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.     

Strains of Xylella fastidiosa Rapidly Distinguished by Arbitrarily Primed-PCR

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0.872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors. Received: 12 October 1999 / Accepted: 16 November 1999     

Specific Detection and Identification of American Mulberry-Infecting and Italian Olive-Associated Strains of Xylella fastidiosa by Polymerase Chain Reaction

Xylella fastidiosa causes bacterial leaf scorch in many landscape trees including elm, oak, sycamore and mulberry, but methods for specific identification of a particular tree host species-limited strain or differentiation of tree-specific strains are lacking. It is also unknown whether a particular landscape tree-infecting X. fastidiosa strain is capable of infecting multiple landscape tree species in an urban environment. We developed two PCR primers specific for mulberry-infecting strains of X. fastidiosa based on the nucleotide sequence of a unique open reading frame identified only in mulberry-infecting strains among all the North and South American strains of X. fastidiosa sequenced to date. PCR using the primers allowed for detection and identification of mulberry-infecting X. fastidiosa strains in cultures and in samples collected from naturally infected mulberry trees. In addition, no mixed infections with or non-specific detections of the mulberry-infecting strains of X. fastidiosa were found in naturally X. fastidiosa-infected oak, elm and sycamore trees growing in the same region where naturally infected mulberry trees were grown. This genotype-specific PCR assay will be valuable for disease diagnosis, studies of strain-specific infections in insects and plant hosts, and management of diseases caused by X. fastidiosa. Unexpectedly but interestingly, the unique open reading frame conserved in the mulberry-infecting strains in the U. S. was also identified in the recently sequenced olive-associated strain CoDiRO isolated in Italy. When the primer set was tested against naturally infected olive plant samples collected in Italy, it allowed for detection of olive-associated strains of X. fastidiosa in Italy. This PCR assay, therefore, will also be useful for detection and identification of the Italian group of X. fastidiosa strains to aid understanding of the occurrence, evolution and biology of this new group of X. fastidiosa strains.     

Xylella fastidiosa Does Not Occur in Lebanon

Xylella fastidiosa has been reported as responsible for a devastating disease on olive trees in Apulia region (south‐eastern Italy), characterized by a quick decline syndrome. In Lebanon, the pathogen was recently associated with leaf scorch symptoms on oleander, and reports on leaf scorch and dieback of olive trees branches by technicians and farmers have shown an increasing trend in the main agricultural areas. To assess the occurrence and distribution of the pathogen in Lebanon, samples of twigs from olive trees (82), olive seedlings (26), grapevine (30), oleander (32) and ornamentals imported from Italy (48) were analysed by isolation on four agarized media, serological techniques (ELISA and DTBIA) using Xylella fastidiosa‐specific antibodies and by PCR, using three specific sets of primers. Results unequivocally demonstrated that all the collected samples were free from the pathogen. As well, both detection protocols and attempts at isolating the pathogen on agarized media demonstrated that oleander samples gathered from American University campus in Beirut, where X. fastidiosa was previously reported, were not infected. Nevertheless, continuous monitoring and rigorous control measures of propagative materials are necessary to prevent the introduction of Xylella fastidiosa in Lebanon.     

Xylella fastidiosa in Europe: From the Introduction to the Current Status

Xylella fastidiosa is xylem-limited bacterium capable of infecting a wide range of host plants, resulting in Pierce’s disease in grapevine, citrus variegated chlorosis, olive quick decline syndrome, peach phony disease, plum leaf scald, alfalfa dwarf, margin necrosis and leaf scorch affecting oleander, coffee, almond, pecan, mulberry, red maple, oak, and other types of cultivated and ornamental plants and forest trees. In the European Union, X. fastidiosa is listed as a quarantine organism. Since its first outbreak in the Apulia region of southern Italy in 2013 where it caused devastating disease on Olea europaea (called olive leaf scorch and quick decline), X. fastidiosa continued to spread and successfully established in some European countries (Corsica and PACA in France, Balearic Islands, Madrid and Comunitat Valenciana in Spain, and Porto in Portugal). The most recent data for Europe indicates that X. fastidiosa is present on 174 hosts, 25 of which were newly identified in 2021 (with further five hosts discovered in other parts of the world in the same year). From the six reported subspecies of X. fastidiosa worldwide, four have been recorded in European countries (fastidiosa, multiplex, pauca, and sandyi). Currently confirmed X. fastidiosa vector species are Philaenus spumarius, Neophilaenus campestris, and Philaenus italosignus, whereby only P. spumarius (which has been identified as the key vector in Apulia, Italy) is also present in Americas. X. fastidiosa control is currently based on pathogen-free propagation plant material, eradication, territory demarcation, and vector control, as well as use of resistant plant cultivars and bactericidal treatments.     

