Effect of xylem fluid from susceptible and resistant grapevines on developmental biology of Xylella fastidiosa

Xylella fastidiosa causes Pierce??s disease (PD), a serious disease in grapevines, and grapevine cultivars vary in susceptibility to X. fastidiosa in the field. The mechanism(s) by which this occurs has not been clearly elucidated. To explore possible mechanisms, X. fastidiosa cells from a PD strain were grown in pure xylem fluid of PD-susceptible grapevines, Vitis vinifera and V. labrusca, versus PD-resistant grapevines, V. champinii and V. smalliana. When grown in xylem fluid from the susceptible species, X. fastidiosa cells formed a heavier biofilm compared to those in xylem fluid from the resistant species. Differential expression of selected genes of X. fastidiosa cultured in the xylem fluids of V. vinifera and V. smalliana was analyzed using a DNA macroarray. Compared with xylem fluid of V. smalliana, xylem fluid of V. vinifera stimulated the expression of X. fastidiosa genes involved in virulence regulation, such as rpfC, gacA, xrvA, gcvR, and cysB, and genes involved in biogenesis of pili and twitching motility, such as pilI, pilU, pilE and pilG. Increased expression of virulence genes likely contributes to the expression of PD symptom in the susceptible grapevines, whereas reduced expression of these genes may lead to limitation of symptoms in resistant grapevines.     

Effect of Vitis vinifera L. cv. Chardonnay xylem fluid on cecropin B activity against Xylella fastidiosa

Cecropin B (CB) is a very efficient antimicrobial agent against Xylella fastidiosa. CB activity decreased after incubation with xylem fluid from Vitis vinifera cv. Chardonnay. SDS-PAGE demonstrated the existence of protein(s) in xylem fluid of V. vinifera cv. Chardonnay that interact with CB and inhibit CB activity. The antimicrobial activity of CB was dependent on incubation time and colony size of X. fastidiosa. X. fastidiosa colonies in periwinkle wilt medium plus (PW⁺), and chemically defined media (3G10-R and CHARD2) showed different patterns in colony size and colony number in these media. After 1 h of incubation in 10 μM CB, only large-sized colonies were observed in the three media. These results suggested that most of the bacterial cells or small aggregates in suspension were killed by CB. Aggregation of X. fastidiosa cells may serve as a mechanism of protection against CB.     

Effects of grape xylem sap and cell wall constituents on <Emphasis Type="Italic">in vitro</Emphasis> growth,biofilm formation and cellular aggregation of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis>

Pierce’s disease (PD) of grapevines is caused by the xylem-limited bacterium Xylella fastidiosa (Xf). All Vitis vinifera-based cultivars are susceptible to Xf infection; however, many grape species from the southern United States (such as V. arizonica, V. shuttleworthii, V. simpsonii, V. smalliana, and Muscadinia rotundifolia) are resistant. In this study, the effects of xylem sap from PD-resistant and PD-susceptible grapes, as well as several free cell wall constituents, on in vitro bacterial growth, biofilm formation, and cellular aggregation were investigated. Media containing xylem sap from PD-susceptible plants provided better support for bacterial growth and biofilm formation than media supplemented with xylem sap from PD-resistant plants. Culturing Xf on media containing various purified cell wall constituents demonstrated that CM-cellulose, xylan, β-D-glucan, k-carrageenan, cello-oligosaccharide and laminarin promoted bacterial growth whereas lichenan suppressed growth. However, only laminarin, xylan, and k-carrageenan promoted biofilm formation in vitro. Lichenan, oligosaccharide, k-carrageenan, laminarin, xylan and β-D-glucan all significantly decreased Xf cellular aggregation in vitro. This study suggests that differences in xylem sap composition and cell wall properties among PD-resistant and PD-susceptible grapes may affect characteristics of Xf growth, biofilm formation and cellular aggregation involved in pathogenesis.     

