Citrus Variegated Chlorosis Strain Research Articles

Selection of host plants for vector transmission assays of citrus variegated chlorosis strains of Xylella fastidiosa subsp. pauca

The vector-borne bacterium Xylella fastidiosa subsp. pauca is the etiological agent of important plant diseases, such as citrus variegated chlorosis (CVC). Biological studies are relatively scarce for CVC, in part because citrus is a low titer woody host, in which X. fastidiosa has a long incubation period and low transmission efficiency by vectors. To optimize transmission bioassays, this study was conducted to: i) identify herbaceous plants that allow rapid multiplication and systemic colonization of CVC strains; and ii) evaluate the herbaceous hosts for feeding and survival of the sharpshooter vector, Bucephalogonia xanthophis (Berg), and as indicators of infection after vector inoculation. Mechanical inoculations showed systemic colonization of X. fastidiosa in seven plant species, with higher rates of infection mainly in Catharanthus roseus. The lowest vector mortality was observed on C. roseus, Medicago sativa and Ocimum basilicum. The highest feeding rate was verified on O. basilicum, followed by M. sativa and C. roseus. Infections after vector inoculation were detected in C. roseus, M. sativa, O. basilicum and S. americanum. Among the plant species tested, C. roseus proved to be the most suitable experimental plant for transmission assays with CVC strains of X. fastidiosa subsp. pauca.

Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus Display Differential Behavior, Secretome, and Plant Virulence.

Xylella fastidiosa colonizes the xylem of various cultivated and native plants worldwide. Citrus production in Brazil has been seriously affected, and major commercial varieties remain susceptible to Citrus Variegated Chlorosis (CVC). Collective cellular behaviors such as biofilm formation influence virulence and insect transmission of X. fastidiosa. The reference strain 9a5c produces a robust biofilm compared to Fb7 that remains mostly planktonic, and both were isolated from symptomatic citrus trees. This work deepens our understanding of these distinct behaviors at the molecular level, by comparing the cellular and secreted proteomes of these two CVC strains. Out of 1017 identified proteins, 128 showed differential abundance between the two strains. Different protein families were represented such as proteases, hemolysin-like proteins, and lipase/esterases, among others. Here we show that the lipase/esterase LesA is among the most abundant secreted proteins of CVC strains as well, and demonstrate its functionality by complementary activity assays. More severe symptoms were observed in Nicotiana tabacum inoculated with strain Fb7 compared to 9a5c. Our results support that systemic symptom development can be accelerated by strains that invest less in biofilm formation and more in plant colonization. This has potential application in modulating the bacterial-plant interaction and reducing disease severity.

Differential Responses of Tobacco to the Citrus Variegated Chlorosis and Coffee Stem Atrophy Strains of Xylella fastidiosa.

Xylella fastidiosa comprises a diverse group of xylem-limited, insect-transmitted bacterial pathogens. In Brazil, the citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) diseases are caused by X. fastidiosa subspecies pauca transmitted by common insect vectors. No simple protocol allowing strain discrimination exists, making epidemiological studies, which are important for devising control measures, difficult to undertake. Here, we show that both strains can easily be distinguished based on the pattern of leaf symptoms that they induce on pin prick-inoculated tobacco seedlings, namely small orange lesions and large necrotic lesions induced by the CVC and CSA strains, respectively. These differential responses allowed us to investigate whether mixed strain infections would occur in citrus or coffee trees in the field. Seedlings were individually inoculated with X. fastidiosa colonies recovered from citrus or coffee plants from various locations at three different times. No mixed infections were detected. In two experiments, the citrus and coffee strains infected only their original hosts as well as tobacco. The usefulness of this tobacco bioassay as a tool to study X. fastidiosa spread was demonstrated. It provided evidence that, over the years, the CVC and CSA pathogens have remained limited to their original hosts, despite crop proximity and the presence of sharpshooter vectors that favor transmission of the bacteria to and between both host species.

Proteomic and Metabolomic Analyses of Xylella fastidiosa OMV-Enriched Fractions Reveal Association with Virulence Factors and Signaling Molecules of the DSF Family.

Xylella fastidiosa releases outer membrane vesicles (OMVs) known to play a role in the systemic dissemination of this pathogen. OMVs inhibit bacterial attachment to xylem wall and traffic lipases/esterases that act on the degradation of plant cell wall. Here, we extended the characterization of X. fastidiosa OMVs by identifying proteins and metabolites potentially associated with OMVs produced by Temecula1, a Pierce's disease strain, and by 9a5c and Fb7, two citrus variegated chlorosis strains. These results strengthen that one of the OMVs multiple functions is to carry determinants of virulence, such as lipases/esterases, adhesins, proteases, porins, and a pectin lyase-like protein. For the first time, we show that the two citrus variegated chlorosis strains produce X. fastidiosa diffusible signaling factor 2 (DSF2) and citrus variegated chlorosis-DSF (likewise, Temecula1) and most importantly, that these compounds of the DSF (X. fastidiosa DSF) family are associated with OMV-enriched fractions. Altogether, our findings widen the potential functions of X. fastidiosa OMVs in intercellular signaling and host-pathogen interactions.

