Assessment of Pierce's disease susceptibility in Vitis vinifera cultivars with different pedigrees

Pierce's disease (PD) of grapevine is caused by the bacterium Xylella fastidiosa. In this study, an integrated approach was applied to assess PD susceptibility among different Vitis vinifera cultivars that incorporated disease severity, bacterial pathogen abundance and loss of stem xylem hydraulic conductivity. It was hypothesized that levels of PD susceptibility in V. vinifera can be attributed in part to the host anatomical features that are shaped by its pedigree background. Two popular wine grape cultivars were initially selected from the occidentalis group, Merlot and Cabernet Sauvignon, and one from the orientalis group, Thompson Seedless. The more recently bred table grape cultivar Scarlet Royal, that has mixed pedigree parentage, was also included. PD susceptibility was compared to the known PD resistant b43-17 V. arizonica/candicans wild grape species from North America. The data indicated that Thompson Seedless was ranked as the most susceptible to PD because it significantly exhibited the most severe disease symptoms at 12 weeks post-inoculation and hosted the highest X. fastidiosa titre of the cultivars, and lost over 90% of its stem hydraulic conductivity. In contrast, the other three cultivars displayed less susceptibility to PD. The way in which the xylem anatomy could impact PD susceptibility in V. vinifera cultivars is discussed, together with how grape pedigrees and their cognate centre of domestication may have influenced xylem anatomical features. This work provides a reference framework to further test the hypothesis that V. vinifera cultivars with wide xylem vessels may be more prone to PD decline.     

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.     

The chemotaxis regulator <Emphasis Type="Italic">pilG</Emphasis> of <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> is required for virulence in <Emphasis Type="Italic">Vitis vinifera</Emphasis> grapevines

Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate the roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant XfΔpilG and complemented strain XfΔpilG-C were generated. While all strains had similar growth curves in vitro, XfΔpliG showed significant reduction in cell-matrix adherence and biofilm production compared with wild-type X. fastidiosa and XfΔpilG-C. The genes pilE, pilU, pilT, and pilS were down-regulated in XfΔpliG when compared with its complemented strain and wild-type X. fastidiosa. Finally, no Pierce’s disease symptoms were observed in grapevines inoculated with XfΔpilG, whereas grapevines inoculated with the wild-type X. fastidiosa and complemented strain of XfΔpilG-C developed typical Pierce’s Disease (PD) symptoms. The results indicate that pilG has a role in X. fastidiosa virulence in grapevines.     

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.     

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.     

Proteomic analysis of grapevine stem in response to Xylella fastidiosa inoculation

Xylella fastidiosa (Xf) is the bacterial causal agent of Pierce’s disease (PD) as well as other economically important diseases in a number of agronomic, horticultural and ornamental plants. The objective of this research was to tentatively identify proteins that are differentially expressed in grapevines and involved in disease development or defense responses to Xf-inoculation. We comparatively analyzed proteins differentially expressed in Xf-inoculated grape stems using a pair of siblings of 9621-67 (highly susceptible) and 9621-94 (highly resistant) from a cross of Vitis rupestris × Vitis arizonica. Total proteins were extracted from the stems of uninoculated controls and Xf-inoculated plants at 1, 6, and 12 weeks after inoculation, separated by a 2D-PAGE system, and spots representing differentially expressed proteins were analyzed and tentatively identified using LC/MS/MS. Protein identification was performed using BLASTp and tBLASTn against NCBI non-redundant protein databases and EST databases, respectively. Ten tentatively identified proteins were differentially expressed at different time points after inoculation. A thaumatin-like protein and the pathogenesis-related protein 10 from both genotypes, and the 40S ribosomal protein S25 from the susceptible genotype were up-regulated in response to Xf-inoculation. Furthermore, the expression of the thaumatin-like protein increased sharply 12 weeks post-inoculation in the PD-resistant genotype only. Three heat shock proteins, 17.9 kDa class II, protein 18 and 21 were highly expressed in healthy tissues compared with those in tissues infected with Xf, and heat shock protein 21 was not detectable in the Xf-inoculated PD-susceptible genotype. In addition, a down-regulated putative ripening related protein was found in the Xf-inoculated PD-susceptible genotype. Glycoprotein and formate dehydrogenase were identified in the PD-resistant genotype and their expression was constant during plant development. A putative GTP-binding protein was down-regulated in the PD-susceptible genotype. Our results revealed that differential expression of proteins in response to Xf-inoculation was genotype and tissue development stage dependent. The specific roles of these candidate proteins in alleviation or aggravation of this disease are under investigation. The information obtained in this study will aid in the understanding of the mechanisms related to the host–pathogen interactions involved in PD.     

