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Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants

非苜蓿轮枝菌在蛇麻植物上的致病性测定

Marko Flajšman Marko Flajšman *
Sebastjan Radišek Sebastjan Radišek *
Branka Javornik Branka Javornik

 (*contributed equally to this work) 发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2171 浏览次数: 10417

评审: Zhaohui LiuValeria LullaTimo Lehti

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Abstract

Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant’s xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this phytopathogenic fungus. Artificial infections of hop plants and disease scoring are prerequisites for studying the pathogen’s virulence/pathogenicity and its interaction with hop plants. In this protocol, we describe the root dipping inoculation method for conducting pathogenicity assay of V. nonalfalfae on hop plants.

Keywords: Verticillium nonalfalfae (非苜蓿轮枝菌) search Pathogenicity assay (病原性测定) search Hop (蛇麻) search Disease symptoms (疾病症状) search Plant-pathogen interactions (植物 - 病原体相互作用) search

Background

Verticillium spp. infects more than 400 different host plants and every species has its own range of host. The primary host of V. nonalfalfae is hop. However, hop has several disadvantages for use as a test plant for pathogenicity assay; e.g., it is a perennial plant and needs to undergo a dormancy phase. Plants can therefore only be used for pathogenicity assay for a few months in the year, from late spring to late summer. Hop varieties are vegetatively propagated as softwood cuttings in a greenhouse or as dormant cuttings from rootstock. Seeds are obtained by crossing female and male plants and are used only for breeding purposes. The root dipping inoculation method has been widely used for pathogenicity assay of Verticillium spp. on other plant hosts, e.g., tomato (Fradin et al., 2009), N. benthamiana (Klosterman et al., 2011) and Arabidopsis thaliana (Ellendorff et al., 2009).

Materials and Reagents

  1. Miracloth (EMD Millipore, catalog number: 475855-1R )
  2. Petri dish (Golias, catalog number: PE01K )
  3. Host plants (hop Humulus lupulus, susceptible cultivar ‘Celeia’)
  4. Fungal conidia (Verticillium nonalfalfae; lethal pathotype PV1 [isolate T2]) (Radisek et al., 2006)
  5. Fertilizer YaraKristalon yellow NPK 13-40-13 + ME [ME - trace elements: B - 0.025%; Cu * - 0.01%; Fe * - 0.07%; Mn * - 0.04%; Mo - 0.004%; Zn * - 0.025%; * - Chelate base] (Yara International ASA)
  6. Fertilizer YaraKristalon special NPK 18-18-18 + ME [ME - trace elements: B - 0.025%; Cu * - 0.01%; Fe * - 0.07%; Mn * - 0.04%; Mo - 0.004%; Zn * - 0.025%; * - Chelate base] (Yara International ASA)
  7. Growing medium
    1. For fungus inoculum preparation: liquid GFM - general fungal medium (Kayser, 1992) (see Recipes)
    2. For plants: soil substrate (soil substrate for growing plants)
    3. For fungus re-isolation: potato dextrose agar + antibiotics (streptomycin sulphate, neomycin, chloramphenicol; each 100 mg/ml) = PDA + A plates (see Recipes)
  8. Sterile distilled water (IDT, catalog number: 231-791-5 )
  9. 96% ethanol
  10. Peptone (Sigma-Aldrich, catalog number: 73049-73-7 )
  11. Yeast extract (AMRESCO, catalog number: J850 )
  12. Glucose (Kemika, catalog number: 07051 )
  13. Potassium nitrate (KNO3) (EMD Millipore, catalog number: 105063 )
  14. Potato dextrose agar (Biolife, catalog number: 4019352 )
  15. Streptomycin sulphate (Duchefa Biochemie, catalog number: S0148 )
  16. Neomycin (Duchefa Biochemie, catalog number: M0135 )
  17. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )

Equipment

  1. 500 ml Erlenmeyer flask (BRAND, catalog number: 92824 )
  2. 2 L plastic cup (BRAND, catalog number: 87822 )
  3. Plastic pots 0.5 L
  4. Rotary shaker (Infrost, catalog number: 29313 )
  5. Growth chamber (Kambič Laboratory Equipment, model: RK-13300 )
  6. Fluorescent grow lamp (Idealo, model: Osram Fluora L 58 W/77 )
  7. Thoma counting chamber (BRAND, Wertheim, Germany)
  8. Wooden sticks for hop’s bine support
  9. Scalpel and tweezers
  10. Light microscope (Nikon Instruments)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Flajšman, M., Radišek, S. and Javornik, B. (2017). Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants. Bio-protocol 7(6): e2171. DOI: 10.21769/BioProtoc.2171.

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分类

植物科学 > 植物免疫 > 病害生物测定

微生物学 > 微生物-宿主相互作用 > 真菌

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