detection and identification methods and new tests as used ... - SIPaV

019_COST(Kaluzna)_S117_COL

15-06-2012

13:07

Pagina 117

Journal of Plant Pathology (2012), 94 (1, Supplement), S1.117-S1.126

Edizioni ETS Pisa, 2012

S1.117

DETECTION AND IDENTIFICATION METHODS AND NEW TESTS AS USED AND DEVELOPED IN THE FRAMEWORK OF COST 873 FOR BACTERIA PATHOGENIC TO STONE FRUITS AND NUTS

Pseudomonas syringae pathovars M. Kaluzna1, J.D. Janse2 and J.M. Young3 1 Research

Institute of Pomology and Floriculture, Department of Plant Pathology, Pomologiczna 18., Skierniewice, Poland 2 Department of Laboratory Methods and Diagnostics, Dutch General Inspection Service, PO Box 1115,8300 BC Emmeloord, The Netherlands 3 49 Allendale Rd, Mt Albert, Auckland 1025, New Zealand

SUMMARY

Pseudomonas syringae is responsible for causing diseases on over 180 plant species including fruit trees, vegetable crops and flowers. Pathovars of main economic importance in Europe are the pvs syringae, morsprunorum, avii and persicae, causing bacterial canker on sweet and sour cherry, plum, peach and apricot as well as in wild cherry. In the framework of COST873 methods and techniques as developed and used in different laboratories for detection and identification have been tested and partly validated in several training schools. They are compiled in this contribution. The methods used for diagnosis and discrimination of pathovars include, among the others, the biochemical tests LOPAT and GATTa, the analysis for production of the phytotoxins coronatine and syringomycin and the siderophore yersiniabactin as well as a pathogenicity test. Molecular tests described concern PCR for phytotoxins, rep-PCR, including BOX, ERIC, REP and IS50, multilocus sequence typing (MLST) and the quite new melting profile PCR (PCR MP). All enable distinction of the pathogens and determination of homogeneous or heterogeneous groups within pathovars and pathovar’s races. Key words: Pseudomonas, syringae, morsprunorum, bacterial canker, diagnosis, diversity.

INTRODUCTION

Pseudomonas rRNA group I (Holt et al., 1994; Palleroni, 1984, 2005). The P. syringae complex (Young, 2010) comprises plant pathogens in eight closely related Pseudomonas spp., including 64 pathovars in P. syringae. These were divided into genomospecies determined by DNA:DNA hybridization (Gardan et al., 1999). P. syringae infecting Prunus spp. include five different pathogens; (i) P. syringae pv. syringae (P.s. syringae) and (ii) P.s. morsprunorum race 1 (Psm1), belonging to genomospecies 1 and 2 respectively; (iii) P.s. morsprunorum race 2 (Psm2); (iv) P.s. avii (Psa) and (v) P.s. persicae (Psp), all three belonging to genomospecies 3. Since then, genomospecies based on multi-locus sequence analysis (MLSA) have been recommended (Stackebrandt et al., 2002) as forming the basis of species, but requiring also comprehensive phenotypic descriptions for formal proposals. Until now further differentiation/reclassification has been difficult and is perhaps impossible to achieve (Gardan et al., 1999; Young, 2010). Training programmes on bacterial canker in stone fruits and on the P. syringae pathovars is an activity of COST 873 that includes the theoretical and practical aspects of classical and molecular diagnostic tests, pathogenicity tests, systematics and phytosanitary issues. Summaries of these programmes and the background (instruction) material can be found on the COST 873 website (www.cost873.ch) under “Activities of Workshops” held in York in 2008, in Belgrade in 2010 and again in Belgrade in 2011. P. syringae pathovars, except for P. syringae pv. persicae, are not quarantine pathogens for the EU or EPPO. No diagnostic protocols exist and symptomatology of the diseases is complex. A description of symptoms and of detailed differential diagnostic steps is included in this contribution.

Pseudomonas syringae is a member of the genus Pseudomonas classified into the Gamma Proteobacteria (Kersters et al., 1996). Strains of P. syringae are Gramnegative, aerobic, motile, straight or slightly curved rods with one or several polar flagella. They belong to the

HOST RANGE

Corresponding author: M. Kaluzna Fax: +48.468345375 E-mail: [email protected]

With the exception of P. syringae pv. syringae, pathovars belonging to P. syringae are usually specific to small numbers of host species and genera (Radbury, 1986; Agrios, 2005).


15-06-2012

13:07

Pagina 118

S1.118 P. syringae pathovars diagnosis and identification methods Journal of Plant Pathology (2012), 94 (1, Supplement), S1.117-S1.126

• Pseudomonas syringae pv. avii: Prunus avium (wild cherry), Ménard et al., 2003. • Pseudomonas syringae pv. morsprunorum: Prunus spp., P. amygdalus (almond), P. armeniaca (apricot), P. avium (sweet and sour cherry), P. cerasifera (P. divaricata), P. cerasoides (P. puddum), Prunus domesticum (plum), P. institia, P. persica, P. pissardi, and P. triloba (Bradbury, 1986). • Pseudomonas syringae pv. persicae: Prunus cerasifera [myrobalan plum (Garrett, Crosse, 1987; Young et al., 1996)], P. persicae (peach) and P. persica var. nucipersica (nectarine) and P. salicina (Japanese plum) (Young, 1988; OEPP/EPPO, 2005). • Pseudomonas syringae pv. syringae has a very wide host range (Bradbury, 1986), which includes the following Prunus spp.: Prunus amygdalus (P. dulcis), P. armeniaca, P. avium, P. cerasifera (P. cerasijera var. divaricata), P. cerasus, P. domestica, P. laurocerasus, P. mahaleb, P. mume, P. persica, P. persica var. nectarina, P. pumila and P. salicina.