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the specific identification of X. fastidiosa strains that cause citrus variegated chlorosis.

Xylella fastidiosa: A Model for Analyzing Agricultural Biosecurity

The threat to national security posed by the hostile release of human pathogens has been extensively documented. However, conclusions regarding the menace that plant pests and pathogens pose to the national economy and food supply are less developed. Here we present a risk analysis for the arrival and spread of foreign plant pathogens into the U.S. agricultural system. Xylella fastidiosa Citrus Variegated Chlorosis strain, an exotic plant pathogenic bacterium listed as a regulated biological agent under the Agricultural Bioterrorism Protection Act of 2002, serves as a model to evaluate the threat that plant diseases pose to crops and agriculture-dependent industries. We assess the nation's capacity to detect, contain, and manage newly introduced plant pathogens, exposing areas that need improvement and limitations of the system. This analysis provides a framework for understanding the risk that exotic plant diseases pose to U.S. agricultural biosecurity and a reference to continue preparing for the possible arrival of these threats.

First Report of Xylella fastidiosa in Avocado in Costa Rica.

Since the late 1990s, chlorotic mottling, marginal scorch, deformation of leaves, defoliation, shortening of internodes, and branch dieback have been observed in avocado trees (Persea americana Mill.) in Costa Rica. The symptoms are not uniformly distributed in the tree, so some branches are symptomatic while others are not. These symptoms are similar to several leaf scorch diseases caused by the bacterium Xylella fastidiosa Wells (2,4). This bacterium has been detected in coffee and citrus plants in Costa Rica. Of 227 avocado trees tested by double-antibody sandwich (DAS)-ELISA with X. fastidiosa specific antiserum (Agdia Inc., Elkhart, IN) from 2000-2004, 188 were positive. Results of ELISA tests of individual trees varied with the season and branches tested. Fifteen greenhouse-grown, ELISA-negative avocado seedlings were grafted with budwood from an ELISA-positive tree. Eight of these developed scorch symptoms and one also showed chlorotic mottling and deformation, showing that the disease is graft transmitted. All of these features are characteristic of diseases caused by X. fastidiosa (2,4). Transmission electron microscopy of leaf petioles from three field trees positive by ELISA, revealed rod-shaped bacilli approximately 1.6 to 2.0 μm long and 0.3 μm in diameter with a rippled cell wall inside xylem vessels and embedded in a matrix; morphology and measurements that are consistent with those reported for X. fastidiosa (2). DNA extraction and PCR attempts have been limited by mucilaginous sap from avocado. Positive PCR results (approximately 472-bp band) were obtained from two of the grafted seedlings and seven field trees from two distinct geographical locations (Alajuela and San José provinces) with DNA extractions from the plant sap using DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) following a modified protocol (1) and nested PCR (3). Four of the PCR products, including one from the grafted seedlings, were cloned and sequenced in duplicate. GenBank sequences EU021997 to EU022000 present 99 to 100% sequence identity to a Pierce's disease strain from California (Temecula1) and 94 to 95% to a citrus variegated chlorosis strain from Brazil (Found-5). Several attempts have been made to isolate the bacterium in 'periwinkle wilt' and buffered cysteine-yeast extract media with negative results, probably because of the rapid production of mucilaginous sap when the avocado tissues were sampled. To our knowledge, this is the first report of X. fastidiosa in avocado trees.

Host colonization differences between citrus and coffee isolates of Xylella fastidiosa in reciprocal inoculation

Citrus variegated chlorosis (CVC) and coffee stem atrophy (CSA) are important diseases in Brazil associated with closely-related strains of Xylella fastidiosa, but little is know about host aoverlappingnd importance of citrus and coffee as inoculum sources of these strains. In this study, reciprocal-inoculation experiments were performed to determine if CVC and CSA isolates are biologically similar within citrus and coffee plants. These two hosts were mechanically inoculated with a CVC and a CSA isolate of X. fastidiosa at four concentrations ranging between10³ and 10(9) colony forming units CFU mL-1. At two, four and eight months after inoculation, the infection efficiency and bacterial populations of the isolates in each host were determined by culturing. The CVC isolate infected both citrus and coffee plants, but developed lower populations in coffee. The CSA isolate did not colonize citrus. Inoculation of coffee plants with the CVC isolate resulted in low rates of infection and required an inoculum concentration ten-fold higher than that necessary to obtain a similar (25%) rate of infection in citrus. The relatively low infection rates and bacterial numbers of the CVC isolate in coffee plants compared with those observed in citrus suggest that coffee is not a suitable host to serve as a source of inoculum of the CVC strain for primary spread to citrus or within coffee plantations.

