Xylella fastidiosa Biologia i epidemiologia

Emili Montesinos Seguí Catedràtic de Producció Vegetal (Patologia Vegetal) Universitat de Girona [email protected] www.youtube.com/watch?v=sur5VzJslcM

Xylella fastidiosa, un patogen que no és nou

Newton B. Pierce (1890s, USA) Agrobacterium tumefaciens Chlamydiae Bartonella bacilliformis Campylobacter coli  Bartonella henselae  CDC Chlamydophila psittaci Campylobacter fetus  Bartonella quintana Bacteroides fragilis CDC Brucella melitensis Bacteroidetes Chlamydophila pneumoniae Campylobacter hyointestinalis  Bacteroides thetaiotaomicron  Campylobacter jejuni CDC Brucella melitensis biovar Abortus CDC Chlamydia trachomatis   Capnocytophaga canimorus  Campylobacter lari  CDC Brucella melitensis biovar Canis  Chryseobacterium meningosepticum  Parachlamydia acanthamoebae Campylobacter upsaliensis  CDC Brucella melitensis biovar Suis  Helicobacter pylori Candidatus Liberibacter africanus CDC Candidatus Liberibacter asiaticus  Borrelia burgdorferi Epsilon Borrelia hermsii  CDC Anaplasma phagocytophilum Borrelia recurrentis Alpha CDC Ehrlichia canis Spirochetes Borrelia turicatae CDC Ehrlichia chaffeensis Eikenella corrodens Leptospira interrogans CDC Ehrlichia ewingii CDC CDC Neisseria gonorrhoeae Treponema pallidum Ehrlichia ruminantium CDC  Neisseria meningitidis CDC Neorickettsia sennetsu Spirillum minus Orientia tsutsugamushi Fusobacterium necrophorum Beta CDC Bordetella pertussis Rickettsia conorii Streptobacillus moniliformis    Burkholderia cepacia Rickettsia felis Burkholderia mallei Rickettsia prowazekii CDC CDC Rickettsia rickettsii Staphylococcus aureus  Burkholderia pseudomallei Gamma Enterotoxin A  Ralstonia mannitolilytica  Rickettsia typhi Enterococcus faecalis Ralstonia pickettii Enterotoxin B  CDC Francisella tularensis Enterococcus faecium Ralstonia solanacearum   Staphylococcus, coagulase-negative Tatlockia micdadei Enterococcus flavescens Staphylococcus epidermidis  Legionella longbeachae Stentrophomonas maltophila acidominimus Staphylococcus haemolyticus CDC Legionella pneumophila Xanthomonas campestris pv. malvacearum Streptococcus agalactiae Staphylococcus intermedius  CDC Xanthomonas oryzae pv. oryzae Streptococcus bovis Staphylococcus saprophyticus Coxiella burnetti Streptococcus canis Xanthomonas oryzae pv. oryzicola ivanovii Streptococcus criceti Acinetobacter baumannii syringae pv. glycinea Xylella fastidiosa CDC Listeria monocytogenes  Streptococcus equi Acinetobacter lwoffii Alteromonas tetraodonis Listeria seeligeri  Streptococcus intermedius Acinetobacter radioresistans Pseudomonas alcaligenes Tetrodotoxin Listeria welshimeri  Streptococcus milleri Moraxella catarrhalis Pseudomonas fluorescens CDC CDC CDC Acinetobacter calcoaceticus Haemophilus influenzae Streptococcus pneumoniae anthracis  Pseudomonas putida Aeromonas sp.  CDC Streptococcus pyogenes Acinetobacter calcoaceticus Pseudomonas stutzeri Haemophilus aegyptius  Streptococcus suis  subsp. Anitratus CDC Haemophilus ducreyi Streptococcus uberis Bacillus subtilis Haemophilus parahaemolyticus Streptococcus viridans CDC Vibrio cholerae Haemophilus parainfluenzae Cholera Toxin Haemophilus paraphrophilus Mycoplasma pneumoniae Vibrio mimicus Pasteurella multocida Erysipelothrix rhusiopathiae Vibrio parahaemolyticus Mycoplasma mycoides Actinobacteria  Mollicutes Vibrio vulnificus Mycoplasma capricolum CDC Mycobacterium tuberculosis  CDC subsp. Capripneumoniae   Mycobacterium abscessus Shiga Toxin  Rhodococcus equi Mycobacterium avium  Salmonella bongori  Actinomyces israelii Citrobacter freundii Salmonella choleraesuis Anaerococcus prevotii Mycobacterium bovis Actinomyces naeslundii  CDC Salmonella enteriditis CDC Clostridium botulinum Mycobacterium fortuitum Propionibacterium acnes Enterobacter aerogenes   Salmonella paratyphi Botulinum toxin Mycobacterium kansasii Rothia dentocariosa Enterobacter cloacae  CDC Salmonella typhimurium Clostridium difficile CDC Mycobacterium leprae Clavibacter michiganensis Klebsiella oxytoca   Klebsiella pneumoniae CDC Salmonella typhi Clostridium perfringens Mycobacterium haemophilum subsp. Sepedonicus  CDC   Nocardia asteroides Morganella morganii Shigella boydii Epsilon toxin Mycobacterium marinum CDC  Nocardia nova Proteus mirabilis Shigella dysenteriae Clostridium septicum Mycobacterium scrofulaceum  CDC Shigella flexneri CDC Corynebacterium amycolatum Proteus vulgaris Clostridium tetani Mycobacterium simiae  CDC Providencia stuartii Shigella sonnei Peptococcus niger Corynebacterium jeikeium Mycobacterium szulgai Serratia marcescens Yersinia enterocolitica anaerobius Corynebacterium xerosis Mycobacterium ulcerans  Yersinia pseudotuberculosis  CDC Corynebacterium diphtheriae Veillonella dispar Mycobacterium xenopi  CDC Yersinia pestis  Diphtheria toxin

