EXTERNAL SCIENTIFIC REPORT

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1 EXTERNAL SCIENTIFIC REPORT APPROVED:22 March 2016 PUBLISHED: 29 March 2016 Pilot project on Xylella fastidiosa to reduce risk assessment uncertainties Institute for Sustainable Plant Protection, National Research Council of Italy, CNR In collaboration with: Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari Aldo Moro (Italy) Centro di Ricerca, Sperimentazione e Formazione in Agricoltura Basile Caramia, Locorotondo, Bari, Italy Authors: Maria Saponari, Donato Boscia, Giuseppe Altamura, Giusy D Attoma, Vincenzo Cavalieri, Stefania Zicca, Massimiliano Morelli, Danilo Tavano Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Unità Organizzativa di Bari (Italy) Giuliana Loconsole, Leonardo Susca, Oriana Potere, Vito Savino, Giovanni P. Martelli Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi di Bari Aldo Moro (Italy) Francesco Palmisano, Crescenza Dongiovanni, Antonia Saponari, Giulio Fumarola, Michele Di Carolo Centro di Ricerca, Sperimentazione e Formazione in Agricoltura Basile Caramia, Locorotondo, Bari, Italy Abstract Xylella fastidiosa (X. fastidiosa) has a very broad host range, including many cultivated and wild plants common in Europe. There is, however, scant information on the potential hosts of X. fastidiosa in the natural European flora, as a wide range of these plants have never been exposed to the bacterium. Investigations carried out in the recent years ( ) in Apulia (southern Italy) where X. fastidiosa was first recorded on olive trees, determined that: (i) the strain associated with this outbreak, denoted CoDiRO, is genetically related to the subspecies pauca, representing a variant classified as sequence type 53 ; (ii) CoDiRO is consistently associated with infections occurring under natural conditions on several hitherto unknown hosts of X. fastidiosa. The aim of the studies conducted in this pilot project was to assess the host range of the Apulian strain of X. fastidiosa by artificial inoculations and exposure to infective vectors, of selected cultivars of major perennial crops and some forest species. Diagnostic tests, isolation and symptom evaluation were performed to assess bacterial colonization in the different hosts and the development of symptoms associated with X. fastidiosa infections. The overall results of molecular assays and bacterial isolation tests carried out up to 14 months post-inoculation clearly differentiated the plant species in which rapid colonization of the plants occurred from those that did not support X. fastidiosa multiplication and movement. Bacterial inoculation of olives, oleanders and Polygala myrtifolia plants resulted in systemic colonization by the bacterium, and symptoms resembling those observed under natural infection conditions were observed. Indeed, the results relative to different olive cultivars confirmed the high susceptibility of this crop to strain CoDiRO and the consistent association of the infections with the appearance of symptoms of dieback and desiccation of the inoculated plants. Conversely, inoculated plants of citrus, grapes and Quercus ilex were never found to be systemically infected nor did they develop any suspicious symptom. In addition, field experiments confirmed, although with a different transmission rate, that infective Philaenus spumarius was able to transmit the bacterium to the host plants used in the field experiment (olive, oleander and Polygala myrtifolia). Specifically, serological and molecular assays readily detected the bacterium in these host plants as soon as six months after caging the infective vectors, when the plants had not yet shown any symptom. In agreement with the results of the artificial inoculation, none of the citrus, grape or Q. ilex plants so far tested positive for X. fastidiosa upon exposure to infective P. spumarius. European Food Safety Authority, EFSA Supporting publication 2016:EN-1013

2 Key words: (CoDiRO, Xylella fastidiosa, olives, pathogenicity, needle inoculations, vector transmission) Question number: EFSA-Q Correspondence: Disclaimer: The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, Acknowledgements: We thank Prof. R. Almeida, Prof. A.H. Purcell, Dr. H. Della Coletta Filho for their scientific advice and input in the development of the work plan of this pilot project. We thank A.PR.O.L. Lecce for their collaboration in the field trials. Suggested citation: Maria Saponari, Donato Boscia, Giuseppe Altamura, Giusy D Attoma, Vincenzo Cavalieri, Giuliana Loconsole, Stefania Zicca, Crescenza Dongiovanni, Francesco Palmisano, Leonardo Susca, Massimiliano Morelli, Oriana Potere, Antonia Saponari, Giulio Fumarola, Michele Di Carolo, Danilo Tavano, Vito Savino, Giovanni P. Martelli, Pilot project on Xylella fastidiosa to reduce risk assessment uncertainties. EFSA supporting publication 2016:EN pp. European Food Safety Authority, 2016 Reproduction of the images contained in this report is prohibited and permission must be sought directly from the Institute for Sustainable Plant Protection, National Research Council of Italy, CNR. 2 EFSA Supporting publication 2016:EN-1013

3 Summary Xylella fastidiosa (X. fastidiosa) was detected in olive trees growing in the Lecce province of Apulia (southern Italy) in October 2013, representing the first documented field outbreak of this quarantine bacterium in the European Union (EU). Subsequently, the pathogen was found in symptomatic plants of almond, sweet cherry, oleander, broom, Polygala myrtifolia, Westringia fruticosa and Acacia saligna (Saponari et al., 2014). X. fastidiosa has a very broad host range ( including monocotyledonous and dicotyledonous species, herbaceous and woody plants, cultivated crops and weeds, native flora, riparian and landscape shade trees. However, the four known subspecies of the bacterium (X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi and X. fastidiosa subsp. pauca) show some host preference, i.e. they may cause disease few host plants, while colonizing many additional hosts without causing significant disease. Known hosts of X. fastidiosa include many cultivated and wild species growing in Europe. To assess the risk of an epidemic spreading of the Apulian strain of the bacterium in the EU territory one of the crucial aspects is to secure information, as accurate as possible, on its host range and epidemiology, both of which are only partly known. The aim of this pilot project was to conduct specific investigations for a better understanding of the susceptibility of some important perennial crop species to the Apulian strain of X. fastidiosa (denoted CoDiRO), by testing the susceptibility of major Mediterranean crops such as grapes, citrus, peach and plum, but also of forest species such as oak or other temperate trees. The experimental approach included needle-inoculations with bacterial suspensions from pure cultures and the exposure of plants to natural infection conditions in the open field with exposure to caged naturally infected vectors (to increase the likelihood of infection in the field). Results from needle inoculations revealed the high susceptibility of olives, oleander and Polygala myrtifolia (high rate of infected plants) to the CoDiRO strain. From these hosts, the bacterium was successfully re-isolated and symptoms resembling those associated to X. fastidiosa (leaf scorching, dieback, discoloration) were recorded during the experiments. Conversely, the experimental plants for the remaining species (citrus, grapes and Q. ilex) did not support bacterial infections nor developed symptoms. All the artificially inoculated plants will be maintained under observation for at least an additional vegetative season, whereas observations in the field experimental plot will be prolonged for 5-10 years depending on the progress of the infections and symptom(s). Although preliminary evidences have been collected for Prunus dulcis and the hybrid GF677, the experiments on Prunus spp. will be completed within the next 6-12 months. Even though limited to few species and cultivars, all together the results achieved in the timeframe of this contract provided critical information on the host response and susceptibility of relevant host species. However, further investigations aiming at exploring the genetic and phenotypic differential responses amongst different cultivars and species should be promoted in order to strengthen the surveillance programmes and the containment strategies to put in place in the demarcated area (EU Commission Implementing Decision 2015/789). 3 EFSA Supporting publication 2016:EN-1013

4 Table of contents Abstract... 1 Summary Introduction Objectives Specific objectives Background and Terms of Reference as provided by the requestor Methodology Artificial inoculation for the evaluation of the host range and pathogenicity Host plants selected for artificial inoculations and controls Inoculation procedure Laboratory tests Set up of the experimental plot in the contaminated area Field surveys Results Artificial inoculations under controlled environmental conditions (greenhouse) Pathogenicity tests Host range evaluation Control plants Artificial inoculations in a screenhouse without temperature control Exposure of different plant species to natural infective P. spumarius in the field Field surveys Discussion on experimental plan to assess the full host range of Xylella fastidiosa strain CoDiRO Standard requirements for the artificial inoculation Diagnostic tests Additional experimental approaches Conclusions References EFSA Supporting publication 2016:EN-1013