SYMPTOMS AND ISOLATION

Field symptoms. P.s. syringae/P.s. morsprunorum. Leaves show small round lesions, light brown changing to dark brown, sometimes surrounded by a yellowish halo in spring. Necrotic spots dehisce causing a characteristic ‘shot-hole’ effect. Gumming cankers and bud die-back occur in spring infections and are confined to nodes. Successful isolation of pathogens can only be made in spring. Blossoms become brown, dry out and often die. Sunken brown-black, irregular or regular lesions occur on immature fruits. Successful isolations can only be made in spring. P.s. persicae. Small irregular, watery brown spots occur on the leaves, and large, regular, oily dark spots on the fruits. Stem damage with cankers and necroses occur on internodes. Gummosis is not usually observed. P.s. avium. Bark cankers form along the trunk and branches with gum exudation (preliminary observations; Ménard et al., 2003). In summer, the branches and entire apricot, peach and nectarine trees wither suddenly. Dissection at the base of the affected organs reveals girdling cankers. These may express the characteristic ‘sour sap’ of longdead tissue. Pathogenic bacteria populations cannot be isolated from cankers, which may be more that one season old. Peach tree short life (PTSL) in the United States (Anonymous, 1978; Yavada and Doud, 1980; Beckman and Nyczepir, 2004), ‘déperissement’ of peach and nectarine in France (Vigouroux and Blache, 1967; Luisetti et al., 1976), ‘decline’ of peach, nectarine and Japanese plum in New Zealand (Young 1987a, 1988), ‘apoplexy’ of apricot in Hungary (Klement et al., 1972,

1974) are the result of infection by particular pathovars that cause this common syndrome. All stone fruits may be subject to the same syndrome induced by one or another pathovar in the right conditions. Always associated with this syndrome are clay pans (USA) or shallow soils over gravels in France (L. Gardan, personal communication), Hungary (S. Süle, personal communication) and New Zealand (Young, 1987b). Shallow soils result in an anaerobic layer forming at the soilgravel or soil-pan interface which kills only the lower root layers so that a superficial investigation does nor reveal the extent of the damage. The syndrome is exacerbated in soils with a low pH and if water sprinklers are used for frost-fighting. Tree growth appears to continue, often for more that one season until a gross imbalances between root and branch development results in a water imbalance leading to a mid-summer collapse of the limb or the whole tree. The cankers are the result of the pathogenic activity that first develops in autumn, reaches a maxinimum in mid-winter and declines in early spring. The severity of infection is dependent on frosts during the infection phase. An extensive range of pictures on symptoms can be found at http://www.cost873.ch/5_activites/meeting_detail.php?ID=24 (contribution by J.M. Young, Cetara, Italy, 2009). Plant specimens. Successful diagnosis of bacterial pathogens depends on processing plant specimens from which the pathogen can be obtained in pure culture. This requires that specimens be received in the laboratory in a fresh condition and be processed expeditiously. Specimens received from individuals who are unaware of appropriate requirements for sample collection commonly exhibit over-developed symptoms from which isolations are difficult or impossible. Success is most likely from specimens in which there are the earliest manifestations of infection. If specimens are shipped by post, then they should be wrapped in moist paper to maintain evaporative cooling during transport. Preliminary examination. Close inspection of specimens and symptom recording may, in some cases, allow for a reliable identification though, in cases such as quarantine control or a new record, formal confirmation is necessary. Preliminary confirmation of bacterial presence can be done by examining wet mounts of tissue using a phase contrast microscope at about 200X. If left for 15-20 min prior to examination, masses of bacteria will usually merge from the exudation at the cut edges of lesions. Motility can be stimulated by mounting in nutrient broth. Tissue. Leaves, stems and fruit. Lesions to select should be those with oily, water-soaked margins, if present. Isolations should always be from the most immature lesions, or their outermost margins, avoiding inclu-


15-06-2012

13:07

Pagina 119


sion of brown, necrotic, saprobe-containing tissue. Secondary woody tissue. Sampling from woody tissue is complicated because the active margin of the lesion is not usually visible externally. By the time when visible symptoms of disease are expressed, as sunken tissue over the lesion, or cankers, or wilting, the disease may be fully developed, tissue are heavily necrotized and populations of the pathogen disappeared. Pathogen activity must be discovered by dissection. Pathogens expressing wilt symptoms are usually present in parenchymas well in advance of visible necrosis. Root tissue. Prior to sampling, root tissue should be surface-sterilized by soaking samples freed of soil in 510% commercial hypochlorite (3% active chlorine) for 15 min or more if necessary, followed by rinsing briefly in clean or sterile water. Primary isolation. Tissue fragments are excised at the margin of the healthy and diseased tissue and crushed in a drop of distilled sterile water or PBS buffer (0.27% Na2HPO4, 0.04% NaH2PO4, 0.8% NaCl) in a Petri dish lid. A flat-ended glass rod will crush the most slippery tissue. After 20 min, the suspension should be plated using a glass spreader, on a surface-dried medium that is then incubated at 25-27ºC for 2-3 days. Use of King’s medium B (King et al., 1954) is advisable because some differentiation can be made based on the form of developing colonies. Sugar-containing media should be avoided because they produce mucoid, spreading colonies and favor fungal contamination. Examination of plates. Most strains belonging to the genus Pseudomonas produce fluorescent pigments on iron-deficient media, e.g. King’s medium B under UV light (Cody and Gross, 1987). P.s. syringae and some P.s. morsprunorum race 2 strains are fluorescent, while most strains of P.s. morsprunorum race 1, P.s. persicae and P.s. avii are non-fluorescent on KB. The first examinations of plates should be made after 24 h. At this stage, visible colonies are invariably saprobes because pathogenic bacteria, with the possible exception of some soft-rotting bacteria, are slow-growing. Colonies of pathogens first appear after 36-48 h. P. syringae strains produce a blue-green or ‘electric-blue’ fluorescence while the fluorescence of fast-growing saprophytes is leaf-green. The presence of large numbers of saprobes, or the presence of more than 3-4 colony types, is a warning that the tissue has been invaded by secondary organisms, that pathogenic populations are in decline or have disappeared, and that identification of pathogens may be extremely difficult if not impossible. Incubation should continue until a population becomes visible as almost pure. Colonies of Pss and Psm are visible after 2 days but Psp and Psa grow more slowly after 3-4 days on King’s Medium B. All pseudomonad pathogens are visible after 72 h. As soon as colonies of putative pathogens

Kaluzna et al. S1.119

are distinguished, representative single colonies should be restreaked to confirm purity and sub-cultured onto storage media.

