MOLECULAR PLANT PATHOLOGY (2012) 13(9), 998–1009 DOI: 10.1111/j.1364-3703.2012.00816.x Pathogen profile Pseudomonas savastanoi pv. savastanoi: Some like it knot Cayo Ramos1, Isabel M. Matas1, Leire Bardaji2, Isabel M. Aragón1, Jesús Murillo2* Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Área de Genética, Facultad de Ciencias, Campus Teatinos s/n, E-29010 Málaga, Spain 2 Departamento de Producción Agraria, ETS Ingenieros Agrónomos, Universidad Pública de Navarra, 31006 Pamplona, Spain 1 * Corresponding author: Jesús Murillo; jesus.murillo@unavarra.es Key words: Pseudomonas syringae pv. savastanoi, olive knot, tumour, genome sequencing, plant disease, control, pathogenicity, virulence, effector. SUMMARY Pseudomonas savastanoi pv. savastanoi is the causal agent of olive (Olea europaea) knot disease and an unorthodox member of the P. syringae complex, causing aerial tumours instead of the foliar necroses and cankers characteristic of most members of this complex. Olive knot is present wherever olive is grown; although losses are difficult to assess, it is assumed that olive knot is one of the most important diseases of the olive crop. The last century has witnessed a good deal of scientific articles describing the biology, epidemiology and control of this pathogen. However, most P. savastanoi pv. savastanoi strains are highly recalcitrant to genetic manipulation, which has effectively left the pathogen out of the scientific progress in molecular biology that has elevated the foliar pathogens of the P. syringae complex to supermodels. A series of studies in the last years have made significant advances in the biology, ecology and genetics of P. savastanoi pv. savastanoi, paving the way for the molecular dissection of its interaction with other nonpathogenic bacteria and their woody hosts. The selection of a genetically pliable model strain was soon followed by the development of rapid methods for virulence assessment with micropropagated olive plants and the analysis of cellular interactions with the plant host. The generation of a draft genome of strain NCPPB 3335 and the closed sequence of its three native plasmids has allowed for functional and comparative genomic analyses for the identification of its pathogenicity gene Pseudomonas savastanoi pv. savastanoi complement. This includes 34 putative type III effector genes and genomic regions, shared with other pathogens of woody hosts, that encode metabolic pathways associated with the degradation of lignin-derived compounds. Now, the time is right to explore the molecular basis of the P. savastanoi pv. savastanoi-olive interaction and to get insights into why some pathovars like it necrotic and why some like it knot. Synonyms: Pseudomonas syringae pv. savastanoi Taxonomy: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; included in genomospecies 2 together with at least P. amygdali, P. ficuserectae, P. meliae and 16 other pathovars from the P. syringae complex (aesculi, ciccaronei, dendropanacis, eriobotryae, glycinea, hibisci, mellea, mori, myricae, phaseolicola, photiniae, sesami, tabaci, ulmi, and certain strains of lachrymans and morsprunorum); when a formal proposal is made for the unification of these bacteria, the species name P. amygdali would take priority over P. savastanoi. Microbiological properties: Gram-negative rods, 0.4-0.8 by 1.0-3.0 µm, aerobic. Motile by one to four polar flagella, rather slow growing, optimal temperatures for growth of 25–30 °C, oxidase negative, arginine dihydrolase negative, elicits the hypersensitive response on tobacco, most isolates are fluorescent and levan negative although some isolates are non-fluorescent and levan positive. Host range: P. savastanoi pv. savastanoi causes tumours in cultivated and wild olive and ash (Fraxinus excelsior). Although strains from olive were reported to infect oleander (Nerium oleander), this is generally not the case; however, strains of P. savastanoi pv. nerii can infect olive. Pathovars fraxini and nerii differentiate from pv. savastanoi mostly in their host range, and were not formally recognized until 1996. Literature previous to about 1996 generally name strains of the three pathovars as P. syringae subsp. savastanoi or P. savastanoi subsp. savastanoi, contributing to confusion about host range and biological properties. Disease symptoms: Symptoms of infected trees include hyperplastic growths (tumorous galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits. Epidemiology: The pathogen can survive and multiply on aerial plant surfaces, as well as in knots, from where it can be dispersed by rain, wind, insects and human activities, entering the plant through wounds. Populations are very unevenly distributed in the plant, and suffer drastic fluctuations throughout the year, with maximum numbers of bacteria occurring during rainy and warm months. Populations of P. savastanoi pv. savastanoi are normally associated to non-pathogenic bacteria, both epiphytically and endophytically, and were demonstrated to form mutualistic consortia with Erwinia toletana and Pantoea agglomerans that could result in increased bacterial populations and disease symptoms. Disease control: Based on preventive measures, mostly sanitary and cultural practices. Integrated control programs benefit from regular applications of copper formulations, which should be maintained at least a few years for maximum benefit. Olive cultivars vary in their susceptibility to olive knot, but there are no known cultivars with full resistance to the pathogen. Useful websites: http://www.pseudomonassyringae.org/; http://genome.ppws.vt.edu/cgibin/MLST/home.pl; ASAP access to the P. savastanoi pv. savastanoi NCPPB 3335 genome sequence https://asap.ahabs.wisc.edu/asap/logon.php. INTRODUCTION Pseudomonas syringae is an economically important pathogen and one of the most relevant models for the study of plant-microbe interactions (e.g., Mansfield, 2009, Mansfield et al., 2012). The species is currently a taxonomic conundrum and has been pulled together with P. amygdali, P. avellanae, P. cannabina, P. caricapapayae, P. ficuserectae, P. meliae, P. savastanoi, P. tremae and P. viridiflava into a group designated as the P. syringae complex, which could correspond to at least nine different species (Gardan et al., 1999, Young, 2010, Parkinson et al., 2011). Pathovars of the P. syringae complex generally exploit the plant apoplast as a parasitic C. Ramos et al. niche and cause foliar necrosis in diverse plant hosts, with a minority of strains causing other types of symptoms, such as vascular diseases on woody plants (Agrios, 2005). A remarkable exception are a few pathovars producing aerial tumours in woody plants, including P. savastanoi pv. savastanoi. P. savastanoi pv. savastanoi is the causal agent of olive (Olea europaea) knot disease, whose symptoms include hyperplastic growths (tumorous galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits (Fig. 1). Olive knot is present worldwide, wherever olive is grown, and it is considered one of the most important diseases of olive (CMI, 1987, Young, 2004, Quesada et al., 2010a). Diverse research groups worldwide have made substantial contributions towards understanding the biology, epidemiology and control of this pathogen; however, most strains of P. savastanoi pv. savastanoi are highly recalcitrant to genetic manipulation (Pérez-Martínez et al., 2007), which has significantly slowed down their molecular analysis. The growing availability of microbial genomes has spurred a new research era in the field of plant-microbe interactions, leading to the identification of potentially comprehensive repertoires of putative virulence genes and the emergence of unified models of interaction between prototypical pathogens and plant hosts (Lindeberg et al., 2008, Mansfield, 2009, Schneider & Collmer, 2010). Extensive recent research efforts have focused on Pseudomonas diseases of herbaceous plants, with knowledge on the virulence and pathogenicity determinants specific for infection of woody plants, including those of tumour-inducing strains, lagging far behind. The selection of strain P. savastanoi pv. savastanoi NCPPB 3335 as a research model (Pérez-Martínez et al., 2007) has opened the door for the