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.     

Host plant identification in the generalist xylem feeder Philaenus spumarius through gut content analysis

The meadow spittlebug, Philaenus spumarius L. (Hemiptera: Aphrophoridae), is the main vector of the phytopathogenic bacterium Xylella fastidiosa in Europe, where the ST53 strain induces the olive quick decline syndrome, causing severe economic damage in southern Italy. The wide range of plant species infected by X. fastidiosa, and the wide host range of P. spumarius suggest that a large number of wild and cultivated plants may become infected by the pathogen following unintentional introduction events. Therefore, it is necessary to detail the host plant preference of the vector, in order to include preferred plants in the field, in pathogen-targeted diagnostic efforts. This would allow the identification of main sources of X. fastidiosa acquisition by P. spumarius; such plant species represent an important target for rational disease management. Here, we investigated the host plants of P. spumarius in north-western Italy, a region where X. fastidiosa is still not present but is regarded as a primary threat. We designed a new molecular diagnostic tool targeting chloroplast DNA, to characterize the gut content of single P. spumarius adults. The newly set up, nested PCR/sequencing-based identification protocol was proven to be useful for retrieving sequences from the last two different host plants used by P. spumarius, even if limited persistence of intact chloroplast DNA was reported in the spittlebug gut. We propose this protocol as a new tool for supporting research on xylem feeder biology that could be particularly useful for highly polyphagous species such as P. spumarius. Furthermore, the method could help monitor X. fastidiosa invasion, and contribute to the study of vector ecology and pathogen epidemiology.     

Isolation and molecular characterization of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> from coffee plants in Costa Rica

Coffee plants exhibiting a range of symptoms including mild to severe curling of leaf margins, chlorosis and deformation of leaves, stunting of plants, shortening of internodes, and dieback of branches have been reported since 1995 in several regions of Costa Rica’s Central Valley. The symptoms are referred to by coffee producers in Costa Rica as “crespera” disease and have been associated with the presence of the bacterium Xylella fastidiosa. Coffee plants determined to be infected by the bacterium by enzyme linked immunosorbent assay (ELISA), were used for both transmission electron microscopy (TEM) and for isolation of the bacterium in PW broth or agar. Petioles examined by TEM contained rod-shaped bacteria inside the xylem vessels. The bacteria measured 0.3 to 0.5 μm in width and 1.5 to 3.0 μm in length, and had rippled cell walls 10 to 40 nm in thickness, typical of X. fastidiosa. Small, circular, dome-shaped colonies were observed 7 to 26 days after plating of plant extracts on PW agar. The colonies were comprised of Gram-negative rods of variable length and a characteristic slight longitudinal bending. TEM of the isolated bacteria showed characteristic rippled cell walls, similar to those observed in plant tissue. ELISA and PCR with specific primer pairs 272-l-int/272-2-int and RST31/RST33 confirmed the identity of the isolated bacteria as X. fastidiosa. RFLP analysis of the amplification products revealed diversity within X. fastidiosa strains from Costa Rica and suggest closer genetic proximity to strains from the United States of America than to other coffee or citrus strains from Brazil.     

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.     

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.     

Vessel occlusion in three cultivars of Olea europaea naturally exposed to Xylella fastidiosa in open field

Xylella fastidiosa is a Gram‐negative, xylem‐limited, bacterium which is responsible, in Italy, for the olive quick decline syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce's disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In this study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analysing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars “Ogliarola salentina” and “Cellina di Nardò,” and the tolerant cultivar “Leccino.” Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.     