Mining new resources for grape resistance against Botryosphaeriaceae: a focus on Vitis vinifera subsp. sylvestris

Botryosphaeria dieback is an important grapevine trunk disease with global impact. Susceptibility differences between grape varieties manifest as different expression of canopy symptoms in the field. However, the cause of these dieback symptoms and their relation with wood necrosis remain only partially understood. As a first step towards future strategies for resistance breeding, wood necrosis was investigated over a large selection of the Vitaceae family members following artificial inoculation of the Botryosphaeriaceae fungi Neofusicoccum parvum and Diplodia seriata into woody internodes. Large variation of resistance levels was found, with good performance in several accessions from V. vinifera subsp. sylvestris, the ancestor of cultivated grapevine. To get insight into the mechanisms of this apparent resistance, expression of defence genes was studied in V. vinifera cv. Chardonnay, Gewürztraminer and different V. vinifera subsp. sylvestris genotypes, in both green and necrotic areas of inoculated woods. Resistance to Botryosphaeriaceae in V. vinifera subsp. sylvestris correlated with earlier and higher induction of some defence genes, both in green and necrotic wood. Moreover, leaves of several V. vinifera subsp. sylvestris accessions were also less susceptible to necrosis induced by treatment with a culture filtrate of Botryosphaeriaceae, compared to commercial cultivars of V. vinifera. The results show that V. vinifera subsp. sylvestris provides interesting genetic resources for breeding new varieties with enhanced resistance to botryosphaeria dieback.     

Susceptibility to flavescence dorée of different Vitis vinifera genotypes from north-western Italy

The aim of this work was to evaluate the susceptibility to flavescence dorée (FD) of 12 Vitis vinifera cultivars grown in Piedmont, and representative of the wine-making tradition of this area. The experiments were conducted under controlled conditions to ensure constant infection pressure. Test plants were ex vitro potted vines, singly inoculated with four Scaphoideus titanus infected by FD-C phytoplasma (FDp), under greenhouse conditions. Vines were tested for FDp at 5 and 8 weeks postinoculation (wpi) and the phytoplasma load was measured in leaves and roots at 8 wpi. Within the 14 V. vinifera accessions (belonging to 12 cultivars), three susceptibility clusters were identified. Cultivars within the low susceptibility group showed low phytoplasma loads and low percentages of infected plants, suggesting a tolerant behaviour. To confirm these results, four Vitis cultivars, representing extremes of FD susceptibility from low to high, were grafted onto Kober 5BB rootstocks and inoculated with laboratory-infected S. titanus, under semi-field conditions. The transmission experiments onto grafted cuttings confirmed that susceptibility to the disease depends on the scion genotype. The data indicated that none of the tested V. vinifera genotypes are resistant to FD, although some cultivars with low susceptibility are available, and can be explored for identifying genetic traits involved in disease tolerance/resistance. Moreover, ranking Vitis genotypes for their susceptibility to FD is in itself a valuable tool to support vine growers in their decision management, by helping them to choose the most appropriate varieties according to their specific FD epidemiological contexts.     

Heritability of Ulmus minor resistance to Dutch elm disease and its relationship to vessel size,but not to xylem vulnerability to drought

Dutch elm disease (DED) is a vascular wilt disease that causes the occlusion and cavitation of xylem vessels. Therefore, it is hypothesized that those elms that are less vulnerable to cavitation by drought might be more resistant to DED. To test this hypothesis, the relationship between xylem vulnerability to cavitation and susceptibility to DED was examined in progenies of crosses between susceptible and resistant individuals of Ulmus minor. Hydraulic conductivity and xylem vulnerability curves were evaluated and anatomical features such as vessel size, length and grouping were measured. Next, elms were inoculated with Ophiostoma novo‐ulmi, the cause of DED, and pre‐dawn and midday water potentials, stomatal conductance and wilting percentages were assessed. Progenies of R × R crosses showed significantly lower mean wilting percentages (30–50%) than the progeny of S × S crosses (75%). Fifty percent conductivity loss was reached at c. ?1 MPa, pointing out a high vulnerability of this species to drought‐induced cavitation. Crown wilting percentage as a result of inoculation and xylem vulnerability to cavitation by water stress did not show any significant correlation. Nevertheless, significant differences in theoretical hydraulic conductivity and vessel size parameters (diameter, length and size distributions) were found among the tested progenies. Susceptible trees had significantly wider and longer vessels. Xylem structure of resistant elms seems to restrict pathogen spread rather than prevent cavitation.     