application of high-throughput molecular tools to the analysis of the molecular basis of bacterial adaptation to woody hosts. TAXONOMY BIOLOGY AND POPULATIONS Despite significant advances in molecular phylogeny and taxonomy, the nomenclature and classification of P. savastanoi pv. savastanoi is still a source of confusion. This bacterium is part of the P. syringae complex, encompassing at least 60 pathovars and several other Pseudomonas species (Bull et al., 2010, Young, 2010). A study limited to a few taxa, formally classified pathovars glycinea, phaseolicola and savastanoi into the new species P. savastanoi (Gardan et al., 1992), to which pathovars fraxini, nerii and retacarpa were later added (Bull et al., 2010). DNA-DNA hybridization distributed P. syringae into at least nine separate genomospecies (Gardan et al., 1999, Young, 2010). P. savastanoi pv. savastanoi was included in genomospecies 2, together with 16 other P. savastanoi-P. syringae pathovars (see Taxonomy, above) and the species P. amygdali, P. ficuserectae and P. meliae; when genomospecies 2 is formally named, however, the species should be designated Pseudomonas amygdali and not P. savastanoi (Gardan et al., 1999). Multilocus sequence analyses show that P. savastanoi pv. savastanoi NCPPB 3335 is evolutionarily closer to P. syringae pathovars aesculi 2250 and NCPPB 3681, tabaci ATCC 11528 and phaseolicola 1448A (genomospecies 2) than to P. syringae pv. tomato DC3000 (genomospecies 3) or P. syringae pv. syringae B728a (genomospecies 1) (Fig. S1) (Sarkar & Guttman, 2004, Parkinson et al., 2011). These studies support the genomospecies 2 grouping and indicate that it might encompass at least 9 further pathovars (broussonetiae, castaneae, cerasicola, cunninghamiae, daphniphylli, fraxini, nerii, rhaphiolepidis and retacarpa) plus P. tremae (Sarkar & Guttman, 2004, Parkinson et al., 2011). Then, what name should be used for this bacterium? Whereas P. savastanoi is being widely used in the literature, the P. syringae designation helps avoid the false idea that this pathogen is a different species than, for example, P. syringae pv. tabaci. Natural isolates of P. savastanoi pv. savastanoi are heterogeneous, both phenotypically and genotypically (Table 1), although they tend to generate clonal populations in colonized areas (e.g., Sisto et al., 2007, Quesada et al., 2008). There is an important variation in virulence, with strains showing either low, intermediate or, most commonly, high virulence to diverse olive cultivars (Penyalver et al., 2006), and also variation in the size and morphology of tumours in artificial inoculations (Pérez-Martínez et al., 2007). Certain isolates in Central Italy are non-fluorescent and produce Pseudomonas savastanoi pv. savastanoi levan, in contrast with the majority of other isolates (Marchi et al., 2005). AFLP data clustered these levan-positive isolates separately from most of the common levan-negative isolates. Arbitrarilyprimed PCR (Scortichini et al., 2004, Krid et al., 2009), AFLP (Sisto et al., 2007), and typing with IS53 (Quesada et al., 2008) revealed high levels of polymorphism; also, AFLP clearly differentiated pv. savastanoi from pvs. fraxini and nerii. In general, genetic variability associates with geographic origin, with strains from the same area having a closer genetic relationship than those from different areas (Sisto et al., 2007, Quesada et al., 2008, Krid et al., 2009, Matas et al., 2009), suggesting a preference for clonal colonization of olive orchards; indeed, the spread of bacteria Fig. 1 Symptoms produced by Pseudomonas savastanoi pv. savastanoi NCPPB 3335 in olive plants and pathogen visualization within knots. In vitro micropropagated olive plants not inoculated (A) and inoculated (B). Knot induced on a two-years-old olive plant 90 days post inoculation (dpi) (C). Real-time monitoring of GFP-tagged P. savastanoi infection of a young micropropagated olive plant at 30 dpi (D) and complementary epifluorescence microscopy image (E). Cross section of the knot exposed in (C) showing necrosis associated with infection of the stem (F). Cross section of a 30 dpi knot, stained with methylene blue-picrofuchsin; asterisks indicate newly formed bundles of xylem vessels (G). Transverse section of a knot, induced by GFPtagged NCPPB 3335, showing GFP emission within the lumen of xylem vessels, in the internal cavities and at the periphery of the tumour tissue (H). Semithin cross-section of a knot stained with toluidine blue. Stained primary and secondary walls show dark and light blue colour, respectively (I). SCLM image of a knot induced by GFP-tagged NCPPB 335 (J). SEM micrograph showing a group of rodshaped P. savastanoi cells (K). TEM micrograph of ultrathin section of a knot showing pathogen cells colonizing the intercellular spaces of the host tissue (L). C. Ramos et al. from inoculated to non-inoculated trees in an olive orchard, where they produced tumours, was documented in less than one year (Quesada et al., 2010a). Typing with IS53 revealed higher diversity than any of the other techniques, and could be used to track strains in the environment because many strains display unique patterns (Quesada et al., 2008). Table 1 Phenotypic and genetic differences among selected pathovars of P. savastanoia Plant hostb Genomic location of hormone biosynthesis genesc P. savastanoi pv. Ash Oleander Olive Spanish broom iaaMH iaaL ptz fraxini c c nd uk uk nerii K K K P P P savastanoi K K Ch Ch Ch retacarpa K uk uk uk a Modified from (Janse, 1982, Iacobellis et al., 1998, Pérez-Martínez et al., 2008). b ash, Fraxinus excelsior; oleander, Nerium oleander; olive, Olea europaea; Spanish broom, Retama sphaerocarpa; c, cankers accompanied by wart-like excrescences; K, knots; -, no visible symptoms. c Symbols indicate that the gene is located in the chromosome (Ch) or in plasmids (P) in at least 70% of the strains examined; nd; not detected; uk, unknown. Genes iaaMH and iaaL are involved in the biosynthesis of indoleacetic acid and indoleacetic acid lysine, respectively, whereas ptz codes for an isopentenyl transferase, involved in the biosynthesis of cytokinins. EPIDEMIOLOGY AND CONTROL P. savastanoi pv. savastanoi does not survive for long in soil, and is normally found as an epiphyte and also endophytically, being able to migrate to produce secondary knots in new wounds (Ercolani, 1978, Penyalver et al., 2006, Quesada et al., 2007). Epiphytic life allows the build-up of populations for plant colonization and also fosters the interaction with other microbial communities in the phyllosphere. The pathogen is usually introduced to new areas through infected plant material. The bacterium can survive and multiply as a saprophyte on plant surfaces (Ercolani, 1978, Quesada et al., 2007), as well as inside knots, from where it could be disseminated by rain, windblown aerosols, insects and cultural practices, such as pruning. It enters the plant through any type of wound, such as leaf scars or those caused by pruning, harvesting, frost and hail. The presence of knots in even a single tree normally leads to the rapid infection of the whole orchard because the pathogen is very rapidly and efficiently disseminated, with significant colonization of healthy trees in as little as a year (Quesada et al., 2010a). The size of P. savastanoi pv. savastanoi populations is highly variable, even of several orders of magnitude between different leaves of the same shoot (Quesada et al., 2007), with the highest populations occurring in rainy months with moderate temperatures (10-20 ºC). A plethora of non-pathogenic bacterial species are found colonizing olive leaves or closely associated to knots produced by P. savastanoi pv. savastanoi, and the sizes of their populations are often positively correlated (Ercolani, 1978, Rojas et al., 2004, Marchi et al., 2006, Quesada et al., 2007, Ouzari et al., 2008, Moretti et al., 2011). Several of these species could synthesize large amounts of IAA, which could favour proliferation of the pathogen and colonization of the plant (Cimmino et al., 2006, Marchi et al., 2006, Ouzari et al., 2008). Pantoea agglomerans is the species most frequently found associated to P. savastanoi pv. savastanoi populations, its growth stimulated in