The current status of the quick decline syndrome of olive in southern Italy

The quick decline syndrome of olive (OQDS) is a disease that appeared all of a sudden some years ago in a restricted area near the city of Gallipoli (Ionian coast of the Salento peninsula, southern-east Italy) and began spreading through the heavily olive-grown countryside of lower Salento. Xylella fastidiosa, a quarantine pathogen of American origin previously undetected in the European Union territory, except for two unconfirmed records from Kosovo and Turkey, proved to be consistently associated with symptomatic trees. X. fastidiosa is a Gram-negative bacterium that invades and multiplies in the xylem vessels of infected hosts, from which it is acquired by xylem-feeding insect vectors (belonging to Auchenorrhyncha, including cicadellids sharpshooter leafhoppers group, Cicadellidae, Cicadellinae), and aphrophorids (cercopids and spittlebugs, Cercopidae) and transferred to other plants. The Salentian strain of X. fastidiosa, denoted CoDiRO, was obtained in axenic culture. Its genome, a DNA molecule ca. 2.5 million base-pairs in size, was sequenced and identified as a genotype of X. fastidiosa subsp. pauca molecularly identical to an isolate of the same subspecies from Costa Rica. In nature, strain CoDiRO infects a number of woody and shrubby hosts but not grapevines and citrus and is mainly transmitted by Philaenus spumarius (meadow spittlebug), a froghopper quite common in the Salento area where it thrives primarily on olive. Since OQDS eradication and sanitation of infected olives are unfeasible, strategies have been envisaged for restraining the spread of the pathogen and its vector within the boundaries of the currently infected zone.     

Involvement of phenolic compounds in the resistance of grapevine callus to downy mildew (Plasmopara viticola)

Callus tissue of different grapevines (Vitis spp.) was inoculated withPlasmopara viticola. Short, highly-branched hyphae with necrosis, and long hyphae with heavy sporulation were observed on resistant and susceptible callus respectively. Thin-layer chromatography and spectrophotometric analysis showed that resistant callus contained greater quantities of gallocatechin derivatives than susceptible callus. Regression analysis between the field disease rating of each variety and its gallocatechin derivatives content indicated 92.2% correlation. Histochemical studies showed that, after infection withP. viticola, flavonoids appeared in the superficial cell walls of the callus, to a lesser degree on susceptible callus than on resistant callus. At a late stage of infection, the superficial cells of resistant callus were suberized, which did not occur in susceptible callus. This study showed that the preformed gallocatechin derivatives, the induced flavonoids and suberized superficial cells might play a role in the resistance of grapevine callus tissue to this fungus.Abbreviations CallusI Callus ofV. riparia var. Gloire de Montpellier - CallusV Callus ofV. vinifera var. Grenache - TLC Thin Layer Chromatography - var. variety - GAD Gallic acid derivatives - GD gallocatechin derivatives - RC resistant callus - SC susceptible callus     

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.     

Vitis Resistance to Pierce's Disease Is Characterized by Differential Xylella fastidiosa Populations in Stems and Leaves

ABSTRACT The pattern of Xylella fastidiosa infection in resistant and susceptible grapevines representing a diverse selection of Vitis spp. was characterized through measurements of X. fastidiosa bacterial movement and accumulation in artificially inoculated greenhouse-grown grapevines. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was optimized for quantification of X. fastidiosa populations and tested on known amounts of X. fastidiosa added to grape tissue extracts. Predicted versus known X. fastidiosa concentrations proved to be highly correlated (R(2) = 0.99). Populations of X. fastidiosa in stem internode, stem node, petiole, and leaf blade samples from the genotypes in this study were measured at 12 weeks postinoculation using the optimized ELISA procedure. Samples from each plant part were taken at eight positions along the inoculated shoots. Systemic infection was detected in both susceptible and resistant genotypes. Resistant genotypes were characterized by significant differences in X. fastidiosa populations between stem internodes and leaves (1.0 x 10(6) and 1.1 x 10(7) cells/g of sample, respectively). In contrast, the susceptible genotypes were characterized by high mean X. fastidiosa populations in both stems and leaves (5.6 x 10(7) and 4.8 x 10(7) cells/g, respectively) the latter of which were not significantly different from the resistant genotypes. A high correlation (R(2) = 0.97) between stem X. fastidiosa numbers to previously characterized field Pierce's disease (PD) performance indicates that the quantitative ELISA measurements of X. fastidiosa in greenhouse-grown grapevines should be a useful tool for predicting PD resistance under field conditions.     

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.     