5 1. Introduction The unexpected emergence of Xylella fastidiosa (X. fastidiosa) in the Salento peninsula of Italy (Fig. 1) has created an alarming situation, because of the dramatic damage suffered by the olive groves where the bacterium has established itself and the alarm that this finding has raised in the European Union and Mediterranean basin (Martelli et al., 2016). Infections to olive by a strain of X. fastidiosa subspecies multiplex were first reported by Krugner et al. (2014) from California (USA) in trees exhibiting leaf scorching and branch dieback symptoms. However, no ultimate conclusions were drawn as to the pathogenicity of this bacterium to olive. In 2013, an alarmingly severe disorder characterized by extensive leaf scorching and desiccation of twigs and branches, denoted Olive quick decline syndrome (OQDS), was recorded in Southern Italy, in the Salento area in the Apulian Region. A strain of X. fastidiosa subspecies pauca, denoted CoDiRO, was found to be consistently associated with declining trees (Saponari et al., 2013; Giampetruzzi et al., 2015). The genome of this bacterial strain proved to be a DNA molecule ca. 2,500,000 bp in size (Giampetruzzi et al., 2015), molecularly identical to that of a bacterial isolate (ST53) from Costa Rica. Following the Italian report, olive diseases strikingly resembling OQDS have been reported from Argentina (Haelterman et al., 2015) and Brazil (Coletta-Filho et al., 2016). In both cases, symptomatic plants are infected by X. fastidiosa strains closely related genetically to the subspecies pauca, but distinct from the Salentinian isolate. Such findings, in three distant regions of the world (Italy, Argentina and Brazil), are indicative of the strong correlation between the olive syndrome and the presence of X. fastidiosa. As to the risks for Europe and the Mediterranean basin represented by the introduction of X. fastidiosa into Italy and more recently in France, it has been predicted (Purcell, 1997; Bosso et al., 2016) how widely the bacterium will spread in the region, should nothing be done to confine it within its current boundaries. Phylogenetic studies support the classification of X. fastidiosa into at least four subspecies (Schaad et al. 2004): (i) X. fastidiosa subsp. fastidiosa, the causal agent of Pierce's Disease of grapevines; (ii) X. fastidiosa subsp. sandyi, associated with oleander leaf scorch; (iii) X. fastidiosa subsp. multiplex, associated with leaf scorching of a number of trees, including almond, ornamental and shade trees, and (iv) the proposed X. fastidiosa subsp. pauca, the agent of Citrus variegated chlorosis and Coffee leaf scorch. There is some host differentiation between these generally accepted subspecies, with regard to symptomatic hosts. There is, however, no information on the potential hosts of X. fastidiosa in the European flora, as a wide range of European wild plant species have never been exposed to the bacterium and it is not known whether they would be hosts, and, if so, whether they would be symptomatic or not (EFSA PLH Panel, 2015). Surveys carried out in the Salento peninsula have revealed that: (i) olive is the primary host in the Italian outbreak; (ii) Polygala myrtifolia, Myrtus communis, Grevillea juniperina, Laurus nobilis, Myoporum insulare and Dodonaea viscosa purpurea represent hitherto unreported hosts of the species X. fastidiosa; (iii) Olea europaea, Nerium oleander, Rosmarinus officinalis, Prunus avium, Prunus dulcis and Rhamnus alaternum, although already reported hosts of the species X. fastidiosa elsewhere, are infected by the CoDiRO strain, representing novel hosts for the subspecies pauca. 5 EFSA Supporting publication 2016:EN-1013

6 Figure 1: Demarcated area in the EU territory based on the EU Commission Implementing Decision 2015/ Objectives The overall goal of this project was to: (i) investigate the host range of X. fastidiosa subsp. pauca strain CoDiRO; (ii) provide an accurate description of the symptoms shown by olive and other known susceptible hosts; (iii) investigate if other economically relevant plant species, like grapevines, citrus and oak, which have not been found infected under natural conditions in the outbreak areas, are susceptible or not to infection by strain CoDiRO; (iv) provide research-based information in view of the implementation of a comprehensive program for the evaluation of the susceptibility of a wider panel of host species. In fact, the European Commission Implementing Decision (EU) 2015/789 includes more than 160 species listed as specified plants known to be susceptible to bacterial isolates. Host plants vary in their susceptibility to X. fastidiosa infection; this variation has been attributed to differences in bacterial concentration and the rate of xylem vessel occlusion (Fry et al., 1990). Thus, the assessment of the susceptibility of relevant species to infections caused by strain CoDiRO is of key importance for containment strategies to be adopted in the affected area Specific objectives of the Contract (from Terms of reference) Based on these general concepts, the specific objectives of this contract were: - Objective 1 is to collect preliminary data on the susceptibility of important tree species in the EU to the Apulian strain of X. fastidiosa by conducting small-scale experiments under controlled and field conditions. These studies include known hosts as positive control and important EU fruit and forest trees that have been reported elsewhere as susceptible to X. fastidiosa pauca and/or to other subspecies; a classical host range experiment by needle inoculation with the Apulian cultured isolate of X. fastidiosa for establishing host susceptibility to the pathogen and the systemic movement of the pathogen within the plant; an observational experiment by growing young plants of target species 6 EFSA Supporting publication 2016:EN-1013

7 under natural inoculum pressure in infected olive orchards in the outbreak area so as to determine the host susceptibility to infection by the local vectors of X. fastidiosa. - Objective 2 is to develop an experimental plan for a future comprehensive evaluation of the full host range of the Apulian strain of X. fastidiosa, based on the results of Objective 1 (see section 5). - Objective 3 is to provide information, field observations and, when relevant, laboratory tests for validation purposes to support a pilot analysis of the spreading pattern of X. fastidiosa in Apulia 2.2. Background of the Contract (from Terms of reference) This contract/grant was awarded by EFSA to: Contractor: Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche Contract title: Pilot project on Xylella fastidiosa to reduce risk assessment uncertainties Contract number: NP.EFSA.ALPHA Background The EFSA Scientific Panel on plant health (PLH Panel) received a mandate from the European Commission DG SANCO (Mandate ) in November 2013 under which a statement of EFSA was delivered on 22 November 2013 on Host plants, entry and spread pathways and risk reduction options for Xylella fastidiosa and a scientific opinion of the EFSA PLH Panel has been released Risk to plant health posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk reduction option (EFSA Journal 2015;13(1):3989). From the uncertainties listed in the published EFSA statement and from the ongoing work of the PLH Panel Working Group on X. fastidiosa it became evident that still very little is known on the host range and epidemiology of the Apulian strain of X. fastidiosa, and this may significantly affect the assessment of the risk for the EU in terms of endangered crops and areas. Considering that the currently known X. fastidiosa subspecies have a very broad range of host plants and vectors, thus potentially threatening many Mediterranean and temperate EU fruit tree crops and forestry species, but that very little epidemiological knowledge is available about the host range of the newly reported Apulian strain of X. fastidiosa, there is a need for a pilot project to collect preliminary experimental data on the susceptibility of the main potential perennial hosts of this pathogen in Apulia. Part of these data will support the current PLH Panel working group in its assessment, whereas this pilot project will also deliver a plan for future experiments to fill the knowledge gap on this emerging bacterial disease in the EU. The aim of this pilot exercise is to increase the EFSA responsiveness for the risk assessment work on this pathogen through reduction of risk assessment uncertainties and development of experimental plans for future research. The aim of this procurement procedure is to conclude a direct contract for the execution of specific tasks over a clearly defined period as defined in these tender specifications. The general objective is to secure a better understanding of the susceptibility of the most important tree species to the Apulian strain of X. fastidiosa which has been genotypically attributed to the subspecies pauca, with an established identity to a pauca isolate from oleander in Costa Rica. From an extensive review of the scientific literature, only ca. 40 host species can be attributed as hosts of the subspecies pauca compared to the ca. 180 hosts of the more extensively studied subspecies fastidiosa. In addition, very little is known about the host range of the oleander isolate from Costa Rica and of the Apulian isolate of X. fastidiosa. In the Apulian outbreak the confirmed hosts up to now are mainly olive, oleander, periwinkle, almond, cherry and some ornamentals. Other species as oak were found positive in the first tests in 2013 but this was not confirmed afterwards. To assess the risk of X. fastidiosa for the EU territory, it is therefore urgent to gain a better knowledge on the host range of the Apulian strain(s) of the bacterium by experimental studies. Given the very large number of hosts and vectors reported in literature, it is important to start first by testing the susceptibility of important Mediterranean crops such as grapes, citrus, peach and plum, but also of forest species such as oak or other temperate trees. The experience and results of this pilot exercise will help developing an experimental plan for a comprehensive evaluation of the full host range of the Apulian strain of X. fastidiosa. 7 EFSA Supporting publication 2016:EN-1013