Ecker et al. BMC Microbiology 2005 5:19 doi:10.1186/1471-2180-5-19 MALALTIES CAUSADES PER Xylella fastidiosa

Malaltia de Pierce (vid) Clorosi variegada dels citrics

http://ucce.ucdavis.edu/files/repository/ calag/img6801p20.jpg http://www.ivia.es/iocv/enfermedades/

Mort sobtada de les oliveres Asurat del baladre (Nereum oleander)

http://www.eppo.int/QUARANTINE/special_topics/ http://orange.ifas.ufl.edu/cfnurseries/pdffiles/ Xylella_fastidiosa/Xylella_fastidiosa.htm plantfacts/Oleander.pdf ULTRAESTRUCTURA

TEM http://www.apsnet.org/edcenter/intropp/lessons/prokaryotes/Pages/ BacterialLeafScorch.aspx

Colonització dels vasos del xilema

http://www.padil.gov.au/pests-and-diseases/pest/main/136652/3760 CULTIU

Medi PCYE

1.0g KH2PO4

1.1g K HPO 2 4

0.25g FeSO4.7H2O

0.4g Mg SO4.7H2O

2.0 g carbó activat 10.0 g extracte de llevat, 1 L aigua destil.lada 8.0 g Gelrite o 10.0 g agar-agar. pH=6.9 con 1 M KOH Autoclavar Colònies en Agar (1-3 mm, 15-30 dies) Lopes y Torres. Current Microbiol. Vol. 53 (2006), pp. 467–469 http://www.apsnet.org/edcenter/intropp/ lessons/prokaryotes/Pages/ BacterialLeafScorch.aspx DIVERSITAT subsp. fastidiosa (xff): causa la malaltia de Pierce en vid en la UE i asurat de la fulla de l’ametller (almond leaf scorch). subsp. multiplex (xfm): produeix asurat (leaf scorch diseases) en una gran nombre de arbres i és endèmica de Nord Amèrica. Afecta a fruiters de pinyol, Quercus i pecans. subsp. pauca (xfp): causa la clorosi variegada dels cítrics (Brasil), Asurat de la fulla del cafeto a Sud Amèrica, del baladre a Costa Rica. subsp. sandyi (xfs): responsable del asurat de fulles del baladre a California y Texas (USA). També afecta a magnolies i xicrandes. Plantes hoste de Xylella fastidiosa

Subespècie Hoste Gènere Espècie fastidiosa 42 138 164 multiplex 28 69 84 pauca 16 30 36 sandyi 5 6 5