DIAGNOSIS

Diagnosis can be initiated using a relatively small number of tests, based on the information available. It is commonly recommended that the first step in diagnosis should be testing for Gram reaction. However, because most of the common pathogens are Gram- negative, this test does not usually reduce possible candidates significantly. A more direct method is to consider colony characteristics, then, for fluorescent pseudomonads, the test series outlined below. At every stage in diagnosis, symptoms and host range should be reconsidered as indicating potential target pathogens. Culture media. King’s Medium B (King et al., 1954). Protease peptone No.3 20 g; K2HPO4 1.5 g, MgSO4x7H2O 1.5 g; glycerol 15 ml, agar 15 g, de-ionized or distilled water 1000 ml. Modified King’s Medium B for electric blue fluorescence by P.s. persicae (L. Gardan, personal communication). Proteose peptone No.3 20 g; MgSO4.7H2O 1.5 g; K3PO4 1.8 g; Glycerol 10 ml; Agar 15 g; distilled water 1000 ml. Adjust to pH 7.2. Alternatively Pseudomonas agar F (Difco, USA) can be used when many saprophytes are expected to be present in the sample: Pseudomonas agar F 38 g, glycerol 10 ml. Sterilization for 20 min at 121°C. Medium 2 agar (modified nutrient agar), (Bultreys and Gheysen, 1999). Yeast extract 2 g, peptone 5 g, NaCl 5 g, KH2PO4 0.45 g, Na2HPO4 0.96 g, agar 8 g; deionized or distilled water 1000 ml. Adjust pH to 7.0 with NaOH and sterilize for 20 min. YPGA for P.s. avii (Ménard et al., 2003). Yeast extract 3 g, peptone 5 g, glucose 5 g, agar 15 g, de-ionized or distilled water 1000 ml. Adjust to pH 7.2. R2A medium (Reasoner and Geldreich, 1985). Proteose peptone, 0.5 g; casamino acids 0.5 g, yeast extract 0.5 g, glucose 0.5 g, soluble starch 0.5 g, K2HPO4 0.3 g, MgSO4x7H2O 0.05 g; sodium pyruvate 0.3 g; agar 15 g. Final pH 7.0.

Long term storage of isolates. Storage of bacteria can be performed as follows: (i) Preservation by freeze-drying and keeping at -4 to -6°C (Benedict et al., 1961; Wagman and Weneck, 1963) or by vacuum-drying (Fletcher and Young, 1997, 1998); (ii) Collected isolates washed from media are stored in a mixture of PBS buffer and glycerol (4:1, v/v). Glycerol should be separately prepared (200 µl) in an 1.5 ml Eppendorf tube and autoclaved. Wash with


15-06-2012

13:07

Pagina 120

S1.120 P. syringae pathovars diagnosis and identification methods Journal of Plant Pathology (2012), 94 (1, Supplement), S1.117-S1.126 Table 1. Identification of some green fluorescent Pseudomonas species by the LOPAT scheme (Lelliott, 1966). LOPAT group Ia

Levan formation +

Oxidase reaction -

Potato rot capability -

Arginine dihydrolase -

Tobacco hypersensitivity +

Species

Ib

-

-

-

-

+

II III IVa IVb Va

-/+ + -

+ + + +

+ + + -

+ + +

+ + -

Vb

+

+

-

+

-

P. syringae pv. savanastoi; P. delphini P. viridiflava P. cichorii P. marginalis P. fluorescens P. tolaasii; saprophytic pseudomonads P. fluorescens; saprophytic pseudomonads

PBS a 48-hour-old bacterial colony from the culture medium and add 800 µl of the suspension to 200 µl glycerol. Vortex to create a homogeneous suspension and store at -75°C. For safety prepare bacteria for storage in two separate tubes. Viability should be checked by plating the bacterial suspension onto a fresh medium.

Fig. 1. Growth of characteristic, Levan-positive, mucoid, convex (dome shaped), shiny colonies on Nutrient Agar with 5% of sucrose of strain RIPF 110 of Pseudomonas syringae pv. syringae, 2 days after inoculation (Photo: M.Kaluzna).

P. syringae

Biochemical tests. Identification tests that proved very useful for the identification of P. syringae patovars are the so-called LOPAT tests, i.e. levan production (polysaccharide) on nutrient agar media supplemented with 5% sucrose (Fig. 1); oxidase reaction (negative for P. syringae), potato rot (variable), production of arginine dehydrolase (negative) and hypersensitivity reaction on tobacco (positive) (see Table 1). Many strains of P. syringae produce a yellow to green to blue diffusible pigment on certain iron-deficient media e.g. King’s medium B, which is especially suitable for demonstrating the production of the siderophore pyoverdin, strongly fluorescent under UV light (Cody and Gross, 1987). The bacterial canker agents Pss and some strains of Psm 2 are fluorescent, almost all strains of Ps morsprunorum race 1, pathovars persicae and avii are not fluorescent. Optimum temperature for growth of these bacteria ranges from 25 to 28°C. For species determination LOPAT tests are used according to Lelliott and Stead (1987). (i) Levan production from sucrose (L): inoculate Petri dishes containing Nutrient Agar (Difco, USA) supplemented with 5% sucrose with bacteria and incubate at 26-28°C for 24-48 h. Formation of characteristic mucoid, convex (dome-shaped), shiny, pearly- white colonies demonstrates levan production (Fig. 1). (ii) Presence of oxidase (O): transfer bacteria grown for 24-48 h on King’s medium B with a sterile tip on a filter paper soaked in 1% w/v solution of N,N,N’,N’- tetramethyl-p-phenylenediamine dihydrochloride. The appearance of a violet colour in less than 10 seconds indicates a positive result. As aerobic bacteria, all pseudomonads have the glycolytic oxidative pathway. Close inspection of ‘oxidase-negative’ results shows that there is a reductive, bleaching reaction that over-rides the sign of oxidase activity.