8 3. Methodology Like other vector-borne diseases those induced by X. fastidiosa are driven by complexes interactions among the host, the pathogen and vector (i.e. vector host choice and its feeding behaviour). Furthermore, environment and climate can greatly affect the way in which this pathogen interacts with both the vector and the host plants. The assessment of host plant susceptibility to the CoDiRO strain was conducted through artificial inoculations and vector-mediated transmission, either under controlled conditions (glasshouse, GH), insect-proof net protection (screenhouse, SH) or under field conditions (Table 1) Artificial inoculation for the evaluation of the host range and pathogenicity Inoculation techniques should ensure infiltration of the bacterial suspension directly into the xylem vessels. According to the different species, several approaches can be used: pinprick stem inoculation, inoculum injection, trunk slicing and pricking, root immersion in a bacterial suspension. However, the most widely used method for X. fastidiosa inoculation is the pin-prink inoculation consisting in needlepunctures made on the stem (at the insertion of a leaf petiole) through a small drop of a bacterial suspension placed at the inoculum point (IP) (Hill and Purcell, 1995; Almeida et al., 2001). The procedure for the experimental inoculation (needle inoculation) was defined in collaboration with Prof. A.H. Purcell and R. Almeida (University of California, Berkeley). Furthermore, technical experience was gained by the Principal Investigator Dr. M. Saponari during a training at the Centro de Citricultura Sylvio Moreira in Brazil (April-May 2014), under the supervision of Dr. H. della Coletta Filho. The list of the host species and their cultivars selected for the mechanical inoculations was discussed and agreed with the requestor during the kick off meeting of the project. The bacterial isolate selected for the inoculations, denoted as De Donno, was recovered from an ancient olive tree growing in the Gallipoli countryside, the heart of the X. fastidiosa outbreak, that showed typical OQDS symptoms (Fig. 2). Prior to recovering this isolate in pure culture, a DNA library was prepared from the total DNA purified from the xylem tissue of the donor and subjected to a metagenomic sequence analyses. Results confirmed that the tree was infected by X. fastidiosa, all reads matching the full length genome of the previously sequenced strain CoDiRO (Giampetruzzi et al., 2015). Isolation from the De Donno tree was attempted in June 2014, following the protocol described by Almeida and Purcell (2003), and selected colonies were triple-cloned by sequential passages (8-10 days each). The recovered triple-cloned colonies were directly used for needle inoculation or stored in glycerol at -80 C in 50% glycerol for subsequent use. Two sets of experiments were carried out respectively in: (A) a glasshouse in the premises of the Bari University Campus, where the CNR-IPSP and the UNIBA laboratories are located. This is an insect-proof facility, with an additional internal layer of an insect-proof net (20/10, mesh 50) with temperature control (26-28 C). The structure has double entrance doors with a vestibule equipped with an air curtain device, to avoid the entrance/exit of air-borne insects (authorization of the Ministry of Agriculture No dated 15/12/2014); (B) a screenhouse located in the heavily contaminated area of Gallipoli (province of Lecce - location 40 1'25.18"N '54.18"E), where inoculated plants were maintained under environmental conditions close to those occurring in the open field. 8 EFSA Supporting publication 2016:EN-1013

9 Table 1: Description of the experiments Exp. Date Location Type of inoculation Host species (number of inoculated plants in parenthesis) Number of inoculum sites or insects caged on each plant A December, 2014 February March, 2015 Bari Artificial inoculation with plant maintained under controlled condition in glasshouse Olea europaea (10 plants for each of the 3 cultivars and 10 seedlings), Nerium oleander (10), Vitis vinifera cv. Cabernet sauvignon (19), Polygala myrtifolia (10), Quercus ilex (12), GF677 (19) Prunus spp. (a total of 90 plants corresponding to 10 different cultivars) 3 sites per plant B October, 2014 February March, 2015 Gallipoli (Lecce) Artificial inoculation with plants maintained under screenhouse O. europaea (10 plants for each of the 3 cultivars and 10 seedlings), N. oleander (10), P. myrtifolia (10) Prunus spp.* (a total of 184 plants of 14 different cultivars) 3 sites per plant C April, 2015 On going Parabita (Lecce) Exposure of the plants to naturally infective vectors under field conditions * see details on the cultivars and map of plot in section 3.2 *O. europaea; N. oleander, V. vinifera, P. myrtifolia, Prunus spp., Quercus ilex 10 insects caged on each single tree for 4 weeks 9 EFSA Supporting publication 2016:EN-1013

10 Figure 2: Symptom of dieback and desiccation on the olive tree denoted De Donno, infected with Xylella fastidiosa subsp. pauca strain CoDiRO Host plants selected for artificial inoculations and controls The host plants listed below (Table 2) were selected for needle inoculations with the CoDiRO strain. The panel of plant species included in the experiments were known hosts of the CoDiRO strain (olive, oleander, Polygala myrtifolia, almond and cherry) and other species whose susceptibility to the CoDiRO strain had not been established (oak, grapes, citrus and other stone fruits). For each host plant species/cultivar, 10 to 20 replicates (for some stone fruits inoculated plants were 6-8, due to the failure of graft take) were inoculated with the bacterial suspension as described in section and 2 to 5 plants were used as non-inoculated controls kept in the same conditions as the inoculated plants. Grafted, self-rooted plants or seedlings of the appropriate size (Fig. 3) were selected and maintained under optimal growing conditions. For Prunus spp., grafted plants were obtained from a nursery by the end of October 2014, exposed for 40 days at 4 C prior to their moving to a glasshouse by mid- December. Once the new shoots reached the right length (10-20 cm) they were used for needle inoculation. For all Prunus spp. the inoculations were completed between February and March All inoculated plants were periodically (at least monthly) inspected for symptoms expression and laboratory assays were performed periodically starting 1-month post-inoculation (mpi) and then every 3 months up to 12 mpi. Three to 6 inoculated plants (according to the host species) were entirely sectioned and stems and leaves from the proximal and distal portions from inoculum points (IP) were used for bacterial isolation and quantification by qpcr assays. Roots were also harvested and subjected the laboratory analysis. Seedlings of Nicotiana tabacum (cv. Havana SR1) and Catharanthus roseus (periwinkle) were needleinoculated with the same bacterial suspension used for the woody test plants. Specifically, seedlings of N. tabacum at the 4-5 leaf stage were needle-inoculated with two drops (10 µl) of the bacterial suspension placed on the main vein of each leaf, then punctured to let the suspension to be adsorbed (Hopkins and Adlerz, 1988). Visual inspections were carried out every week and laboratory assays were done one mpi by sampling 1-2 inoculated leaves and six mpi by testing the entire plant EFSA Supporting publication 2016:EN-1013