Font: EFSA Journal 2015;13(1):3989. Xylella fastidiosa pest risk assessment ALTRES PLANTES HOSTE DE X. fastidiosa

http://www.cnr.berkeley.edu/xylella/index.html I MOLTS MÉS … GENOMA CARÁCTERÍSTIQUES DEL GENOMA DE X. fastidiosa

Soques seqüenciades:

9a5c (taronger, 1992) Ann-1 (baladre) Temecula-1 (vid, 1998) Dixon (ametller) 2.390-2.730 Kb 1600 gens (varis plàsmids)

• absència de gens d’avirulència (avr) • toxines (hemolisines, bacteriocines, poliquètids…) • exomucopolisacàrids (xantans!). Agregació, taponament. • fímbries (adhesió, biofilm) • sistema secretor Tipus II (enzims) • betaglucanases (PG, CEL). Migració xilema • proteïnes de transport (ions, aa, ch, etc.). Nutrició • metabolisme oxidatiu (glucòlisi, Krebs, ETS) AÏLLAT D’APULIA (ITÀLIA) EN OLIVERES

crossmark

Draft Genome Sequence of the Xylella fastidiosa CoDiRO Strain

Annalisa Giampetruzzi,a Michela Chiumenti,a Maria Saponari,a Giacinto Donvito,b Alessandro Italiano,b Giuliana Loconsole,a Donato Boscia,a Corrado Cariddi,c Giovanni Paolo Martelli,c Pasquale Saldarellia Institute for Sustainable Plant Protection, National Research Council (CNR), Bari, Italya; Department of Physics, University of Bari Aldo Moro, Bari, Italyb; Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italyc A.G. and M.C. contributed equally to this work.

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.

Received 22 December 2014 Accepted 5 January 2015 Published 12 February 2015 Citation Giampetruzzi A, Chiumenti M, Saponari M, Donvito G, Italiano A, Loconsole G, Boscia D, Cariddi C, Martelli GP, Saldarelli P. 2015. Draft genome sequence of the Xylella fastidiosa CoDiRO strain. Genome Announc 3(1):e01538-14. doi: 10.1128/genomeA.01538-14. Copyright © 2015 Giampetruzzi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Annalisa Giampetruzzi, [email protected].