the presence of active populations of the pathogen (Marchi et al., 2006, Quesada et al., 2007). Their interaction is not completely understood, and it can apparently lead to either an increase in virulence or a decrease of the pathogen populations (Marchi et al., 2006, Hosni et al., 2011). As described below (see “Other virulence factors”), a recent study demonstrated that both Erwinia toletana and P. agglomerans can form stable communities in planta (Hosni et al., 2011). The literature is elusive regarding crop losses caused by olive knot, which greatly Pseudomonas savastanoi pv. savastanoi depend on geographical location and olive cultivar, although it is generally accepted that it is one of the most important diseases affecting the olive crop (Young, 2004). Tree vigour, growth and yield can be moderately or severely reduced, as well as the size and quality of fruits (Schroth et al., 1973, Quesada et al., 2010a). Olive knot cannot be eradicated once it is established in a tree or orchard, and therefore its control is based on preventive measures, mostly sanitary and cultural practices (Young, 2004, Quesada et al., 2010b, Quesada et al., 2010a). Methods should aim to avoid introduction and dissemination of the pathogen, for instance by using certified pathogen-tested trees and rootstocks to start new olive groves (EPPO, 2006), by minimizing wounding of trees and by reducing epiphytic populations of the pathogen. Detection and diagnosis of the pathogen can be done using diverse rapid and highly sensitive PCR methodologies (see Table S1), some of which allow differentiation of pathovars fraxini, nerii and savastanoi. Pivotal for an efficient disease management is a carefully planned and executed pruning, which should always start with the healthy trees and be avoided in wet weather. Chemical control with copper compounds has been traditionally used both in nurseries and in the field (Teviotdale & Krueger, 2004, Young, 2004). An extensive and systematic study (Quesada et al., 2010b), reported a significant reduction of pathogen populations from the very first application of copper compounds, either copper oxychloride or cuprocalcic sulphate plus mancozeb. Nevertheless, treatments should be part of an appropriate integrated control program that includes the regular application of two copper treatments per year. This schedule produced the greatest differences with respect to the untreated control in the third year, after five copper treatments, resulting in a significant reduction in the average number of knots per plant (Quesada et al., 2010b). Conversely, acibenzolar-S-methyl treatments did not result in a significant reduction of the disease symptoms. Reduction of host susceptibility, including the use of resistant cultivars, is a most effective method for the integrated control of plant diseases; unfortunately, there are no known olive cultivars that are completely resistant to the pathogen. Early comparative studies showed a considerable degree of phenotypic variation among olive cultivars, ranging from high susceptibility to a certain resistance (reviewed in Young, 2004). A larger assay evaluated the effect on symptom development of diverse variables— cultivar, plant age, development of secondary knots, inoculum dose and strain virulence—, and proposed a standardized method to assess cultivar susceptibility (Penyalver et al., 2006). These authors demonstrated large differences in disease response with small variations in the inoculum dose, which might explain discrepancies in cultivar assessment among different studies, and classified 29 cultivars in three categories of high, medium and low susceptibility to the pathogen. PSEUDOMONAS SAVASTANOI PV. SAVASTANOI: LIFE INSIDE THE KNOT P. savastanoi pathogenicity and virulence is generally tested on 1- to 3-year-old olive plants (Glass & Kosuge, 1988, Iacobellis et al., 1994, Sisto et al., 2004, Penyalver et al., 2006, PérezMartínez et al., 2007, Hosni et al., 2011). Aside from the space required, this often results in large variability in the size and number of knots that develop. In vitro techniques are widely used to study pathogenicity and virulence of animal bacterial pathogens and can also be conveniently applied in plant pathology. Several techniques have been described for micropropagation of a vast number of fruit trees, including several olive varieties, facilitating mass production of clonal and disease-free plants that can easily be maintained under controlled conditions in growth chambers. The use of in vitro micropropagated olive plants has been established as a fast and inexpensive method to study pathogenicity and virulence of P. savastanoi strains isolated from olive and oleander knots (Rodríguez-Moreno et al., 2008). As previously observed with older olive plants, symptom development in micropropagated olive plants is highly dependent on both the olive variety and the strain. Nevertheless, histological modifications observed in in vitro olive plants after infection by P. savastanoi pv. savastanoi strains (RodríguezMoreno et al., 2008, Marchi et al., 2009, Rodríguez-Moreno et al., 2009) are very similar to those in older olive plants (Smith, 1920, Surico, C. Ramos et al. 1977, Temsah et al., 2008), further confirming the suitability of this model system. Tagging of P. savastanoi pv. savastanoi NCPPB 3335 with the green fluorescent protein (GFP), in combination with the use of in vitro olive plants and epifluorescence microscopy, allows real-time monitoring of disease development at the whole-tumour level, as well as the monitoring of bacterial localization inside knots at the singlecell level by scanning confocal electron microscopy (SCLM). Additionally, scanning and transmission electron microscopy (SEM and TEM, respectively) can be used for a detailed ultrastructural analysis of tumour histology, as well as for the visualization of the P. savastanoi pv. savastanoi lifestyle within knot tissues (Fig. 1) (Rodríguez-Moreno et al., 2009). A combination of these microscopy techniques was used for in vivo analysis of P. savastanoi pv. savastanoi NCPPB 3335 mutants affected in virulence (Pérez-Martínez et al., 2010, Bardaji et al., 2011). GENOMIC INSIGHTS INTO P. SAVASTANOI PV. SAVASTANOI PATHOGENICITY AND VIRULENCE In this section we review how the recent sequencing of the P. savastanoi pv. savastanoi NCPPB 3335 draft genome and the complete sequence of its three plasmid complement, allowed the identification of the virulence gene Fig. 2 Unrooted NJ trees of iaaL nucleotide sequences from strains of the P. syringae complex. See Fig. S1 for methodology and Table S3 for accession numbers. Only pathovar name and strain designation are shown; all strains belong to P. syringae, except P. cannabina pv. alisalensis ES4326, which was previously designated as P. syringae pv. maculicola. complement of this tumour-inducing pathogen of woody hosts (Rodríguez-Palenzuela et al., 2010, Bardaji et al., 2011). Phytohormones In P. savastanoi, indoleacetic acid (IAA) is synthesized from tryptophan in two steps catalysed by the products of genes iaaM (tryptophan monooxygenase) and iaaH (indoleacetamide hydrolase) (Comai & Kosuge, 1982, Palm et al., 1989). Additionally, P. savastanoi pv. nerii (oleander isolates) also converts IAA to IAA-lysine through the action of the iaaL gene (Glass & Kosuge, 1988), which is also present in most P. syringae complex pathovars (Glickmann et al., 1998). Although P. savastanoi pv. savastanoi strains contain two iaaL alleles (Matas et al., 2009), IAA-Lys has not been detected in culture filtrates of P. savastanoi strains isolated from olive (Evidente et al., 1986, Glass & Kosuge, 1988). Two chromosomally encoded iaaM, iaaH and iaaL alleles were also found in the genome of P. savastanoi pv. savastanoi NCPPB 3335; however, the iaaM-2 and iaaH-1 alleles appeared to be pseudogenes (Rodríguez-Palenzuela et al., 2010). Resequencing of these two loci has recently confirmed that, in fact, iaaM-2 is a pseudogene whereas iaaH-1 encodes a complete CDS. Gene iaaL is widely distributed within the P. syringae complex (Glickmann et al., 1998), and its phylogeny (Fig. 2) is largely congruent with the phylogeny deduced from housekeeping genes (Fig. S1), suggesting that iaaL