15-06-2012

13:07

Pagina 121


(iii)Ability to cause rot on potato tubers (P) and showing pectolytic activity. Bacteria grown for 24-48h on King’s medium B are stab-inoculated in the centre of 1-inch diameter sterile potato slices placed in a sterile Petri dish on moist sterile filter paper. The appearance of softness and soft rot on potato slices after 24-48 hours of incubation at 26-28°C confirms pectolytic ability. Because the quality of stored potatoes is variable, some with incipient soft-rot, it is essential to include negative controls for every tuber. (iv) Presence of arginine dihydrolase (A) is checked by introducing bacteria grown for 24-48h on King’s medium B into two tubes of a semi-liquid medium containing arginine 0.01% peptone, 0.5% NaCl, K2HPO4, 0.001% phenol red, L (+) arginine, 1% agar). One tube is covered with sterile mineral oil after inoculation. Variations in colour, from orangered to magenta-amarant after 2-5 days in the anaerobic tube prove the positive reaction. (v) Hypersensitive reaction (HR) on tobacco plants (T). Introduce a thick suspension (ca. 108 cells.ml-1) of bacteria grown for 24-48 h on King’s medium B into the mesophyll of a fully grown tobacco leaf (e.g. cv. Samsun). Rapid glassy to white necrosis of the infiltrated area 24 h after inoculation indicates the ability to induce a hypersensitivity reaction. For distinguishing pathovars within P. syringae the so called GATTa tests (Lelliot and Stead, 1987) and the Llactate utilisation test are commonly used. (i) Gelatine hydrolysis: introduce a 24-h-old bacterial culture into a tube with solidified medium containing 0.3% yeast extract, peptone 0.5%, gelatine 12%. Characteristic liquefaction of gelatine after 714 days incubation at 18°C, is the indication of a positive result (Lelliott and Stead, 1987). (ii) Aesculin hydrolysis (A): bacteria grown for 24-48h on NA are inoculated into a semi-solid medium containing peptone 1%, esculine 0.1%, 0.05% ferric citrate, agar 2%. Brown colour of the medium after 24-48 h incubation at 26-28°C proves presence of the β-glucosidase enzyme. (iii)Tyrosinase activity (T): inoculate bacteria grown for 24-48 h on NA into a semi-solid medium containing 0.5% sucrose, 1% casamino acid, L-tyrosine 0.1%, 0.05% potassium phosphate, magnesium sulphate heptahydrate 0.0125%, agar 2%, pH 7.2. A colour change to red of the medium after 7-10 days incubation at 26-28°C shows the presence of tyrosinase. (iv) Tartrate (Ta) utilization: inoculate bacteria grown for 24-48 h on NA into a liquid medium pH 7.0 containing 0.1% ammonium dihydrogen phosphate, potassium chloride 0.02%, magnesium sulfate heptahydrate 0.02%, 1 ml of 4% alcohol bromothymol blue solution. A colour change of the medium from green to blue is a positive test result (Lelliott and Stead, 1987)


(v) L-lactate utilization test: bacteria grown for 24-48 h on NA are inoculated into a semi-solid medium containing ammonium diphosphate 0.1%, 0.02% potassium chloride, magnesium sulfate 0.02%, 0.004% alcoholic solution of bromothymol blue, 2% agar and 1% lactate. A positive result is indicated by a change in colour of the medium from yellow green to blue (Lattore and Jones, 1979). Other features. Ability to produce syringomycin. It is a reliable method for the identification of all pathovars in the “genomospecies” syringae and can be tested by biological and molecular methods. In the first case, syringomycin production is commonly evaluated on PDA medium by checking the ability of isolates to inhibit the yeast Rhodotorula pilimanae MUCL 30397 or the fungus Geotrihum candidum MUCL 31566 (Bultreys and Gheysen, 1999) or Aspergillus niger (Hu et al., 1998). The simplest method is to reactivate a faintly turbid suspension of commercial baker’s yeast and spread it on surface-dried PDA or other fungal medium, followed by loops of bacterial culture on the plate. Clear zones will appear around bacteria after 24-48 h (Fig. 2). For molecular determination, detection by PCR of the genes syrB and syrD (Sorensen et al., 1998; Takemoto, 1992) encoding syringomycin production is carried out. In the course of the COST project it was found that a slight modification of the annealing temperature/time to 71°C for 45 sec gave better results for syrB detection. Ability to produce the yersiniabactin and/or coronatine. For detection of the genes responsible for coronatine and yersiniabactin production, two primer pairs are available and useful. For coronatine, primers cfl1 and cfl2 are used for amplification, yielding a specific 650bp product (Berenswill, 1994). Primers for

Fig. 2. Inhibition of growth of yeast Rhodotorula pilimanae MUCL 30397 on peptone-glucose-NaCl agar medium by strain RIPF 227 of P. syringae 2 days after seeding the plate with the yeast cells (Photo: M. Kaluzna)


15-06-2012

13:07

Pagina 122

S1.122 P. syringae pathovars diagnosis and identification methods Journal of Plant Pathology (2012), 94 (1, Supplement), S1.117-S1.126

Fig. 3. Symptoms on immature sweet cherry fruits of cv. Napoleon 4 days after inoculation with Pseudomonas syringae strains. From left: fruits inoculated with strains of Pss RIPF 68 (black-brown sunken necroses), fruits inoculated with strains of Psm (races 1 and 2; brownish water soaked superficial lesions), fruits inoculated with sterile water (negative control) (Photo: M. Kaluzna).

yersiniabactin detection are PSYE2 and PSYE2R which amplify a product of 943bp (Bultreys et al., 2006). Amplifications were conducted according to the methods reported by the above-mentioned authors with slight modifications, i.e annealing temperature of 70°C for 45 sec for cfl primers and of 66°C for 45 sec for PSYE primers. Demonstration of the presence of persicomycin. This is an exotoxin produced only by strains of pv. persicae (Ballio et al., 1994; Barzic and Guittet, 1996) Presence of ice nucleation activity (INA). Many P. syringae patovars are INA-positive for they freeze supercooled liquids (Maki et al., 1974; Arny et al., 1976; Paulin and Luisetti, 1978; Lindow et al., 1982; Kozloff, 1983). A simple method for INA detection involves the preparation of a bath of saturated salt (NaCl) and addition of ice until the temperature is lowered to about -10°C. Bacterial suspension added to pre-cooled, clean

test tubes placed in the bath will produce ice nuclei through the tube (and therefore freezing of the suspension) almost instantly. Serological tests. These have rarely been used and often present cross-reactions between pathovars (Garrett et al., 1966; Latorre and Jones, 1979; Zamze et al., 1986; Vicente et al., 2004). Control strains that express positive and negative reactions should always be included in all phenotypic tests.

DNA-BASED TECHNIQUES

DNA isolation. DNA can be extracted from bacterial suspensions from 24 to 48-hour-old colonies growing on King’s medium B heated at 95°C for 10-15 min, then cooled on ice for 5 min and freed from cell debris by centrifuging at 14,000 rpm for 10 min.

