Draft Genome Sequence Resources of Three Strains (TOS4, TOS5, and TOS14) of Xylella fastidiosa Infecting Different Host Plants in the Newly Discovered Outbreak in Tuscany, Italy.

An outbreak of Xylella fastidiosa was discovered in late 2018 in northern Italy affecting several plant species. Multilocus sequence typing analyses detected the presence of strains clustering in X. fastidiosa subsp. multiplex and harboring a hitherto uncharacterized sequence type, ST87. Three cultured strains (TOS4, TOS5, and TOS14) were subjected to high-throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated that they belong to the subspecies multiplex. The genetic information generated for these newly discovered strains further supports the evidence that sequence types are associated with the emergence of X. fastidiosa in Europe, posing major challenges for predicting the main threatened European and Mediterranean crops and plant species.

Draft Genome Resources of Two Strains ("ESVL" and "IVIA5901") of Xylella fastidiosa Associated with Almond Leaf Scorch Disease in Alicante, Spain.

An outbreak of Xylella fastidiosa subsp. multiplex sequence type ST6 was discovered in 2017 in mainland Spain affecting almond trees. Two cultured almond strains, "ESVL" and "IVIA5901," were subjected to high throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated they belong to the subspecies multiplex, and pairwise comparisons of the chromosomal genomes showed an average nucleotide identity higher than 99%. Interestingly, the two strains differ for the presence of the plasmids pXF64-Hb_ESVL and pUCLA-ESVL detected only in the ESVL strain. The availability of these draft genomes contribute to extend the European genomic sequence dataset, a first step toward setting new research to elucidate the pathway of introduction and spread of the numerous strains of this subspecies so far detected in Europe.

Genome-Wide Analysis Provides Evidence on the Genetic Relatedness of the Emergent Xylella fastidiosa Genotype in Italy to Isolates from Central America.

Xylella fastidiosa is a plant-pathogenic bacterium recently introduced in Europe that is causing decline in olive trees in the South of Italy. Genetic studies have consistently shown that the bacterial genotype recovered from infected olive trees belongs to the sequence type ST53 within subspecies pauca. This genotype, ST53, has also been reported to occur in Costa Rica. The ancestry of ST53 was recently clarified, showing it contains alleles that are monophyletic with those of subsp. pauca in South America. To more robustly determine the phylogenetic placement of ST53 within X. fastidiosa, we performed a comparative analysis based on single nucleotide polymorphisms (SNPs) and the study of the pan-genome of the 27 currently public available whole genome sequences of X. fastidiosa. The resulting maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pan-genome analysis are consistent with previously described X. fastidiosa taxonomy, distinguishing the subsp. fastidiosa, multiplex, pauca, sandyi, and morus. Within the subsp. pauca, the Italian and three Costa Rican isolates, all belonging to ST53, formed a compact phylotype in a clade divergent from the South American pauca isolates, also distinct from the recently described coffee isolate CFBP8072 imported into Europe from Ecuador. These findings were also supported by the gene characterization of a conjugative plasmid shared by all the four ST53 isolates. Furthermore, isolates of the ST53 clade possess an exclusive locus encoding a putative ATP-binding protein belonging to the family of histidine kinase-like ATPase gene, which is not present in isolates from the subspecies multiplex, sandyi, and pauca, but was detected in ST21 isolates of the subspecies fastidiosa from Costa Rica. The clustering and distinctiveness of the ST53 isolates supports the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade within subsp. pauca.

Transcriptome profiling of two olive cultivars in response to infection by the CoDiRO strain of Xylella fastidiosa subsp. pauca.

BACKGROUND: The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show much milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To determine whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. RESULTS: A global quantitative transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. CONCLUSIONS: Collectively these findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response.

Infectivity and transmission of Xylellua fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae) in Apulia, Italy.