is ancestral to the complex. However, clustering of iaaL from P. syringae pv. oryzae 1_6 (genomospecies 4) with genomospecies 2 (Fig. 2) evidences horizontal transfer. This is not surprising because iaaL is often found in several copies and located in plasmids (Glickmann et al., 1998, Matas et al., 2009), although the transfer appears to preferentially occur within the P. syringae complex (not shown). Conversely, highly conserved iaaMH alleles are present in only a handful of P. syringae complex strains (Table S2) (Glickmann et al., 1998); nevertheless, diverse pathovars contain CDSs (Baltrus et al., 2011) whose deduced products show very low identity to those of iaaMH (e.g. PSPTO_0518/PSPTO_4204; 29.3/29.7% aa identity), but high identity with putative monooxygenase and amidase genes common in the P. syringae complex (e.g. 99/89% aa identity with PSA3335_4651/PSA3335_4172 from NCPPB 3335), and whose role in IAA biosynthesis has not been demonstrated. The limited data available also suggests horizontal transfer of iaaMH within the P. syringae complex, which are also less related to the corresponding genes of other organisms (Table S2). Genes for phytohormone biosynthesis have a disparate genomic localization in different tumour-inducing strains of P. savastanoi (Table 1), with those for the biosynthesis of cytokinins (CKs) preferentially located in plasmids of the pPT23A-family in P. savastanoi pv. savastanoi (Macdonald et al., 1986, Silverstone et al., 1993, Pérez-Martínez et al., 2008). The ptz gene, encoding an isopentenyl transferase and characterized by a low G+C content (43.4% G+C), was found in a potential genomic island located in plasmid pPsv48A of P. savastanoi pv. savastanoi NCPPB 3335 (Bardaji et al., 2011). Knots induced in olive plants by P. savastanoi strains cured of plasmids containing ptz are smaller (Iacobellis et al., 1994, RodríguezMoreno et al., 2008, Bardaji et al., 2011) and show a lower presence of spiral vessels (Bardaji et al., 2011) than those induced by wild-type strains. Another gene putatively involved in the biosynthesis of CKs, gene ipt, encoding a putative isopentenyl-diphosphate deltaisomerase, was found in plasmid pPsv48C of P. savastanoi pv. savastanoi NCPPB 3335; however, its role in virulence has not been tested, since derivatives lacking pPsv48C are not yet available (Bardaji et al., 2011). Apparently, P. savastanoi pv. savastanoi does not belong to the group of 2-oxoglutaratedependent ethylene producers, a pathway dependent on gene efe in several P. syringae pathovars (Weingart et al., 1999). First, no homology to an efe probe was found by hybridization analysis of 32 different P. savastanoi pv. savastanoi plasmids (PérezMartínez et al., 2008). Second, proteins homologous to ethylene forming enzymes from P. syringae pv. phaseolicola, pv. glycinea and pv. pisi have not been found in the P. savastanoi pv. savastanoi NCPPB 3335 genome (RodríguezPalenzuela et al., 2010). Fig. 3 Updated and corrected comparison of the type III effector gene complements of P. savastanoi pv. savastanoi (Psv) NCPPB 3335 and other sequenced plant-pathogenic pseudomonads. Pph, P. syringae pv. phaseolicola; Pta, P. syringae pv. tabaci. Gene hopAF1-2, plasmid encoded in NCPPB 3335, shows 73%-74% amino acid identity with hopAF1 from Psy B728a, Pph 1448A and Pto DC3000. Psv NCPPB 3335 effectors included in the Hop database (http://www.pseudomonassyringae.org/pst_func_gen2.htm) are indicated in boldface. #, Plasmid-encoded gene; asterisks indicate putative pseudogenes; hop genes truncated by a frameshift or a premature stop codon are indicated by a single quotation mark (Lindeberg et al., 2005). C. Ramos et al. Type III secretion system and effectors Cluster analysis of HrpS protein sequences (Inoue & Takikawa, 2006) showed that P. savastanoi pv. savastanoi NCPPB 3335 belongs to group I, which comprises exclusively proteins from P. syringae pathovars from genomospecies 2 (Gardan et al., 1999). In relation to HrpA, P. savastanoi pv. savastanoi NCPPB 3335 contains a hrpA2 gene, which is highly similar to those of P. syringae pvs. phaseolicola, glycinea and tabaci (Pérez-Martínez et al., 2010). In agreement with Sisto et al. (2004), a T3SS mutant of strain NCPPB 3335 was also unable to multiply in olive tissues and induce the formation of knots in woody olive plants. Interestingly, tumours induced by the T3SS mutant on young micropropagated olive plants did not show the necrosis and internal open cavities observed in knots induced by the wild-type strain (PérezMartínez et al., 2010). Bioinformatic analysis of the P. savastanoi pv. savastanoi NCPPB 3335 genome sequence (Rodríguez-Palenzuela et al., 2010) allowed a prediction of hop genes, including 19 putative T3SS effectors with amino acid identities of 6580% to previously described effectors. Additionally, a further 11 candidate genes do not share sequence similarity with known effectors (Rodríguez-Palenzuela et al., 2010). A later revision of this genome sequence identified four new candidate effectors, AvrPto1, HopAT1’, HopAZ1 and HopF4 (Hops Database, http://www.pseudomonassyringae.org/home.html) (Fig. 3). Furthermore, sequencing of the three-plasmid complement of this strain revealed that two of the T3SS effector genes are plasmid encoded, hopAF1 (plasmid pPsv48A) and hopAO1 (plasmid pPsv48B) (Bardaji et al., 2011). Figure 3 shows an updated and corrected comparison of the T3SS effector gene complements of P. savastanoi pv. savastanoi NCPPB 3335 and other sequenced plant-pathogenic pseudomonads. Translocation analysis of the T3SS effector repertoire of P. savastanoi pv. savastanoi NCPPB 3335 is currently in progress. Other virulence factors Pathogenicity of P. savastanoi pv. savastanoi in olive critically depends on quorum sensing (QS) regulation. The QS system of P. savastanoi pv. savastanoi strain DAPP-PG 722 consist of a luxI homolog (pssI) and a luxR homolog (pssR) (Hosni et al., 2011). However, the lack of signal production in a pssI mutant of this pathogen has been shown to be complemented in planta by the presence of wild-type Erwinia toletana, a nonpathogenic bacterium that is very often found associated with the olive knot pathogen (Hosni et al., 2011). E. toletana produced the same N-acylhomoserine lactone molecules than P. savastanoi pv. savastanoi; moreover, populations of E. toletana significantly declined over time after inoculation in olive tissues, but increased upon co-inoculation with a strain of P. savastanoi pv. savastanoi. This relationship is mutualistic, because the populations of P. savastanoi pv. savastanoi also increased significantly when the pathogen was co-inoculated with E. toletana; additionally, the size of knots also increased, reflecting an increase in virulence (Hosni et al., 2011). The mechanism underlying this relationship is not fully clear, but it appears to result at least in part from sharing QS signalling mediated by N-acyl-homoserine lactones. Other known virulence determinants in plantpathogenic Pseudomonas include phytotoxins, cell wall-degrading hydrolytic enzymes, extracellular polysaccharides, iron-uptake systems, resistance to plant-derived antimicrobials, adhesion, as well as the general processes of motility and chemotaxis. Annotation of the P. savastanoi pv. savastanoi NCPPB 3335 draft genome revealed the existence of 551 genes potentially involved in several processes that could contribute to virulence, most of which are conserved in P. syringae pv. phaseolicola 1448A. However, the subset of P. savastanoi pv. savastanoi NCPPB 3335-specific genes (not found in 1448A), includes a cellulase, a pectate lyase and a putative filamentous hemagglutinin (Rodríguez-Palenzuela et al., 2010). Genes for levansucrase, the enzyme responsible for biosynthesis of the exopolysaccharide levan, are found in the genome of all sequenced P. syringae strains, although their numbers vary from three to one (O'Brien et al., 2011). Only a single levansucrase-coding gene (PSA3335_2033) was identified in P. savastanoi pv. savastanoi NCPPB 3335 (Rodríguez-Palenzuela et al., 2010), probably because P. savastanoi pv. savastanoi strains are in general levan-negative whereas the Pseudomonas savastanoi pv. savastanoi P. syringae pathovars of LOPAT