11 Seedlings of periwinkle were inoculated when they were cm tall. Three drops (10 µl) of the bacterial suspension were placed on the stem and allowed to be absorbed after pricking through the drops 4-5 times with a syringe needle. Plants were inspected weekly for symptom development, whereas laboratory assays were performed starting one mpi. Table 2: Host species and cultivars used for artificial inoculation Olive cultivars Leccino (widely grown in Italy and elsewhere, for which there is preliminary field and laboratory evidence of a resistant response to X. fastidiosa infections) (grafted and self-rooted plants) Frantoio (widely grown in Italy and elsewhere) (self-rooted plants) Coratina (one of the major Apulian cvs) (self-rooted plants) Cellina di Nardò (one of the two predominant local cvs, widely grown in the contaminated area and severely affected by OQDS) (grafted plants) Seedlings from olive seeds (commonly used in the nurseries as rootstocks) Prunus spp. GF677 (Prunus amygdalus x P. persica, micropropagated plantlets) Prunus avium cv. Ferrovia (grafted plants) Prunus cerasus cv. Visciola (grafted plants) Prunus armeniaca cv. Monaco Bello (grafted plants) Prunus persica cv. Laure (grafted plants) Prunus salicina cv. Santa Rosa (grafted plants) Prunus dulcis cv. Tuono (grafted plants) Prunus domestica cv. President (grafted plants) Prunus domestica cv. Stanley (grafted plants) Prunus avium cv. Bigarreau Moreau (grafted plants) Prunus armeniaca cv. Errani (grafted plants) Prunus persica cv. Caldesi (grafted plants) Prunus persica cv. Baby Gold 6 (grafted plants) Prunus salicina cv. Friar (grafted plants) Prunus dulcis cv. Genco (grafted plants) Nerium oleander Polygala myrtifolia Vitis vinífera Cabernet Sauvignon (susceptible to X. fastidiosa subsp. fastidiosa, self-rooted cuttings) Citrus spp (seedlings) Mandarin cv. Comune Sweet orange cv. Madame Vinous Citrange Carrizo Citrange Troyer Citrange selection C35 Grapefruit cv. Duncan Quercus ilex (predominant landscape tree amongst potentially susceptible hosts) (seedlings) Controls Tobacco (Nicotiana tabacum Havana SR1) (seedlings) Periwinkle (Catharanthus roseus) (seedlings) 11 EFSA Supporting publication 2016:EN-1013

12 Figure 3: Plant used for the artificial inoculations. A. Self-rooted plants of Cabernet Sauvingon; B. Self-rooted plants of Polygala myrtifolia; C. Grafted plants of olive cv. Cellina di Nardò; D. Olive seedlings; E. Seedlings of Duncan grapefruit; F. C35 seedlings. Continue EFSA Supporting publication 2016:EN-1013

13 Figure 3: Plant used for the artificial inoculations. G and H. Grafted plants of different Prunu spp.; I. Seedlings of Quercus ilex; L. Self-rooted cuttings of oleander Inoculation procedure Inoculation of test plants was done by prick inoculation with a culture of X. fastidiosa strain CoDiRO. A drop of a turbid suspension of the bacterium in phosphate-buffered saline (PBS 1X) was placed on the stem at the insertion of leaf petioles, and a sterile needle was inserted through the droplet into the plant tissues. The bacterial suspension was prepared from colonies of the CoDiRO strain grown on BCYE solid medium for 8-10 days. Bacterial suspensions consisted of PBS-aliquots standardized to an OD 600 value of and an estimated cell concentration of 2x10 8 to 10 9 CFU/ml. A 10 µl drop of the suspension was placed on the young stem of the test plants, and the tissue was pricked through the drop 5 times with a no. 0 entomological pin (Hill and Purcell, 1995). Three sites per plant were inoculated (3 contiguous nodes at the base of the plant) (Fig.4). Inoculation points were marked in order to follow the bacterial multiplication/movement afterwards EFSA Supporting publication 2016:EN-1013

14 Laboratory tests Quantitative PCR assay (qpcr). The inoculated plants were tested periodically by sampling the leaves at different distance from the IPs. Specifically, tests were started 1 mpi by sampling the leaf next to the top (first) IP and one leaf from the node above the 3rd (bottom) IP. Three months later, sampling was repeated at the node above the one sampled in the previous tests. At the sites that had tested negative in the previous assay, additional leaf samples were taken. This process, was then repeated every 3 months. One year post inoculation 3 replicates of each plant were dissected in 10 cm pieces and the stem and leaves of each portion tested separately. Samples were also taken from the roots. Extractions from leaf tissues were performed using leaf petioles excised from the blade, diced to small pieces and homogenised in CTAB buffer using a Mixer Mill MM 300 apparatus (Loconsole et al., 2010). The recovered total nucleic acids were used as template for the qpcr tests performed according to Harper et al. (2010). Similarly, the roots or the xylem tissues (ca. 0.5g) recovered from the stem portions were grounded in CTAB buffer by using the semi-automated homogenizer Homex 6 (Bioreba, Switzerland). Purified DNA was subjected to qpcr following the same procedure mentioned above. In addition to molecular tests, which would detect the presence of X. fastidiosa regardless of whether the bacterial cells were in active multiplication or dead, attempts were made to re-isolate the bacterium in axenic culture from the stem portions and leaf tissues collected from the sectioned plants. In this case, isolation and qpcr assays were performed on the same plant tissues. Specifically, the leaves collected from each portion were split in two subsamples, and processed either for DNA extraction or for bacterial isolation; stems were first processed for isolation, then the same squeezed stems were used to recover the xylem tissue from which DNA was extracted. The bacterial titre in inoculated plants was assessed through qpcr assays based on a standard curve generated by using a serial 10-fold dilution prepared with a bacterial suspension concentrated 10 8 CFU/ml. Re-isolation of the bacterium from inoculated plants, was preceeded by surface sterilization of tissues in 2% solution of NaOCl and 70% ethanol for 2 min each. Two distinct procedures were followed for recovering bacterial colonies from leaves and stems. Isolation from leaves. After surface sterilization, tissues were ground in PBS buffer at the ratio of 1:10 (w:v) using the homogenizer Homex 6. An aliquot of recovered sap (100 μl) was added to 900 μl of PBS, and used to prepare a serial 10-fold dilution (up 10-5 ). Aliquots of 10-2, 10-3, 10-4 dilutions were then plated on culture media, incubated at approximately 28 C, and monitored for colony development over six weeks. Isolation from the stem. Stems were cut into pieces of 10 cm, keeping separated the portion comprising the inoculation points and the distal portions above and below. After surface sterilization each piece was cut in the middle, the internal cut ends were squeezed with a pair of pliers and the sap blotted onto BCYE plates, incubated and monitored as described above EFSA Supporting publication 2016:EN-1013

15 Figure 4: Needle inoculation. A. Bacterial suspension placed on the stem of a grapevine plant; B. Inoculated shoot with the marked inoculum point (stem punctured 5-6 times); C. Inoculated olive shoot with three contiguous inoculum points Set up of the experimental plot in the contaminated area In April 2015 an experimental plot was established in the contaminated area, in the municipality of Parabita (in Lecce province, Apulian region) in collaboration with the local olive producer organization A.PR.O.L. Lecce. The number of olive cultivars to be tested was increased from 4 to 10 upon the request of A.PR.O.L. Twenty-four replicates for each of the selected species/cultivar were planted in four randomized 15 EFSA Supporting publication 2016:EN-1013

16 blocks of six plants each, except for Polygala myrtifolia, Nerium oleander and Quercus ilex planted in a single row surrounding the borders of the experimental plot. Olive cultivars Grapevine cultivars Coratina Cima di Melfi Chardonnay Leccino Frantoio Negramaro Arbosana Don Carlo Primitivo Koroneiki Arbequina Cellina di Nardò FS17 Italia Stone fruits and citrus Prunus avium cv. Ferrovia Prunus avium cv. Bigarreau Moreau Prunus dulcis cv. Supernova Prunus dulcis cv. Tuono Citrus sinensis Navelina Citrus trifoliata Citrange Troyer Citrus clementina Hernandina Other species Quercus ilex Nerium oleander Polygala myrtifolia To increase the vector transmission rate from the surrounding environment, in July 2015 all plants were caged and naturally infective specimens of Philaenus spumarius collected in diseased olive groves were placed inside each cage. The insects were collected during July when, based on previous findings, P. spumarius populations had the highest infectivity potential. Briefly, cages were placed on the trees so as to cover the entire canopy. Then, 8 to 10 P. spumarius adults collected in the surrounding Xylella-affected olive groves were placed inside each cage (Fig. 5). Plants of the olive selections Don Carlo and FS17 were not caged due to their small size. To determine the percentage of infective P. spumarius, more than 70 specimens, representative of the different sampling locations, were placed in ethanol and transferred to the laboratory for qpcr assays. The cages were maintained on the plants for 4 weeks. Plants of P. myrtifolia, oleander and Quercus ilex located at the border of the experimental plot were not caged so as to be exposed to infection by free-living vectors. Symptom inspection and sampling were conducted in January 2016, six months after experimental vector transmission (i.e. 9 months after planting). Samples consisted of 4-5 cuttings collected at random from the canopy of each of the evergreen species (olive, oleander, P. myrtifolia, Q. ilex, and citrus), from which 8-10 leaves were excised and used for DNA extraction and qpcr testing (Harper et al., 2010; Loconsole et al., 2014). For grapevines and Prunus spp., tests were performed on xylem tissues recovered from the dormant cuttings. To avoid removal of the shoots with a potential initial infection, none of the plants was pruned in the first year (grapevines included) EFSA Supporting publication 2016:EN-1013