Differential colonization patterns of Xylella fastidiosa infecting citrus genotypes

Xylella fastidiosa is a phytopathogenic bacterium that causes disease in many different crops worldwide. In Brazil, X. fastidiosa subsp. pauca causes citrus variegated chlorosis (CVC), which is a disease responsible for economic losses in the citrus agribusiness. Variable host responses to bacterial colonization and disease development have been observed. This work studies the colonization processes of a pathogenic GFP‐labelled X. fastidiosa citrus strain in sweet orange (susceptible) and tangor (resistant) parents and two resulting hybrids that exhibited contrasting responses to CVC. Xylella fastidiosa showed increased populations and movement in the susceptible genotypes, but slower compared to other hosts such as grapevine. Scanning electron microscopy revealed that the predominant pitted stem morphology in citrus makes the bacterial movement difficult. In susceptible genotypes X. fastidiosa can move from the primary to the secondary xylem, whilst it is confined to the primary xylem in resistant plants. Associated with this is an induction of lignification that occurs earlier in the resistant genotypes when in the presence of the pathogen, and represents a genetic mechanism that leads to formation of a physical barrier, impairing bacterial colonization.     

Assessment of the genetic diversity of Xylella fastidiosa in imported ornamental Coffea arabica plants

A study was performed in order to assess the presence of Xylella fastidiosa in imported ornamental plants, among them Olea europaea, Coffea arabica and Nerium oleander. Positive results were only obtained from C. arabica, where 15 plant samples tested positive for X. fastidiosa by PCR, nine from Costa Rica and six from Honduras. Transmission electron microscopy observations indicated that rod‐shaped bacterial cells exhibiting the characteristics of X. fastidiosa cells were present in the xylem vessels of leaf petioles obtained from the infected C. arabica plants. Diversity of X. fastidiosa in C. arabica plants was assessed through a multilocus sequence typing (MLST) analysis of seven housekeeping genes (leuA, petC, lacF, cysG, holC, nuoL and gltT) and compared with X. fastidiosa infecting different host plants worldwide. Based on this MLST analysis, the prevalence of different sequence types (STs) of X. fastidiosa in the C. arabica ornamental plants was demonstrated and related to different X. fastidiosa subspecies, underlining the risk of introducing additional genetic diversity for X. fastidiosa to Europe. ST53, related to X. fastidiosa subsp. pauca, was frequently found in these C. arabica samples. A second ST related to X. fastidiosa subsp. pauca, ST73, has been assessed in coinfection with ST53 in one individual plant. Additionally, ST72 and ST76, related to X. fastidiosa subsp. fastidiosa, have been recorded. Next to these previously described STs, a novel ST, namely ST77 has been revealed, related to X. fastidiosa subsp. fastidiosa. Isolation of X. fastidiosa from leaf petioles and midribs of infected C. arabica plants was successfully performed only after the application of an additional ultrasonication step during the extraction procedure. Based on this approach, a number of X. fastidiosa isolates were obtained and further characterized.     

Characterization of Cytospora isolates from wood cankers of declining grapevine in North America,with the descriptions of two new Cytospora species

Cytospora species are ubiquitous pathogens of numerous woody plants, causing dieback and wood cankers in agronomic crops, timber trees and wildland trees (e.g. Prunus, Eucalyptus and Salix, respectively). Cytospora chrysosperma, C. cincta and C. leucostoma have been reported from grapevines in Iran showing symptoms of one or more recognized trunk diseases (esca, botryosphaeria‐, eutypa‐ and phomopsis diebacks); however, only C. chrysosperma was shown to be pathogenic to grapevine. To understand the potential role of Cytospora species in the grapevine trunk‐disease complex, 21 Cytospora isolates were examined that were recovered from dieback and wood cankers of Vitis vinifera and Vitis interspecific hybrids in seven northeastern U.S. states and two Canadian provinces. Phylogenetic analyses of ITS and translation elongation factor 1‐α identified two novel species: Cytospora vinacea sp. nov. and Cytospora viticola sp. nov. Differences in culture morphology and conidial dimensions also distinguished the species. When inoculated to the woody stems of potted V. vinifera ‘Thompson Seedless’ in the greenhouse, both species were pathogenic, based on development of wood lesions and fulfilment of Koch's postulates. Cytospora viticola was the most virulent based on lesion length at 12 months post‐inoculation. As cytospora canker shares some of the same general dieback‐type symptoms as botryosphaeria‐, eutypa‐ and phomopsis diebacks, it may be considered part of the grapevine trunk‐disease complex in eastern North America.     

