Two different Xylella fastidiosa strains circulating in Italy: phylogenetic and evolutionary analyses

Xylella fastidiosa, a bacterial species infecting a broad range plants, includes five subspecies, fastidiosa, multiplex, pauca, mulberry and sandyi. In Europe, Xylella was isolated in olive trees in southern Italy (Apulia region) during the year 2013. The aim of the present study was to apply phylogenetic and evolutionary analysis to trace the possible origin and way of the entrance of Xylella fastidiosa in Italy. All the genomes available for Xylella fastidiosa spp were downloaded from NCBI. A phylogeographic analysis was performed using BEAST. X. fastidiosa strains belonging to X. fastidiosa subsp. pauca and subsp. sandyi have been reported to infect olive trees and coffee plants, respectively. The phylogeographic analysis also revealed and confirmed these two different ways of provenience X. fastidiosa subsp. pauca from Costa Rica and X. fastidiosa subsp sandyi from California Phylogeny have been an important tool to validate and support the recent hypothesis for X. fastidiosa pauca provenience.     

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.     

Retention Sites for <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> in Four Sharpshooter Vectors (Hemiptera: Cicadellidae) Analyzed by Scanning Electron Microscopy

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis (CVC), Pierce’s disease of grapevine, and leaf scald of coffee and plum and many other plant species. This pathogen is vectored by sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) and resides in the insect foregut. Scanning electron microscopy was used to determine the retention sites of X. fastidiosa for the most common vector species in Brazilian citrus groves, Acrogonia citrina, Bucephalogonia xanthophis, Dilobopterus costalimai, and Oncometopia facialis. After a 48-h acquisition access period on infected citrus or plum, adult sharpshooters were kept on healthy citrus seedlings for an incubation period of 2 weeks to allow for bacterial multiplication. Then the vector heads were incubated for 24 h in a fixative and transferred into a cryoprotector liquid. Bacterial rod cells exhibiting similar X. fastidiosa morphology were found laterally attached to different regions inside the cibarial pump chamber (longitudinal groove, lateral surface, cibarial diaphragm and apodemal groove) of A. citrina, O. facialis, and D. costalimai, and polarly attached to the precibarium channel of O. facialis. Polymerase chain reactions of vector’s heads were positive for the presence of X. fastidiosa. No X. fastidiosa-like cells were detected in B. xanthophis. A different type of rod-shaped bacterium was found on B. xanthophis cibarium chamber and images suggest that the cibarium wall was degraded/digested by these bacteria. Colonization patterns of X. fastidiosa in their vectors are fundamental aspects to be explored toward understanding acquisition, adhesion, and transmission mechanisms for development of X. fastidiosa control strategies.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Endophytic Methylobacterium extorquens expresses a heterologous β-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa

Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens—X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.     

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.     

The Complex Biogeography of the Plant Pathogen Xylella fastidiosa: Genetic Evidence of Introductions and Subspecific Introgression in Central America

The bacterium Xylella fastidiosa is a plant pathogen with a history of economically damaging introductions of subspecies to regions where its other subspecies are native. Genetic evidence is presented demonstrating the introduction of two new taxa into Central America and their introgression into the native subspecies, X. fastidiosa subsp. fastidiosa. The data are from 10 genetic outliers detected by multilocus sequence typing (MLST) of isolates from Costa Rica. Six (five from oleander, one from coffee) defined a new sequence type (ST53) that carried alleles at six of the eight loci sequenced (five of the seven MLST loci) diagnostic of the South American subspecies Xylella fastidiosa subsp. pauca which causes two economically damaging plant diseases, citrus variegated chlorosis and coffee leaf scorch. The two remaining loci of ST53 carried alleles from what appears to be a new South American form of X. fastidiosa. Four isolates, classified as X. fastidiosa subsp. fastidiosa, showed a low level of introgression of non-native DNA. One grapevine isolate showed introgression of an allele from X. fastidiosa subsp. pauca while the other three (from citrus and coffee) showed introgression of an allele with similar ancestry to the alleles of unknown origin in ST53. The presence of X. fastidiosa subsp. pauca in Central America is troubling given its disease potential, and establishes another route for the introduction of this economically damaging subspecies into the US or elsewhere, a threat potentially compounded by the presence of a previously unknown form of X. fastidiosa.     

