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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   

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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), Pathogenicity assay(病原性测定), Hop(蛇麻), Disease symptoms(疾病症状), Plant-pathogen interactions(植物 - 病原体相互作用)

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

  1. Preparation of host plants
    1. For inoculation, use two-month-old, well-rooted plants that have been grown in 0.5 L plastic pots and have been vegetatively produced in a greenhouse from hop mother plants as soft wood cuttings. Plants should be directly produced by a professional grower.
    2. Grow plants in a greenhouse on mist benches. Water the plants every 2 days and fertilize once per week using foliar fertilizers that contain macro and micro elements (e.g., 0.1% solution of YaraKristalon yellow NPK 13-40-13 + ME).
    3. To maintain the appropriate health status, spray the plants once per week against pests and foliar diseases using protective plant protection products.
    4. Use the susceptible cultivar ‘Celeia’.

  2. Inoculum preparation
    1. Place a small piece of fungal mycelium (of the size of a 2 mm ‘ball’) from stock PDA plates (which should be maintained in a fridge) in each of four 500 ml Erlenmeyer flasks filled with 250 ml of liquid GFM, supplemented with 100 mg/ml streptomycin sulphate and 100 mg/ml chloramphenicol.
    2. Incubation takes approximately 5 days at 100 rpm on a rotary shaker at room temperature and in the dark (place the shaker in a dark room) (Figure 1).


      Figure 1. A picture of fungal culture grown after 5 days. White fungal mycelium in a sphere shape can be seen. The medium with fungal isolates that produce a lot of spores is duller.

    3. Prepare the conidia inoculum (spore size 5 to 7 μm) by filtration of mycelia and the spore through miracloth (typical pore size is 22-25 µm):
      1. Place a non-autoclaved piece of miracloth (20 x 20 cm) on top of a 2 L plastic cup, hand held, and filter each flask separately, one by one.
      2. Wash spores and suspend them in sterile distilled water. There is no need to spin down the spores after washing.
      3. If the concentration is low, all the filtration material is needed.
    4. Use a Thoma counting chamber to determine the spore concentration. Determine the spore concentration four times for one filtrated suspension and calculate an average.
    5. Adjust the spore concentration to 5 x 106 conidia/ml with sterile distilled water.
    6. The final volume of inoculum with a concentration of 5 x 106 conidia/ml for artificial infection should be 1 L. Use a maximum of 12 plants per one volume of inoculum.

  3. Artificial infection
    1. Uproot hop plants that have been grown in 0.5 L plastic pots. Use as much soil substrate as possible from the roots by hand.
    2. Rinse the roots in sterile water (just gently dip for a few seconds).
    3. Then dip the roots for 10 min in 1 L of inoculum, poured into a 2 L plastic cup.
    4. Inoculate a minimum of 10 plants per treatment.
    5. Dispose of the remaining fungal inoculum after autoclaving.
    6. Treat control plants similarly but dip their roots in sterile water for 10 min.
    7. Pot the plants in new pots of size 0.5 L.
    8. Grow the plants as a single bine in a growth chamber (RK-13300, Kambič) under a 12-h photoperiod of fluorescent light (L 58 W/77; Fluora, Osram) at a temperature of 22 °C and relative humidity of 65% during the light period and 20 °C and 70% during the dark period. During growth in the chamber, water twice a week (every Monday and Friday) and fertilize once per week using foliar fertilizer containing a higher quantity of nitrogen (e.g., 0.2% solution of YaraKristalon special NPK 18-18-18 + ME).

  4. Symptoms monitoring
    1. The first disease symptoms occur approximately 20 days post inoculation (dpi). The first symptoms can be seen as pale yellowing of some leaves (Figure 2 – section 1). At that time no wilting is observed.


      Figure 2. The 0-5 scale used to evaluate foliar symptoms of Verticillium wilt of hop. 0 indicates no leaf symptoms, 1 = 1 to 20% leaf area wilted, 2 = 21 to 40% leaf area wilted, 3 = 41 to 60% leaf area wilted, 4 = 61 to 80% leaf area wilted and 5 = 81 to 100% leaf area wilted.