subgroup 1a are all levan-positive (Lelliott & Stead, 1987). The relevance of all these putative virulence factors in P. savastanoi has not been reported to date. Fig. 4 Unrooted UPGMA tree based upon nutrient utilization data of P. savastanoi pv. savastanoi (Psv) and other pseudomonads. Metabolic activities of Psv strains, tested using Biolog GN2 plates, were compared with carbon utilization data reported for P. syringae strains and non-plant pathogenic species of Pseudomonas (Rico & Preston, 2008). The tree was constructed using MEGA5 (Tamura et al., 2011) and is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the dendro-gram. Distances were computed using the Maximum Composite Likelihood method and are in the units of the number of substrate utili-zation per site. Abbrevia-tions are P. syringae patho-vars syringae, Psy; tomato, Pto; tabaci, Pta; phaseo-licola, Pph; and P. putida, Ppu; P. entomophila, Pme; P. fluorescens, Pfl; and P. aeruginosa, Pae. METABOLIC VERSATILITY AND ADAPTATION TO WOODY HOSTS P. syringae pathovars are nutritionally specialized for growth in the plant environment relative to non-pathogenic pseudomonads (Rico et al., 2011). The Biolog GN2 MicroPlate technology (Bochner et al., 2001) revealed that the carbon utilization profiles of five different P. savastanoi pv. savastanoi strains, including NCPPB 3335, are almost identical. However, comparative analysis with previously reported data for P. syringae pathovars and non-pathogenic pseudomonads (Rico & Preston, 2008, Mithani et al., 2011) shows that P. savastanoi pv. savastanoi metabolic activities are more similar to those shown by P. syringae pv. tabaci ATCC 11528, P. syringae pv. tomato DC3000 and P. syringae pv. syringae B728a than to those observed for P. syringae pv. phaseolicola 1448A (Fig. 4), in spite that both strains ATCC 11528 and 1448A cluster together with P. savastanoi pv. savastanoi NCPPB 3335 by multilocus sequence analysis of housekeeping genes (Group 3, Fig. S1). Thus, nutritional divergence does not mirror phylogenetic divergence, possibly due to hostspecific features, or pathogen evolutionary history. Production of phenolic compounds, which provide a natural defence against pathogen attack, is greatly increased in olive knots induced by P. savastanoi pv. savastanoi (Cayuela et al., 2006), suggesting that bacterial resistance to phenols could be of paramount importance in pathogenicity. The P. savastanoi pv. savastanoi NCPPB 3335 genome (Rodríguez-Palenzuela et al., 2010) encodes a region of about 15 Kb, named VR8 (60.1% G+C), which is absent in all sequenced P. syringae strains infecting herbaceous plants but shared with P. syringae pathovars infecting woody hosts, such as aesculi (Green et al., 2010), morsprunorum and actinidiae (Fig. 5), which are pathogenic to chestnut, cherry and kiwi, respectively. Among other genes encoded in this region, the antABC and catBCA operons are involved in the degradation of anthranilate and catechol, respectively, and could offer a selective advantage for growth in woody hosts. In fact, the antABC cluster is homologous to the anthranilate degradation genes found on plasmid pCAR1 of Pseudomonas resinovorans (Nojiri et al., 2002, Urata et al., 2004), a bacterium commonly found in the lubricating oils of wood mills. Other metabolic pathways involving the cat and/or ant genes included in the KEGG Pathway Database (http://www.genome.jp/kegg/pathway.html) are those related to the degradation of benzoate, fluorobenzoate, toluene, chlorocyclohexane and chlorobenzene. In P. savastanoi pv. savastanoi NCPPB 3335 and all other strains encoding VR8, the genetic content and chromosomal location of this region is identical (Fig. 5). However, genetic elements suggesting its possible acquisition by C. Ramos et al. horizontal transfer were not found bordering VR8 in P. savastanoi pv. savastanoi NCPPB 3335 (Rodríguez-Palenzuela et al., 2010). Fig. 5 Schematic map of variable region 8 (VR8) in the genomes of P. savastanoi pv. savastanoi NCPPB 3335 and other sequenced P. syringae pathovars. A) P. savastanoi pv. savastanoi NCPPB 3335, P. syringae pathovars aesculi strains 2250 and NCPPB 3681, morsprunorum MAFF302280, actini-diae MAFF302091; B) P. syringae pathovars tabaci ATCC 11528, mori 301020, phaseolicola 1448A, glycinea race 4, lachrymans MAFF302278, and japo-nica MAFF301072; C) P. syringae pathovars syringae B728a, tomato DC3000, and oryzae 1_6. Black and grey arrows indicate genes flanking VR8 in P. savastanoi pv. savastanoi NCPPB 3335 which are present (PSA3335_3197 and PSA3335_3214) or not (PSA3335_3198), respecttively, in the genome of all the strains analysed. Pink and orange arrows indicate genes involved in the catabolism of catechol (catBCA) or anthranilate (antABC and antR), respectively. PSA3335_3197, outer membrane protein; PSA3335_3198, ribosomal protein S5p alanine acetyltransferase (rimJ); PSA3335_3206, aerotaxis receptor; PSA3335_3207, nitrilotriacetate monooxygenase component B, flavin reductase; PSA3335_3208, protein involved in meta-pathway of phenol degradation; PSA3335_3209, putative oxygenase subunit; PSA3335_3210, short-chain alcohol dehydrogenase/reductase; PSA3335_3211, dienelactone hydrolase; PSA3335_3212, hypothetical protein; PSA3335_3214, voltage-dependent potassium channel protein. PLASMID GENETICS AND BIOLOGY Plasmids are the main agents in the horizontal exchange of DNA among bacteria, and the P. syringae complex contains a significant horizontal gene pool distributed in diverse native plasmids (Jackson et al., 2011). Strains of P. savastanoi pv. savastanoi usually contain one to six plasmids (around 10 to >100 kb) (Murillo & Keen, 1994, Pérez-Martínez et al., 2008). Most of these belong to the pPT23A-like family of plasmids (PFP), characterized for sharing a highly conserved replication module (Gibbon et al., 1999), although strains might contain from zero to four non-PFP plasmids. As usual in the P. syringae complex, plasmid profiles are highly variable and often strain-specific, offering a simple way for strain tracking (Pérez-Martínez et al., 2007, Pérez-Martínez et al., 2008). Nevertheless, plasmid profiles of P. syringae complex strains are dynamic and often change in response to repeated subculture or interaction with the host (e.g. Lovell et al., 2011). PFP plasmids carry a panoply of genes involved in pathogenicity, virulence and adaptation to the environment, such as genes for T3SS effectors, type IV secretion systems, phytotoxins, phytohormones, and resistance to antibiotics and heavy metals, as well as an array of insertion sequences (Sundin, 2007). Similar types of genes were found in 32 native plasmids from 10 P. savastanoi pv. savastanoi strains using a macroarray containing 135 different genes, albeit with a limited presence of genes rulAB, for UV radiation tolerance. This could be significant because rulAB genes appear to often control the expression of integrases, and are predicted to facilitate the dispersal of associated T3SS effector genes (Jackson et al., 2011). Native plasmids from P. savastanoi pv. savastanoi contain diverse virulence genes carried indistinctly by PFP and non-PFP plasmids (Pérez-Martínez et al., 2008), although PFP plasmids have been traditionally recognized as the main, or the only, repository of valuable genes in the P. syringae complex. At least eight T3SS effector genes (Jackson et al., 2002, Pérez-Martínez et al., 2008) are frequently found on P. savastanoi pv. savastanoi plasmids. Other relevant virulence genes are those involved in the biosynthesis of phytohormones, which were the first plasmid-borne pathogenicity genes found in Pseudomonas spp. (Comai & Kosuge, 1980). Unlike pv. nerii, most strains of pv. savastanoi carry chromosomal copies of genes for the biosynthesis of IAA and CKs (Table 1). The complete sequences of the three PFP plasmid complement of strain NCPPB 3335 (pPsv48A, 78 kb; pPsv48B, 45 kb; pPsv48C, 42 kb) (Bardaji et al., 2011) contain 152 predicted coding sequences (CDSs); the majority (38 CDSs) were annotated as hypothetical proteins, followed by 37 CDSs involved in DNA metabolism, including plasmid replication and Pseudomonas savastanoi pv. savastanoi maintenance. Each of the plasmids contained at least one putative