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

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

Proteomic analysis of grapevine stem in response to Xylella fastidiosa inoculation

Xylella fastidiosa (Xf) is the bacterial causal agent of Pierce’s disease (PD) as well as other economically important diseases in a number of agronomic, horticultural and ornamental plants. The objective of this research was to tentatively identify proteins that are differentially expressed in grapevines and involved in disease development or defense responses to Xf-inoculation. We comparatively analyzed proteins differentially expressed in Xf-inoculated grape stems using a pair of siblings of 9621-67 (highly susceptible) and 9621-94 (highly resistant) from a cross of Vitis rupestris × Vitis arizonica. Total proteins were extracted from the stems of uninoculated controls and Xf-inoculated plants at 1, 6, and 12 weeks after inoculation, separated by a 2D-PAGE system, and spots representing differentially expressed proteins were analyzed and tentatively identified using LC/MS/MS. Protein identification was performed using BLASTp and tBLASTn against NCBI non-redundant protein databases and EST databases, respectively. Ten tentatively identified proteins were differentially expressed at different time points after inoculation. A thaumatin-like protein and the pathogenesis-related protein 10 from both genotypes, and the 40S ribosomal protein S25 from the susceptible genotype were up-regulated in response to Xf-inoculation. Furthermore, the expression of the thaumatin-like protein increased sharply 12 weeks post-inoculation in the PD-resistant genotype only. Three heat shock proteins, 17.9 kDa class II, protein 18 and 21 were highly expressed in healthy tissues compared with those in tissues infected with Xf, and heat shock protein 21 was not detectable in the Xf-inoculated PD-susceptible genotype. In addition, a down-regulated putative ripening related protein was found in the Xf-inoculated PD-susceptible genotype. Glycoprotein and formate dehydrogenase were identified in the PD-resistant genotype and their expression was constant during plant development. A putative GTP-binding protein was down-regulated in the PD-susceptible genotype. Our results revealed that differential expression of proteins in response to Xf-inoculation was genotype and tissue development stage dependent. The specific roles of these candidate proteins in alleviation or aggravation of this disease are under investigation. The information obtained in this study will aid in the understanding of the mechanisms related to the host–pathogen interactions involved in PD.     

Differential colonization patterns of Xylella fastidiosa infecting citrus genotypes

Xylella fastidiosa is a phytopathogenic bacterium that causes disease in many different crops worldwide. In Brazil, X. fastidiosa subsp. pauca causes citrus variegated chlorosis (CVC), which is a disease responsible for economic losses in the citrus agribusiness. Variable host responses to bacterial colonization and disease development have been observed. This work studies the colonization processes of a pathogenic GFP‐labelled X. fastidiosa citrus strain in sweet orange (susceptible) and tangor (resistant) parents and two resulting hybrids that exhibited contrasting responses to CVC. Xylella fastidiosa showed increased populations and movement in the susceptible genotypes, but slower compared to other hosts such as grapevine. Scanning electron microscopy revealed that the predominant pitted stem morphology in citrus makes the bacterial movement difficult. In susceptible genotypes X. fastidiosa can move from the primary to the secondary xylem, whilst it is confined to the primary xylem in resistant plants. Associated with this is an induction of lignification that occurs earlier in the resistant genotypes when in the presence of the pathogen, and represents a genetic mechanism that leads to formation of a physical barrier, impairing bacterial colonization.     

Estimating the epidemiology of emerging Xylella fastidiosa outbreaks in olives

Xylella fastidiosa is an important insect-vectored bacterial plant pathogen with a wide host range, causing significant economic impact in the agricultural and horticultural industries. Once restricted to the Americas, severe European outbreaks have been discovered recently in Italy, Spain, France, and Portugal. The Italian outbreak, detected in Puglia in 2013, has spread over 100 km, killing millions of olive trees, and is still expanding. To date, quantified assessment of important epidemiological parameters useful for risk assessment and management, such as transmission rates, symptomless periods, and time to death in field populations, has been lacking. This is due to the emergent and novel nature of the outbreak and length of time needed to monitor the course of disease progression. To address this, we developed a Bayesian method to infer epidemiological parameters by fitting and comparing compartmental epidemiological models to short snapshots of disease progression observed in multiple field plots. We estimated that each infected tree with symptoms is able to infect around 19 trees per year (95% credible range 14–26). The symptomless stage was estimated to have low to negligible infectivity and to last an average of approximately 1.2 years (95% credible range 1.0–1.3 years). Tree desiccation was estimated to occur approximately 4.3 years (95% credible range 4.0–4.6 years) after symptom appearance. However, we were unable to estimate the infectiousness of desiccated trees from the data. Our method could be used to make early estimates of epidemiological parameters in other emerging disease outbreaks where symptom expression is slow.     