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.     

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.     

Transmission of Xylella fastidiosa by naturally infected Philaenus spumarius (Hemiptera,Aphrophoridae) to different host plants

The recent establishment of Xylella fastidiosa subspecies pauca in the southern Italian region of Apulia threatens agricultural crops and the environment. Olive is an important and widespread ancient crop in Italy and, so far, the most impacted host. The meadow spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae) has been identified as a vector of X. fastidiosa in southern Italy; this species is one of the most common potential vectors in Europe. To generate disease management strategies, data on X. fastidiosa transmission by P. spumarius are necessary. Therefore, we carried out transmission experiments by using field‐collected spittlebugs in 2014 and 2015 (5 and 11 collection dates, respectively), and transferring groups of insects immediately on to recipient plants. Various host plant species were tested: olive, oleander, sweet orange, grapevine and the stone fruit rootstock GF677 (Prunus persica × Prunus amygdalus). Xylella fastidiosa was detected in all the host plants after insect plant access except for grapevine; infections to sweet orange and stone fruit were not systemic. In 2015, estimates of insect X. fastidiosa infectivity were obtained; the number of PCR‐positive P. spumarius on each plant was positively correlated with the plant infection status. The proportion of P. spumarius infected with X. fastidiosa ranged from 25% to 71% during the entire survey period. The number of X. fastidiosa cells detected in P. spumarius heads ranged from 3.5 × 10 to 4.0 × 10² (CFU equivalents), which is lower than that reported for leafhopper vectors in the Americas. These data show that field‐collected P. spumarius have high rates of X. fastidiosa infection and are competent vectors.     

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.     

Biological Traits of Xylella fastidiosa Strains from Grapes and Almonds

Xylella fastidiosa is a xylem-limited bacterium that causes various diseases, among them Pierce's disease of grapevine (PD) and almond leaf scorch (ALS). PD and ALS have long been considered to be caused by the same strain of this pathogen, but recent genetic studies have revealed differences among X. fastidiosa isolated from these host plants. We tested the hypothesis that ALS is caused by PD and ALS strains in the field and found that both groups of X. fastidiosa caused ALS and overwintered within almonds after mechanical inoculation. Under greenhouse conditions, all isolates caused ALS and all isolates from grapes caused PD. However, isolates belonging to almond genetic groupings did not cause PD in inoculated grapes but systemically infected grapes with lower frequency and populations than those belonging to grape strains. Isolates able to cause both PD and ALS developed 10-fold-higher concentrations of X. fastidiosa in grapes than in almonds. In the laboratory, isolates from grapes overwintered with higher efficiency in grapes than in almonds and isolates from almonds overwintered better in almonds than in grapes. We assigned strains from almonds into groups I and II on the basis of their genetic characteristics, growth on PD3 solid medium, and bacterial populations within inoculated grapevines. Our results show that genetically distinct strains from grapes and almonds differ in population behavior and pathogenicity in grapes and in the ability to grow on two different media.     

Evaluation of Beauveria bassiana (Ascomycota: Hypocreales) as a control of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) emerging from fallen,infested coffee berries on the ground

The effects of Beauveria bassiana strains on coffee berry borers (CBB), which emerge from infested berries left on soil, and its impact on the infestation of coffee berries on tree branches were evaluated at two Experimental Stations (Naranjal-Caldas and Paraguaicito-Quindio) in the Colombian coffee zone. Using a completely randomized design with 10 repetitions, 50 coffee berries artificially infested with CBB were placed on the base of a coffee tree. Four treatments including B. bassiana strain Bb9205, a mixture of Cenicafé strains (Bb9001, Bb9024 and Bb9119), a commercial formulation of B. bassiana and a control (water) were sprayed with 1×10⁹ conidia per tree. After 30 days, all fungal strains lowered the infestation levels of the coffee berries on the trees at both locations. The mixture of Cenicafé strains decreased the tree infestation between 50 and 30% at both locations. In the berries dissected from each treated tree, insect mortality was about 40% at both locations compared to 15% in the control. B. bassiana strains also decreased the insect population inside the newly infested berries on the trees by 55–75%. The mixture of Cenicafé strains was the most effective for decreasing insect populations. B. bassiana significantly decreased CBB populations that emerged from fallen, infested, coffee berries and reduced future insect generations.     