toxin-antitoxin system, involved in plasmid maintenance, which is probably why we could not obtain derivatives cured of the three plasmids (Bardaji et al., 2011). The three plasmids contain seven putative virulence genes, five of which are putative type III effectors preceded by a Hrp-box: pPsv48A contains a chimeric copy of gene hopAF1, included in the transposon effector ISPsy30, plus three copies of a large CDS found in many plant-associated proteobacteria, whereas pPsv48B contains gene hopAO1 (avrPphD2). Additionally, two genes putatively involved in CKs biosynthesis, ptz (PSPSV_A0024) and ipt (PSPSV_C0024), were also found in plasmids A and C, respectively. Plasmids are very plastic and dynamic molecules, facilitating the exchange of sequences among them and with the chromosome (Ma et al., 2007, Sundin, 2007, Jackson et al., 2011). This is illustrated by plasmids pPsv48B and pPsv48C, which probably arose from a duplication event because their replication gene, repA, is 98.6% identical. However, they only share around 10 kb with at least 80% identity, implying they participated in an active exchange of DNA. Indeed, pPsv48B contains a complete type IVA secretion system and a well conserved origin of conjugational transfer, suggesting that it might be conjugative; additionally, pPsv48C also contains an origin of transfer and could be mobilizable by pPsv48B. Although, in principle, plasmids can be transferred to very distant organisms, they tend to propagate within a specific host clade (Jackson et al., 2011). A phylogenetic analysis of the repA gene from diverse PFP plasmids, and of other genes carried by them, indicate that they are actively exchanging DNA and moving amongst P. syringae complex pathovars (Ma et al., 2007). The role of native plasmids in the life cycle of P. savastanoi pv. savastanoi has not been assessed in detail due to the difficulties for its genetic manipulation and for plasmid curing. Nevertheless, in diverse strains of P. savastanoi pv. savastanoi and pv. nerii certain native plasmids are essential for the expression of wild type symptoms, to reach high population densities in planta and for competitive fitness, all of which was related to the presence in those plasmids of genes for IAA and/or CKs biosynthesis (Silverstone et al., 1993, Iacobellis et al., 1994, Rodríguez-Moreno et al., 2008, Bardaji et al., 2011). Since these effects are very drastic, they could conceivably have obscured more subtle roles in the pathogenic process of other plasmid-borne genes; however, the current availability of genetically tractable strains and plasmid sequences shall facilitate a more detailed analysis of their potential role. FUTURE PROSPECTS Diseases of woody plants caused by pathovars of the P. syringae complex are of major concern in fruit producing areas and nurseries worldwide and result in considerable economic losses (Kennelly et al., 2007). Undoubtedly, advances in the understanding of diseases caused by P. syringae pathovars on herbaceous plants, including the model plant Arabidopsis, are relevant to our understanding of fruit tree diseases, and vice versa. However, there is a pressing need for appropriate research model systems facilitating the identification and analysis of specific determinants involved in bacterial interactions with trees and shrubs. A series of works in the last years made significant advances in the biology, ecology, genetics, and genomics of P. savastanoi pv. savastanoi, which emerged as a powerful and uniquely valuable model for the study of the molecular basis of disease production and tumour formation in woody hosts. Analysis of the P. savastanoi-olive interaction, and comparison with the model systems of herbaceous plants, can provide insights into the interactions of other bacterial pathogens with woody hosts and address relevant unresolved questions, such as: What is the role of the T3SS system and its effectors during infection of woody tissues? Are there differences in the metabolic network required by bacterial pathogens for survival in woody hosts and herbaceous hosts? What virulence determinants are singularly required for infection of woody tissues? What factors are involved in tumour induction by P. savastanoi and what evolutionary advantage derives from producing them instead of necroses? To what degree do bacterial consortia influence disease incidence and severity, and can they be targeted for disease control? What traits govern host specificity in P. savastanoi pathovars? Comparative genomics among P. C. Ramos et al. syringae and P. savastanoi pathovars is generating workable hypotheses to critically investigate these questions. However, a great deal of research remains to establish genomewide approaches allowing functional characterization of bacterial interactions with woody hosts and to develop effective control strategies for Pseudomonas diseases. Genetic dissection of the P. savastanoi pv. savastanoiolive pathosystem is technically very challenging and requires the analysis of the always unfriendly woody plants but, as Osgood wisely summarized in the delightful Billy Wilder film, “Well, nobody’s perfect”. ACKNOWLEDGEMENTS Supported by Spanish Plan Nacional I+D+i grants AGL2008-05311-C02-01, AGL2008-05311-C02-02, AGL2011-30343-C02-01 and AGL2011-30343-C0202 (Ministerio de Economía y Competitividad), cofinanced by FEDER, and by grant P08-CVI-03475 from the Junta de Andalucía, Spain (http://www.juntadeandalucia.es). I.M.M. and I.M.A. were supported by the Ramón Areces Foundation (Spain) and by a FPU fellowship from the Ministerio de Economía y Competitividad (Spain), respectively. We thank L. Rodríguez-Moreno for confocal and electron microscopy images and T. Osinga for help with the English language. REFERENCES Agrios, G.N. (2005) Plant pathology. San Diego, USA: Elsevier Academic Press. Baltrus, D.A., Nishimura, M.T., Romanchuk, A., Chang, J.H., Mukhtar, M.S., Cherkis, K., Roach, J., Grant, S.R., Jones, C.D. and Dangl, J.L. (2011) Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 Pseudomonas syringae isolates. PLoS Pathog., 7, e1002132. Bardaji, L., Pérez-Martínez, I., Rodríguez-Moreno, L., Rodríguez-Palenzuela, P., Sundin, G.W., Ramos, C. and Murillo, J. (2011) Sequence and role in virulence of the three plasmid complement of the model tumorinducing bacterium Pseudomonas savastanoi pv. savastanoi NCPPB 3335. PLoS ONE, 6, e25705. Bertolini, E., Olmos, A., López, M.M. and Cambra, M. (2003) Multiplex nested reverse transcription-polymerase chain reaction in a single tube for sensitive and simultaneous detection of four RNA viruses and Pseudomonas savastanoi pv. savastanoi in olive trees. Phytopathology, 93, 286-292. Bochner, B.R., Gadzinski, P. and Panomitros, E. (2001) Phenotype microarrays for high-throughput phenotypic testing and assay of gene function. Genome Res., 11, 1246-1255. Bull, C.T., De Boer, S.H., Denny, T.P., Firrao, G., Fischer-Le Saux, M., Saddler, G.S., Scortichini, M., Stead, D.E. and Takikawa, Y. (2010) Comprehensive list of names of plant pathogenic bacteria, 1980-2007. J. Plant Pathol., 92, 551-592. Bull, C.T., Clarke, C.R., Cai, R., Vinatzer, B.A., Jardini, T.M. and Koike, S.T. (2011) Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley. Phytopathology, 101, 847-858. Cayuela, J.A., Rada, M., Rios, J.J., Albi, T. and Guinda, A. (2006) Changes in phenolic composition induced by Pseudomonas savastanoi pv. savastanoi infection in olive tree: Presence of large amounts of verbascoside in nodules of tuberculosis disease. J. Agric. Food Chem., 54, 5363-5368. Cimmino, A., Andolfi, A., Marchi, G., Surico, G. and Evidente, A. (2006) Phytohormone production by strains of Pantoea agglomerans from knots on olive plants caused by Pseudomonas savastanoi pv. savastanoi. Phytopathol. Mediterr., 45, 247-252. CMI (1987) Pseudomonas syringae pv. savastanoi (E.F. Smith) Young, Dye & Wilkie. CMI Distribution Maps of Plant Diseases, Map 135, 4th Edn. Wallingford, UK: CAB International. Comai, L. and Kosuge, T. (1980) Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi. J. Bacteriol., 143, 950-957. Comai, L. and Kosuge, T. (1982) Cloning and characterization of iaaM, a virulence determinant of Pseudomonas savastanoi. J. Bacteriol., 149, 40-46. EPPO (2006) Pathogen-tested