Table 2. Discrimination of the P. syringae pathovars syringae (Pss), morsprunorum (Pmp) race 1, morsprunorum race 2 pv. avii (Psa) and persicae (Psp) by the GATTa tests and other tests (Crosse and Garrett, 1966; Schaad, 2001; Vicente and Roberts, 2007; Ménard et al., 2003; Gilbert et al., 2009; Young, 1987). Test Fluorescence on King’s Medium B Fluorescence on CSGM Gelatin hydrolysis (G) Aesculin hydrolysis (A) Tyrosinase activity (T) Utilization of tartaric acid L (+) (Ta) Utilization of lactic acid Syringomycin production Ice nucleation activity

Pss + + + + + + +

Psm race 1 + + + -

Psm race 2 -/+ + + -/+ -/±/+ -/+ -

Psa nd + nd nd -

Psp + nd nd +


15-06-2012

13:07

Pagina 123


Commercial kits for DNA extraction can also be used, according to manufacturer instructions. DNA isolation without centrifugation steps, using columns and gravitation principles, avoiding damage of DNA e.g. the Genomic Mini AX bacteria isolation kit (as realised in A&A Biotechnology, Poland) is used in the Polish laboratory. A modified Aljanabi and Martinez (1997) method was successfully used in the Polish laboratory. Suspend loopful of a 24-48 h King’s medium B culture in 500 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), vortex and centrifuge for 5 min at 5,000 rpm. Suspended the pellet in 400 µl extraction buffer (400 mM NaCl, 10 mM Tris HCl, 1 mM EDTA, pH 8.0). Add 8 µl of Proteinase K (20 mg ml-1) and 40 µl of 20% SDS solution and mix thoroughly. Incubate at 55-60°C for 1.5 h, then transfer for 5 min into ice, adding during this incubation 360 µl of 5M NaCl. Vortex for 30 sec and centrifuge for 30 min at 14,000 rpm. Transfer the supernatant to a new tube, add 1 µl of RNAse (10 mg ml-1) and incubate for 1 h at 37°C in a water bath. Precipitate DNA by adding 650 µl of cold (-20°C) isopropanol. Centrifuge (15,000 rpm for 30 min), wash pellet with 70% ethanol, centrifuge again (15,000 rpm or 5 min) remove ethanol, dry in a vacuum concentrator (Eppendorf, Germany) and dissolve in 200 µl TE buffer. Repetitive PCR. Genetic fingerprints of P. syringae patovars can be obtained by analysing repetitive elements via rep-PCR, using ERIC1R and ERIC2, BOX, IS50 and/or REP1R and REP2I primers (Louws et al., 1994; Weingart and Völksch, 1997; Versalovic et al., 1991, 1994). In the Polish laboratory the following protocol used separately for each set of primers is applied. The standard reaction mixture contain in the final concentration: 1X Dream Taq Green buffer (Fermentas, Lithuania) for ERIC, BOX and REP, 1X Go Taq Flexi buffer™ Green (Promega, USA) for IS50, 1.25 mM deoxyribonucleoside triphosphate, 4.5 and 6.5 mM of MgCl2 for IS50, ERIC, BOX and REP, respectively, 60 pmol of each primer, 2U of polymerase and 40 ng of DNA. The following amplification conditions are applied: initial denaturation at 95°C for 7 min for ERIC, BOX and REP, and 3 min for IS50, 35 cycles at 94°C for 1 min, 40, 52, 53 and 38°C for 1 min for REP, ERIC, BOX and IS50 respectively, and 65°C for 8 min ERIC, BOX and REP and 72°C for 3.5 min for IS50, and the final elongation step of 65°C for 8 min and 72°C for IS50. The results of repetitive PCRs enable differentiation of strains at pathovar and race level for Pss, Psm1 and Psm2, Psa and Psp). In the case of strains within Pss, heterogeneity is quite high and rep-PCR cannot be used as the only method for identification. For P. syringae pathovars from stone fruit trees and nuts BOXPCR has been found by several laboratories participating in COST873 to be more suitable for determining


the genetic diversity of the strains in comparison with ERIC-PCR. Melting Profile PCR Required ingredients for melting profile PCR (Masny and Plucienniczak, 2003) are: total bacterial DNA (100-200 ng), restriction enzyme for DNA digestion e.g PstI, HindIII, two oligonucleotides binding to restriction sites, [for PstI – PstIa 5’TGTACGCAGTCTAC-3’ and PstIIa 5’-TCGTAGACTGCGTACATGCA-3’ Waugh et al. (1997); for HindIII – POWIE 5’-CTCACTCTCACCAACGTCGAC-3’ and HINLIG 5’-AGCTGTCGACGTTGG-3’ (Masny and Plucienniczak, 2003)] and one non selective primer (Pst0 5’-GACTGCGTACATGCAG-3’ and POWAGCTT primer 5’-CTCACTCTCACCAACGTCGACAGCTT-3’ for PstI and HindIII. In the Polish laboratory the following materials and conditions are used: 15 µl ligation mixture containing 8 µl of DNA digested according to manufacturer’s instructions, 5 mM of each of two specific oligonucleotides, 1X ligation buffer, 1 U of T4 DNA ligase (Invitrogen, USA) incubation at 37°C for 3 h followed by incubation for 20 min at 65°C. After ligation, products are diluted with H2O up to 30-50 µl. Amplifications are performed in a total volume of 15 µl. Amplification mixture consists of 1 µl of diluted ligation reaction products, 0.4 U of GoTaq DNA polymerase (Promega, USA), 1X GoTaq reaction buffer, 0.2 mM concentration of each dNTPs and 1 mM of nonselective primer. The PCR conditions are an initial step at 72ºC for 5 min, 30 cycles of denaturation at temperatures of 86.5ºC (for PstI) for 40 sec, 55ºC for 40 sec, 72ºC for 1.5 min, and the final step at 72ºC for 10 min. PCR products are separated on a 1.5% agarose gel with 0.5 TBE buffer and visualized by staining with (0.5 mg·l-1) ethidium bromide (Kaluzna et al., 2010b). Multi-locus sequence typing (MLST). This assay can be used for discrimination of strains and proved to be more specific than 16S rRNA sequence analysis especially when analysing a few of housekeeping genes of the bacterial core genome (Maiden et al., 1998; Enright and Spratt, 1999; Hwang et al., 2005; Wang et al., 2007; Sarkar and Guttman, 1999; Stackebrant et al., 2002). During the COST project primers for amplification of four housekeeping genes, rpoD, gyrB, gltA (also known as cts) and gapA were successfully used following the protocol of Sarkar and Guttman (2004). Small modifications of the PCR protocol were made, i.e. annealing temperature of 62ºC, 52°C, 73°C and 76ºC for gyrB, gapA, gltA and rpoD instead of 63ºC, 62°C, 56°C and 63ºC, respectively (Kaluzna et al., 2010a). As with rep-PCR, the PCR MP and MLST methods enable differentiation of strains according to pathovars and races. However due to heterogeneity of Pss these assays cannot be used as a single method for identification.