Specific Detection and Identification of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Strains Causing Oleander Leaf Scorch Using Polymerase Chain Reaction

A pair of PCR primers, QH-OLS05/QH-OLS08 specific for strains of Xylella fastidiosa causing oleander leaf scorch was developed. The primers were designed according to a DNA sequence of a randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) product unique to oleander strains. The PCR assay using primer pair QH-OLS05/QH-OLS08 allowed quick and simple detection and identification of oleander strains in cultured bacterium and infected plant samples. The assay also can be applied to insect samples. Specific detection and identification of oleander strains of X. fastidiosa by PCR is useful for epidemiologic and etiologic studies of oleander leaf scorch by identifying what plants and insect vectors harbor or carry this particular strain of X. fastidiosa, especially in locations where mixed natural infections by oleander and other strains of X. fastidiosa occur.     

Bacteria causing important diseases of citrus utilise distinct modes of pathogenesis to attack a common host

In this review, we summarise the current knowledge on three pathogens that exhibit distinct tissue specificity and modes of pathogenesis in citrus plants. Xanthomonas axonopodis pv. citri causes canker disease and invades the host leaf mesophyll tissue through natural openings and can also survive as an epiphyte. Xylella fastidiosa and Candidatus Liberibacter are vectored by insects and proliferate in the vascular system of the host, either in the phloem (Candidatus Liberibacter) or xylem (X. fastidiosa) causing variegated chlorosis and huanglongbing diseases, respectively. Candidatus Liberibacter can be found within host cells and is thus unique as an intracellular phytopathogenic bacterium. Genome sequence comparisons have identified groups of species-specific genes that may be associated with the particular lifestyle, mode of transmission or symptoms produced by each phytopathogen. In addition, components that are conserved amongst bacteria may have diverse regulatory actions underpinning the different bacterial lifestyles; one example is the divergent role of the Rpf/DSF cell–cell signalling system in X. citri and X. fastidiosa. Biofilm plays a key role in epiphytic fitness and canker development in X. citri and in the symptoms produced by X. fastidiosa. Bacterial aggregation may be associated with vascular occlusion of the xylem vessels and symptomatology of variegated chlorosis.     

Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.     

Development and Application of Monoclonal Antibodies Against Xylella fastidiosa, the Causal Bacterium of Pear Leaf Scorch

Ten stable hybridoma cell lines, M As l -10, secreting monoclonal antibodies specific to the causal bacterium of pear leaf scorch (PLS), Xylella fastidiosa. were produced. The monoclonal antibodies can detect 3 × 10⁵ PLS-bacterium cells by indirect enzyme-linked immunosorbent assay (ELISA). In the antibody titer determination by indirect ELISA, hybridoma-culture supernatant from clone MA4 had the highest titer of 20480. In the antibody specificity tests, nine of the 10 monoclonal antibodies did not cross-react with 14 other bacterial strains belonging to nine genera. Only the antibody from hybridoma clone MAI cross-reacted with Xanthomonas campestris pv. cam-pestris and X. campestris pv. vesicatoria. In western blot analysis, all the monoclonal antibodies recognized the major 46.9-kDa polypeptide from all 12 X. fastidiosa strains and a distinct 21.5-kDa polypeptide only from PLS bacterium. In tissue-blotting detection, the PLS bacteria were specifically detected in blots of tissue sections from infected pear with the antibodies developed.     

Detection and Diversity Assessment of Xylella fastidiosa in Field-Collected Plant and Insect Samples by Using 16S rRNA and gyrB Sequences

The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.     

Transmission efficiency of Xylella fastidiosa by sharpshooters (Hemiptera: Cicadellidae) in coffee and citrus

Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner) is a bacterial pathogen transmitted by several sharpshooters in two tribes of Cicadellinae (Proconiini and Cicadellini). Here, we compared the transmission efficiency of X. fastidiosa in coffee (Coffea arabica L.) and citrus [Citrus sinensis (L.) Osbeck] by Cicadellini [Bucephalogonia xanthophis (Berg) and Dilobopterus costalimai Young] and Proconiini [Homalodisca ignorata Melichar and Oncometopia facialis (Signoret)] sharpshooters that occur in both crops. At different seasons, healthy adults of each species were submitted to a 48-h acquisition access period on citrus or coffee source plants infected with X. fastidiosa isolates that cause Citrus variegated chlorosis (CVC) and Coffee leaf scorch (CLS), respectively, and then confined on healthy seedlings of the corresponding host plant for a 48-h inoculation access period. No significant effect of inoculation season was observed when comparing infection rates of citrus or coffee plants inoculated by vectors at different times of the year. In citrus, the transmission rate by single insects was significantly higher for H. ignorata (30%) in relation to B. xanthophis (5%) and O. facialis (1.1%), but there was no difference among vector species in coffee, whose transmission rates ranged from 1.2 to 7.2%. Comparing host plants, H. ignorata was more effective in transmitting X. fastidiosa to citrus (30%) in relation to coffee (2.2%), whereas the other vectors transmitted the bacterium to both hosts with similar efficiencies. Despite these variations, vector efficiency in coffee and citrus is lower than that reported in other hosts.     

