A Triply Cloned Strain of Xylella fastidiosa Multiplies and Induces Symptoms of Citrus Variegated Chlorosis in Sweet Orange

Xylella fastidiosa isolate 8.1.b obtained from a sweet orange tree affected by citrus variegated chlorosis in the state of S?o Paulo, Brazil, and shown in 1993 to be the causal agent of the disease, was cloned by repeated culture in liquid and on solid PW medium, yielding triply cloned strain 9a5c. The eighth and the 16th passages of strain 9a5c were mechanically inoculated into sweet orange plants. Presence of X. fastidiosa in sweet orange leaves of shoots having grown after inoculation (first-flush shoots) was detected by DAS-ELISA and PCR. Thirty-eight days after inoculation, 70% of the 20 inoculated plants tested positive, and all plants gave strong positive reactions 90 days after inoculation. Symptoms first appeared after 3 months and were conspicuous after 5 months. X. fastidiosa was reisolated from sweet orange leaves, 44 days after inoculation. These results indicate that X. fastidiosa strain 9a5c, derived from pathogenic isolate 8.1.b by triply cloning, is also pathogenic. Strain 9a5c is now used for the X. fastidiosa genome sequencing project undertaken on a large scale in Brazil. Received: 1 February 1999 / Accepted: 1 April 1999     

Potential vectors of Xylella fastidiosa: a study of leafhoppers and treehoppers in citrus agroecosystems affected by Citrus Variegated Chlorosis

This study investigated the predominant leafhopper and treehopper (Hemiptera, Auchenorrhyncha) species in Citrus Variegated Chlorosis (CVC)‐affected citrus agroecosystems in Argentina, their seasonal fluctuation, and their potential role as vectors of Xylella fastidiosa Wells et al., using molecular methods for detection. More than 6 000 Auchenorrhyncha were collected from three citrus agroecosystems over a period of 3 years using yellow sticky traps and entomological nets. Cicadellidae and Membracidae were the most abundant families. Of the 43 species identified, five were predominant in citrus orchards, and three were predominant in weeds surrounding citrus plants. All predominant species and another four non‐predominant species tested positive for X. fastidiosa in PCR and real‐time PCR assays. In a transmission assay, Dechacona missionum (Berg), Tapajosa rubromarginata (Signoret), and Cyphonia clavigera (Fabricius) transmitted X. fastidiosa successfully. Scaphytopius bolivianus Oman and Frequenamia spiniventris (Linnavuori) populations increased once (during the summer), possibly due to favorable weather conditions, and Bucephalogonia xanthophis (Berg), Molomea lineiceps Young, and T. rubromarginata populations increased twice a year: once in summer and once in winter, coinciding with the increase in early citrus shoots (flush). Among the X. fastidiosa‐positive species, those with the higher population densities during the sprouting period, where trees are highly susceptible to infection, must be considered as most relevant vectors of CVC in the citrus‐growing areas in Argentina.     

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.     

Insertional Transposon Mutagenesis in the <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Citrus Variegated Chlorosis Strain with Transposome

To overcome the difficulty in obtaining mutants of the citrus strains of Xylella fastidiosa, we evaluated mutagenesis using the transposome system as a tool for the isolation of a large number of mutants. Electroporation of a commercial transposome system in X. fastidiosa CVC (Citrus Variegated Chlorosis) strain J1a12 yielded an efficiency of 1.2 × 10³ kanamycin (Km)-resistant clones per g of DNA. Southern blot analysis demonstrated that the transposon was randomly inserted, and nucleotide sequence analysis indicated the presence of 9 bp direct repeats flanking the transposon insertion site. Analysis by PCR of one of the insertion mutants (clone J15) showed that the transposon was stable after eight passages in solid media. These results show that the transposome system can be used to generate a random mutant library of Xylella fastidiosa CVC strain.     

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

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

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.     

Assessment of the diagnostic potential of Immmunocapture-PCR and Immuno-PCR for Citrus Variegated Chlorosis

Xylella fastidiosa causes significant losses in many economically important crops. An efficient pathogen detection system is critical for epidemiology studies, particularly when large sample size is involved. In this study we report the development of immunomolecular assays like Immmunocapture-PCR and Immuno-PCR for direct detection of X. fastidiosa without DNA isolation. Whereas the reactivity of ELISA and PCR ranged from 10(6) to 10(4) bacterial cells, the IC-PCR sensitivity was up to 10(3) and the detection limit of I-PCR was up to 10(1) bacterial cells. These methods can use either plant sample extracts or cultivated media, and show no cross reaction for any other endophytic citrus-bacteria. Therefore, IC-PCR and I-PCR assays provide an alternative for quick and very sensitive methods to screening X. fastidiosa, with the advantage of not requiring any concentration or DNA purification steps while still allowing an accurate diagnosis of CVC.     