Discovery of Xylella fastidiosa from olive trees with "Olive quick decline syndrome" in October 2013 on the west coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-fluid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophoridae). Adult P. spumarius, collected in November 2013 from ground vegetation in X. fastidiosa-infected olive orchards, were 67% (40 out of 60) positive for X. fastidiosa by polymerase chain reaction (PCR) assays. Euscelis lineolatus Brullé were also collected but tested negative for the pathogen. Transmission tests with P. spumarius collected from the Salento area were, therefore, conducted. After a 96-h inoculation access period with 8 to 10 insects per plant and a 30-d incubation period, PCR results showed P. spumarius transmitted X. fastidiosa to two of five periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected field trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy.

A model for predicting the phenology of Philaenus spumarius.

The design and implementation of Philaenus spumarius control strategies can take advantage of properly calibrated models describing and predicting the phenology of vector populations in agroecosystems. We developed a temperature-driven physiological-based model based on the system of Kolmogorov partial differential equations to predict the phenological dynamics of P. spumarius. The model considers the initial physiological age distribution of eggs, the diapause termination process, and the development rate functions of post-diapausing eggs and nymphal stages, estimated from data collected in laboratory experiments and field surveys in Italy. The temperature threshold and cumulative degree days for egg diapause termination were estimated as 6.5 °C and 120 DD, respectively. Preimaginal development rate functions exhibited lower thresholds ranging between 2.1 and 5.0 °C, optimal temperatures between 26.6 and 28.3 °C, and upper threshold between 33.0 and 35 °C. The model correctly simulates the emergence of the 3rd, 4th, and 5th nymphal instars, key stages to target monitoring actions and control measures against P. spumarius. Precision in simulating the phenology of the 1st and 2nd nymphal stages was less satisfactory. The model is a useful rational decision tool to support scheduling monitoring and control actions against the late and most important nymphal stages of P. spumarius.

Update of the Xylella spp. host plant database - systematic literature search up to 30 June 2022.

This scientific report provides an update of the Xylella spp. host plant database, aiming to provide information and scientific support to risk assessors, risk managers and researchers dealing with Xylella spp. Upon a mandate of the European Commission, EFSA created and regularly updates a database of host plant species of Xylella spp. The current mandate covers the period 2021-2026. This report is related to the seventh version of the database published in Zenodo in the EFSA Knowledge Junction community, covering literature published from 1 January 2022 up to 30 June 2022, and recent Europhyt outbreak notifications. Informative data have been extracted from 30 selected publications. Fifteen new host plants were identified and added to the database. Those plant species were reported from Brazil, France, Italy, Portugal and Spain, and infected by subsp. multiplex, pauca or unknown (i.e. not reported). No additional data were retrieved for X. taiwanensis. Two new STs (namely ST88 and ST89) belonging to subspecies multiplex were identified in host plants in natural conditions, and new information on the tolerant/resistant response of plant species to X. fastidiosa infection were added to the database. The overall number of Xylella spp. host plants determined with at least two different detection methods or positive with one method (between sequencing and pure culture isolation) reaches now 423 plant species, 194 genera and 68 families. Such numbers rise to 679 plant species, 304 genera and 88 families if considered regardless of the detection methods applied.

Exploring the xylem-sap to unravel biological features of Xylella fastidiosa subspecies pauca ST53 in immune, resistant and susceptible crop species through metabolomics and in vitro studies.

Xylella fastidiosa subsp. pauca ST53 (Xfp) is a pathogenic bacterium causing one of the most severe plant diseases currently threatening the olive-growing areas of the Mediterranean, the Olive Quick Decline Syndrome (OQDS). The majority of the olive cultivars upon infections more or less rapidly develop severe desiccation phenomena, while few are resistant (e.g. Leccino and FS17), being less impacted by the infections. The present study contributes to elucidating the basis of the resistance phenomenon by investigating the influence of the composition of the xylem sap of plant species on the rate of bacterial multiplication. Xylem saps from Xfp host and non-host species were used for growing the bacterium in vitro, monitoring bacterial growth, biofilm formation, and the expression of specific genes. Moreover, species-specific metabolites, such as mannitol, quinic acid, tartaric acid, and choline were identified by non-targeted NMR-based metabolomic analysis in olive, grapevine, and citrus. In general, the xylem saps of immune species, including grapevine and citrus, were richer in amino acids, organic acids, and glucose. The results showed greater bacterial growth in the olive cultivar notoriously susceptible to Xfp (Cellina di Nardò), compared to that recorded in the resistant cultivar Leccino. Conversely, higher biofilm formation occurred in Leccino compared to Cellina di Nardò. Using the xylem saps of two Xfp-immune species (citrus and grapevine), a divergent bacterial behavior was recorded: low planktonic growth and biofilm production were detected in citrus compared to the grapevine. A parallel evaluation of the expression of 15 genes showed that Xfp directs its molecular functions mainly to virulence. Overall, the results gained through this multidisciplinary study contribute to extending the knowledge on the host-pathogen interaction, while confirming that the host response and resistance mechanism have a multifactorial basis, most likely with a cumulative effect on the phenotype.