Grapevine phenolic compounds in xylem sap and tissues are significantly altered during infection by Xylella fastidiosa

Pierce's disease of grapevine (PD), caused by the bacterial pathogen Xylella fastidiosa, remains a serious problem for grape production in California and elsewhere. This research examined induction of phenolic compounds in grapevines ('Thompson Seedless') infected with X. fastidiosa over a 6-month period. Two months postinoculation with X. fastidiosa, catechin, digalloylquinic acid, and astringin were found at greater levels in xylem sap; multiple catechins, procyanidins, and stilbenoids were found at greater levels in xylem tissues; and precursors to lignin and condensed tannins were found at greater levels in xylem cell walls. However, such large-scale inductions of phenolic compounds were not observed 4 months after inoculation. Six months after inoculation, infected plants had significantly reduced phenolic levels in xylem sap and tissues when compared with control plants, including lowered levels of lignin and condensed tannins. At 6 months, PD symptoms were severe in infected plants and most photosynthetic tissue was abscised. These results suggest that, even though grapevine hosts may initially respond to X. fastidiosa infections with increased production of phenolic compounds, ultimately, PD causes grapevines to enter a state of decline whereby diseased hosts no longer have the resources to support secondary metabolite production, including defense-associated phenolic compounds.     

Identification and characterization of Phoma tracheiphila mutants impaired in pathogenicity following Agrobacterium-mediated mutagenesis

Mal secco disease, caused by the pathogenic fungus Phoma tracheiphila, is a devastating disease of susceptible citrus species, especially lemon. To study the molecular interactions between the pathogen and its host, a method for identifying the genes involved in pathogenicity is needed. This work describes the transformation of P. tracheiphila phialoconidia by Agrobacterium tumefaciens, and the generation of mutated P. tracheiphila isolates exhibiting reduced virulence on rough lemon seedlings. A rapid, replicable, and reliable method for screening P. tracheiphila mutants to assess their virulence by using rough lemon seedlings was developed. Among 2263 transformants obtained, three were non-virulent and 43 displayed reduced virulence. In addition, one of the transformants, which exhibited virulence similar to that of the wild type, was used for in planta visualization of the fungus progression through the plant xylem. To our knowledge, this is the first report of A. tumefaciens-mediated transformation of P. tracheiphila, and subsequent screening of the transformants to identify non-virulent mutants.     

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.     

Bacterial inflorescence rot of grapevine caused by Pseudomonas syringae pv. syringae

Molecular sequencing (rpoB) and standard pathological and microbiological methods identified Pseudomonas syringae pv. syringae (Pss) as the causal agent of bacterial inflorescence rot of grapevines (Vitis vinifera) in three vineyards in Tumbarumba, NSW, Australia in 2006 and 2007. Pss strains from shrivelled berries and necrotic inflorescences of diseased grapevines were used to inoculate leaves and inflorescences of potted cv. Semillon grapevines. Pss caused disease symptoms similar to those experienced in the field, including angular leaf lesions, longitudinal lesions in shoot tissues and rotting of inflorescences from before flowering until shortly after fruit set. High humidity promoted symptom severity. The necrotic bunch stem and leaf lesions were susceptible to the development of Botrytis cinerea infections. Cryo‐scanning electron microscopy (cryoSEM) indicated that Pss entered leaves and inflorescence tissues via distorted, open, raised stomata surrounded by folds of tissue that appeared as ‘star‐shaped’ callose‐rich complexes when viewed by UV light microscopy. In necrotic tissues, cryoSEM revealed Pss within petiole parenchyma cells and air‐filled rachis xylem vessels. This is the first report of inflorescence and hence fruit loss caused by Pss in grapevines. The disease is described as ‘bacterial inflorescence rot’ and regarded as one that expands the previously reported pathology of grapevines caused by P. syringae. This study also indicated that infection by Pss might promote destructive B. cinerea infections when the fungus is already present but latent, although further experimentation is needed to prove such an interaction.     

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.     

Characterization of acephate resistance in the diamondback moth Plutella xylostella

Decreased acetylcholinesterase (AChE) sensitivity and metabolic detoxification mediated by glutathione S-transferases (GSTs) were examined for their involvement in resistance to acephate in the diamondback moth, Plutella xylostella. The resistant strain showed 47.5-fold higher acephate resistance than the susceptible strain had. However, the resistant strain was only 2.3-fold more resistant to prothiofos than the susceptible strain. The resistant strain included insects having the A298S and G324A mutations in AChE1, which are reportedly involved in prothiofos resistance in P. xylostella, showing reduced AChE sensitivity to inhibition by methamidophos, suggesting that decreased AChE1 sensitivity is one factor conferring acephate resistance. However, allele frequencies at both mutation sites in the resistant strain were low (only 26%). These results suggest that other factors such as GSTs are involved in acephate resistance. Expression of GST genes available in P. xylostella to date was examined using the resistant and susceptible strains, revealing no significant correlation between the expression and resistance levels.