17 Figure 5: Experimental plot realized in the infected area. A. Trees planted in April 2015; B. trees caged with insects in July 2015; C and D Trees sampled in January Field surveys Two olive groves, identified as Site 1 and Site 2, were selected in the contaminated area in which visual inspections were conducted twice a year and the presence of branches or twigs with dieback and desiccation was recorded and scored (from 0 = symptomless; 1 = few desiccated branches affecting a limited part of the canopy; 2 = desiccation interesting a large part of the canopy; 3 = canopy with desiccated branches uniformly distributed; 4 = severe tree decline). In one of the plot, laboratory testing by ELISA (Loconsole et al., 2014) were performed on single trees to confirm the presence of bacterial infection and the correlation with symptom severity scored on the trees. Site 1, located in the municipality of Ugento (39 55'2.49"N '6.13"E), was selected because of the low incidence of infected symptomatic plants (3 of 112) in November Almost all plants present in the plot are of cv. Ogliarola salentina. Site 2, located in the municipality of Alliste (39 55'8.86" - N 18 6'34.49"E) is a multivarietal (Nocellara, Carolea, Picholine, Gioconda) olive grove of 257 trees that were inspected in March, July and December Scores were assigned as previously reported EFSA Supporting publication 2016:EN-1013

18 4. Results 4.1. Artificial inoculations under controlled environmental conditions (greenhouse) Pathogenicity tests Olives: X. fastidiosa strain CoDiRO was successfully prick-inoculated into the olive stem. Multiplication and movement of occurred in plants of all cultivars inoculated, regardless of whether they were grafted (cv. Cellina di Nardò), self-rooted (cvs Leccino, Coratina and Frantoio), or seedlings. One month post-inoculation, the percentage of plants that tested positive in qpcr assays at the point of inoculations (leaf petiole next to the IP) ranged between 40 and 100% (Table 3) with the highest detection rate in the plants of cv. Cellina di Nardò and in the seedlings. Attempt to detect the bacterium at the inoculum points was repeated 3 mpi if the first test were negative. Time-course qpcr assays for monitoring the multiplication and movement of the bacterium from the inoculum points showed that the plants of cv. Cellina di Nardò and the seedlings were more rapidly colonized by the bacterium than the other cultivars (Fig. 6-9 at months). In almost all inoculated plants (9 out of 10) of cv. Cellina di Nardò at 9 mpi the bacterium was detected up to the 6 th internode above the inoculum points (ca. 18 cm). Whereas in the other cultivars the number of plants in which the bacterium had reached the same distance from the IP was as low as two for Coratina, three for Leccino and Frantoio, four for the seedlings. In the remaining plants of these cultivars the bacterium remained confined between the 1 st and the 5 th internode (ca cm above the IPs). Interestingly, 12 mpi when roots were also tested for the presence of X. fastidiosa, in all analyzed plants of cv. Cellina di Nardò (3 plants) and olive seedlings (5 plants) the bacterium was clearly detectable in the roots (Table 4). Conversely, in cvs Coratina, Leccino and Frantoio, the presence of the bacterium in the roots was not consistently detected in the analyzed plants whose canopy was systemically infected Systemic colonization of the olive plants was further confirmed 12 mpi by testing the leaves collected along the plant stem and from the shoot apex. The overall results collected at 12 mpi indicated that, regardless of the cultivar, the plants that at 9 mpi had shown detectable levels of bacterial concentrations between the 4 th and 6 th node above the IP, were entirely colonized by the bacterium up to the top, suggesting that in these plants X. fastidiosa has continuously multiplied and spread systemically. However, comparing the time course assays (at 3, 9 and 12 months), it is evident a different colonization rate in the different cultivars (Fig. 6-9), indicating that cv. Cellina di Nardò and olive seedlings are the most susceptible to bacterial colonization. In the remaining cultivars a few plants escaped infection (consistent negative results in the different sampling period) and in a few of them the bacterium was still localized at the inoculation point (Fig. 6-9). Bacterial quantification in systemically infected plants was achieved through qpcr assays in the leaves and stem portions. The results clearly showed significant differences in the concentration of the bacterium among the cultivars examined (Fig. 10), with an overall higher concentration in the plant of cv. Cellina di Nardò. Indeed, a clear distinctive feature was observed in cvs Cellina di Nardò, Coratina and in the olive seedlings, in which an higher concentration of the bacterium was detected in the stem than in the leaf petioles; in contrast with what was observed in cvs Leccino and Frantoio. The samples collected from the non-inoculated controls gave always negative results in qpcr assays and no symptoms have appeared in these non-inoculated controls (Fig. 11). Bacteria re-isolation was also successfully accomplished from plants cv. Cellina di Nardò, Leccino, Frantoio and from the olive seedling (Fig. 12). Colonies were recovered from all sections of the stems, with the highest number of colonies per spot recovered from the distal portions above the inoculum points. Conversely, isolations failed from the leaf petioles harvested from the same portions. These 18 EFSA Supporting publication 2016:EN-1013

19 results are in agreement with those obtained from field trees, from which the bacterium has successfully been cultured only from twigs (M. Saponari, unpublished information). Periodical visual inspections did not show any clear evidence of bacterial-related reactions up to 12 mpi, when initial symptoms of desiccation and dieback were first recorded in two of the inoculated plants of cv. Cellina di Nardò. During the following couple of months (13-14 mpi) these symptoms rapidly progressed and developed on the remaining plants of cv. Cellina di Nardò, with the exception of the plant ID2-1, which was the only plant of cv. Cellina in which, at month 9, the bacterium was still confined to the IP. At the same time symptoms began to appear on 2 and 3 plants of cvs Frantoio and Leccino, respectively. None of the plants of cv. Coratina showed symptoms up to 14 mpi. So far only one inoculated seedling displayed desiccation of apical part of the inoculated shoot. For the olive cultivars, a strict correlation was found amongst the presence of the systemic bacterial infection at 9 mpi and the appearance of symptoms at mpi (Fig. 6-9, Table 3), indicating that in these young plantlets symptoms expression occurred not earlier than 3-5 months after the inoculated shoots were colonized by the bacterium. The majority of the inoculated plants of Cellina di Nardo were found to be systemically infected at 9 mpi and most of them were symptomatic at month Conversely, the data recovered for the other cultivars showed that only 2-3 plants were systemically infected at 9 mpi and only 2-3 plants started then to show symptoms at mpi. The presence of systemic bacterial infections significantly affected the growth of inoculated plants (Fig. 13), with major impact in Cellina di Nardo where symptoms developed more rapidly than in the other cultivars. Symptoms in most of the symptomatic plants started from the apical portion of the inoculated shoots and progressed toward the base. The evolution of the symptoms was similar to what was observed in the field on infected olive trees: leaves are first yellow and show signs of wilting, then they turn rapidly brown and desiccate (Fig ). Generally, one month after the appearance of initial symptoms, desiccation progresses in the inoculated shoots from apex to the base. Ornamentals (Nerium oleander and Polygala myrtifolia): Mechanical inoculation of X. fastidiosa in both host resulted in a high rate of systemic infections and symptom development. In oleander, the bacterium was readily detected at the IP of all of the ten inoculated plants.subsequent qpcr assays showed that in eight of these plants the bacterium had spread rapidly colonizing the entire plant between 9 and 12 mpi. Mild stunting and delayed flowering was observed only on inoculated plants (Fig. 22) followed by marginal chlorotic patterns parallel to the main vein on some of the inoculated plants at 10 mpi and later on the rest of them (Fig. 22). These alterations are similar to those observed on naturally infected oleanders, where chlorotic and yellow leaf pattern develop prior to the appearance of the typical leaf necrosis (Fig. 23). In oleander, like in cv. Cellina di Nardò, the bacterium was found in the roots of the plants tested at 12 mpi. Currently all the 7 plants remained after the diagnostic tests show the above described symptoms. Likewise, systemic infections associated with symptoms were found in artificially inoculated plants of P. myrtifolia. In this species, leaf scorching appeared as soon as at 4-6 mpi (Fig ) in the medium-basal part of the plants. More severe leaf scorching and leaf dessication of the entire apical shoots were observed starting at 10 mpi, when all inoculated plants were infected and all strongly symptomatic (Fig. 25). X. fastidiosa was also detected in the roots of all plants. Re-isolation from leaf petioles and stems of symptomatic plants has been successfully obtained EFSA Supporting publication 2016:EN-1013