Update of the Xylella spp. host plant database - systematic literature search up to 31 December 2021.

This Scientific report provides an update of the Xylella spp. host plant database, aiming to provide information and scientific support to risk assessors, risk managers and researchers dealing with Xylella spp. Upon a mandate of the European Commission, EFSA created and regularly updated a database of host plant species of Xylella spp. The current mandate covers the period 2021-2026. This report is related to the sixth version of the database published in Zenodo in the EFSA Knowledge Junction community, covering literature published from 1 July 2021 up to 31 December 2021, and recent Europhyt outbreak notifications. Informative data have been extracted from 29 selected publications. Eleven new host plants were identified and added to the database: six plant species naturally infected by subsp. multiplex of X. fastidiosa in the EU (France, Italy and Portugal) and five plant species artificially infected by different X. fastidiosa subspecies (multiplex, pauca, fastidiosa and sandyi). No additional data were retrieved for X. taiwanensis. New information on the tolerant/resistant response of plant species to X. fastidiosa infection were added, while no new STs have been identified worldwide compared to the previous update published in January 2022. The overall number of Xylella spp. host plants determined with at least two different detection methods or positive with one method (between: sequencing, pure culture isolation) reaches now 412 plant species, 190 genera and 68 families. Such numbers rise to 664 plant species, 299 genera and 88 families if considered regardless of the detection methods applied.

Fast and Reliable Electronic Assay of a Xylella fastidiosa Single Bacterium in Infected Plants Sap.

Pathogens ultra-sensitive detection is vital for early diagnosis and provision of restraining actions and/or treatments. Among plant pathogens, Xylella fastidiosa is among the most threatening as it can infect hundreds of plant species worldwide with consequences on agriculture and the environment. An electrolyte-gated transistor is here demonstrated to detect X. fastidiosa at a limit-of-quantification (LOQ) of 2 ± 1 bacteria in 0.1 mL (20 colony-forming-unit per mL). The assay is carried out with a millimeter-wide gate functionalized with Xylella-capturing antibodies directly in saps recovered from naturally infected plants. The proposed platform is benchmarked against the quantitave polymerase chain reaction (qPCR) gold standard, whose LOQ turns out to be at least one order of magnitude higher. Furthermore, the assay selectivity is proven against the Paraburkholderia phytofirmans bacterium (negative-control experiment). The proposed label-free, fast (30 min), and precise (false-negatives, false-positives below 1%) electronic assay, lays the ground for an ultra-high performing immunometric point-of-care platform potentially enabling large-scale screening of asymptomatic plants.

Analysis of the epitope structure of Plum pox virus coat protein.

Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

Introduction and adaptation of an emerging pathogen to olive trees in Italy.

The invasive plant pathogen Xylella fastidiosa currently threatens European flora through the loss of economically and culturally important host plants. This emerging vector-borne bacterium, native to the Americas, causes several important diseases in a wide range of plants including crops, ornamentals, and trees. Previously absent from Europe, and considered a quarantine pathogen, X. fastidiosa was first detected in Apulia, Italy in 2013 associated with a devastating disease of olive trees (Olive Quick Decline Syndrome, OQDS). OQDS has led to significant economic, environmental, cultural, as well as political crises. Although the biology of X. fastidiosa diseases have been studied for over a century, there is still no information on the determinants of specificity between bacterial genotypes and host plant species, which is particularly relevant today as X. fastidiosa is expanding in the naive European landscape. We analysed the genomes of 79 X. fastidiosa samples from diseased olive trees across the affected area in Italy as well as genomes of the most genetically closely related strains from Central America. We provided insights into the ecological and evolutionary emergence of this pathogen in Italy. We first showed that the outbreak in Apulia is due to a single introduction from Central America that we estimated to have occurred in 2008 [95 % HPD: 1930-2016]. By using a combination of population genomic approaches and evolutionary genomics methods, we further identified a short list of genes that could play a major role in the adaptation of X. fastidiosa to this new environment. We finally provided experimental evidence for the adaptation of the strain to this new environment.

