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.     

An Evolutionary Perspective of Pierce's Disease of Grapevine,Citrus Variegated Chlorosis,and Mulberry Leaf Scorch Diseases

Xylella fastidiosa causes diseases on a growing list of economically important plants. An understanding of how xylellae diseases originated and evolved is important for disease prevention and management. In this study, we evaluated the phylogenetic relationships of X. fastidiosa strains from citrus, grapevine, and mulberry through the analyses of random amplified polymorphic DNAs (RAPDs) and conserved 16S rDNA genes. RAPD analysis emphasized the vigorous genome-wide divergence of X. fastidiosa and detected three clonal groups of strains that cause Pierce's disease (PD) of grapevine, citrus variegated chlorosis (CVC), and mulberry leaf scorch (MLS). Analysis of 16S rDNA sequences also identified the PD and CVC groups, but with a less stable evolutionary tree. MLS strains were included in the PD group by the 16S rDNA analysis. The Asiatic origins of the major commercial grape and citrus cultivars suggest the recent evolution of both PD and CVC disease in North and South America, respectively, since X. fastidiosa is a New World organism. In order to prevent the development of new diseases caused by X. fastidiosa, it is important to understand the diversity of X. fastidiosa strains, how strains of X. fastidiosa select their hosts, and their ecological roles in the native vegetation. Received: 7 February 2002 / Accepted: 7 March 2002     

Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis

By cloning and sequencing specific randomly amplified polymorphic DNA (RAPD) products, we have developed pairs of PCR primers that can be used to detect Xylella fastidiosa in general, and X. fastidiosa that cause citrus variegated chlorosis (CVC) specifically. We also identified a CVC-specific region of the X. fastidiosa genome that contains a 28-nucleotide insertion, and single base changes that distinguish CVC and grape X. fastidiosa strains. When using RAPD products to develop specific PCR primers, we found it most efficient to screen for size differences among RAPD products rather than presence/absence of a specific RAPD band.     

Expression of Green Fluorescent Protein in <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Is Affected by Passage Through Host Plants

Xylella fastidiosa, a Gram-negative bacterial plant pathogen, causes Pierces disease of grapevine in North America. In South America the pathogen causes citrus variegated chlorosis, which is widespread in Brazil. We have introduced into Xylella fastidiosa a mini-Tn5 transposon that encodes a green fluorescent protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was expressed well and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. The transconjugants were able to colonize the plants and were subsequently isolated from points distal to the inoculation sites. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared with that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly increased the level of expression of the GFP. The increased level of expression of GFP was transient, and was lost upon further culture in vitro. Xylella fastidiosa forms biofilms in planta which are believed to represent a metabolically differentiated state. The increased expression of GFP observed after passage through plants may be accounted for by this phenomenon.     

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.     

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.     

Global gene expression under nitrogen starvation in <Emphasis Type="Italic">Xylella fastidiosa</Emphasis>: contribution of the σ<Superscript>54</Superscript> regulon

Background  

Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat.     

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.     

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     

Influence of Xylella fastidiosa infection of citrus on host selection by leafhopper vectors

Infection of plants by pathogens can influence their attractiveness and suitability to insect vectors and other herbivores. Here we examined the effects of Citrus sinensis (L.) Osbeck (Rutaceae) infection by the bacterium Xylella fastidiosa, which causes citrus variegated chlorosis (CVC), on the feeding preferences of two sharpshooter vectors, Dilobopterus costalimai Young and Oncometopia facialis (Signoret) (Homoptera: Cicadellidae). Experiments were performed inside observation chambers, in which a healthy plant and an infected one (with or without CVC symptoms) were supplied to a group of 40 sharpshooters. The number of insects that selected each treatment was recorded at several time intervals in 48 h. In another experiment, the ingestion rate on healthy and infected (symptomatic or not) plants was evaluated by measuring the liquid excretion of sharpshooters that were confined on branches of each plant for 72 h. Both sharpshooter species preferred healthy plants to those with CVC symptoms. However, O. facialis did not discriminate between healthy citrus and symptomless infected plants. Feeding by D. costalimai was markedly reduced when confined on CVC‐symptomatic plants, but not on asymptomatic infected ones. The ingestion rate by O. facialis was not affected by the presence of CVC symptoms. The results suggest that citrus trees with early (asymptomatic) infections by X. fastidiosa may be more effective as inoculum sources for CVC spread by insect vectors than those with advanced symptoms.     

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.     

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.     