20 Table 3: Summary of the olive plants testing positive and showing symptoms upon the needle inoculation with Xylella fastidiosa strain CoDiRO. Cultivar Cellina Nardò di Number of plants yielding positive qpcr reactions at the inoculum points/total plants inoculated Plants infected up to the 5-6 th node above the inoculation point Months post inoculation Plants systemically infected Plants showing symptoms/total number of plants currently under observation /10 10/ /8 Coratina 4/10 4/ /7 Frantoio 5/10 6/ /7 Leccino 6/10 6/ /7 Seedlings 10/10 10/ /5 Table 4: Detection of Xylella fastidiosa by qpcr in the roots of the plants selected for each host/cultivar. Species and cultivars No. of plants in which X. fastidiosa was detected in the roots/no. of plants tested Olea europaea cv. Cellina di Nardò 3/3 Olea europaea cv. Coratina 0/3 Olea europaea cv. Frantoio 1/3 Olea europaea cv. Leccino 1/3 Olea europaea seedlings 4/5 Nerium oleander 3/3 Polygala myrtifolia 3/ EFSA Supporting publication 2016:EN-1013

21 Figure 6: Schematic representation of the differential bacterial movement in inoculated olive plants, at 3 months post-inoculation, based on qpcr assays. In red, the portions of the plants from which leaf tissues gave positive reactions in qpcr assays. Yellow arrows point to the areas site of inoculation (IP) with the number of plants testing positive. The number of plants testing positive above the IP is also reported on the corresponding distal nodes EFSA Supporting publication 2016:EN-1013

22 Figure 7: Schematic representation of bacterial colonization (red sections) in cv. Cellina di Nardò at 9 and 12 month post inoculation. The red color shows the portion of the plants from which leaf tissues gave positive reactions in qpcr assays. Numerical digits on the pots indicate the number of plants having the same level of bacterial colonization, over the total number of inoculated plants EFSA Supporting publication 2016:EN-1013

23 Figure 8: Schematic representation of the bacterial colonization (red sections) of cvs Coratina and Leccino at 9 and 12 month post inoculation. The red colour shows the portion of the plants from which leaf tissues gave positive reactions in qpcr assays. Numerical digits on the pots indicate the number of plant(s) with the same level of bacterial colonization, over the totality of inoculated plants (10). The cultivar is indicated in the text box below the pots EFSA Supporting publication 2016:EN-1013

24 Figure 9: Schematic representation of bacterial colonization (red sections) in cv. Frantoio and olive seedlings at 9 and 12 month post-inoculation. The red colour shows the portion of the plants from which the leaf tissues gave positive reactions in qpcr assays. Numerical digits on the pots indicate the number of plants with the same level of bacterial colonization, over the totality of the inoculated plants (10) EFSA Supporting publication 2016:EN-1013

25 Figure 10: Quantification of Xylella fastidiosa expressed in CFU/ml in systemically infected plants of different cultivars S= stem; L=leaf petioles 25 EFSA Supporting publication 2016:EN-1013

26 Figure 11: Non-inoculated controls for the olive cultivars used in the pathogenicity study. A. cv Cellina di Nardò; B. cv Coratina; C. cv. Frantoio; D. cv Leccino EFSA Supporting publication 2016:EN-1013

27 Figure 12: Fifteen-day-old Xylella fastidiosa colonies recovered from the stem portion above the point of inoculation of the artificially inoculated olive seedlings. Figure 13: Relationship between systemic bacterial infection and reduction of growth observed in the inoculated shoots of olive and oleander plants EFSA Supporting publication 2016:EN-1013

28 Figure 14: Cellina di Nardò. A. Plants at months post-inoculation (mpi), ID2-7=inoculated plant; Xf (-)= non-inoculated control. The red arrow indicates the inoculated and symptomatic shoot. B. The inoculated plant ID 2-7: the left hand panel shows initial chlorosis of the apical shoot 13 mpi. (January 8, 2016). The right hand panel show the same plant one month afterwards (February 10, 2016) EFSA Supporting publication 2016:EN-1013

29 Figure 15: Cellina di Nardò. A. Plants at months post-inoculation, ID2-10=inoculated plant; Xf (-)= non-inoculated control. B. The inoculated plant ID 2-10: the left hand panel shows chlorosis and wilting of the apical shoot (January 8, 2016); the right hand panel shows the same shoot in an advanced stage of desiccation (February 10, 2016) EFSA Supporting publication 2016:EN-1013

30 Figure 16: Cellina di Nardò. A. Plants at 14 months post-inoculation, ID2-5=inoculated plant; Xf (-) = non-inoculated control. B. The inoculated plant ID 2-5: the left hand panel shows a shoot with chlorosis and wilting of the apical shoot (January 8, 2016); the right hand panel shows the same plant, one month after the appearance of the symptoms (February 10, 2016) EFSA Supporting publication 2016:EN-1013

31 Figure 17: Leccino. A. Plants at 14 months post-inoculation, ID2-9=inoculated plant; Xf (-) = non-inoculated control. The red arrow indicates the inoculated and symptomatic shoot. B. The close-up on the symptomatic shoot, with mild chlorosis and wilting progressing over the time EFSA Supporting publication 2016:EN-1013

32 Figure 18: Leccino. A. Plants at 14 months post-inoculation, ID2-7=inoculated plant; Xf (-) = non-inoculated control. The red arrows indicate two symptomatic shoots. B. A closeup on the symptomatic shoots showing wilting and desiccation progressing over the time EFSA Supporting publication 2016:EN-1013

33 Figure 19: Frantoio. A. Plants at 14 months post-inoculation, ID2-8=inoculated plant; Xf (-) = non-inoculated control. The red arrow indicates the symptomatic shoot. B. A close-up on the symptomatic shoots showing wilting and desiccation progressing over the time EFSA Supporting publication 2016:EN-1013

34 Figure 20: Frantoio. A. Plants at 14 months post-inoculation, ID2-1=inoculated plant; Xf (-) = non-inoculated control. The red arrow indicates the desiccated inoculated shoot. B. A close-up on the desiccated shoot, the blue triangles indicate the three-inoculum points EFSA Supporting publication 2016:EN-1013

35 Figure 21: Olive seedlings. Plants at 14 months post inoculation. ID2-1=inoculated plant showing desiccation; Xf (-) = non-inoculated control EFSA Supporting publication 2016:EN-1013

36 Figure 22: Oleander. A. stunting and flowering delay on the inoculated plants (ID 2-4 and ID 2-2) 6 month post-inoculation; Xf (-) = non-inoculated control. B. Chlorotic leaf pattern and initial necrosis observed on the mature leaves after months post-inoculation EFSA Supporting publication 2016:EN-1013

37 Figure 23: Oleander naturally infected by Xylella fastidiosa A. Initial symptoms of marginal chlorosis; B. Leaves in an advanced stage of infection with marginal leaf necrosis. Figure 24: Inoculated plants of Polygala myrtifolia. A. Initial leaf scorching 5-6 months postinoculation; B. Heavily symptomatic plants 12 months post inoculation EFSA Supporting publication 2016:EN-1013

38 Figure 25: Plants of Polygala myrtifolia. A and B. Desiccated shoots 12 months post inoculation; C. Non-inoculated (control) symptomless plants 12 months post-inoculation EFSA Supporting publication 2016:EN-1013