Comparative seed germination study across alien grapes (Vitis,Vitaceae) in Europe

Alien grape (Vitis, Vitaceae) species and recently described hybrids are recognised as major environmental weeds in southern Europe, based on their tendency to spread into natural and semi‐natural habitats, often behaving as invasive species. Despite a high number of agronomic, genetic and phytopathological studies on Vitis cultivars, regeneration from seed has been poorly investigated, although it plays a key role in plant distribution and evolution. To this end, in this study we exposed seeds of eight wild grapes in Europe (three species and five nothospecies, including the native Vitis vinifera), to different temperature treatments in the laboratory. Fresh seeds from all studied taxa were dormant and germinated only after cold and/or warm plus cold stratification, but showing significant differences between the species. Seeds of V. vinifera showed an intermediate complex morphophysiological dormancy, while all other tested Vitis taxa exhibited a deep complex morphophysiological dormancy. Dark conditions reduced the germination percentage in Vitis labrusca, Vitis riparia and V. vinifera and their hybrids, and alternating temperatures elicited the highest germination percentages in all populations. Our results demonstrated for the first time that Vitis hybrids are capable of regeneration by seed, even in the absence of dispersal by animals. The germination processes studied here help understand the current expansion of alien Vitis taxa in Europe outside the viticultural areas. Consequently, germination requirements contribute to a better understanding of the mechanisms underlying alien Vitis taxa establishment and invasion.     

Phloem and xylem modifications of Vitis vinifera stems in response to flavescence dorée phytoplasma infection

Anatomical modifications of xylem and phloem tissues of grapevine (Vitis vinifera) stems of shoots infected by the flavescence dorée phytoplasma (FDp) were first observed and described in the 1960s, but never quantified in detail. In this paper, we describe and quantify the impact of FDp on grapevine stem tissues, and relate it to the level of expression of symptoms and to cultivar-specific FDp susceptibility. For this purpose, we measured and quantified the anatomical parameters of xylem and phloem tissues of a tolerant (Merlot) and a susceptible (Chardonnay) cultivar. For each cultivar, thin sections of eight shoots with symptoms from FDp-infected grapevines, eight symptomless shoots from the same FDp-infected grapevines, and eight symptomless shoots from symptomless grapevines (control) were compared. Results showed general inhibition of xylem growth and proliferation of phloem tissues (hyperplasia) with lack or irregular arrangement of the fibre-sclereids in the axial phloem of the stems from shoots with symptoms, irrespective of the cultivar. Xylem vessels of infected Merlot shoots were partly occluded by tyloses and a higher number of smaller vessels were produced than in control plants. Thus, the anatomical responses confirmed the detrimental effect of FDp on stems of infected grapevine shoots, including impaired stem development and lack of periderm formation. Statistically significant differences were found between the two cultivars with different levels of susceptibility to FDp infection.     