Utilization of the Etest Assay for Comparative Antibiotic Susceptibility Profiles of Citrus Variegated Chlorosis and Pierce’s Disease Strains of Xylella fastidiosa

Xylella fastidiosa has a wide host range. Isolates of this bacterium that cause diseases in citrus (CVC) and grapes (PD) share 98% genome homology, and 95.7% amino acid identity. Drug resistance genes show a higher level of divergence and may be involved in the X. fastidiosa–host interaction. Antibiotic susceptibility of CVC and PD strains were compared utilizing the Etest strip method (AB Biodisk). Etest is applicable for fastidious slow-growing organisms due to its reproducibility. Results showed that the CVC strain was resistant to bacitracin, cefotaxime, and trimethoprim, and susceptible to chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, and tetracycline. The PD strain was susceptible to all tested antibiotics, except kanamycin and trimethoprim. Both isolates produced a class C β-lactamase. These data support previous antibiotic studies and gene discrepancies found in the sequencing data of PD and CVC strains. These results demonstrate the efficacy of utilizing Etest assays for X. fastidiosa strains.     

A Suitable <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> CVC Strain for Post-Genome Studies

The genome sequence of the pathogen Xylella fastidiosa Citrus Variegated Chlorosis (CVC) strain 9a5c has revealed many genes related to pathogenicity mechanisms and virulence determinants. However, strain 9a5c is resistant to genetic transformation, impairing mutant production for the analysis of pathogenicity mechanisms and virulence determinants of this fastidious phytopathogen. By screening different strains, we found out that cloned strains J1a12, B111, and S11400, all isolated from citrus trees affected by CVC, are amenable to transformation, and J1a12 has been used as a model strain in a functional genomics program supported by FAPESP (São Paulo State Research Foundation). However, we have found that strain J1a12, unlike strains 9a5c and B111, was incapable of inducing CVC symptoms when inoculated in citrus plants. We have now determined that strain B111 is an appropriate candidate for post-genome studies of the CVC strain of X. fastidiosa.     

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.     

Assessment of the genetic diversity of Xylella fastidiosa isolated from citrus in Brazil by PCR-RFLP of the 16S rDNA and 16S-23S intergenic spacer and rep-PCR fingerprinting

The genetic diversity among twenty three strains of Xylella fastidiosa, isolated from sweet orange citrus, was assessed by RFLP analysis of the 16S rDNA and 16S-23S intergenic spacer and by rep-PCR fingerprinting together with strains isolated from coffee, grapevine, plum and pear. The PCR products obtained by amplification of the 16S rDNA and 16S-23S spacer region were digested with restriction enzymes and a low level of polymorphism was detected. In rep-PCR fingerprinting, a relationship between the strains and their hosts was observed by using the BOX, ERIC and REP primers. Two major groups were obtained within the citrus cluster and relationships to the geographic origin of the strains revealed. Citrus strains isolated from the States of São Paulo and Sergipe formed one group and strains from the Southern States formed another group. Distinct origins of X. fastidiosa in the Southern and Southeastern States is postulated. The pear isolate was distantly related to all of the other X. fastidiosa strains.     

Genetic Discovery in Xylella fastidiosa Through Sequence Analysis of Selected Randomly Amplified Polymorphic DNAs

Xylella fastidiosa causes many important plant diseases including Pierces disease (PD) in grape and almond leaf scorch disease (ALSD). DNA-based methodologies, such as randomly amplified polymorphic DNA (RAPD) analysis, have been playing key roles in genetic information collection of the bacterium. This study further analyzed the nucleotide sequences of selected RAPDs from X. fastidiosa strains in conjunction with the available genome sequence databases and unveiled several previously unknown novel genetic traits. These include a sequence highly similar to those in the phage family of Podoviridae. Genome comparisons among X. fastidiosa strains suggested that the phage is currently active. Two other RAPDs were also related to horizontal gene transfer: one was part of a broadly distributed cryptic plasmid and the other was associated with conjugal transfer. One RAPD inferred a genomic rearrangement event among X. fastidiosa PD strains and another identified a single nucleotide polymorphism of evolutionary value.     

Leaf Symptoms on Plum, Coffee and Citrus and the Relationship with the Extent of Xylem Vessels Colonized by Xylella fastidiosa

Leaf petioles of plum, coffee and sweet orange were examined using scanning electron microscopy (SEM). The presence of Xylella fastidiosa in the samples was confirmed by polymerase chain reaction and gel electrophoresis. The number of vessels colonized by X. fastidiosa was determined by SEM in petiole areas that were transversally sectioned under liquid nitrogen. The percentage of colonized vessels in petioles of coffee was higher than in petioles of plum and citrus whether trees were exhibiting mild symptoms (MS) or severe symptoms (SS). The percentage of vessels colonized varied from 10.9 (MS) to 38.0% (SS), 26 (MS) to 51.6% (SS), and 8 (MS) to 11.8% (SS) for plum, coffee and citrus, respectively, and did not vary by position within the petiole. Severity of symptoms consistently reflected higher proportion of colonized vessels in coffee and plum, but not in citrus.     

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.     

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.