Causal Role of Xylella fastidiosa in Oleander Leaf Scorch Disease

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

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

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

Genome-based PCR Primers for Specific and Sensitive Detection and Quantification of Xylella fastidiosa

Xylella fastidiosa is an important pathogen of many commercial crops. Detection of X. fastidiosa is difficult due to low concentrations of the bacteria in insects and asymptomatic plant tissue, and non-uniform distribution in infected plants. A dual purpose conventional PCR and quantitative PCR (TaqMan™) system was developed for the generic detection of X. fastidiosa strains. Primers HL5 and HL6, designed to amplify a unique region common to the sequenced genomes of four Xylella strains, amplified a 221 bp fragment from strains associated with Pierce’s disease of grapes, almond leaf scorch, and oleander leaf scorch disease and from DNA from an Xf strain associated with citrus variegated chlorosis. Standard curves were obtained using concentrations of Xylella ranging from 5 to 10⁵ cells per reaction in water and grape extracts and 10–10⁵ cells in insect DNA. Regression curves were similar, with correlation coefficients of r ² > 0.97. In quantitative PCR, C_t values ranged between 20 and 36 cycles for 5–10⁵ bacterial cells per reaction. No amplicons were obtained with several non-Xf bacterial strains tested including related plant pathogenic, grape endophytic bacteria and endosymbiotic bacteria isolated from glassy-winged sharpshooters. The method was evaluated for clinical diagnosis of Xf in grapes, almonds and insect vectors. The procedure described is reliable for detection of the pathogen with a high degree of sensitivity and specificity.     

SDS-PAGE: a tool to discriminate Xylella fastidiosa from other endophytic grapevine bacteria*

There is a real risk of introducing Xylella fastidiosa into the EPPO region where this quarantine pest has not yet been found and constitutes a serious threat to the Mediterranean grapevine industry. Direct isolation of endophytic bacteria from imported propagation material, combined with one-dimensional SDS-PAGE of the total soluble proteins of the bacterial cell envelope, can be considered as a test to be used in grapevine certification schemes. The gel electrophoretic pattern of Xylella fastidiosa is characterized by a constant distribution of 14 major diagnostic bands. Even without gel scanning, this relatively simple technique allows discrimination between grapevine strains of X. fastidiosa and other xylem-inhabiting, slow-growing bacteria.     

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

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

Growth- and leaf-temperature effects on photosynthesis of sweet orange seedlings infected with Xylella fastidiosa

The effects of growth and leaf temperature on photosynthesis were evaluated in sweet orange seedlings ( Citrus sinensis cv. Pera) infected with Xylella fastidiosa (the bacterium that causes citrus variegated chlorosis, CVC). Measurements of leaf gas exchange and chlorophyll  a fluorescence were taken at leaf temperatures of 25, 30, 35 and 40°C in healthy and infected (without visible symptoms) seedlings submitted to two temperature regimes (25/20 or 35/20°C, day/night), not simultaneously. The CO₂ assimilation rates ( A ) and stomatal conductance ( g _s) were higher in healthy plants in both temperature regimes. Values for A and g _s of infected and healthy plants were higher in the 35/20°C regime, decreasing with leaf temperature increase. In addition, differences between healthy and infected plants were higher at 35/20°C, while no differences in chlorophyll  a fluorescence parameters were observed except for potential quantum efficiency of photosystem II, which was higher in infected plants. Low A values in infected plants were caused by low g _s and probably by biochemical damage to photosynthesis. The high alternative electron sink of infected plants was another effect of reduced A . Both high growth and high leaf temperatures increased differences in A between healthy and infected plants. Therefore this feature may be partially responsible for lower growth and/or productivity of CVC-affected plants in regions with high air temperature.     

Populations of Xylella fastidiosa in Plants Required for Transmission by an Efficient Vector

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium that causes Pierce's disease (PD) of grapevine and other diseases, is transmitted efficiently by xylem-feeding leafhoppers. Acquisition of a PD strain of X. fastidiosa by the blue-green sharpshooter (BGSS) from five plant host species-grapevine (Vitis vinifera), Himalayan blackberry (Rubus discolor), California mugwort (Artemisia douglasiana), watergrass (Echinochloa crus-galli), and Bermuda grass (Cynodon dactylon)-was tested at various time intervals after vector inoculation. The minimum incubation periods in plant hosts before BGSS acquired X. fastidiosa were 4, 22, 29, and 25 days for grapevine, blackberry, mugwort, and watergrass, respectively. There were no transmissions by vectors or recoveries of X. fastidiosa by culturing from Bermuda grass in 133 attempts, including 80 attempts with the green sharpshooter, Draeculacephala minerva. The first acquisitions and subsequent transmissions by BGSS occurred after X. fastidiosa multiplied to a population of about 10(4) CFU/g of stem tissue. Higher populations of bacteria in plants resulted in higher rates of transmission. In grapevine, the rate of transmission increased over time (4.5% in the first 10 days to 55% after day 25) as the maximum number of viable CFU of X. fas-tidiosa recovered by culturing also increased (from 5 x 10(5) CFU/g during the first 10 days to 5 x 10(8) after day 25).     

