoms. The DinJ/RelE toxin-antitoxin system is characterized here for a role in controlling bacterial proliferation and population size during plant colonization. The DinJ/RelE locus is transcribed from two separate promoters, allowing for coexpression of antitoxin DinJ with endoribonuclease toxin RelE, in addition to independent expression of RelE. The ratio of antitoxin/toxin expressed is dependent on bacterial growth conditions, with lower amounts of antitoxin present under conditions designed to mimic grapevine xylem sap. A knockout mutant of DinJ/RelE exhibits a hypervirulent phenotype, with higher bacterial populations and increased symptom development and plant decline. It is likely that DinJ/RelE acts to prevent excessive population growth, contributing to the ability of the pathogen to spread systemically without completely blocking the xylem vessels and increasing probability of acquisition by the insect vector.

Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

A Temperature-Independent Cold-Shock Protein Homolog Acts as a Virulence Factor in Xylella fastidiosa.

Xylella fastidiosa, causal agent of Pierce's disease (PD) of grapevine, is a fastidious organism that requires very specific conditions for replication and plant colonization. Cold temperatures reduce growth and survival of X. fastidiosa both in vitro and in planta. However, little is known regarding physiological responses of X. fastidiosa to temperature changes. Cold-shock proteins (CSP), a family of nucleic acid-binding proteins, act as chaperones facilitating translation at low temperatures. Bacterial genomes often encode multiple CSP, some of which are strongly induced following exposure to cold. Additionally, CSP contribute to the general stress response through mRNA stabilization and posttranscriptional regulation. A putative CSP homolog (Csp1) with RNA-binding activity was identified in X. fastidiosa Stag's Leap. The csp1 gene lacked the long 5' untranslated region characteristic of cold-inducible genes and was expressed in a temperature-independent manner. As compared with the wild type, a deletion mutant of csp1 (∆csp1) had decreased survival rates following cold exposure and salt stress in vitro. The deletion mutant also was significantly less virulent in grapevine, as compared with the wild type, in the absence of cold stress. These results suggest an important function of X. fastidiosa Csp1 in response to cellular stress and during plant colonization.

A Large Repetitive RTX-Like Protein Mediates Water-Soaked Lesion Development, Leakage of Plant Cell Content and Host Colonization in the Pantoea stewartii subsp. stewartii Pathosystem.

Pantoea stewartii subsp. stewartii is the etiological agent of Stewart's wilt and is a serious bacterial pathogen affecting sweet corn. During the leaf blight phase, P. stewartii colonizes the leaf apoplast and causes a characteristic water-soaked lesion. The Hrp type III secretion system has been implicated in the water-soaking phenotype, and the goal of this study was to investigate other potential factors that contribute to the plant cellular disruption associated with these lesions. The P. stewartii genome contains a gene encoding a large repetitive RTX toxin, designated rtx2. RTX toxins comprise a large family of pore-forming proteins, which are widely distributed among gram-negative bacteria. These cytotoxins usually lyse their target host cells and cause significant tissue damage as a consequence. We hypothesized that this RTX-like toxin plays a role in the water-soaking phase of infection due to its predicted cytolytic properties. Based on the data reported here, we conclude that RTX2 contributes significantly to the development of water-soaked lesions and leakage of plant cellular contents and is an important pathogenicity factor for P. stewartii.

Role for Rhizobium rhizogenes K84 cell envelope polysaccharides in surface interactions.

Rhizobium rhizogenes strain K84 is a commercial biocontrol agent used worldwide to control crown gall disease. The organism binds tightly to polypropylene substrate and efficiently colonizes root surfaces as complex, multilayered biofilms. A genetic screen identified two mutants in which these surface interactions were affected. One of these mutants failed to attach and form biofilms on the abiotic surface although, interestingly, it exhibited normal biofilm formation on the biological root tip surface. This mutant is disrupted in a wcbD ortholog gene, which is part of a large locus predicted to encode functions for the biosynthesis and export of a group II capsular polysaccharide (CPS). Expression of a functional copy of wcbD in the mutant background restored the ability of the bacteria to attach and form normal biofilms on the abiotic surface. The second identified mutant attached and formed visibly denser biofilms on both abiotic and root tip surfaces. This mutant is disrupted in the rkpK gene, which is predicted to encode a UDP-glucose 6-dehydrogenase required for O-antigen lipopolysaccharide (LPS) and K-antigen capsular polysaccharide (KPS) biosynthesis in rhizobia. The rkpK mutant from strain K84 was deficient in O-antigen synthesis and exclusively produced rough LPS. We also show that strain K84 does not synthesize the KPS typical of some other rhizobia strains. In addition, we identified a putative type II CPS, distinct from KPS, that mediates cell-surface interactions, and we show that O antigen of strain K84 is necessary for normal cell-cell interactions in the biofilms.