Histopathological aspects of mango resistance to the infection process of Ceratocystis fimbriata

Mango wilt, caused by Ceratocystis fimbriata, is one of the most important diseases affecting mango yields in Brazil. Information regarding the infection process of C. fimbriata in the stem tissues of mango from different cultivars and the basis of host resistance to the pathogen is rare in the literature. Thus, the objective of the study was to investigate how infection by two isolates of C. fimbriata can be affected by mango cultivar‐specific mechanisms of resistance. Disease progress on the inoculated stem tissues of the mango cultivars was evaluated and stem sections were obtained from the site of inoculation and prepared for histopathological observations using light microscopy. The factors mango cultivars and C. fimbriata isolates and their interaction were significant for all measures of disease development. Plants from the cultivars Espada, Haden and Palmer inoculated with isolates of C. fimbriata were more susceptible, whereas plants from the cultivars Tommy and Ubá were moderately resistant and resistant, respectively. Histopathologically, fungal isolates apparently massively colonized the stem tissues of plants from the susceptible cultivars Espada, Haden and Palmer, starting from the collenchyma and moving in the direction of the cortical parenchyma, xylem vessels and pith parenchyma. By contrast, on stem tissues of plants from the resistant cultivars Tommy Atkins and Ubá, most of the cells reacted to C. fimbriata infection by accumulating amorphous material. The results from the present study strongly indicated the importance of phenolic compounds for mango cultivar resistance against infection by Brazilian C. fimbriata isolates.     

A combined agar-absorption and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrus

Application of the polymerase chain reaction (PCR) to disease diagnosis is limited in part by the presence of PCR inhibitors. Inhibition can be overcome and sensitivity increased by culturing bacteria on agar media prior to PCR (termed BIO-PCR). However, Xylella fastidiosa grows slowly, requiring 10–14 days for visible colonies to appear. In this study an agar-absorption BIO-PCR method for detecting X. fastidiosa in grape and citrus plants was developed. Optimum lengths of time for absorption of inhibitors by the agar medium or enrichment of bacteria on the medium were determined for Pierce's disease of grape and citrus variegated chlorosis. When petioles of grape and citrus leaves with symptoms were spotted onto agar media, the spots washed after various time intervals and assayed for X. fastidiosa by real-time PCR, 97% (31 out of 32) and 100% (six out of six) of spots were positive after 2 days and 4 h for grape and citrus, respectively. With direct PCR, only 12·5% (four out of 32) and 33% (two out of six) of spots were positive, respectively, and visible X. fastidiosa colonies were evident after 10 and 14 days, respectively. In a separate experiment with samples from a different vineyard, 46% (13 out of 28) of the grape samples (agar spots) were positive after 1 day and 93% (26 out of 28) after 5 days using agar-absorption PCR. In contrast, all samples were negative by direct PCR. Viable X. fastidiosa were recovered from all samples after 14 days. Further tests with eight randomly selected grape petioles from three Texas vineyards known to have Pierce's disease resulted in 50% being positive by a simple 24 h agar-absorption PCR assay, whereas none was positive by direct PCR. Overall, 10 out of 16 (63%) vines from five vineyards (two in California and three in Texas) were positive after the 24 h agar-absorption PCR assay. In contrast, only one vine was positive by direct PCR. This simple agar absorption-based PCR assay protocol should prove useful for the routine detection of X. fastidiosa and other slow-growing bacteria in the presence of PCR inhibitors.     

Is there need for leaf-galling grape phylloxera control? Presence and distribution of Dactulosphaira vitifoliae in Swiss vineyards

Grape phylloxera (Dactulosphaira vitifoliae, FITCH) was a major pest in Vitis vinifera grape production in the late 19th and early 20th century in Europe. Roots of V. vinifera varieties are susceptible to D. vitifoliae attack whereas the leaves are not affected. The pest has been controlled by using American grapevine varieties as rootstocks. However, in recent years, grape producers have reported higher incidences of leaf galls induced by D. vitifoliae on V. vinifera leaves. Research on the occurrence of D. vitifoliae on grapevine leaves and roots has shown that D. vitifoliae is present in all wine-growing regions of Switzerland. In more than 80% of vineyards claiming to be infested, D. vitifoliae was found on leaves of varieties with interspecific crosses in their parentage. Three locations with leaf-galling individuals on V. vinifera were detected. D. vitifoliae individuals were found emerging from soil potentially associated with the commonly used rootstock varieties, which are assumed to be phylloxera tolerant. As climate change may necessitate the relocation of some viticultural areas to new wine-growing regions, there will be a greater need to control D. vitifoliae on leaves of interspecific crosses and the inspected planting material to limit further spread of the pest.     

