PM 7/120 (2) <i>Pseudomonas syringae</i> pv. <i>actinidiae</i>

EPPO BulletinVolume 51, Issue 3 p. 549-567 STANDARD - DIAGNOSTICSFree Access PM 7/120 (2) Pseudomonas syringae pv. actinidiae Correction(s) for this article Corrigendum Volume 52Issue 2EPPO Bulletin pages: 513-513 First Published online: July 11, 2022 published: 25 November 2021 https://doi.org/10.1111/epp.12782Citations: 1 1Use of brand names chemicals or equipment in these Standards implies no approval them to the exclusion others that may also be suitable. AboutSectionsPDF ToolsRequest permissionExport citationAdd favoritesTrack citation ShareShare Give accessShare full text full-text accessPlease review our Terms and Conditions Use check box below share version article.I have read accept Wiley Online Library UseShareable LinkUse link a with your friends colleagues. Learn more.Copy URL Share linkShare onFacebookTwitterLinkedInRedditWechat Abstract Specific scope This Standard describes diagnostic protocol actinidiae. It should used conjunction 7/76 Diagnostic Protocols.1 amendment Approved 2014–09. Revised 2021–06. Authors contributors are given Acknowledgements section. INTRODUCTION is causal agent bacterial canker Actinidia spp., most damaging severe disease cultivated kiwifruits. spp. only known host plant species P. Wild reported hosts Alternanthera philoxeroides, Paulownia tomentosa Setaria viridis (EPPO, 2020). Since its first report Japan 1989 on (Serizawa et al., 1989; Takikawa 1989) has been different countries where grown 2020), including major producing such as Italy New Zealand. populations show genetic variability. Five biovars, present areas world, characterized by differences virulence (Chapman 2012; Vanneste 2013; Sawada & Fujikawa, 2019). Biovar groups strains associated initial epidemics observed (1984–1989) (1992). 2 isolated South Korea. corresponds highly virulent population, responsible outbreaks since 2008. 4, which originally included low strains, was subsequently removed new pathovar actinidifoliorum established (Cunty 2015). Strains differing from four existing biovars were found local classified 5 (Fujikawa Sawada, 2016) 6 (Sawada 2014). provides information diagnosis 1, 3. When available provided, but limited distribution. The tests allow distinction actinidifoliorum. Flow diagrams describing procedure presented Figure 1a,b. FIGURE 1Open figure viewerPowerPoint (a) diagram detection identification (Psa) samples symptomatic (b) asymptomatic IDENTITY Name: (Takikawa 1989). Synonyms: None. Taxonomic position: Proteobacteria, Gamma subdivision, Order Pseudomonadales, Family Pseudomonadaceae. Code: PSDMAK. Phytosanitary categorization: A2 list no. 370. EU emergency measures 2020/885 June 2020. DETECTION can detected both symptomless, latently infected, aerial parts. pathogen occasionally roots plants (Mazzaglia 2010; Abelleira contaminated pollen, it survive epiphytically (Vanneste 2011a; Stefani Giovanardi, 2011; Gallelli 2011b). Commodities infected planting (including plantlets cuttings), bud chips pollen. Harvested fruits very rarely happen when harvested heavily orchards (Gallelli 2011b; 2011). 3.1 Symptoms caused easily parts, trunks, leaders, canes, leaves, flowers fruits. Mazzaglia al. (2010) browning darkening vascular tissues underlying root cortex observed. On leaders cankers autumn, late winter throughout growing season. Cankers moist spring, ooze exudates sap. trunks turns white reddish-brown colour. Infected canes shoots wilting blight leaves. Lenticels affected usually larger more elevated than symptomless plants. symptoms appear small, angular water-soaked areas, later become necrotic turn dark brown; chlorotic halo sometimes around foliar spots. Similar might leaves syringae, common several worldwide fruit crops, especially stone pome Another bacterium, viridiflava, cause leaf spots, often coalescing not showing haloes. Affected buds brown, then fall ground (Balestra 2008). necrotising syringae. fruitlets misshapen, smaller size healthy develop apex; they during spring early summer manually detached thrown away pre-harvest selection. Fruits collapse consequence branches; marketable. A description according period year at https://upload.eppo.int/download/745oaa282c610. 