15-06-2012

13:07

Pagina 124

S1.124 P. syringae pathovars diagnosis and identification methods Journal of Plant Pathology (2012), 94 (1, Supplement), S1.117-S1.126 PATHOGENICITY TESTS

REFERENCES

In the Polish laboratory a pathogenicity test using sweet cherry fruitlets of cv. Napoleon was developed and it is employed routinely (Garrett et al., 1966; Endert and Ritchie, 1984; Sobiczewski et al., 1980; Kaluzna and Sobiczewski, 2009). Freshly collected immature sweet cherry fruits are disinfected by dipping in 50% ethanol for 3 min rinsed three times with sterile distilled water, and blotted dry with tissue paper. Ten fruitlets per bacterial strain are used. Each fruitlet is inoculated by pricking in two places to the depth of 2 mm with sterile needle previously immersed in a water suspension of a particular P. syringae strain (24-48 h culture grown on King’s B). After inoculation, fruitlets are immediately placed on moist filter paper in a sterile Petri dish and incubated at 22°C for 4 days. Reference strains and sterile distilled water should be included as positive and negative control, respectively. After 24, 48 and 96 h of incubation the symptoms developing around inoculation wounds are observed (Fig. 3). Their severity is evaluated separately for each wound, using scales depending on the type of symptoms. For details on the scoring of results see Sobiczewski et al. (1980) and Kaluzna and Sobiczewski (2009). Other tests on different host plants can also be used e.g. inoculation of lilac leaves and plantlets (Young, 1991; Vicente et al., 2004; Gilbert et al., 2009), sweet cherry leaf scars (Crosse and Garret, 1966; Freigoun and Crosse, 1975), wild cherry plantlets (Vicente et al., 2004; Liang et al., 1994), apricot leaves (Donmez et al., 2010) and lemon fruits (Garrett et al., 1966).

Aljanabi S.M., Martinez I., 1997. Universal and rapid salt extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research 25: 4692-4693. Anonymous, 1978. Managing peach tree short life in the Southwest. Agricultural Extension Services of Georgia, Alabama, North Carolina, South Carolina, and the United Stataes Department of Agriculture, Circular No. 585. Arny D.C., Lindow S.E., Upper C.D., 1976. Frost sensitivity of Zea mays increased by application of Pseudomonas syringae. Nature (London) 262: 282-284. Ballio A., Bossa F., Di Giorgio D., Ferranti P., Paci M., Pucci P., Scalonia A., Segred A., Strobel G.A., 1994. Novel bioactive lipodepsipeptides from Pseudomonas syringae: the pseudomycins. FEBS Letters 355: 96-100. Ballio A., Bossa F., Collina A., Gallo M., Iacobellis N.S., Paci M., Pucci P., Scaloni A., Segre A., Simmaco M., 1990. Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae. FEBS Letters 269: 377380. Barzic M-R., Guittet E., 1996. Structure and activity of persicomycins, toxins produced by a Pseudomonas syringae pv. persicae/Prunus persica isolate. European Journal of Biochemistry 239: 702-709 Beckman T.G., Nyczepir A.P., 2004. Peach tree short life. In: Horton D., Johnson D. (eds) Southeastern Peach Growers Handbook, pp. 199-205. University of Georgia, College of Agricultural and Environmental Sciences. Cooperative Extension Service, Athens, GA, USA. Bender C.L., 1999. Chlorosis-inducing phytotoxins produced by Pseudomonas syringae. Plant Pathology 105: 1-12. Bender C.L., 2004. Regulation of coronatine biosynthesis in Pseudomonas syringae. In: Iacobellis N.S., Collmer A., Hutcheson S.W., Mansfield J.W., Morris C.E., Murillo J., Schaad N.W., Stead D.E., Surico G., Ullrich M.S. (eds). Pseudomonas syringae and Related Pathogens. Biology and Genetics, pp 285-292. Kluwer Academic Publishers, Dordrecht, The Netherlands. Benedict R.G., Sharpe E.S., Corman J., Meyers G.B., Baer E.F., Hall H.H., Jackson R.W., 1961. Preservation of microorganisms by freeze-drying. II. The destructive action of oxygen. Additional stabilizers for Serratia marcescens. Experiments with other microorganisms. Applied Microbiology 9: 256-262. Bereswill S., Bugert P., Volksch B., Ullrich M., Bender C.L., Geider K., 1994. Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR. Analysis and sequence determination of the amplification products Applied and Environmental Microbiology 60: 2924-2930. Bradbury J.F., 1986. Guide to Plant Pathogenic Bacteria. CAB International Mycological Institute, Kew, U.K. Bultreys A., Gheysen I., 1999. Biological and molecular detection of toxic lipodepsipeptide-producing Pseudomonas syringae strains and PCR identification in plants. Applied and Environmental Microbiology 65: 1904-1909. Bultreys A., Kaluzna M., 2010. Bacterial cankers caused by Pseudomonas syringae on stone fruit species with special emphasis on the pathovars syringae and morsprunorum race 1 and race 2. Journal of Plant Pathology 92: 21-33.

TYPE/PATHOTYPE STRAINS

In all tests reference strains should be included: Pseudomonas syringae pv. syringae ICMP 3023; LMG 1247. Pseudomonas syringae pv. morsprunorum race 1 LMG 2222, LMG 5075. Pseudomonas syringae pv. morsprunorum race 2 CFBP 3800. Pseudomonas syringae pv. avii CFBP 3846; ICMP 14479; NCPPB 4290. Pseudomonas syringae pv. persicae ICMP 5846; LMG 5184.

ACKNOWLEDGEMENTS

This work was carried out within the framework of COST Action 873 “Bacterial diseases of stone fruits and nuts” and was supported by the Polish Ministry of Science and Higher Education grant No118/N-COST/ 2008/0.