Antagonistic activity of olive endophytic bacteria and of Bacillus spp. strains against Xylella fastidiosa.

Strains of Xylella fastidiosa subsp. pauca characterized by a specific genotype, the so called sequence type "ST53", have been associated with a severe disease named Olive Quick Decline Syndrome (OQDS). Despite the relevant research efforts devoted to control the disease caused by X. fastidiosa, so far there are no therapeutic means able to cure the infected host plants. As such, the aim of this study was the identification of antagonistic bacteria potentially deployable as bio-control agents against X. fastidiosa. To this end, two approaches were used, i.e. the evaluation of the antagonistic activity of: i) endophytic bacteria isolated from olive trees located in an infected area but showing mild or no symptoms, and ii) Bacillus strains, as they are already known as bio-control agents. Characterization of endophytic bacterial isolates revealed that the majority belonged to different species of the genera Sphingomonas, Methylobacterium, Micrococcus and Curtobacterium. However, when they were tested in vitro against X. fastidiosa ST53 none of them showed antagonistic activity. On the contrary, when strains belonging to different species of the genus Bacillus were included in these tests, remarkable antagonistic activities were recorded. Some B. velezensis strains also produced culture filtrates with inhibitory activity against X. fastidiosa ST53. Taking also into account that two of these B. velezensis strains (namely strains D747 and QST713) are already registered and commercially available as bio-control agents, our results pave the way for further studies aimed at the development of a sustainable bio-control strategy of the OQDS.

Ionomic Differences between Susceptible and Resistant Olive Cultivars Infected by Xylella fastidiosa in the Outbreak Area of Salento, Italy.

Olive quick decline syndrome (OQDS) is a devastating disease of olive trees in the Salento region, Italy. This disease is caused by the bacterium Xylella fastidiosa, which is widespread in the outbreak area; however, the "Leccino" variety of olives has proven to be resistant with fewer symptoms and lower bacterial populations than the "Ogliarola salentina" variety. We completed an empirical study to determine the mineral and trace element contents (viz; ionome) of leaves from infected trees comparing the two varieties, to develop hypotheses related to the resistance of Leccino trees to X. fastidiosa infection. All samples from both cultivars tested were infected by X. fastidiosa, even if leaves were asymptomatic at the time of collection, due to the high disease pressure in the outbreak area and the long incubation period of this disease. Leaves were binned for the analysis by variety, field location, and infected symptomatic and infected asymptomatic status by visual inspection. The ionome of leaf samples was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) and compared with each other. These analyses showed that Leccino variety consistently contained higher manganese (Mn) levels compared with Ogliarola salentina, and these levels were higher in both infected asymptomatic and infected symptomatic leaves. Infected asymptomatic and infected symptomatic leaves within a host genotype also showed differences in the ionome, particularly a higher concentration of calcium (Ca) and Mn levels in the Leccino cultivar, and sodium (Na) in both varieties. We hypothesize that the ionome differences in the two varieties contribute to protection against disease caused by X. fastidiosa infection.

Update of the Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory.