    2. Subsequently, assess the plants visually for the appearance of foliar symptoms using a 0-5 scale (Radisek et al., 2003) (Figure 2) at 7-day intervals.
    3. There are 3 to 5 assessment time points. Photograph the plants at the last time point, when the disease symptoms are the most severe. Photographing at earlier time points does not show the biggest difference between fungal inoculated and mock inoculated plants.
    4. At the end of visual disease assessments, carry out mycological re-isolation of the pathogen to confirm the presence of the pathogen in the inoculated plants.
      1. Cut the plants at ground level and transfer them to a laminar. Spray the cut stems with 96% ethanol and place very briefly on an open flame in order to sterilise the surface.
      2. Perform re-isolation by using xylem sections that have been sterilely harvested from the stem of inoculated plants and placed on PDA + A plates.
      3. The xylem section is actually the whole inner part of the stem, which should be cut into 1-2 cm long pieces. Place three to six pieces from each plant on PDA + A plates.
      4. Fungal outgrowth can be detected after the plates have been incubated for 3-5 days in the dark at 24 °C. Examine the emerging mycelium (if any) is examined by light microscopy.
    5. Only infected plants should be considered and used for further analysis.
    6. Calculate the disease severity index (DSI) for every time point for each plant and determine the average disease severity index (Equation 1). Also present DSI results graphically (Figure 3).
      Equation 1:
      Average disease severity index = sum of assessments of infected plants for last assessment time points/number of infected plants (Jakse et al., 2013).


      Figure 3. Example of graphic presentation of DSI index. DSI curves of hop plants infected with wild type and two mutants of V. nonalfalfae, which had impaired virulence, are indicated by symptom evaluation of infected plants at five different time points.

    7. The area under the disease progress curve (AUDPC) is calculated as indicated by Campbell and Madden (1990) (Equation 2) and expressed as relative AUDPC - rAUDPC (Simko and Piepho, 2012) (Equation 3).
      Equation 2:

      Where,
      n = total number of observations,
      y = DSI index for each plant,
      t = number of days from inoculation.

      Equation 3:
      rAUDPC = actual AUDPC value/maximum potential AUDPC value

Data analysis

Figure 4 shows a representative example of the results of pathogenicity assay.


Figure 4. Results of pathogenicity assay. A. Disease symptoms of hop plants; left are mock inoculated asymptomatic plants, right are disease symptoms of hop plants infected with wild type. Plants were imaged 31 days after inoculation. B. Mycological re-isolation of one infected hop plant. Fungal outgrowth is shown 3 days after plating of xylem sections on PDA + A plates at room temperature. White, fluffy mycelium is typical of V. nonalfalfae. However, outgrowth of some other endophytic fungi can also appear, as is clearly seen with the first xylem section in the first row and to a lesser extent with the first and second xylem sections in the second row. Other microorganisms can also arise from xylem sections (see Notes). V. nonalfalfae mycelium prefers to grow on the plant material (xylem sections are completely overgrown by mycelium) and not on the PDA medium.

Statistical analysis
Perform statistical analysis of results of pathogenicity assay on rAUDPC values in the following order:

  1. Firstly, use Levene’s test to evaluate the difference in variance values.
  2. Secondly, if there is not a significant difference among variances of rAUDPC values of treatments, subject rAUDPC values calculated for each treatment to standard one-way analysis of variance followed by Dunnett’s test, which compares average rAUDPC values of mutant-infected plants with average rAUDPC values of wild-type-infected plants.
  3. However, if there is a significant difference among variances of rAUDPC values of treatments, the Kruskal-Wallis test of non-parametrical statistical analysis should be used, which compares the ranks of rAUDPC values.
  4. If differences among ranks exist, perform Dunn’s test in order to compare the average rank value of mutant-infected plants with the average rank value of wild-type-infected plants.
  5. Results are presented in a dot graph (Figure 5).


    Figure 5. Example of graphic presentation of rAUDPC values. Mean rAUDPC values of wild type and two mutants, which had impaired virulence, are presented. The asterisks show significant differences (***P < 0.001; **P < 0.01). Error bars indicate standard error of the mean.