Xylella fastidiosa subsp. pauca ST53 exploits pit membranes of susceptible olive cultivars to spread systemically in the xylem

Xylella fastidiosa subspecies pauca strain De Donno (XfDD) ST53 is the causal agent of olive quick decline syndrome, a severe disease first described in Apulia, Italy. Although the two local cultivars Cellina di Nardò and Ogliarola Salentina showed high susceptibility, traits of resistance to the bacterium were found in the cultivar Leccino. Previous studies in field-grown olives suggested that vascular occlusions and anatomophysiological properties of the different cultivars played a role in the olive response to XfDD. The present investigation reports observations at the early stage of the infection on artificially inoculated olives. Electron microscope studies showed that XfDD exploits the pit membranes (PMs) of the susceptible cultivar Cellina di Nardò to spread systemically. In this cultivar, PMs were degraded upon XfDD infection, suggesting activity of bacterial cell wall-degrading enzymes. Moreover, occluded vessels contained an amorphous electrondense matrix resembling gum. Conversely, in Leccino, occluded vessels were mainly filled by callose-like granules that tightly entrapped XfDD cells. In addition, PMs from Leccino had a compact undegraded structure that was not permeable to XfDD. Our study suggests that exploitation of PMs is a key event in the infection process of X. fastidiosa subsp. pauca ST53 in susceptible olive cultivars.     

Novel methodologies in screening and selecting olive varieties and root-stocks for resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

An innovative inoculation process, involving the drilling of a trunk hole in 3 year-old olive trees and injecting a dense conidial suspension of Verticillium dahliae, was developed to study differentiation in foliar symptom expression between olive cultivars tolerant or susceptible to the pathogen. It was demonstrated that V. dahliae conidia could be translocated and colonize the xylem at the same distance above and below the point of trunk injection in both cultivars. However, the pathogen could be subsequently isolated at statistically significant percentages in susceptible cv. Amphissis compared to the tolerant cv. Kalamon, indicating operation of resistance mechanisms in the vascular phase of the disease. Consequently symptom development in the susceptible cultivar was at least sixfold more intensive compared to the tolerant cultivar, 6–11 months after trunk inoculation. Perennial olive orchard experiments, aimed at selecting Verticillium-resistant root-stocks, were conducted by applying the novel method in 2–3 year-old root-stock suckers of Amphissis olive trees and in the tolerant cvs Lianolia of Corfu and Koroneiki. It was indicated that potentially resistant root-stocks could be obtained following the trunk drilling technique. Resistance differentiation between cvs Amphissis and Kalamon was further verified through root inoculation by various V. dahliae microsclerotial concentrations and demonstrated that the trunk drilling inoculation procedure is equally efficient in resistance evaluation of olives to Verticillium wilt. The trunk inoculation procedure could be useful in selecting and screening root-stocks for resistance to V. dahliae and other vascular pathogens and could elucidate resistance mechanisms in woody plants against vascular wilt diseases.     

Insights into the epidemiology of Pierce's disease in vineyards of Mallorca,Spain

Xylella fastidiosa (Xf), the causal agent of Pierce's disease (PD), has long been considered a major threat to prosperous European viticulture. In May of 2017, PD was officially detected on a grapevine in Mallorca. To better understand the epidemiology of the first established outbreak of PD in Europe, the disease incidence and severity were assessed, vector transmission experiments were performed, and cultivar responses to Xf infections both in the field and in inoculation experiments were monitored. The genetic structure of Xf local populations was also investigated by multilocus sequence typing analysis, and their whole genome sequences and phylogenetic positions compared with respect to PD strains worldwide. Disease incidence was moderately low (0.07) and highly variable, ranging from 0.0 to 0.99. Its economic impact on wine production was considered low. Vineyards managed under conventional viticulture were significantly less infected than organic farming. Under greenhouse conditions, the insect Philaenus spumarius efficiently transmitted the pathogen from infected to healthy grapevines. In the inoculation experiments, Xf infected, to some degree, 29 of the 30 cultivar × rootstock combinations (n = 239). Cultivars Viura, Gorgollasa and Sauvignon Blanc were the most susceptible ones. All Xf isolates from grapevines belonged to sequence type ST1; they phylogenetically clustered within the Californian ST1 clade, with which they shared a 99.94% identity and the same plasmid. In addition, ST1 caused almond leaf scorch disease, where notably its incidence (0.78) and severity was much higher than PD. The likely reasons for these wide differences are discussed in the context of a 20-year Xf introduction scenario.     