3.2 Screening 3.2.1 Detection Direct isolation (section 3.2.1.2) molecular 3.2.1.3) screening material symptoms. positive results obtained two tests. pest an area, single test performed conclude if sample. Two performance studies showed lower analytical sensitivity (Loreti 2014, 2018). 3.2.1.1. Test sample requirements Plant include trunk leader shoots, those other lesions, buds, spots lesions. field glasshouse processed soon possible after collection. After reception laboratory, kept 4–8°C until analysis. Freshly prepared extracts necessary successful pathogen. remaining cold stored (as mentioned above) up 9 days additional verification. Extraction procedures Appendix 1. 3.2.1.2. Isolation relatively easy because high number culturable bacteria present. However, difficult advanced environmental conditions infection favourable multiplication (e.g. collected middle summer) copper treatment recently orchard; cases preferable use In order avoid growth too many saprophytes contaminants, overgrow agar plates within 36–48 h plating, preferably attempted semi-selective medium, particular modified nutrient sucrose (NSA) King's B medium (KB) (Mohan Schaad, 1987) NSA KB supplemented boric acid antibiotics: cycloheximide cephalexin. Media described 2. Colonies grow slowly media containing antibiotics, compared same without them. done dilution plating 30–50 μL extract solution bleeding sap indicated 10- 100-fold dilutions onto (modified and/or KB). Plates 3–6 approximately 24°C incubator. As control, 10–15 µL suspension control strain (see reference material) concentration 104–105 cfu mL−1 plated medium. days, pale, pinhead colonies plates, sixth day plating. Suspect require further purification step re-streaking before proceeding identification. Colony morphology smooth, convex, round entire margins, pearly whitish colour (Figure 2a). shiny fresh, becoming pale ivory whitish. distinguish just visual observation days. 2Open Morphology antibiotics antibiotics. (Courtesy: E. Stefani, Universita di Modena e Reggio Emilia, IT) flat, slightly lobed whitish-yellowish colour, 4–5 mm wide tiny, spot centre colony 2b). 3.2.1.3. Molecular recommended selected based study Loreti (2018) except LAMP Ruinelli (2017). DNA extraction together Table TABLE Conventional duplex PCR (2011a) 4 X Xa (2017) PSA PSA3 Real-time (2014) Andersen 7 simplex 8 Based silico analysis detect 6. Although characteristics (2017), unlike section interlaboratory comparison performed. 5). evaluated TPS 2018) widely region, (along range biovars. Tests detecting biovar presence suspected following developed specifically (the biovar): 7. specific 5. 8. multiplex 2013) nested (Biondi gave undetermined false-positive colonies. real-time kits Qualiplante (2011a, 2014), respectively. verification (data published) original publications (C. Chatillon, pers. comm., 3.2.1.4. Serological rapid serological (immunochromatographic strips) polyclonal antiserum effector Hopz5 (Chen validation data there experience region test. Performance publication. Analytical reach 2.2 × 103 mL−1. specificity. Exclusivity 100% (based 10 species: tomato, theae, putida, fluorescens, koreensis, Bacillus subtilis, megaterium, Ralstonia solanacearum, Erwinia rhapontici, syringae). Inclusivity (evaluated 3). sensitivity: 84%, 28 result out 33 total tested (leaf limb). 3.2.2 3.2.2.1. (latent infection) requires composite enhances Dormant cuttings, budwood, twigs, vitro micro propagated (vitroplants) susceptible Sampling allowed nurseries, would needed establish optimal sampling. erratic Pollen carry disseminate into kiwi demonstrated spread (Tontou 2014) tested. Detailed descriptions materials Concentration concentrated increase viable cells. (usually 1.5–3 mL) divided parts: one perform second confirmation analyses –80°C adding 15–25% sterile glycerol. 3.2.2.2. Section 3.2.1.3 extract. IDENTIFICATION five typical using least below. case critical (4.1 4.2) biological principles. At declared identified. 4.1 used. 4.2 sequencing Assignment multilocus sequence (MLSA) housekeeping genes (gapA, gltA, gyrB rpoD) MLSA scheme seven (acnB, cts, gapA, gyrB, pfk, pgi (Sarkar Guttman, 2004) discriminate all Psa 4.3 Other 4.3.1 Biochemical physiological Arbutin aesculin hydrolysis (both positive) negative) performing (Lelliot Stead, 1987). biochemical metabolic (1989) American Society Microbiology Committee Bacteriological Technic, 1957). 