39 4.1.2 Host range evaluation Citrus: For all citrus species used in the tests the percentage of plants that tested positive at the IP (leaf petiole next to the IP) 3 mpi, was comprised between 20 and 100%. At 3 mpi, the bacterium was also recovered from the node above ( cm) the IP in some plants. Further qpcr assays performed at 6, 9 and 12 mpi, showed that the bacterium could not be detected above the first 2-3 internodes, ca. 10 cm, from the inoculum point (Fig. 26). At 12 mpi, when 6 plants were entirely sectioned and subjected to isolation and DNA extraction for qpcr assays, the bacterium could not be re-isolated from the stem or leaf petiole (Table 5, Fig. 27). Similarly, most of the samples produced negative qpcr reactions either using xylem tissues recovered from the stems or the leaf petioles (Table 5). None of the citrus plants yielded positive reactions when the roots were tested. The overall results obtained on citrus suggest that initial multiplication of the bacterium may occur in the plants soon after the inoculation but also that the further colonization and systemic infection of the inoculated plants fails. Inoculated citrus plants did not display any leaf alteration resembling those typical of citrus variegated chlorosis (CVC). Six plants of each citrus species/cultivar were sectioned and used for isolation and qpcr assays at 12 mpi, whereas the four plants left over are kept in the greenhouse for further tests and observations (Fig. 28). Grapevine. Two set of self-rooted cuttings of cv. Cabernet sauvignon were inoculated in two independent experiments (10 plants for each experiment). The overall testing performed periodically using leaf petioles collected after needle inoculations in both set of experiments, showed that bacterial establishment at the inoculum point was limited as only 40% and 50% of the plants gave positive qpcr reactions. Subsequent tests performed collecting leaf petioles above the inoculation points were never positive. When 12 plants out of 20 were sectioned and tested, the overall results indicated that in 9 of these plants the bacterium could be detected only at the inoculation point. All the leaves and the stem portions above the IP consistently gave negative qpcr reactions (Table 5). Isolation from the stem (including the portions with the IP) and from the leaf petioles still available on the plants (mainly in the upper portion) failed to yield bacterial colonies in axenic culture (Fig. 27). None of the inoculated plants showed leaf reactions resembling those associated with Pierce s disease (Fig. 29) during the experiments EFSA Supporting publication 2016:EN-1013

40 Figure 26: Schematic representation of the differential bacterial movement in inoculated citrus plants, at 12 months post-inoculation (mpi), based on qpcr assays performed on the stems and leaf petioles of 6 sectioned plants for each genotype. In red, the portions of the plants which gave positive reactions in qpcr assays. Yellow arrows point to the areas site of inoculation (IP) with the number of plants testing positive 12 mpi. The number of plants testing positive above the IP is also reported on the corresponding distal nodes and the distance from the IP indicated in centimetres (cm) EFSA Supporting publication 2016:EN-1013

41 Figure 27: Fifteen days old BCYE plate imprinted with the stem portions of different inoculated plants 14 months post-inoculation. A. imprints from olive seedlings showing white colonies of Xylella fastidiosa strain CoDiRO, B. Imprints from citrus (Duncan grapefruit) inoculated plants; C. imprints from Cabernet Sauvignon plants. No colonies were observed in the imprints from citrus (B) and grape (C) EFSA Supporting publication 2016:EN-1013

42 Figure 28: Symptomless Citrus spp. plants 14 months post-inoculation. A B Figure 29: Symptomless Vitis vinifera cv. Cabernet Sauvignon, 6 (A) and 12 (B) months postinoculation EFSA Supporting publication 2016:EN-1013

43 Prunus spp. Experiments have been concluded for the hybrid GF677 from which a full panel of data has been produced; whereas due to the vegetative cycle (leaves were not available for testing in some period of the year) and to the delayed inoculation period, further data need to be collected from the other species included in the experiment. On the two sets of GF677 (19 inoculated plants), qpcr assays at 1-3 mpi showed that 6 and 9 out of 10 plants yielded positive reactions at the IP. qpcr assays, performed periodically up to 12 mpi using leaf petioles collected above the IP, failed to detect the bacterium, except for one plant in which a positive reactions was obtained from the first node (2-2.5 cm) above the IP. qpcr assays on sectioned plants confirmed that the bacterium could be detected only in the stem portion site of inoculation (Table 5). Isolation attempts from the stem that gave qpcr positive reactions failed to recover cultivable bacteria cells. For almond, the overall results of inoculation experiments confirmed the finding relative to naturally infected almond trees that had been analyzed. Specifically, systemic infections were detected in inoculated plants of cvs Genco and Tuono, both grafted on GF677. Indeed, at 12 mpi systemic infections were detected in the scion of all plants up to cm above the inoculum points. By contrast, negative results were obtained when rootstocks and roots were tested. No leaf scorching was observed during the vegetative period. However, the remaining plants (those not sacrificed for testing) will be maintained till October 2016 for symptom evaluation and bacterial isolation. The results on cherry are partial as they refer to detection of the bacterium at the inoculum point only. The grafted (on Prunus mahaleb) cherry plants had a reduced and stunted growth and thus it was not possible to collect samples from the distal portion(s) of the inoculated shoots. The plants were not sacrificed and are kept for symptom evaluation, qpcr assays and isolation to be performed in the coming vegetative season (April-October 2016). As to the other Prunus spp. used in the experiments, qpcr assays detected X. fastidiosa only at the inoculum point (1-3 month post inoculation) of few inoculated plants (Table 6). Yet, the bacterium was not detected in any of the shoot portions sampled above the inoculum points, nor in the rootstock or in the roots of any of inoculated plants. Quercus ilex. A total of 12 plants of Q. ilex were inoculated with the CoDiRO strain. When the leaf petioles adiacent to the inoculum points were tested at 1 and 3 months post inoculation, a total of 8 of 12 plants gave positive qpcr reactions at the IP. Following qpcr assays on the leaves above the inoculum points gave consistently negative reactions for all the 12 plants, at 3, 6, 9 and 12 months post inoculation. qpcr assays performed on three sectioned plants, produced negative results when the total DNA recovered from the stem portions, leaves and roots were used to recover the DNA template for amplification. So far none of the inoculated plants has shown any sign of leaf scorching (Fig. 30). Further tests and isolation attempts will be performed in the next few months on the remaining plants EFSA Supporting publication 2016:EN-1013

44 Table 5: Plants yielding positive reaction in qpcr assay at 3 and 12 months post inoculation for citrus, grapes, Quercus ilex and GF months 12 months Species/cultivar Number of plants yielding positive qpcr reactions at the inoculation points Number of plants yielding positive qpcr reactions at the node above the inoculation point Number of plants with positive qpcr reactions (details about the positive samples are reported in parenthesis) Citrus spp. Madam Vinous 9/10 3/10 1 (stem at IP)/6 Duncan grapefruit 9/10 2/10 1 (stem and leaves at IP.), 4* (10 cm above IP)/6 Carrizo 9/10 5/10 2 (stem at IP)/6 Citrange troyer 10/10 4/10 4 (stem at IP)/6 C35 2/10 1/10 Mandarin 7/10 n. d. 2 (stem at IP), 1 (10 cm above IP.)/6 4 (stem at IP), 1 (10 cm above IP)/6 Vitis vinifera Cabernet sauvignon 8/19 0/19 9 (stem at IP)/19 Quercus ilex 8/12 0/12 0/3 Prunus amygdalus x P. persica (GF677) 10/19 1/19 5 (stem at IP)/6 1 (10 cm above IP)/6 *For 3 of the 4 plants, positive qpcr reactions were obtained only on the leaf petioles whereas the corresponding stems gave negative results. Table 6: Percentage of plants that gave positive reaction in qpcr assays from leaves collected at the inoculation points Stone fruit species/cultivars Number of plants yielding positive qpcr reactions at the inoculation points/total plants inoculated Prunus amygdalus x Prunus persica (GF677) 14/17 Prunus avium cv. Bigarreau Moreau 2/6 Prunus avium cv. Ferrovia 5/8 Prunus persica cv. Baby Gold 6 2/3 Prunus domestica cv. President 4/8 Prunus salicina cv. Santa Rosa 3/8 Prunus amygdalus cv. Genco 7/ EFSA Supporting publication 2016:EN-1013