Molecular evidence for host-adapted races of the fungal endophyte Epichloë bromicola after presumed host shifts

Abstract.— Host shifts of plant‐feeding insects and parasites promote adaptational changes that may result in the formation of host races, an assumed intermediate stage in sympatric speciation. Here, we report on genetically differentiated and host‐adapted races of the fungal endophyte Epichloë bromicola, which presumably emerged after a shift from the grass Bromus erectus to other Bromus hosts. Fungi of the genus Epichloë (Ascomycota) and related anamorphs of Neotyphodium are widespread endophytes of cool‐season grasses. Sexually reproducing strains sterilize the host by formation of external fruiting structures (stromata), whereas asexual strains are asymptomatic and transmitted via seeds. In E. bromicola, strains infecting B. erectus are sexual, and strains from two woodland species, B. benekenii and B. ramosus, are asexual and seed transmitted. Analyses of amplified fragment length polymorphism fingerprinting and of intron sequences of the tub2 and tef1 genes of 26 isolates from the three Bromus hosts collected at natural sites in Switzerland and nearby France demonstrated that isolates are genetically differentiated according to their host, indicating that E. bromicola does not form a single, randomly mating population. Phylogenetic analyses of sequence data did not unambiguously resolve the exact origin of asexual E. bromicola strains, but it is likely they arose from within sexual populations on B. erectus. Incongruence of trees derived from different genes may have resulted from recombination at some time in the recent history of host strains. Reciprocal inoculations of host plant seedlings showed that asexual isolates from B. benekenii and B. ramosus were incapable of infecting B. erectus, whereas the sexual isolates from B. erectus retained the assumed ancestral trait of broad compatibility with Bromus host seedlings. Because all isolates were interfertile in experimental crosses, asexual strains may not be considered independent biological species. We suggest that isolates infecting B. benekenii and B. ramosus represent long‐standing host races or incipient species that emerged after host shifts and that may evolve through host‐mediated reproductive isolation toward independent species.     

Initial Genetic Analysis of Xylella fastidiosa in Texas

Xylella fastidiosa is the causative agent of Pierce’s Disease of grape. No published record of X. fastidiosa genetics in Texas exists despite growing financial risk to the U.S. grape industry, a Texas population of the glassy-winged sharpshooter insect vector (Homalodisca vitripennis) now spreading in California, and evidence that the bacterium is ubiquitous to southern states. Using sequences of conserved gyrB and mopB genes, we have established at least two strains in Texas, grape strain and ragweed strain, corresponding genetically with subsp. piercei and multiplex, respectively. The grape strain in Texas is found in Vitis vinifera varieties, hybrid vines, and wild Vitis near vineyards, whereas the ragweed strain in Texas is found in annuals, shrubs, and trees near vineyards or other areas. RFLP and QRT PCR techniques were used to differentiate grape and ragweed strains with greater efficiency than sequencing and are practical for screening numerous X. fastidiosa isolates for clade identity.     

Phylogeographic studies on natural populations of the South American fruit fly, <Emphasis Type="Italic">Anastrepha fraterculus</Emphasis> (Diptera: Tephritidae)

Anastrepha fraterculus is an important pest of commercial fruits in South America. The variability observed for morphological and behavioural traits as well as genetic markers suggests that A. fraterculus represents a complex of synmorphic species rather than a single biological species. We studied the correlation between geographical distribution and genetic variation in natural populations from Argentina and south Brazil. Fragments of the mitochondrial gene COII were sequenced in 28 individuals. The matrix of aligned sequences was phylogenetically analysed by parsimony, yielding a cladogram of haplotypes. Based on Templeton’s nested method, no clade showed any geographic pattern for the gene COII, indicating lack of significant association between haplotypic variability and geographic distribution. The analysis of nucleotide substitution distances by Neighbour-Joining algorithm showed that geographically distant populations exhibit low genetic distances. The corresponding trees clustered the populations without showing any geographic pattern. This result suggests that the populations studied are not reproductively isolated.