EFSA was asked to update the 2015 EFSA risk assessment on Xylella fastidiosa for the territory of the EU. In particular, EFSA was asked to focus on potential establishment, short- and long-range spread, the length of the asymptomatic period, the impact of X. fastidiosa and an update on risk reduction options. EFSA was asked to take into account the different subspecies and Sequence Types of X. fastidiosa. This was attempted throughout the scientific opinion but several issues with data availability meant that this could only be partially achieved. Models for risk of establishment showed most of the EU territory may be potentially suitable for X. fastidiosa although southern EU is most at risk. Differences in estimated areas of potential establishment were evident among X. fastidiosa subspecies, particularly X. fastidiosa subsp. multiplex which demonstrated areas of potential establishment further north in the EU. The model of establishment could be used to develop targeted surveys by Member States. The asymptomatic period of X. fastidiosa varied significantly for different host and pathogen subspecies combinations, for example from a median of approximately 1 month in ornamental plants and up to 10 months in olive, for pauca. This variable and long asymptomatic period is a considerable limitation to successful detection and control, particularly where surveillance is based on visual inspection. Modelling suggested that local eradication (e.g. within orchards) is possible, providing sampling intensity is sufficient for early detection and effective control measures are implemented swiftly (e.g. within 30 days). Modelling of long-range spread (e.g. regional scale) demonstrated the important role of long-range dispersal and the need to better understand this. Reducing buffer zone width in both containment and eradication scenarios increased the area infected. Intensive surveillance for early detection, and consequent plant removal, of new outbreaks is crucial for both successful eradication and containment at the regional scale, in addition to effective vector control. The assessment of impacts indicated that almond and Citrus spp. were at lower impact on yield compared to olive. Although the lowest impact was estimated for grapevine, and the highest for olive, this was based on several assumptions including that the assessment considered only Philaenus spumarius as a vector. If other xylem-feeding insects act as vectors the impact could be different. Since the Scientific Opinion published in 2015, there are still no risk reduction options that can remove the bacterium from the plant in open field conditions. Short- and long-range spread modelling showed that an early detection and rapid application of phytosanitary measures, consisting among others of plant removal and vector control, are essential to prevent further spread of the pathogen to new areas. Further data collection will allow a reduction in uncertainty and facilitate more tailored and effective control given the intraspecific diversity of X. fastidiosa and wide host range.

Complete Genome Sequence of the Olive-Infecting Strain Xylella fastidiosa subsp. pauca De Donno.

We report here the complete and annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. pauca strain De Donno. This strain was recovered from an olive tree severely affected by olive quick decline syndrome (OQDS), a devastating olive disease associated with X. fastidiosa infections in susceptible olive cultivars.

Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy.

The recent introduction of Xylella fastidiosa in Europe and its involvement in the Olive Quick Decline Syndrome (OQDS) in Apulia (Salento, Lecce district, South Italy) led us to investigate the biology and transmission ability of the meadow spittlebug, Philaenus spumarius, which was recently demonstrated to transmit X. fastidiosa to periwinkle plants. Four xylem-sap-feeding insect species were found within and bordering olive orchards across Salento during a survey carried out from October 2013 to December 2014: P. spumarius was the most abundant species on non-olive vegetation in olive orchards as well as on olive foliage and was the only species that consistently tested positive for the presence of X. fastidiosa using real-time PCR. P. spumarius, whose nymphs develop within spittle on weeds during the spring, are likely to move from weeds beneath olive trees to olive canopy during the dry period (May to October 2014). The first X. fastidiosa-infective P. spumarius were collected in May from olive canopy: all the individuals previously collected on weeds tested negative for the bacterium. Experiments demonstrated that P. spumarius transmitted X. fastidiosa from infected to uninfected olive plants. Moreover, P. spumarius acquired X. fastidiosa from several host plant species in the field, with the highest acquisition rate from olive, polygala and acacia. Scanning electron microscopy (SEM) revealed bacterial cells resembling X. fastidiosa in the foreguts of adult P. spumarius. The data presented here are essential to plan an effective IPM strategy and limit further spread of the fastidious bacterium.

Draft Genome Sequence of CO33, a Coffee-Infecting Isolate of Xylella fastidiosa.

The draft genome sequence of Xylella fastidiosa CO33 isolate, retrieved from symptomatic leaves of coffee plant intercepted in northern Italy, is reported. The CO33 genome size is 2,681,926 bp with a GC content of 51.7%.

Draft Genome Sequence of the Xylella fastidiosa CoDiRO Strain.

We determined the draft genome sequence of the Xylella fastidiosa CoDiRO strain, which has been isolated from olive plants in southern Italy (Apulia). It is associated with olive quick decline syndrome (OQDS) and characterized by extensive scorching and desiccation of leaves and twigs.