45 Figure 30: Inoculated plants of Quercus ilex at 14 months post inoculation EFSA Supporting publication 2016:EN-1013

46 4.1.3 Control plants Systemic infections were recorded at one mpi in all periwinkle plants used in each inoculation experiment, although no symptoms were observed. Similar results were obtained with control tobacco plants, all of which tested positive at the IP, as well as in the roots of the plants sectioned for testing. Specifically, 6 mpi the plants were cut in pieces and the main stem and axillary shoots were tested. The results showed that only the apical part (5 cm) of each plant was qpcr-negative, whereas the rest of the stem was colonized. Systemic infections were detected five to six months post-inoculation, when foliar alterations resembling leaf scorching were also recorded (Fig. 31) in some of the infected plants. Figure 31: Inoculated Nicotiana tabacum SR1 plants with leaf scorching. (A) Healthy control (left) inoculated plant (right). (B) A close-up of a symptomatic leaf EFSA Supporting publication 2016:EN-1013

47 4.2. Artificial inoculations in a screenhouse without temperature control Needle inoculations of olives, oleander and P. myrtifolia were performed in October 2014 and the plants maintained under natural temperature conditions for the entire period of the experiment. Thus, the inoculated plants were exposed to the low (winter) and high (summer) temperature which occurred in the period covered by the experiments (October ). Olives. qpcr results obtained from the samples collected soon after inoculation (1-3 months) from the IP and from the node immediately above it, were similar to those obtained from the panel of plants kept under controlled conditions, ranging from 50 to 70% with the highest detection rate in cv. Cellina di Nardò. However, when the results on bacterial colonization of the plants maintained in the two experimental conditions (controlled and uncontrolled temperature) were compared, the effect of the different environmental conditions was clear-cut. Bacterial colonization of olive plants exposed to winter and summer temperature was significantly lower, likely because of reduced bacterial multiplication under unfavourable temperature conditions. For example, for cv. Cellina di Nardò, only three of the seven plants that tested positive at the IP were systemically infected at 12 mpi. Moreover, in none of these plants the bacterium could be detected in the apical portion of the shoots (Fig. 32) and in the roots. This situation was even more evident with the remaining olive cultivars and seedlings, because the results of the analysis of the different parts of the plants, showed that the bacterium was confined to the inoculum points or was erratically distributed (i.e. for the olive seedlings and cv. Leccino) or, as it occurred in cvs Frantoio and Coratina, was undetected in the samples collected from the entire plants. Visual inspections revealed a few plants (4 of 10) of cv. Cellina di Nardò with symptoms of leaf scorching that appeared in early spring (end of March) (Fig. 33), but did not progress over the season and remained confined to a few leaves, while the new vegetation that developed in the following months was symptomless. None of these plants displayed wilting or desiccated shoots such as those observed in the experiments conducted in Bari under controlled conditions. Quantification assays, although referring to a very limited number of positive samples (Fig. 34), showed that the bacterial concentration in these plants was lower than the concentration obtained in the experiment carried out under controlled conditions. All together, these observations suggest that the low and high temperatures occurring in the area where the experiment was performed significantly affected the progression of the infections and the bacterial colonization of the artificially inoculated young plants. The slow rate of bacterial colonization was associated with the lack of symptom and with no reduction in plant growth (Fig. 34). A similar infection pattern was obtained on oleanders and P. myrtifolia (data not shown), for both hosts no symptoms were so far observed under the screenhouse conditions EFSA Supporting publication 2016:EN-1013

48 Figure 32: Comparison of the bacterial presence in the olive plants of cv. Cellina di Nardò. GH indicates the plants maintained in glasshouse under controlled temperature; SH indicates the plants maintained in the screen-house without control of the temperature. Bacterial colonization is indicated with red sections. Numerical digits on the pot indicate the inoculated plants in which the bacterium reached the shown level of host colonization. Figure 33: Inoculated plants of the cultivar Cellina di Nardò showing symptoms resembling those of bacterial leaf scorching 48 EFSA Supporting publication 2016:EN-1013

49 CFU/ml Height (cm) Host range and pathogenicity of the CoDiRO strain A B Figure 34: A. Height (expressed in cm) of cv. Cellina di Nardò plants grown in a glasshouse (GH) or screenhouse (SH), infected systemically by Xylella fastidiosa. B. Quantification of Xylella fastidiosa (CFU/ml) in the plants maintained in different environmental conditions EFSA Supporting publication 2016:EN-1013

50 4.3. Exposure of different plant species to natural infective P. spumarius in the field The first run of diagnostic tests on the young plants of the experimental plot established in April 2015 in the infected area, and caged in July 2015 with infective P. spumarius, confirmed that the natural spread infectivity of local vector populations. The bacterium was detectable as soon as 6 months after the exposure to infective vectors, while the plants were still symptomless. The data of Table 7 indicate that a different transmission rate occurred on the different olive cultivars (16-83%). Conversely, none of the citrus and grapevine plants tested positive in qpcr assays. Transmission occurred also in plants of oleander and P. myrtifolia exposed to the natural inoculum (without caging infective P. spumarius). The high percentage of P. myrtifolia plants that were infected, shows that other than being susceptible to the CoDiRO strain, this plant is also an excellent host for the vector. No transmission by vector has occurred so far on Q. ilex. Whereas, for Prunus spp. the test perfomed on the dormant plants showed only one cherry plant with a detectable level of the bacterium. Further observations will be carried out periodically to monitor the incidence and the progression of the infections and, most importantly, to monitor the development of X. fastidiosa-associated symptoms on the different host plants. The experimental plot will be maintained for additional 5-10 years depending on the progression of the infections and symptoms development. Table 7: Species/Cultivar Incidence of Xylella fastidiosa infections in the experimental plot located in the infected area. Number of plants tested Olive Number of qpcr positive plants % of infected plants Cellina di Nardò Cima di Melfi Frantoio Arbequina Koroneki Leccino Arbosana Coratina Citrus Navelina Grapevine Negramaro Chardonnay Italia Primitivo Prunus spp. Prunus dulcis Prunus avium Other species* Quercus ilex Nerium oleander Polygala myrtifolia * The plants of these species were not caged with infective specimens of P. spumarius EFSA Supporting publication 2016:EN-1013

51 4.4. Field surveys Surveys conducted in the olive grove identified as Site 1 included 112 trees. Table 8 provides the data from the visual inspections conducted in November 2013, April 2015 and July In July 2015 the plants were also sampled and tested by ELISA for X. fastidiosa (Loconsole et al., 2014). The data show that from the initial 3 symptomatic plants (2.7% of the total trees) the occurence of the disease increased rapidly over the 2-year period, affecting 59 trees (52.7%) within 20 months, with a substantial increase of symptomatic trees from April to July A positive correlation was found among the trees showing mild and severe symptoms (score 2-4) and the presence of X. fastidiosa infections (Table 8). As shown in Figure 35 the distribution of the infected trees in the plot does not follow any specific pattern and does not appear to be correlated to the first three symptomatic trees present in the plot. A similar pattern of symptoms progression was recorded in the second selected plot ( Site 2 ) (Table 9, Fig. 36). In this case, the plants with symptoms increased from 1 to 66 (July 2015), reaching 220 in December Serological and molecular tests are underway to monitor the progression of the infected plants and its impact on the different olive cultivars present in this plot. Table 8: Results of the survey conducted in the plot Site 1. Trees are categorized based on the presence and severity of desiccation and dieback symptoms (score 0-4). The percentage of the infected plants is the results of the ELISA tests performed in July Class of symptoms (score 0-4) November 2013 April 2015 July 2015 No. of trees No. of trees No. of trees % of infected trees for each class of symptoms Tot Table 9: Class of symptoms (score 0-4) Results of the survey conducted in the plot Site 2. Trees are categorized based on the presence and severity of desiccation and dieback symptoms (score 0-4). March 2015 July 2015 December 2015 No. of tree(s) No. of trees No. of trees Tot EFSA Supporting publication 2016:EN-1013

52 dec Figure 35: Map of the plot Site 1, yellow squares indicate the position of the olive cv. Ogliarola salentina; in green, trees of cv. Leccino; in purple, trees of cv. Nocellara. The trees circled in green are those showing symptoms in November 2013; those circled in blue were identified in April 2015; and those in orange were identified in July The numbers indicate the symptom score; those in red correspond to ELISA-positive trees; whereas in those in black indicate ELISA- negative trees. Figure 36: Map of the plot Site 2, each square indicates the position of the olive trees, whose cultivar is shown on the right panel of the map. The numbers indicate the symptom score. The tree circled in yellow isthe first symptomatic plant observed in March Symptomatic trees found in July 2015 are circled in red and those marked in blue were identified in December EFSA Supporting publication 2016:EN-1013