olive trees and rootstocks. EPPO Bull., 36, 77-83. Ercolani, G.L. (1978) Pseudomonas savastanoi and other bacteria colonizing surface of olive leaves in the field. J. Gen. Microbiol., 109, 245-257. Evidente, A., Surico, G., Iacobellis, N.S. and Randazzo, G. (1986) -N-acetyl-indole-3-acetyl--L-lysine: A metabolite of indole-3-acetic-acid from Pseudomonas syringae pv. savastanoi. Phytochemistry, 25, 125-128. Gardan, L., Bollet, C., Abu Ghorrah, M., Grimont, F. and Grimont, P.A.D. (1992) DNA relatedness among the pathovar strains of Pseudomonas syringae subsp. savastanoi Janse (1982) and proposal of Pseudomonas savastanoi sp. nov. Int. J. Syst. Bacteriol., 42, 606-612. Gardan, L., Shafik, H., Belouin, S., Broch, R., Grimont, F. and Grimont, P.A.D. (1999) DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Sutic and Dowson 1959). Int. J. Syst. Bacteriol., 49, 469-478. Gibbon, M.J., Sesma, A., Canal, A., Wood, J.R., Hidalgo, E., Brown, J., Vivian, A. and Murillo, J. (1999) Replication regions from plant-pathogenic Pseudomonas syringae plasmids are similar to ColE2-related replicons. Microbiology, 145, 325-334. Glass, N.L. and Kosuge, T. (1988) Role of indoleacetic acid lysine synthetase in regulation of indoleacetic-acid Pseudomonas savastanoi pv. savastanoi pool size and virulence of Pseudomonas syringae subsp. savastanoi. J. Bacteriol., 170, 2367-2373. Glickmann, E., Gardan, L., Jacquet, S., Hussain, S., Elasri, M., Petit, A. and Dessaux, Y. (1998) Auxin production is a common feature of most pathovars of Pseudomonas syringae. Mol. Plant-Microbe Interact., 11, 156-162. Green, S., Studholme, D.J., Laue, B.E., Dorati, F., Lovell, H., Arnold, D., Cottrell, J.E., Bridgett, S., Blaxter, M., Huitema, E., Thwaites, R., Sharp, P.M., Jackson, R.W. and Kamoun, S. (2010) Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum. PLoS ONE, 5, e10224. Hosni, T., Moretti, C., Devescovi, G., Suárez-Moreno, Z.R., Fatmi, M.B., Guarnaccia, C., Pongor, S., Onofri, A., Buonaurio, R. and Venturi, V. (2011) Sharing of quorum-sensing signals and role of interspecies communities in a bacterial plant disease. ISME J., 5, 1857-1870. Iacobellis, N.S., Sisto, A., Surico, G., Evidente, A. and DiMaio, E. (1994) Pathogenicity of Pseudomonas syringae subsp. savastanoi mutants defective in phytohormone production. J. Phytopathol., 140, 238-248. Iacobellis, N.S., Caponero, A. and Evidente, A. (1998) Characterization of Pseudomonas syringae ssp. savastanoi strains isolated from ash. Plant Pathol., 47, 73-83. Inoue, Y. and Takikawa, Y. (2006) The hrpZ and hrpA genes are variable, and useful for grouping Pseudomonas syringae bacteria. J. Gen. Plant Pathol., 72, 26-33. Jackson, R.W., Mansfield, J.W., Ammouneh, H., Dutton, L.C., Wharton, B., Ortiz-Barredo, A., Arnold, D.L., Tsiamis, G., Sesma, A., Butcher, D., Boch, J., Kim, Y.J., Martin, G.B., Tegli, S., Murillo, J. and Vivian, A. (2002) Location and activity of members of a family of virPphA homologues in pathovars of Pseudomonas syringae and P. savastanoi. Mol. Plant Pathol., 3, 205216. Jackson, R.W., Vinatzer, B., Arnold, D.L., Dorus, S. and Murillo, J. (2011) The influence of the accessory genome on bacterial pathogen evolution. Mob. Genet. Elem., 1, 55-65. Janse, J.D. (1982) Pseudomonas syringae subsp. savastanoi (ex Smith) subsp. nov., nom. rev., the bacterium causing excrescences on Oleaceae and Nerium oleander L. Int. J. Syst. Bacteriol., 32, 166-169. Kennelly, M.M., Cazorla, F.M., de Vicente, A., Ramos, C. and Sundin, G.W. (2007) Pseudomonas syringae diseases of fruit trees. Progress toward understanding and control. Plant Dis., 91, 4-17. Krid, S., Rhouma, A., Quesada, J.M., Penyalver, R. and Gargouri, A. (2009) Delineation of Pseudomonas savastanoi pv. savastanoi strains isolated in Tunisia by random-amplified polymorphic DNA analysis. J. Appl. Microbiol., 106, 886-894. Lelliott, R.A. and Stead, D.E. (1987) Methods for the diagnosis of bacterial diseases of plants. Oxford: Blackwell Scientific Publications. Lindeberg, M., Myers, C.R., Collmer, A. and Schneider, D.J. (2008) Roadmap to new virulence determinants in Pseudomonas syringae: insights from comparative genomics and genome organization. Mol. Plant-Microbe Interact., 21, 685-700. Lovell, H.C., Jackson, R.W., Mansfield, J.W., Godfrey, S.A.C., Hancock, J.T., Desikan, R. and Arnold, D.L. (2011) In planta conditions induce genomic changes in Pseudomonas syringae pv. phaseolicola. Mol. Plant Pathol., 12, 167-176. Ma, Z., Smith, J.J., Zhao, Y., Jackson, R.W., Arnold, D.L., Murillo, J. and Sundin, G.W. (2007) Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae. Appl. Environ. Microbiol., 73, 1287-1295. Macdonald, E.M.S., Powell, G.K., Regier, D.A., Glass, N.L., Roberto, F., Kosuge, T. and Morris, R.O. (1986) Secretion of zeatin, ribosylzeatin, and ribosyl-1''methylzeatin by Pseudomonas savastanoi: plasmidcoded cytokinin biosynthesis. Plant Physiol., 82, 742-747. Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M.A.X., Verdier, V., Beer, S.V., Machado, M.A., Toth, I.A.N., Salmond, G. and Foster, G.D. (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol. Plant Pathol., doi: 10.1111/j.1364-3703.2012.00804.x. Mansfield, J.W. (2009) From bacterial avirulence genes to effector functions via the hrp delivery system: an overview of 25 years of progress in our understanding of plant innate immunity. Mol. Plant Pathol., 10, 721-734. Marchi, G., Viti, C., Giovannetti, L. and Surico, G. (2005) Spread of levan-positive populations of Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot, in central Italy. Eur. J. Plant Pathol., 112, 101-112. Marchi, G., Sisto, A., Cimmino, A., Andolfi, A., Cipriani, M.G., Evidente, A. and Surico, G. (2006) Interaction between Pseudomonas savastanoi pv. savastanoi and Pantoea agglomerans in olive knots. Plant Pathol., 55, 614-624. Marchi, G., Mori, B., Pollacci, P., Mencuccini, M. and Surico, G. (2009) Systemic spread of Pseudomonas savastanoi pv. savastanoi in olive explants. Plant Pathol., 58, 152-158. Matas, I.M., Pérez-Martínez, I., Quesada, J.M., Rodriguez-Hervá, J.J., Penyalver, R. and Ramos, C. (2009) Pseudomonas savastanoi pv. savastanoi contains two iaaL paralogs, one of which exhibits a variable number of a trinucleotide (TAC) tandem repeat. Appl. Environ. Microbiol., 75, 1030-1035. Mithani, A., Hein, J. and Preston, G.M. (2011) Comparative analysis of metabolic networks provides insight into the evolution of plant pathogenic and nonpathogenic lifestyles in Pseudomonas. Mol. Biol. Evol., 28, 483-499. Moretti, C., Hosni, T., Vandemeulebroecke, K., Brady, C., De Vos, P., Buonaurio, R. and Cleenwerck, I. (2011) Erwinia oleae sp. nov., isolated from olive knots caused by Pseudomonas savastanoi pv. savastanoi. Int. J. Syst. Evol. Microbiol., 61, 2745-2752. Murillo, J. and Keen, N.T. (1994) Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 C. Ramos et al. share a large amount of repeated DNA, including replication sequences. Mol. Microbiol., 12, 941-950. Nojiri, H., Maeda, K., Sekiguchi, H., Urata, M., Shintani, M., Yoshida, T., Habe, H. and Omori, T. (2002) Organization and transcriptional characterization of catechol degradation genes involved in carbazole degradation by Pseudomonas resinovorans strain CA10. Biosci. Biotechnol. Biochem., 66, 897-901. O'Brien, H.E., Thakur, S. and Guttman, D.S. (2011) Evolution of plant pathogenesis in Pseudomonas syringae: a genomics perspective. Annu. Rev. Phytopathol., 49, 269-289. Ouzari, H., Khsairi, A., Raddadi, N., Jaoua, L., Hassen, A., Zarrouk, M., Daffonchio, D. and Boudabous, A. (2008) Diversity of auxin-producing bacteria associated to Pseudomonas savastanoi-induced olive knots. J. Basic Microbiol., 48, 370-377. Palm, C.J., Gaffney, T. and Kosuge, T. (1989) Cotranscription of genes encoding indoleacetic-acid production in Pseudomonas syringae subsp. savastanoi. J. Bacteriol., 171, 1002-1009. Parkinson, N., Bryant, R., Bew, J. and Elphinstone, J. (2011) Rapid phylogenetic identification of members of the Pseudomonas syringae species complex using the rpoD locus. Plant Pathol., 60, 338-344. Penyalver, R., García, A., Ferrer, A., Bertolini, E. and López, M.M. (2000) Detection of Pseudomonas savastanoi pv. savastanoi in olive plants by enrichment and PCR. Appl. Environ. Microbiol., 