An Effective and Low-Cost Culture Medium for Isolation and Growth of Xylella fastidiosa from Citrus and Coffee Plants

Buffered charcoal–yeast extract medium (BCYE) has been used for isolation of Xylella fastidiosa from citrus (Citrus sinensis) and coffee (Coffea arabica) plants affected by citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). BCYE is composed of ACES (2-[2-amino-2oxoethyl) amino]-ethanesulfonic acid) buffer, activated charcoal, yeast extract, L-cysteine, ferric pyrophosphate, and agar. ACES buffer is costly and not always commercially available in Brazil, and the L-cysteine and ferric pyrophosphate need to be filter sterilized in 0.22-μm pore membranes before inclusion in the medium. Omission of L-cysteine, addition of magnesium sulfate, and replacements of ACES and ferric pyrophosphate for potassium phosphate and ferrous sulfate resulted in an effective, less expensive, and entirely autoclavable medium, named phosphate buffered charcoal-yeast extract medium (PCYE). The final cost of PCYE was approximately one tenth that of BCYE. Its effectiveness was tested for the isolation of X. fastidiosa from symptomatic leaves collected from 52 citrus plants affected by CVC and 43 coffee plants affected by CLS. PCYE was as effective as BCYE and has been used routinely in our and other laboratories for isolation, growth, and quantification of X. fastidiosa from plant tissues.     

Transmission of <Emphasis Type="Italic">Methylobacterium mesophilicum</Emphasis> by <Emphasis Type="Italic">Bucephalogonia xanthophis</Emphasis> for paratransgenic control strategy of Citrus variegated chlorosis

Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.     

Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data

Genetic relationships among 11 Xylella fastidiosa strains isolated from mulberry, almond, ragweed, grape, plum, elm, and citrus were determined by random amplified polymorphic DNA (RAPD). Twenty-two 10-base primers amplified a total of 77 discrete polymorphic bands. Phenetic analysis based on a similarity matrix corresponded well with previous reports on X. fastidiosa RFLP-based similarity relationships, indicating that RAPD-PCR amplification products can be used as a reliable indicator of genetic distance in X. fastidiosa. Cladistic analysis suggests the existence of five groups of X. fastidiosa: the citrus group, the plum-elm group, the grape-ragweed group, the almond group, and the mulberry group.     

Use of 16S rDNA Sequences as Signature Characters to Identify Xylella fastidiosa

The nucleotide sequences of 16S rDNAs (coding for the small subunit ribosomal RNAs) were used to identify Xylella fastidiosa, a nutritionally fastidious plant pathogenic bacterium. The near-complete 16S rDNAs from nine strains of Xyl. fastidiosa, including seven pathotypes and one strain of Xanthomonas campestris pv. campestris, were amplified through PCR with two conserved primers (forward primer 5′-AGA GTT TGA TCC TGG CTC AG-3′ and reverse primer 5′-AAG GAG GTG ATC CAG CC-3′) and sequenced. The 16S sequences were compared with all eukaryote and prokaryote DNA entries in GenBank database. A Xyl. fastidiosa 16S rDNA sequence, M26601, was determined to be the most similar to all the near-complete (1537 bp) and partial 5′ end sequences from Xyl. fastidiosa, but not those from the Xanthomonas strain. A 20-bp oligonucleotide (5′-TTG GTA GTA ATA CCA TGG GT-3′) was found to be highly characteristic of Xyl. fastidiosa. Since the 16S rDNA of Xyl. fastidiosa strains are highly homologous and characteristically different from other bacteria, including the most closely related Xanthomonas, 16S rDNA sequences can be used as signature characters to identify this bacterium. Received: 22 June 1999 / Accepted: 2 August 1999     

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.     

Indirect Immunofluorescence Microscopy for Direct Detection of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> in Xylem Sap

The plant pathogen Xylella fastidiosa is the causative agent of a number of diseases of economically important crops, including Pierces disease that affects grapevines. Using a commercially available antibody specific for X. fastidiosa, we have established a protocol for microscopic identification of the bacterium by indirect immunofluorescence. This antibody clearly labels an uncharacterized antigen concentrated at a single pole of X. fastidiosa cells, but does not react with a non-Xylella control. This technique was also performed successfully on xylem exudates from several different plant genera and correlated well with standard enzyme-linked immunosorbent assay tests. These results establish a novel method for in situ assessment of X. fastidiosa infection from host plants.