15-06-2012

13:07

Pagina 125

Journal of Plant Pathology (2012), 94 (1, Supplement), S1.117-S1.126 Bultreys A., Gheysen I., Hoffmann E., 2006. Yersiniabactin production by Pseudomonas syringae and Escherichia coli, and description of a second yersiniabactin locus evolutionary group. Applied and Environmental Microbiology 72: 3814-3825. Bultreys A., Gheysen I., Wathelet B., Maraite H., Hoffmann E., 2003. High-performance liquid chromatography analyses of pyoverdin siderophores differentiate among phytopathogenic fluorescent Pseudomonas species Applied and Environmental Microbiology 69: 1143-1153. Cody Y.S., Gross D.C., 1987. Characterization of pyoverdin, the fluorescent siderophore produced by Pseudomonas syringae pv. syringae. Applied and Environmental Microbiology 53: 928-934. Crosse J.E., Garrett C.M.E., 1966. Bacterial canker of stone fruits. VII. Infection experiments with Pseudomonas morsprunorum and P. syringae. Annals of Applied Biology 58: 31-41. Donmez M.F., Karlidag H., Esitken A., 2010. Identification of resistance to bacterial canker (Pseudomonas syringae pv. syringae) disease on apricot genotypes grown in Turkey. European Journal of Plant Pathology 126: 241-247. Endert E., Ritchie D.F., 1984. Detection of pathogencity, measurement of virulence, and determination of strain variation in Pseudomonas syringae pv. syringae. Plant Disease 68: 677-680. Fletcher M.J., Young J.M., 1997-8. Studies on vacuum-drying for the preservation of plant pathogenic bacteria. Journal of Culture Collections 2: 21-25 Freigoun S.O., Crosse J.E., 1975. Host relations and distribution of a physiological and pathological variant of Pseudomonas morsprunorum. Annals of Applied Biology 81: 317-330. Fukuchi, N., Isogai A., Nakayama J., Suzuki A., 1990. Structure of syringotoxin B, a phytotoxin produced by citrus isolates of Pseudomonas syringae pv. syringae. Agricultural and Biological Chemistry 54: 3377-3379. Gardan L., Shafik H., Belouin S., Broch R., Grimont F., 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). International Journal of Systematic Bacteriology 49: 469-478. Garrett C.M.E., Crosse J.E., 1966. Preliminary note on a bacterium associated with a disease of Prunus cerasifera. Report of the East Malling Research Station for 1966: 153-154. Gilbert V., Legros F., Maraite H., Bultreys A., 2009. Genetic analyses of Pseudomonas syringae isolates from Belgian fruit orchards reveal genetic variability and isolate-host relationships within the pathovar syringae, and help identify both races of the pathovar morsprunorum. European Journal of Plant Pathology 124: 199-218. Holt J.G., Krieg N.R., Sneath P.H.A., Staley J.T., Williams S.T., 1994. Bergey’s Manual of Determinative Bacteriology. 9th Ed. Williams & Wilkins, Baltimore, MD, USA. Hu F.-P., Young J.M., Fletcher M.J., 1998. Preliminary description of biocidal (syringomycin) activity in fluorescent plant pathogenic Pseudomonas species. Journal of Applied Microbiology 85: 365-371. Hwang M.S.H., Morgan R.L., Sarkar S.F., Wang P.W., Guttman D.S., 2005. Phylogenetic characterization of viru-


lence and resistance phenotypes of Pseudomonas syringae. Applied and Environmental Microbiology 71: 5182-5191. Kaluzna M., Sobiczewski P., 2009. Virulence of Pseudomonas syringae pathovars and races originating from stone fruit trees. Phytopathologia 54: 71-78. Kaluzna M., Ferrante P., Sobiczewski P., Scortichini M., 2010a. Characterization and genetic diversity of Pseudomonas syringae from stone fruits and hazelnut using repetitive-PCR and MLST. Journal of Plant Pathology 92: 781-787. Kaluzna M., Pulawska J., Sobiczewski P., 2010b. The use of PCR melting profile for typing of Pseudomonas syringae isolates from stone fruit trees. European Journal of Plant Pathology 126: 437-443. Kersters K., Ludwig W., Vancanneyt M., De Vos P., Gillis M., Schleifer K-H., 1996. Recent changes in the classification of the Pseudomonads: an overview. Systematic and Applied Microbiology 19: 465-477. King E.O., Raney M.K., Ward D.E., 1954. Two simple media for the demonstration of pyocianin and fluorescin. Journal of Laboratory and Clinical Medicine 44: 301-307. Klement Z., Rozsnyay D.S., Visnyovszky E., 1972. Apoplexy of apricots. I. Bacterial dieback and development of the disease. Acta Phytopathologica Academiae Scientiarum Hungaricae 7: 3-12. Klement Z., Rozsnyay D.S., Arsenijevic M., 1974. Apoplexy of Apricots. III. Relationship of winter frost and the bacterial canker and die-back of apricots. Acta Phytopathologica Academiae Scientiarum Hungaricae 9: 35-45. Klement Z., 1977. Bacterial canker and diease dieback of apricots (Pseudomonas syringae). Bulletin OEPP/EPPO Bulletin 7: 57-68. Kozloff L.M., Schofield M.A., Lute M., 1983. Ice nucleating activity of Pseudomonas syringae and Erwinia herbicola. Journal of Bacteriology 153: 222-231. Lelliott R.A., Stead D.E., 1987. Methods for the diagnosis of bacterial diseases of plants. In: Preece T.F. (ed.). Methods in Plant Pathology, Vol. 2., p. 216. Blackwell Scientific Press, London, UK. Lelliott R.A., Billing E., Hayward A.C., 1966. A determinative scheme for the fluorescent plant pathogenic Pseudomonads. Journal of Applied Bacteriology 29: 470-489. Liang L.Z., Sobiczewski P., Paterson J.M., Jones A.L., 1994. Variation in virulence, plasmid content, and genes for coronatine synthesis between Pseudomonas syringae pv. morsprunorum and P. s. syringae from Prunus. Plant Disease 78: 389-392. Lindow S.E., Arny D.C., Upper Ch.D., 1982. Bacterial ice nucleation: A factor in frost injury to plants. Plant Physiology 70: 1084-1089. Louws F.J., Fulbright D.W., Stephens C.T., de Bruijn F.J., 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology 60: 2286-2295. Luisetti J., Prunier J.P., Gardan L., Gaignard J.L., Vigouroux A., 1976. Le dépérissement bactérien du pêcher. Institut National pour la Vulgarization. Invuflec, Paris, France. Maiden M.C., Bygraves J.A., Feil E., Morelli G., Russell J.E., Urwin R., Zhang Q., Zhou J., Zurth K., Caugant D.A., Feavers I.M., Achtman M., Spratt B.G., 1998. Multilocus