Specific characters of 16S rRNA gene and 16S–23S rRNA internal transcribed spacer sequences of Xylella fastidiosa pear leaf scorch strains

Pear leaf scorch, the only Xylella fastidiosa-induced disease reported from Taiwan, was found in area where the variety Hengshan (Pyrus pyrifolia) was grown. Strains of pear leaf scorch Xyl. fastidiosa (XF-PLS) shared similarities to strains of other host origins in the requirement of complex medium and the exhibition of rippled cell walls, however, recent serological and molecular biology studies showed difference among them. Five strains of XF-PLS were compared with 20 other strains originally isolated from almond, oleander, pecan, plum, peach, mulberry, grapes, citrus, coffee, and sycamore by sequence analyses of the 16S rRNA gene and 16S–23S rRNA internal transcribed spacer region (ITS). When sequences of 16S rRNA gene based on fragment size of 1,537–1,540 bp were compared, the similarity index among 5 XF-PLS strains was 99.3–99.8%, whereas it was 97.8–98.6% between XF-PLS strains and strains from other hosts. When sequences of 16S–23S rRNA ITS based on fragment size of 510–540 bp were compared, the similarity index among 5 XF-PLS strains was 99.0–100%, whereas it was 80.7–82% between XF-PLS strains and strains from other hosts. Multiple sequence alignments led to the identification of 5 polymorphic nucleotides in the 16S rRNA gene among the 25 Xyl. fastidiosa strains, and there were considerable variations in the nucleotide sequences of 16S–23S rRNA ITS between XF-PLS and the other 20 Xyl. fastidiosa strains. The phylogenetic trees revealed that XF-PLS strains were separated from strains of other hosts. Strains of other hosts were divided into four subgroups: strains from (1) oleander, (2) grape, almond M23 and mulberry, (3) citrus and coffee, and (4) pecan, peach, plum, sycamore and almond M12. Results indicate that XF-PLS strains were not closely related to the above-mentioned strains from other hosts and could possibly belong to a new subspecies of Xyl. fastidiosa.     

Several subspecies and sequence types are associated with the emergence of Xylella fastidiosa in natural settings in France

Xylella fastidiosa is a plant pathogenic bacterium emerging in Europe. In France its emergence has been demonstrated through interceptions of contaminated coffee plants and, in 2015, by a survey of natural settings. The first French focus of contamination was detected in 2015 in Corsica; since then, almost 300 foci have been found and nearly 30 plant species have been declared contaminated, with Polygala myrtifolia remaining the principal host, suffering from severe leaf scorch. This study reports on the diversity of X. fastidiosa identified in France in 2015. Multilocus sequence analysis/typing revealed the presence of mainly X. fastidiosa subsp. multiplex sequence types (STs) ST6 and ST7. A focus of X. fastidiosa subsp. pauca ST53 was identified in mainland France; one sample contaminated by X. fastidiosa subsp. sandyi ST76, one novel recombinant, and coinfections of different isolates in individual samples were also identified, but could not be confirmed by successive samplings, indicating limited or transient contamination. Koch's postulates were fulfilled for two isolates of X. fastidiosa subsp. multiplex on P. myrtifolia, one being ST6 and the other ST7. Comparative genomics of the genome sequences of three French isolates (one ST6 and two ST7) with available sequences revealed that, unlike the American Dixon strain, the French ST6 and ST7 strains are devoid of a plasmid encoding a complete type IV secretion system. Other differences regarding phage sequences were highlighted. Altogether, the results suggest that the emergence of X. fastidiosa in France is linked to several introduction events of diverse strains from different subspecies.