Etiology of three recent diseases of citrus in São Paulo State: Sudden death, variegated chlorosis and huanglongbing

The state of São Paulo (SSP) is the first sweet orange growing region in the world. Yet, the SSP citrus industry has been, and still is, under constant attack from various diseases. In the 1940s, tristeza-quick decline (T-QD) was responsible for the death of 9 million trees in SSP. The causal agent was a new virus, citrus tristeza virus (CTV). The virus was efficiently spread by aphid vectors, and killed most of the trees grafted on sour orange rootstock. Control of the disease resided in replacing sour orange by alternative rootstocks giving tolerant combinations with scions such as sweet orange. Because of its drought resistance, Rangpur lime became the favourite alternative rootstock, and, by 1995, 85% of the SSP sweet orange trees were grafted on this rootstock. Therefore, when in 1999, many trees grafted on Rangpur lime started to decline and suddenly died, the spectre of T-QD seemed to hang over SSP again. By 2003, the total number of dead or affected trees was estimated to be over one million. The new disease, citrus sudden death (CSD), resembles T-QD in several aspects. The two diseases have almost the same symptoms, they spread in time and space in a manner strikingly similar, and the pathological anatomy of the bark at the bud union is alike. Transmission of the CSD agent by graft-inoculation has been obtained with budwood inoculum taken not only on CSD-affected trees (grafted on Rangpur lime), but also on symptomless trees (grafted on Cleopatra mandarin) from the same citrus block. This result shows that symptomless trees on Cleopatra mandarin are tolerant to the CSD agent. Trees on rootstocks such as Sunki mandarin or Swingle citrumelo are also tolerant. Thus, in the CSD-affected region, control consists in replacing Rangpur lime with compatible rootstocks, or in approach-grafting compatible rootstock seedlings to the scions of trees on Rangpur lime (inarching). More than 5 million trees have been inarched in this way. A new disease of sweet orange, citrus variegated chlorosis (CVC), was observed in 1987 in the Triangulo Mineiro of Minas Gerais State and the northern and north-eastern parts of SSP. By 2000, the disease affected already 34% of the 200 million sweet orange trees in SSP. By 2005, the percentage had increased to 43%, and CVC was present in all citrus growing regions of Brazil. Electron microscopy showed that xylem-limited bacteria were present in all symptomatic sweet orange leaves and fruit tissues tested, but not in similar materials from healthy, symptomless trees. Bacteria were consistently cultured from twigs of CVC-affected sweet orange trees but not from twigs of healthy trees. Serological analyses showed the CVC bacterium to be a strain of Xylella fastidiosa. The disease could be reproduced and Koch's postulates fulfilled, by mechanically inoculating a pure culture of X. fastidiosa isolate 8.1.b into sweet orange seedlings. The genome of a CVC strain of X. fastidiosa was sequenced in SSP in the frame of a project supported by FAPESP and Fundecitrus. X. fastidiosa is the first plant pathogenic bacterium, the genome of which has been sequenced. Until recently, America was free of huanglongbing (HLB), but in March 2004 and August 2005, symptoms of the disease were recognized, respectively in the State of São Paulo (SSP) and in Florida, USA. HLB was known in China since 1870 and in South Africa since 1928. Because of its destructiveness and its rapid spread by efficient psyllid insect-vectors, HLB is probably the most serious citrus disease. HLB is caused by a phloem sieve tube-restricted Gram negative bacterium, not yet available in culture. In the 1990s, the bacterium was characterized by molecular techniques as a member of the alpha proteobacteria designated Candidatus Liberibacter africanus for the disease in Africa, and Candidatus Liberibacter asiaticus for HLB in Asia. In SSP, Ca. L. asiaticus is also present, but most of the trees are infected with a new species, Candidatus Liberibacter americanus.

Transmission of Xylella fastidiosa, Causal Agent of Citrus Variegated Chlorosis, by the Glassy-Winged Sharpshooter, Homalodisca coagulata.