15-06-2012

13:07

Pagina 126

S1.126 P. syringae pathovars diagnosis and identification methods Journal of Plant Pathology (2012), 94 (1, Supplement), S1.117-S1.126 sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proceedings of the National Academy of Sciences USA 95: 3140-3145. Maki L.R., Galyan E.L., Chang-Chien M.-M., Caldwell D.R., 1974. Ice nucleation induced by Pseudomonas syringae. Applied Microbiology 28: 456-459. Masny A., Plucienniczak A., 2003. Ligation mediated PCR performed at low denaturation temperatures—PCR melting profiles. Nucleic Acids Research 31: 114. Ménard M., Sutra L., Luisetti J., Prunier J.P., Gardan L., 2003. Pseudomonas syringae pv. avii (pv. nov.), the causal agent of bacterial canker of wild cherries (Prunus avium) in France. European Journal of Plant Pathology 109: 565-576. Palleroni N.J., 1984. Family I. Pseudomonadaceae. In: Krieg N.R., Holt J.G. (eds). Bergey’s Manual of Systematic Bacteriology: 141-199. William & Wilkins, Baltimore, MD, USA. Palleroni N.J., 2005. Genus I. Pseudomonas. In: Brenner D.J., Krieg N.R., Staley J.T., Garrity G.M. (eds). Bergey’s Manual of Systematic Bacteriology. 2nd Ed. The Proteobacteria, Part B Gammaproteobacteria Vol. 2, pp. 323-379. Springer, New York, NY, USA. Paulin J.-P., Luisetti J., 1978. Ice nucleation activity among phytopathoenic bacteria. Proceedings 4th International Conference for Plant Pathogenic Bacteria, Angers, France: 725731. Pelludat C., Rakin A., Jacobi C.A., Schubert S., Heesemann J., 1998. The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation. Journal of Bacteriology 180: 538-546. Reasoner D.J., Geldreich E.E., 1985. A new medium for the enumeration and subculture of bacteria from potable water Applied and Environmental Microbiology 49: 1-7. Scholz-Schroeder B.K., Soule J.D., Gross D.C., 2003. The sypA, sypB, and sypC synthetase genes encode twenty-two modules involved in the nonribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv. syringae B301D. Molecular Plant-Microbe Interactions 16: 271-280. Sobiczewski P., 1984. Etiology of sour cherry bacterial canker in Poland. Fruit Science Report 11: 169-179. Sobiczewski P., Lisiecka A., Millikan D.F., 1980. The use of green cherry fruit to evaluate chemicals for controlling bacterial canker of stone fruits. Phytopatologische Zeitschrift 98: 268-271. Sorensen K.N., Kim K-H., Takemoto J.Y., 1998. PCR Detection of cyclic lipodepsinonapeptide-producing Pseudomonas syringae pv. syringae and similarity of strains. Applied and Environmental Microbiology 64: 226-230. Stackebrandt E., Frederiksen W., Garrity G.M., Grimont P.A.D., Kampfer P., Maiden M.C.J., Nesme X., Rosselló Mora R., Swings J., Truper H.G., Vauterin L., Ward A.C.,Whitman W.B., 2002. Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. International Journal of Systematic and Evolutionary Microbiology 52: 1043-1047.

Versalovic J., Koeuth T., Lupski J.R., 1991. Distribution of repetitive DNA-sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Research 19: 6823-6831. Versalovic J., Schneider M., de Bruijn F.J., Lupski J.R., 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods in Molecular and Cellular Biology 5: 25-40. Vicente J., Roberts S., 2007. Discrimination of Pseudomonas syringae isolates from sweet and wild cherry using repPCR. European Journal of Plant Pathology 117: 383-392. Vigouroux A., Blache M., 1967. Un nouveau dépérissement de pêcher dans l’Ardèche. Phytoma 192: 34-45. Wagman J., Weneck E.J., 1963. Preservation of bacteria by circulating-gas freeze drying. Applied Microbiology 11: 244248. Wang P.W., Morgan R.L., Scortichini M., Guttman D.S., 2007. Convergent evolution of phytopathogenic Pseudomonads onto hazelnut. Microbiology 153: 20672073. Weingart H., Völksch B., 1997. Genetic fingerprinting of Pseudomonas syringae pathovars using ERIC-, REP-, and IS50-PCR. Journal of Phytopathology 145: 339-345. Yadava U.L., Doud S.L., 1980. The short life and replant problems of deciduous fruit trees. Horticultural reviews 2: 1-116. Young J.M., 1987a. New plant disease record in New Zealand: Pseudomonas syringae pv. persicae from nectarine, peach and Japanese plum. New Zealand Journal of Agricultural Research 230: 235-237. Young J.M., 1987b. Orchard management and bacterial diseases of stonefruit. New Zealand Journal of Experimental Agriculture 15: 257-266. Young J.M., 1988. Pseudomonas syringae pv. persicae from nectarine, peach, and Japanese plum in New Zealand. Bulletin OEPP/EPPO 18: 141-151. Young J.M., 1991. Pathogenicity and identification of the lilac pathogen, Pseudomonas syringae pv. syringae van Hall 1902. Annals of Applied Biology 118: 283-298. Young J.M., 1995. Bacterial decline. In: Ogawa J.M., Zehr E.I., Bird G.W., Ritchie D.F., Uriu K., Uyemoto J.K. (eds). Compendium of Stone Fruit Diseases., p. 50. APS Press, St. Paul, MN, USA. Young J.M., 2010. Taxonomy of Pseudomonas syringae. Journal of Plant Pathology 92: 5-14. Young J.M., Jones D.S., Gillings M., 1996. Relationships between populations of Pseudomonas syringae pv. persicae determined by restriction fragment analysis. Plant Pathology 45: 350-357. Zamze S.E., Smith A.R.W., Hignett R.C., 1986. Composition of lipopolysaccharides from Pseudomonas syringae pv. syringae and a serological comparison with lipopolysaccharide from P. syringae pv. morsprunorum. Journal of General Microbiology 132: 3393-3401.