66, 2673-2677. Penyalver, R., García, A., Ferrer, A., Bertolini, E., Quesada, J.M., Salcedo, C.I., Piquer, J., PérezPanadés, J., Carbonell, E.A., del Río, C., Caballero, J.M. and López, M.M. (2006) Factors affecting Pseudomonas savastanoi pv. savastanoi plant inoculations and their use for evaluation of olive cultivar susceptibility. Phytopathology, 96, 313-319. Pérez-Martínez, I., Rodríguez-Moreno, L., Matas, I.M. and Ramos, C. (2007) Strain selection and improvement of gene transfer for genetic manipulation of Pseudomonas savastanoi isolated from olive knots. Res. Microbiol., 158, 60-69. Pérez-Martínez, I., Zhao, Y., Murillo, J., Sundin, G.W. and Ramos, C. (2008) Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids. J. Bacteriol., 190, 625-635. Pérez-Martínez, I., Rodríguez-Moreno, L., Lambertsen, L., Matas, I.M., Murillo, J., Tegli, S., Jiménez, A.J. and Ramos, C. (2010) Fate of a Pseudomonas savastanoi pv. savastanoi type III secretion system mutant in olive plants (Olea europaea L.). Appl. Environ. Microbiol., 76, 3611-3619. Quesada, J.M., García, A., Bertolini, E., López, M.M. and Penyalver, R. (2007) Recovery of Pseudomonas savastanoi pv. savastanoi from symptomless shoots of naturally infected olive trees. Int. Microbiol., 10, 77-84. Quesada, J.M., Pérez-Martínez, I., Ramos, C., López, M.M. and Penyalver, R. (2008) IS53: an insertion element for molecular typing of Pseudomonas savastanoi pv. savastanoi. Res. Microbiol., 159, 207-215. Quesada, J.M., Penyalver, R., Pérez-Panadés, J., Salcedo, C.I., Carbonell, E.A. and López, M.M. (2010a) Dissemination of Pseudomonas savastanoi pv. savastanoi populations and subsequent appearance of olive knot disease. Plant Pathol., 59, 262-269. Quesada, J.M., Penyalver, R., Pérez-Panadés, J., Salcedo, C.I., Carbonell, E.A. and López, M.M. (2010b) Comparison of chemical treatments for reducing epiphytic Pseudomonas savastanoi pv. savastanoi populations and for improving subsequent control of olive knot disease. Crop Prot., 29, 1413-1420. Rico, A. and Preston, G.M. (2008) Pseudomonas syringae pv. tomato DC3000 uses constitutive and apoplastinduced nutrient assimilation pathways to catabolize nutrients that are abundant in the tomato apoplast. Mol. Plant-Microbe Interact., 21, 269-282. Rico, A., McCraw, S.L. and Preston, G.M. (2011) The metabolic interface between Pseudomonas syringae and plant cells. Curr. Opin. Microbiol., 14, 31-38. Rodríguez-Moreno, L., Barceló-Muñoz, A. and Ramos, C. (2008) In vitro analysis of the interaction of Pseudomonas savastanoi pvs. savastanoi and nerii with micropropagated olive plants. Phytopathology, 98, 815822. Rodríguez-Moreno, L., Jiménez, A.J. and Ramos, C. (2009) Endopathogenic lifestyle of Pseudomonas savastanoi pv. savastanoi in olive knots. Microb. Biotechnol., 2, 476-488. Rodríguez-Palenzuela, P., Matas, I., Murillo, J., LópezSolanilla, E., Bardaji, L., Pérez-Martínez, I., RodríguezMoskera, M.E., Penyalver, R., López, M.M., Quesada, J.M., Biehl, B.S., Perna, N.T., Glasner, J.D., Cabot, E.L., Neeno-Eckwall, E. and Ramos, C. (2010) Annotation and overview of the Pseudomonas savastanoi pv. savastanoi NCPPB 3335 draft genome reveals the virulence gene complement of a tumour-inducing pathogen of woody hosts. Environ. Microbiol., 12, 16041620. Rojas, A.M., García de los Rios, J.E., Saux, M.F.-L., Jimenez, P., Reche, P., Bonneau, S., Sutra, L., Mathieu-Daudé, F. and McClelland, M. (2004) Erwinia toletana sp. nov., associated with Pseudomonas savastanoi-induced tree knots. Int. J. Syst. Evol. Microbiol., 54, 2217-2222. Sarkar, S.F. and Guttman, D.S. (2004) Evolution of the core genome of Pseudomonas syringae, a highly clonal, endemic plant pathogen. Appl. Environ. Microbiol., 70, 1999-2012. Schneider, D.J. and Collmer, A. (2010) Studying plantpathogen interactions in the genomics era: beyond molecular Koch's postulates to systems biology. Annu. Rev. Phytopathol., 48, 457-479. Schroth, M.N., Osgood, J.W. and Miller, T.D. (1973) Quantitative assessment of the effect of the olive knot disease on olive yield and quality. Phytopathology, 63, 1064-1065. Scortichini, M., Rossi, M.P. and Salerno, M. (2004) Relationship of genetic structure of Pseudomonas savastanoi pv. savastanoi populations from Italian olive trees and patterns of host genetic diversity. Plant Pathol., 53, 491-497. Silverstone, S.E., Gilchrist, D.G., Bostock, R.M. and Kosuge, T. (1993) The 73-kb pIAA plasmid increases Pseudomonas savastanoi pv. savastanoi competitive fitness of Pseudomonas syringae subspecies savastanoi in oleander. Can. J. Microbiol., 39, 659-664. Sisto, A., Cipriani, M.G. and Morea, M. (2004) Knot formation caused by Pseudomonas syringae subsp. savastanoi on olive plants is hrp-dependent. Phytopathology, 94, 484-489. Sisto, A., Cipriani, M.G., Tegli, S., Cerboneschi, M., Stea, G. and Santilli, E. (2007) Genetic characterization by fluorescent AFLP of Pseudomonas savastanoi pv. savastanoi strains isolated from different host species. Plant Pathol., 56, 366-372. Smith, E.F. (1920) Bacterial diseases of plants. Philadelphia: W.B. Saunders Company. Studholme, D.J. (2011) Application of high-throughput genome sequencing to intrapathovar variation in Pseudomonas syringae. Mol. Plant Pathol., 12, 829-838. Sundin, G.W. (2007) Genomic insights into the contribution of phytopathogenic bacterial plasmids to the evolutionary history of their hosts. Annu. Rev. Phytopathol., 45, 129151. Surico, G. (1977) Osservazioni istologiche sui tubercoli della “rogna” dell’Olivo. Phytopathol. Mediterr., 16, 109125. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol., 28, 2731-2739. Tegli, S., Cerboneschi, M., Libelli, I. and Santilli, E. (2010) Development of a versatile tool for the simultaneous differential detection of Pseudomonas savastanoi pathovars by end point and real-time PCR. BMC Microbiol., 10, 156. Temsah, M., Hanna, L. and Saad, A.T. (2008) Anatomical pathogenesis of Pseudomonas savastanoi on olive and genesis of knots. J. Plant Pathol., 90, 225-232. Teviotdale, B.L. and Krueger, W.H. (2004) Effects of timing of copper sprays, defoliation, rainfall, and inoculum concentration on incidence of olive knot disease. Plant Dis., 88, 131-135. Urata, M., Miyakoshi, M., Kai, S., Maeda, K., Habe, H., Omori, T., Yamane, H. and Nojiri, H. (2004) Transcriptional regulation of the ant operon, encoding two-component anthranilate 1,2-dioxygenase, on the carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10. J. Bacteriol., 186, 6815-6823. Weingart, H., Völksch, B. and Ullrich, M.S. (1999) Comparison of ethylene production by Pseudomonas syringae and Ralstonia solanacearum. Phytopathology, 89, 360-365. Young, J.M. (2004) Olive knot and its pathogens. Australas. Plant Pathol., 33, 33-39. Young, J.M. (2010) Taxonomy of Pseudomonas syringae. J. Plant Pathol., 92, S5-S14. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at: http://onlinelibrary.wiley.com/wol1/doi/10.1111/j.1 364-3703.2012.00816.x/suppinfo Fig. S1 Evolutionary relationships of P. savastanoi pv. savastanoi and selected P. syringae pathovars. Tree was constructed by multilocus sequence analysis using a concatenated data set (exactly 12000 nt) of acnB, fruK, gapA, gltA, gyrB, pgi, recA and rpoD genes. Phylogenetic groups 1, 2, 3 and 4 (Sarkar & Guttman, 2004, Studholme, 2011) correspond to genomospecies (Gsp) 3, 1, 2 and 4 (Gardan et al., 1999), respectively. Sequence alignment using Muscle, determination of the optimal nucleotide substitution model and phylogenetic tree construction were done using MEGA5 (Tamura et al., 2011); all positions containing gaps and missing data were eliminated using the option of complete deletion. Bootstrap values (1,000 repetitions) are shown on branches. Similar or identical topologies were obtained by maximum likelihood. The scale bar represents nucleotide substitutions per site. Table S1 Primers used for the detection of Pseudomonas savastanoi pv. savastanoi. Table S2 Comparison of the deduced products of iaaM-1 (PSA3335_1475) and iaaH-1 (PSA3335_1476), from Pseudomonas savastanoi pv. savastanoi NCPPB 3335, with their homologues in selected organisms. Table S3 Accession numbers and coordinates of the nucleotide sequences used for the construction of the neighbour-joining tree shown in Fig. 2.