4.3.2 Pathogenicity pathogenicity required completion procedure, e.g. cases, see 2018), conveniently carried chinensis (cultivars Erica, Hort16A) deliciosa (cultivar Hayward Soreli). vivo Inoculation spraying appropriate size. Information provided 9. Details REFERENCE MATERIAL Pathotype strain: NCPPB 3739 = ICMP 9617 CFBP 4909 – Japanese isolate. ISF 8.43 Italian isolate, type. controls 7285, 7286, 7287 CRA-PAV 1530 (all isolates). collections provide strains: National Collection Pathogenic Bacteria (NCPPB), FERA, York, GB (https://www.fera.co.uk/ncppb) International Center Microbial Resources French Plant-associated (CIRM-CFBP), IRHS–INRAE Beaucouzé (FR) (https://www6.inrae.fr/cirm/CFBP-Bacteries-associees-aux-Plantes). authenticity guaranteed directly culture collections. REPORTING AND DOCUMENTATION Guidance reporting documentation 7/77 (1) Documentation diagnosis. PERFORMANCE CRITERIA criteria available, Validation Database Expertise (http://dc.eppo.int) consult database detailed specificity, reports, etc.). FURTHER INFORMATION Further organism from: Emilia (IT), e-mail: [email protected]. S. Loreti, Research Centre Protection Certification (CREA-DC), via C.G. Bertero 22, 00156 Roma [email protected]. M. Scortichini, CREA, Olive, Fruit Trees Citrus, 81100 Caserta [email protected]. A. Cunty, ANSES, Laboratoire pour la Santé des Végétaux, rue Jean Dixméras, 44049 Angers Cedex (FR), [email protected]. FEEDBACK ON THIS DIAGNOSTIC If you any feedback concerning Standard, included, standard wish share, please contact [email protected]. REVISION An annual process place identify need revision Standards. identified needing marked website. errata corrigenda press will ACKNOWLEDGEMENTS drafted (Department Life Sciences, University reviewed (CRA, IT). J. Costa (IPN, PT), Cunty (ANSES, FR), (University J.F. Pothier (ZHAW, CH), Panel Diagnostics Bacteriology. APPENDIX buffers) 1.1 Symptomatic Bleeding cankers: taken collecting few drops cotton swab. swab dipped tube mL saline water shaken seconds. direct 3.2.1.2). Old (generally rusty red brown) suitable effective isolation. Lesions (margins canker, flowers, fruits): Three small pieces tissue aseptically removed, put 2–5 water, left soak minutes. Alternatively, fragments mortar (or water) 2–3 min. From large, generally isolation: preference halo. These excised scalpel either comminuted shredded 1–2 5–10 1.2 (particularly useful end winter/early spring): About removing distal cm segment branch, previously sterilized ethanol, open wound 15 tube. Samples maintained container till centrifugation 000g 20 min recover pellet cells, resuspended distilled water. 2.1 For dormant twigs hereafter 2.1.1 cuttings Different sampling occurs. here adopted Italy. One hundred 30 length crushed Crushing stomacher hammer. Fifteen millilitres phosphate buffered (PBS) added again Again, PBS followed last crushing (three steps min). fluid filtered through gauze centrifuged 000 g 4°C. supernatant discarded PBS. 2.1.2 Nursery laboratory consists shoots/twigs randomly selected; disinfection cutting tools (i.e. quaternary ammonium salts 70% ethanol) between cultivar each separately. Leaves shoot/twig cut (total 100 about cm). 300 PBS-Tween Erlenmeyer flasks rotary shaker 125 rpm 1.5 room temperature. washing resulting suspended way. above). three jars (15–25 jar). Ten chosen make sample, basal part upper finely chopped flask volume cover tissue. Flasks (125 rpm) 60 2.1. 2.3 Plantlets composed lot 000. base, thoroughly washed under tap rinsed deionized blotted dry. plantlet stem segments. Segments roughly sealed bag solution. crushing, centrifugation, discharged 2.4 pollen lots 500 g. 50 vial added. 120 settle 180 gently clean Buffers All buffers autoclaving 121°C unless stated otherwise. Sterile 0.90 w/v NaCl. Dissolve NaCl L Sterilize autoclaving. mM KCl 0.2 Na2HPO4·12 H2O 2.9 KH2PO4 Distilled To Adjust pH 7.2 autoclave. Tween (0.1%) add shake buffer Nutrient (Crosse, 1959) broth Sucrose 18 prepare 900 autoclave, cool 50°C 1.5% aqueous solution, mg ethanol With store month refrigerated. (King 1954), Mohan Schaad (1987). Proteose peptone N. Glycerol K2HPO4 MgSO4·7H2O glucose (NGA) (Schaad Forsters, 1985), Glucose 2.5 achieved commercial kits. Several validated European laboratories DNeasy Mini Kit-based (Qiagen), Macherey-Nagel NucleoSpin II, etc. Manufacturer's instructions followed. Single purposes putative actinidiae, fresh pure grade 0.9 0.1 0.5 M NaOH boiled approx. 95°C imm