Citrus variegated chlorosis (CVC) is an economically important disease of citrus in Brazil and Argentina. The causal pathogen is a strain of Xylella fastidiosa transmitted by several sharpshooter species. The glassy-winged sharpshooter (GWSS), Homalodisca coagulata, has become an important new pest of citrus and grapevines in California, where it transmits X. fastidiosa strains to several crops including grapes, oleander, and almonds. Transmission studies over a 3-year period at the USDA BSL3-P containment facility at Fort Detrick, MD, utilizing California field-collected GWSS, a Brazilian strain of CVC, and Madam Vinous sweet orange seedlings, have shown a consistent although low level of transmission of CVC. Test plants were observed for CVC symptoms, analyzed by polymerase chain reaction using species-specific primers for X. fastidiosa, membrane entrapment immunofluorescence, and scanning electron microscopy. X. fastidiosa was not detected in field-collected GWSS but was detected in GWSS following feeding on CVC-infected source plants. Transmission of the CVC strain of X. fastidiosa by GWSS increases the risk of establishment of CVC in the United States if it were introduced.

An Experimental Inoculation System to Study Citrus-Xylella fastidiosa Interactions.

Difficulties in reproducing the citrus variegated chlorosis (CVC) disease symptoms in experimental plants have delayed implementation of studies to better understand the essential aspects of this important disease. In an extensive study, cultivars of sweet orange (Citrus sinensis) were inoculated with Xylella fastidiosa using procedures that included root immersion, and stem absorption, pricking, or infiltration of the inoculum into plants of different ages. Inoculum consisted of 5-day-old cultures or cell suspensions of CVC strain 9a5c diluted in phosphate-buffered saline. Inoculated plants and controls were grown, or transferred just after inoculation, to 5-liter pots or 72-cell foam trays. Approximately 4, 5, 9, and 12 months after inoculation, leaves were collected and processed for polymerase chain reaction analysis or X. fastidiosa isolation on BCYE agar medium. Root immersion and stem inoculation of 4- and 6-month-old plants resulted in low percentages of symptomatic (0 to 7%) and plants positive by isolation (0 to 9%). Pinpricked or injected stems of 1-month-old seedlings resulted in high percentages of plants symptomatic (29 and 90% in Pera Rio, 75, 59, and 83% in Valencia, and 77% in Natal) or positive by isolation (26 and 93% in Pera Rio, 98, 96, and 83% in Valencia, and 77% in Natal). In foam trays, the seedlings grew less, the incubation period was shorter, and disease severity was higher than in pots. This system allows testing of higher numbers of plants in a reduced space with a more precise reproduction of the experimental conditions.

Laboratory-Based Monitoring of an Insect-Transmitted Plant Pathogen System

winged sharpshooter, Homalodisca coagulata (Say), is challenging to study, not only because the bacterium is fastidious, but also because the insect does not survive well in captivity, and it will not feed from an artificial diet (i.e., parafilm sachet). We developed a simple and efficient transmission cycle for the study of X. fastidiosa transmission by H. coagulata, allowing collection of sufficient transmission data in 1 week. Specific numbers of cells can be detected both in the plant tissue and within the insect vector by realtime PCR (7). Real-time PCR is more sensitive than traditional PCR and is a single step process, thus reducing the possibility of error and speeding up detection time. A real-time PCR protocol for detecting the citrus variegated chlorosis strain of X. fastidiosa has already been established as a superior technique compared to traditional PCR (8). Real-time PCR was performed in a Rotor-Gene™ 3000 (Corbett Research, Mortlake, NSW, Australia) using iQ™ Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in 20-µL reactions with 525 nM of each X.

A Suitable Xylella fastidiosa 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.

Insertional transposon mutagenesis in the Xylella fastidiosa 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 x 10(3) kanamycin (Km)-resistant clones per microg 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.

Weeds as Alternative Hosts of the Citrus, Coffee, and Plum Strains of Xylella fastidiosa in Brazil.