ylella fastidiosa is a xylem-restricted Gram-negative bacterium resulting in the identification of 6 rRNA genes, 49 tRNA loci, 2,053 Xand the agent of diseases of a wide range of hosts (1). Currently, protein-encoding genes, and 2 noncoding RNAs. A plasmid of Genomaseven X. fastidiosa genomes: 2.508 have been completelyKb (+ sequenced, pXf35.3), in- 35,318 bp was 2053 also found sharing proteines 98% similarity in the tra and trb cluding citrus-variegated chlorosis strain 9a5c, Pierce’s disease loci with the conjugative plasmid pXF-RIV5 (10), but differing strains Temecula 1 and GB514, almond leaf scorch strains M12 from it for the accessory module containing genes of a toxin- and M23, oleander strain Ann1, and mulberry strain MUL0034. antitoxin system. In 2013, the first confirmed outbreak of X. fastidiosa in the Comparative analyses showed that (i) variations (insertion/ European Union was reported from southern Italy (Apulia), the deletion of nucleotide) exist in genes encoding important viru- infection occurring on olive trees affected by olive quick decline lence factors (rpf cluster gene, polygalacturonase-pglA gene) that syndrome (OQDS) and characterized by extensive scorching and are likely to be involved with recognition of specific host factors, Similaritatdesiccation of leaves and: twigssubsp. (2). Investigations pauca were therefore ambwhich aillat may influence de host Centre specificity, infectivity, Amèrica, and/or develop- initiated to determine the taxonomic allocation, host range, ment of virulence capacity; and (ii) CoDiRO is genetically related peròand vector(s)no amb of the bacterial Xf strainaïllada associated withd’olivera OQDS, with X. fastidiosaen California.subsp. pauca isolates with the highest similarity which was denoted CoDiRO, the abbreviation of the Italian with an isolate from Central America but not with the X. fastidiosa name of the disease. strain infecting olives in California (11). Genomic DNA was recovered from a pure X. fastidiosa CoD- Nucleotide sequence accession numbers. This whole-genome iRO culture from infected periwinkle. A paired-end DNA library shotgun project has been deposited at DDBJ/EMBL/GenBank un- was constructed and sequenced using Illumina technology, which der the accession number JUJW00000000. The version described resulted in 9,008,814 reads and 345ϫ coverage. in this paper is version JUJW01000000. Reads were assembled de novo using EDENA, Velvet, and SOAPdenovo (3–5) with different k-mers. The best contig assem- ACKNOWLEDGMENTS blies from each program were merged using CISA (6) and scaf- This work was supported by grants from the Regional Plant Health Service folded with SSPACE (7) on the Orione instance of Galaxy (8). This of Apulia and from CNR CISIA-“Valorizzazione delle risorse genetiche di reconstruction resulted in a final assembly of 12 scaffolds with colture mediterranee attraverso approcci di metagenomica, tracrittomica sizes ranging from 1,790 to 678,618 bp and an average scaffold size e analisi funzionale per la caratterizzazione di germoplasma autoctono, of 211,911 bp. Scaffolds were ordered on the backbone of the endofiti, agenti di biocontrollo e fitopatogeni (METARGERM)” and was reference genome sequence of X. fastidiosa subsp. pauca 9a5c, conducted using equipment of “Rete di Laboratori Pubblici SELGE- Re- gione Puglia (cod. 14).” which, according to multilocus sequence typing, was the most related strain. Where necessary, PCR and Sanger sequencing were REFERENCES performed by primer walking to fix the position of contigs show- 1. Hopkins DL, Purcell AH. 2002. Xylella fastidiosa: cause of Pierce’s disease ing conflicting ordering information. of grapevine and other emergent diseases. Plant Dis 86:1056–1066. http:// The draft genome of X. fastidiosa CoDiRO consisted of a total dx.doi.org/10.1094/PDIS.2002.86.10.1056. of 2,507,614 bp (with a GC content of 51.8%), likely representing 2. Saponari M, Boscia D, Nigro F, Martelli GP. 2013. Identification of Ͼ95% of the full genomic sequence since other X. fastidiosa DNA sequences related to Xylella fastidiosa in oleander, almond and olive trees exhibiting leaf scorch symptoms in Apulia (Southern Italy). J Plant strains have genomes ranging from 2.39 to 2.73 Mbp (9). Pathol 95:659–668. http://dx.doi.org/10.4454/JPP.V95I3.035. The draft genome was annotated through submission to the NCBI 3. Hernandez D, François P, Farinelli L, Osterås M, Schrenzel J. 2008. De Prokaryotic Genome Automatic Annotation Pipeline (PGAAP), novo bacterial genome sequencing: millions of very short reads assembled

January/February 2015 Volume 3 Issue 1 e01538-14 Genome Announcements genomea.asm.org 1 UNA NOVA SUBESPÈCIE?

Arbre filogenètic de la soca CoDiRO segons la tècnica MLST amb seqüencies de set genes “housekeeping” (Saponari, M. CNR,Bari, Itàlia) Font: EFSA Journal 2015;13(1):3989. Xylella fastidiosa pest risk assessment DISTRIBUCIÓ MUNDIAL (Font EPPO/EFSA) DETECCIÓ