Gene disruption by homologous recombination in the Xylella fastidiosa citrus variegated chlorosis strain

Mutagenesis by homologous recombination was evaluated in Xylella fastidiosa by using the bga gene, coding for beta-galactosidase, as a model. Integration of replicative plasmids by homologous recombination between the cloned truncated copy of bga and the endogenous gene was produced by one or two crossover events leading to beta-galactosidase mutants. A promoterless chloramphenicol acetyltransferase gene was used to monitor the expression of the target gene and to select a cvaB mutant.     

Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

Evaluation of the Genetic Structure of Xylella fastidiosa Populations from Different Citrus sinensis Varieties

Xylella fastidiosa was isolated from sweet orange plants (Citrus sinensis) grown in two orchards in the northwest region of the Brazilian state of São Paulo. One orchard was part of a germ plasm field plot used for studies of citrus variegated chlorosis resistance, while the other was an orchard of C. sinensis cv. Pêra clones. These two collections of strains were genotypically characterized by using random amplified polymorphic DNA (RAPD) and variable number of tandem repeat (VNTR) markers. The genetic diversity (H_T) values of X. fastidiosa were similar for both sets of strains; however, H_TRAPD values were substantially lower than H_TVNTR values. The analysis of six strains per plant allowed us to identify up to three RAPD and five VNTR multilocus haplotypes colonizing one plant. Molecular analysis of variance was used to determine the extent to which population structure explained the genetic variation observed. The genetic variation observed in the X. fastidiosa strains was not related to or dependent on the different sweet orange varieties from which they had been obtained. A significant amount of the observed genetic variation could be explained by the variation between strains from different plants within the orchards and by the variation between strains within each plant. It appears, therefore, that the existence of different sweet orange varieties does not play a role in the population structure of X. fastidiosa. The consequences of these results for the management of sweet orange breeding strategies for citrus variegate chlorosis resistance are also discussed.     

Evaluation of the genetic structure of Xylella fastidiosa populations from different Citrus sinensis varieties

Xylella fastidiosa was isolated from sweet orange plants (Citrus sinensis) grown in two orchards in the northwest region of the Brazilian state of S?o Paulo. One orchard was part of a germ plasm field plot used for studies of citrus variegated chlorosis resistance, while the other was an orchard of C. sinensis cv. Pêra clones. These two collections of strains were genotypically characterized by using random amplified polymorphic DNA (RAPD) and variable number of tandem repeat (VNTR) markers. The genetic diversity (H(T)) values of X. fastidiosa were similar for both sets of strains; however, H(T)(RAPD) values were substantially lower than H(T)(VNTR) values. The analysis of six strains per plant allowed us to identify up to three RAPD and five VNTR multilocus haplotypes colonizing one plant. Molecular analysis of variance was used to determine the extent to which population structure explained the genetic variation observed. The genetic variation observed in the X. fastidiosa strains was not related to or dependent on the different sweet orange varieties from which they had been obtained. A significant amount of the observed genetic variation could be explained by the variation between strains from different plants within the orchards and by the variation between strains within each plant. It appears, therefore, that the existence of different sweet orange varieties does not play a role in the population structure of X. fastidiosa. The consequences of these results for the management of sweet orange breeding strategies for citrus variegate chlorosis resistance are also discussed.     

Diversity of Endophytic Bacterial Populations and Their Interaction with Xylella fastidiosa in Citrus Plants

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.     

Gene Knockout of the Intracellular Amylase Gene by Homologous Recombination in Streptococcus bovis

Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular α-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15–20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium. Received: 26 August 1996 / Accepted: 18 December 1996     

Large-Scale Intersubspecific Recombination in the Plant-Pathogenic Bacterium Xylella fastidiosa Is Associated with the Host Shift to Mulberry

Homologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low within-type genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.     

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.     

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.     

影响动物细胞同源重组发生与基因打靶效率的分子机制

真核细胞的基因打靶是基因结构与功能研究的一种非常有价值的技术,也是可应用于基因治疗的具有潜力的工具。有2个限制因素束缚真核细胞基因打靶的发展,即同源重组(HR)率非常低而随机整合率非常高。通过特定基因的过表达或表达干涉,使一些参与DNA重组的蛋白表达水平瞬间改变,可能会增加HR率,降低随机整合率。本文列举了一些与HR相关的候选基因,详细介绍了其中的Rad52上位簇基因,还讨论了打靶载体的设计与修饰、DNA转染方法的有效性等。