In Brazil, Xylella fastidiosa is present in citrus (Citrus sinensis), coffee (Coffea arabica), and plum (Prunus sp.) crops, causing the citrus variegated chlorosis (CVC), coffee leaf scorch (CLS), and plum leaf scald (PLS). Also present in these crops and infesting weeds, which ultimately could serve as sources of inoculum for the cultivated trees, are diverse populations of xylem-feeding leafhopper vectors. In order to assess host range of X. fastidiosa among weeds and to better understand their role in epidemics, field surveys, mechanical inoculations, and insect transmission tests were conducted. Polymerase chain reaction (PCR) and culture plating were used to detect the pathogen from plant tissues. X. fastidiosa was detected in 10 out of 23 species of the weed plants sampled in two citrus groves affected by CVC. None of the weed plants showed external symptoms. In the greenhouse, the average percentages of infection on plants mechanically inoculated with the CVC, CLS, and PLS strains of X. fastidiosa were, respectively, 25, 10, 0 in Medicago sativa; 70, 45, 20 in Echinochloa crus-galli; 45, 30, 0 in Brachiaria decumbens; 72, 70, 40 in Brachiaria plantaginea; 13, 10, 0 in Digitaria horizontalis; 31, 30, 0 in Solanum americanum; and 17, 0, 0 in Bidens pilosa. Symptoms were observed only in S. americanum and citrus and only when inoculated with the CVC strain. In insect transmission tests, the grass leafhopper Ferrariana trivittata was first caged on citrus plants showing CVC symptoms and then on healthy citrus and on the four most common weeds. No plants tested positive by PCR or culture, or showed symptoms for at least 4 months after inoculation. The amount of X. fastidiosa cells that may accumulate in weeds inoculated by leafhoppers is probably under insect acquisition thresholds, a factor that would limit their importance to the CVC epidemics, as studies on spatial distribution of diseased citrus trees over time indicate.

Genetic differences between two strains of Xylella fastidiosa revealed by suppression subtractive hybridization.

Suppression subtractive hybridization was used to rapidly identify 18 gene differences between a citrus variegated chlorosis (CVC) strain and a Pierce's disease of grape (PD) strain of Xylella fastidiosa. The results were validated as being highly representative of actual differences by comparison of the completely sequenced genome of a CVC strain with that of a PD strain.

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.

Stable transformation of the Xylella fastidiosa citrus variegated chlorosis strain with oriC plasmids.

Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.

Diversity of a Xylella fastidiosa Population Isolated from Citrus sinensis Affected by Citrus Variegated Chlorosis in Brazil

Summary Diversity of a population of Xylella fastidiosa isolated from sweet orange plants showing citrus variegated chlorosis (CVC) symptoms was assessed by PCR-based techniques. Thirty-seven strains were isolated throughout the 1997 year in the orange belt of Sao Paulo State, Brazil. Strains isolated from coffee, grape, oleander and plum were also included as outgroup reference strains. PCR amplification of the spacer sequence between the 16-23S rDNA yielded one fragment of 1.2 kb. Digestion with restriction enzymes, Dde I, Hinf I or Sau3 AI generated identical RFLP patterns for citrus and coffee strains, which could be distinguished from the strains isolated from the other hosts. Eight RAPD primers were also used and the results showed similarity from 80 to 100% within the CVC population. Three prevalent haplotypes comprising 24 CVC strains showed a high level of similarity (95%). Strains from the other hosts clustered apart from CVC strains, forming distinct groups.

Coffee Leaf Scorch Bacterium: Axenic Culture, Pathogenicity, and Comparison with Xylella fastidiosa of Citrus.

Symptoms of coffee leaf scorch (CLS) appear on young flushes of field plants as large marginal and apical scorched areas on recently mature leaves. Affected leaves drop, shoot growth is stunted, and apical leaves are small and chlorotic. Symptoms may progress to shoot dieback. Only scorched leaves which could not be related to other known agents consistently contained bacteria and bacterial agglomerates when observed with light microscopy. Only plants with these symptoms were positive in enzyme-linked immunosorbent assay (ELISA) tests using antiserum to Xylella fastidiosa Wells et al. The bacterium Xylella fastidiosa Wells et al. was isolated in November 1995 from coffee (Coffea arabica) leaves with scorch symptoms on supplemented periwinkle wilt medium. Colonies were circular, dome-shaped, white, and 0.5 to 1.5 mm in diameter. Two of 10 young coffee seedlings stem-inoculated with a suspension of the isolated X. fastidiosa in January 1996 showed leaf scorch symptoms 3 to 5 months later, contained bacteria in xylem extracts, and reacted positively in ELISA using antiserum to the citrus variegated chlorosis (CVC) strain of X. fastidiosa. ELISA-positive bacteria were reisolated from this plant. None of the symptomless plants, including controls, revealed bacteria on microscopic examinations, ELISA, or isolation attempts. Antisera developed against cultured bacteria from both CLS and CVC plants reacted positively against plant extracts of both diseases in dot immunobinding assays (DIBA). The level of detection was about 5 × 105 bacteria ml-1 for both homologous and heterologous reactions. The polymerase chain reaction amplification products produced by CLS and CVC strains of X. fastidiosa were indistinguishable. Geographical distribution of these strains is not the same. CLS is widespread and usually occurs if coffee is adjacent to CVC-affected citrus. However, CVC does not always occur when citrus is grown adjacent to CLS-affected coffee. The bacteria are closely related, if not identical.