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

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

Effect of xylem fluid from susceptible and resistant grapevines on developmental biology of Xylella fastidiosa

Xylella fastidiosa causes Pierce??s disease (PD), a serious disease in grapevines, and grapevine cultivars vary in susceptibility to X. fastidiosa in the field. The mechanism(s) by which this occurs has not been clearly elucidated. To explore possible mechanisms, X. fastidiosa cells from a PD strain were grown in pure xylem fluid of PD-susceptible grapevines, Vitis vinifera and V. labrusca, versus PD-resistant grapevines, V. champinii and V. smalliana. When grown in xylem fluid from the susceptible species, X. fastidiosa cells formed a heavier biofilm compared to those in xylem fluid from the resistant species. Differential expression of selected genes of X. fastidiosa cultured in the xylem fluids of V. vinifera and V. smalliana was analyzed using a DNA macroarray. Compared with xylem fluid of V. smalliana, xylem fluid of V. vinifera stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as rpfC, gacA, xrvA, gcvR, and cysB, and genes involved in biogenesis of pili and twitching motility, such as pilI, pilU, pilE and pilG. Increased expression of virulence genes likely contributes to the expression of PD symptom in the susceptible grapevines, whereas reduced expression of these genes may lead to limitation of symptoms in resistant grapevines.     

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

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

Assessment of the genetic diversity of Xylella fastidiosa in imported ornamental Coffea arabica plants

A study was performed in order to assess the presence of Xylella fastidiosa in imported ornamental plants, among them Olea europaea, Coffea arabica and Nerium oleander. Positive results were only obtained from C. arabica, where 15 plant samples tested positive for X. fastidiosa by PCR, nine from Costa Rica and six from Honduras. Transmission electron microscopy observations indicated that rod‐shaped bacterial cells exhibiting the characteristics of X. fastidiosa cells were present in the xylem vessels of leaf petioles obtained from the infected C. arabica plants. Diversity of X. fastidiosa in C. arabica plants was assessed through a multilocus sequence typing (MLST) analysis of seven housekeeping genes (leuA, petC, lacF, cysG, holC, nuoL and gltT) and compared with X. fastidiosa infecting different host plants worldwide. Based on this MLST analysis, the prevalence of different sequence types (STs) of X. fastidiosa in the C. arabica ornamental plants was demonstrated and related to different X. fastidiosa subspecies, underlining the risk of introducing additional genetic diversity for X. fastidiosa to Europe. ST53, related to X. fastidiosa subsp. pauca, was frequently found in these C. arabica samples. A second ST related to X. fastidiosa subsp. pauca, ST73, has been assessed in coinfection with ST53 in one individual plant. Additionally, ST72 and ST76, related to X. fastidiosa subsp. fastidiosa, have been recorded. Next to these previously described STs, a novel ST, namely ST77 has been revealed, related to X. fastidiosa subsp. fastidiosa. Isolation of X. fastidiosa from leaf petioles and midribs of infected C. arabica plants was successfully performed only after the application of an additional ultrasonication step during the extraction procedure. Based on this approach, a number of X. fastidiosa isolates were obtained and further characterized.     

The importance of multilocus sequence typing: cautionary tales from the bacterium Xylella fastidiosa

Microbial identification methods have evolved rapidly over the last few decades. One such method is multilocus sequence typing (MLST). MLST is a powerful tool for understanding the evolutionary dynamics of pathogens and to gain insight into their genetic diversity. We illustrate the importance of accurate typing by reporting on three problems that have arisen in the study of a single bacterial species, the plant pathogen Xylella fastidiosa. Two of these were particularly serious since they concerned contamination of important research material that has had detrimental consequences for Xylella research: the contamination of DNA used in the sequencing of an X. fastidiosa genome (Ann-1) with DNA from another X. fastidiosa strain, and the unrecognized mislabeling of a strain (Temecula1) distributed from a culture collection (ATCC). We advocate the routine use of MLST to define strains maintained in culture collections and emphasize the importance of confirming the purity of DNA submitted for sequencing. We also present a third example that illustrates the value of MLST in guiding the choice of taxonomic types. Beyond these situations, there is a strong case for MLST whenever an isolate is used experimentally, especially where genotypic differences are suspected to influence the outcome.