Notes

  1. As mentioned in the section ‘Procedure’ (part ‘B. Inoculum preparation’, step 6), a maximum 12 plants per one volume of inoculum (which is 1 L with a concentration of 5 x 106 conidia/ml) should be used. During root dipping of these plants, the inoculum gets diluted. If there are more plants to be inoculated with the same inoculum, the concentration of used inoculum must therefore be recovered to 5 x 106 conidia/ml by adding fresh conidia. It is even better to prepare new inoculum with a concentration of 5 x 106 conidia/ml.
  2. In relation to disease symptom assessments, we strongly advise that the same person always evaluates disease symptoms. Any mistake of assessment will thus be smaller or, at least equal for all assessments.
  3. In relation to mycological re-isolation: although the re-isolation medium contains 300 mg/ml antibiotics and re-isolation is done in a sterile environment, many different microorganisms can arise after plating xylem sections on media. Incubation of plates at room temperature should therefore be no longer than 5 days.
  4. Emerging mycelium on re-isolation plates must be subjected to morphological analysis using light microscopy in order to confirm the presence of V. nonalfalfae. Aerial mycelium of V. nonalfalfae is generally abundant, floccose to pruinose, hyphae are smooth-walled and 1.5-3 µm wide. Conidiophores are erect or slanted, generally determinate, branched or unbranched, formed disjointedly throughout the colonies and hyaline.

Recipes

  1. GFM
    2 g peptone
    2 g yeast extract
    20 g glucose
    1 g KNO3
    Mix in 1,000 ml distilled water and autoclave for 20 min at 121 °C
  2. PDA with 300 mg/ml antibiotics
    1. Add 35 g of potato dextrose agar in 1,000 ml distilled water
    2. Shake and mix the powder and autoclave it for 20 min
    3. Cool down the PDA broth to 55 °C
    4. Add 1 ml of 100 mg/ml streptomycin sulphate, 1 ml of 100 mg/ml neomycin and 1 ml of 100 mg/ml chloramphenicol and mix well
    5. Pour in 90 x 15 mm Petri dishes

Acknowledgments

This protocol is adapted from previously published papers (Radisek et al., 2006; Jakše et al., 2013; Cregeen et al., 2015; Mandelc et al., 2013; Flajsman et al., 2016). We acknowledge the Slovenian Research Agency, research program P4-0077, for funds.