Mètodes independents del culu Mètodes basats en el culu

Basats en àcids nucleics

Medis selecus

DNA/RNA

Culu en Agar PD2/ PW/CS20 Immunodetecció rtPCR

(serologia) PCR LAMP

ELISA Xylella fastidiosa pest risk assessment

documented (Redak et al., 2004). Most of the vector species spread in subtropical and tropical ecosystems and therefore develop and breed throughout the year. However, some North American species, e.g. Draeculacephala minerva, GraphocephalaDISPERSIÓ atropunctata, Xyphon fulgida and Homalodisca vitripennis, are known to overwinter as adult (http://www.cnr.berkeley.edu/xylella/insectVector/insectVector.html) and therefore X. fastidiosa can survive uthe winter A curta in the vector, distància as well as in thees infected transmet plants. per insectes vectors, principalment cicadèlids i The only X. fastidiosa vector species with a record of invasive potential is H. vitripennis. Originally from the southcercòpids,-west of the USA, hemípters H. vitripennis was xucladors first detected in quesouthern s’alimenten California in the late de xilema. 1980s, leading to an epidemic of Pierce’s disease in the late 1990s and early 2000s (Hopkins and Purcell, 2002).Subordre Very large populations Auchenorryncha of H. vitripennis have, been de reported, les up to families two millions per Aphrophoridae, Cercopidae, hectare (Coviella et al., 2006). After its introduction into California, H. vitripennis also moved to the archipelagosCicadidae of French Polynesia i Cicadellidae. and Hawaii where it wasEl reported més to reach eficient high populations és Homalodisca vitripennis. La (Grandgirard et al., 2006). In these two latter cases, it was suggested that the was introduced togethertransmissió with plant shipments. de Biological la control proved pel to bevector successful no in controlling requereix H. vitripennis periode de latència. in both French Polynesia and Hawaii (Grandgirard et al., 2008, 2009). It is not known why only H. vitripennis, among all the other vector species endemic to the Americas, is invasive. The widespread distributionu of A H. llarga vitripennis distància in tropical reg ionsés asel wellmaterial as the US vegetal Gulf and south contaminat.-west regions suggests that European regions with mild temperate climates, such as those in the Mediterranean, are at risk of colonisation by this insect, as previously suggested (Hoddle, 2004).

Table 3: Vectors of X. fastidiosa in the Americas: main insect groups and most important vector species

Insect group Most important Distribution Role as Role as vector: criteria species vector

Sharpshooters Bucephalogonia Neotropical: Argentina, High in Common, abundant on (Cicadellidae, xanthophis (Berg) Bolivia, Brazil, citrus ornamental plants, citrus ): 38 Paraguay and nursery stocks spp. Dilobopterus Neotropical: Brazil High in Common, abundant on costalimai Young citrus ornamental plants and citrus Graphocephala USA and Central High in Common in diverse atropunctata America grapevine ecosystems, on grapevine (Signoret) and ornamental plants Homalodisca USA (southern states), High in Common and abundant in vitripennis Mexico (northern part), grapevine diverse ecosystems, on (Germar) French Polynesia, grape, ornamentals, citrus Easter Island and nursery stock

Spittlebugs Philaenus USA Including Hawaii, Low Not associated with disease (Cercopoidea): spumarius L. Mexico, Tahiti epidemics http://agronotizie.imagelinenetwork.com/difesa-e-diserbo/ six species 2013/12/18/la-sindrome-del-disseccamento-rapido-degli- Cicadas Diceroprocta Mexico, Arizona, Utah, Doubtful Missing information on ulivi-del-salento/36014 (Cicadoidea): two apache Davis Nevada, California transmission capacity species Dorisiana viridis (Olivier)

3.1.4.3. Potential European vectors of X. fastidiosa FollowingFont: EFSA Frazier Journal (1944) and 2015;13(1):3989. Purcell (1989), all Xylellathe xylem fastidiosa fluid feeders pest should risk beassessment considered to be potential vectors. With the exception of Philaenus spumarius (Aphrophoridae), an Old World species introduced in North America and identified as a vector of X. fastidiosa in California (Purcell, 1980), all the American vector species are absent from Europe according to the Fauna Europaea database (de Jong, 2013). X. fastidiosa has never previously established in Europe and, in the case of the current

EFSA Journal 2015;13(1):3989 31 EFSA Journal 2015;13(1):3989

SCIENTIFIC OPINION

Scientific Opinion on the risk to plant health posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk reduction options1

EFSA Panel on Plant Health (PLH)2,3

European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT The EFSA Panel on Plant Health conducted a pest risk assessment and an evaluation of risk reduction options for Xylella fastidiosa. X. fastidiosa has been detected in olive in the EU with a distribution restricted to the regionEUROPEAN of COMMISSION Apulia in Italy and is under official control. X. fastidiosa has a very broad host range, including many commonDIRECTORATE -GENERAL FOR RESEARCH & INNOVATION cultivated and wild plants. All xylem fluid-feeding in Europe are considered to be potential vectors. Philaenus spumarius (: Aphrophoridae), a polyphagous spittlebug widespread in the whole riskDirectorate F - Bioeconomy assessment area, has been identified as a vector in Apulia. The probability of entry of X. fastidiosa fromF.3 - Agri-Food Chain countries where X. fastidiosa is reported is very high with plants for planting and moderate with infectious insect vectors carried with plant commodities or travelling as stowaways. Establishment and spread in the EU is very likely. The consequences are considered to be major because yield losses and other damage would be high and require costly control measures. The systematic use of insecticides for vector controlCONFERENCE may create environmental "Health Checks and Smart Treatments for Our Plants" impacts. With regard to risk reduction options, strategies for the prevention of introduction and for the containment of outbreaks should focus on the two main pathways (plants for planting and infectious insect vectors) and combine the most effective options in an integrated approach. For plants forand planting, WORKSHOP these could be "Xylella fastidiosa: Options for its control" pest-free production areas, surveillance, certification, screened greenhouse production, vector control and testing for infection and, for some plant species, treatments (e.g. thermotherapy). To prevent entry of the infectious vectors, insecticide treatments and inspection of consignments and production sites are required. The Panel15th has July 2015 EXPO Milan - EU PAVILION also reviewed the effectiveness of risk reduction options for X. fastidiosa and its vectors listed in Directive 2000/29/EC and in the EU emergency measures. The Panel recommends the continuation and intensification of research on the host range, epidemiology and control of the Apulian outbreak. Xyllela Workshop

© European Food Safety Authority, 2015 DRAFT AGENDA

EXPO site opens its doors at 10:00

1 On request from the European Commission, Question No EFSA-Q-2013-00891,10:30 adopted - by11:00 written procedureRegistration on 30 /Welcome coffee December 2014. 2 Panel members: Richard Baker, Claude Bragard, David Caffier, Thierry Candresse, Gianni Gilioli, Jean-Claude Grégoire, Imre Holb, Michael John Jeger, Olia Evtimova Karadjova, Christer Magnusson, David Makowski, Charles Manceau, Maria Navajas, Trond Rafoss, Vittorio Rossi, Jan Schans, Gritta Schrader, Gregor Urek, Irene Vloutoglou, Stephan Winter and Wopke van der Werf. Correspondence: [email protected] 11:00 – 11:10 Introduction by Moderator 3 The Panel wishes to thank the members of the Working Group on Xylella fastidiosa: Rodrigo Almeida, Domenico AnnetteBosco, Schneegans, European Commission, DG AGRI, Unit H5 – Claude Bragard, David Caffier, Jean-Claude Grégoire and Stephen Parnell for the preparatory work on this scientific opinion; and the hearing experts Maria Saponari and Donato Boscia, the EFSA staff Gabor Hollo, Ewelina CzwienczekResearch, and Innovation Olaf Mosbach Schulz and Giuseppe Stancanelli and the JRC staff Daniele De Rigo and Giovanni Strona for the support provided to this scientific opinion. 11:00 – 11:20 Title Suggested citation: EFSA PLH Panel (EFSA Panel on Plant Health), 2015. Scientific Opinion on the risks to plantRodrigo health Almeida, University of Berkeley, California, USA posed by Xylella fastidiosa in the EU territory, with the identification and evaluation of risk reduction options. EFSA Journal 2015;13(1):3989, 262 pp., doi:10.2903/j.efsa.2015.3989 11:20 – 11:40 Epidemiology and Management of Pierce’s Disease in California, Available online: www.efsa.europa.eu/efsajournal USA © European Food Safety Authority, 2015 Rodrigo Krugner, Agricultural Research Service, USDA, California, USA

11:40 – 12:00 Xylella fastidiosa in citrus in Brasil – distribution, damages and management practices Silvio Lopes, Fundecitrus, Sao Paulo, Brazil

12:00 – 12:20 The epidemic of Xylella in Puglia: the state of the art" Marina Barba, Plant Pathology Research Centre, Rome, Italy Donato Boscia, National Research Council of Italy, Bari Unit, Italy

12:20 – 13:30 Discussions

13:30-14:10 Lunch in the catering area

14:10-15:40 Discussions and recommendations

15:40-16:10 Coffee at the terrace