References

  1. Campbell, C. L. and Madden, L. V. (1990). Introduction to plant disease epidemiology. John Wiley & Sons.
  2. Cregeen, S., Radisek, S., Mandelc, S., Turk, B., Stajner, N., Jakse, J. and Javornik, B. (2015). Different gene expressions of resistant and susceptible hop cultivars in response to infection with a highly aggressive strain of Verticillium albo-atrum. Plant Mol Biol Report 33(3): 689-704.
  3. Ellendorff, U., Fradin, E. F., De Jonge, R. and Thomma, B. P. (2009). RNA silencing is required for Arabidopsis defence against Verticillium wilt disease. J Exp Bot 60(2): 591-602.
  4. Flajsman, M., Mandelc, S., Radisek, S., Stajner, N., Jakse, J., Kosmelj, K. and Javornik, B. (2016). Identification of novel virulence-associated proteins secreted to xylem by Verticillium nonalfalfae during colonization of hop plants. Mol Plant Microbe Interact 29(5): 362-373.
  5. Fradin, E. F., Zhang, Z., Juarez Ayala, J. C., Castroverde, C. D., Nazar, R. N., Robb, J., Liu, C. M. and Thomma, B. P. (2009). Genetic dissection of Verticillium wilt resistance mediated by tomato Ve1. Plant Physiol 150(1): 320-332.
  6. Jakše, J., Cerenak, A., Radisek, S., Satovic, Z., Luthar, Z. and Javornik, B. (2013). Identification of quantitative trait loci for resistance to Verticillium wilt and yield parameters in hop (Humulus lupulus L.). Theor Appl Genet 126(6): 1431-1443.
  7. Kayser, T. (1992). Protoplasten fusion sowie elektrophoretische Chromosomentrennung und Genkartierung bei filamentösen Pilzen: Penicillium janthinellum, Absidia glauca und Cochliobolus heterostrophus. Dissertation.
  8. Klosterman, S. J., Subbarao, K. V., Kang, S., Veronese, P., Gold, S. E., Thomma, B. P., Chen, Z., Henrissat, B., Lee, Y. H., Park, J., Garcia-Pedrajas, M. D., Barbara, D. J., Anchieta, A., de Jonge, R., Santhanam, P., Maruthachalam, K., Atallah, Z., Amyotte, S. G., Paz, Z., Inderbitzin, P., Hayes, R. J., Heiman, D. I., Young, S., Zeng, Q., Engels, R., Galagan, J., Cuomo, C. A., Dobinson, K. F. and Ma, L. J. (2011). Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog 7(7): e1002137.
  9. Mandelc, S., Timperman, I., Radisek, S., Devreese, B., Samyn, B. and Javornik, B. (2013). Comparative proteomic profiling in compatible and incompatible interactions between hop roots and Verticillium albo-atrum. Plant Physiol Biochem 68: 23-31.
  10. Radisek, S., Jakse, J. and Javornik B. (2006). Genetic variability and virulence among Verticillium albo-atrum isolates from hop. Eur J Plant Pathol 116: 301-314.
  11. Radisek, S., Jakse, J., Simoncic, A. and Javornik, B. (2003). Characterization of Verticillium albo-atrum field isolates using pathogenicity data and AFLP analysis. Plant Dis 87: 633-638.
  12. Simko, I. and Piepho, H. P. (2012). The area under the disease progress stairs: calculation, advantage, and application. Phytopathology 102(4): 381-389.

简介

苜蓿轮枝菌是一种土壤传播的植物病原体,通过根感染其宿主。它传播在植物的木质部,并通过分泌不同的毒力因子引起枯萎病症状。 Hop(umulus lupulus)是V主要的主机。非苜蓿,因此它被用作研究这种植物病原真菌的示范植物。啤酒植物的人工感染和疾病评分是研究病原体的毒力/致病性及其与啤酒花植物的相互作用的先决条件。在该方案中,我们描述了用于进行V的致病性测定的根浸渍接种方法。非苜蓿植物。

背景 轮枝孢属 spp。感染400多种不同的宿主植物,每个物种都有自己的宿主范围。主要的主持人。非苜蓿是跳。然而,跳跃具有用作致病性测定的测试植物的几个缺点;例如,它是一种多年生植物,需要经历休眠阶段。因此,从春季到夏末,植物只能用于几年的致病性测定。 Hop品种在温室中作为软木切片繁殖繁殖,或作为从砧木的休眠扦插。种子是通过交叉雌性和雄性植物获得的,并且仅用于育种目的。根浸渍接种方法已被广泛应用于轮枝孢属菌株的致病性测定。在其他植物宿主上,例如,番茄(Fradin等人,2009),N。 (Klosterman等人,2011)和拟南芥拟南芥(Ellendorff等人,2009)。

关键字:非苜蓿轮枝菌, 病原性测定, 蛇麻, 疾病症状, 植物 - 病原体相互作用

材料和试剂

  1. Miracloth(EMD Millipore,目录号:475855-1R)
  2. 培养皿(Golias,目录号:PE01K)
  3. 寄主植物(hop Hum ulus ulus ulus ulus ulus ulus>>>>>>>>>>>>>>>>>>>>>>>>>>>
  4. 真菌分生孢子(非苜蓿轮枝孢霉属);致死性病原型PV1 [分离株T2])(Radisek等人,2006)
  5. 肥料YaraKristalon黄色NPK 13-40-13 + ME [微量元素:B - 0.025%; Cu * - 0.01%; Fe * - 0.07%; Mn * -0.04%; Mo - 0.004%; Zn * -0.025%; * - 螯合基地(Yara International ASA)
  6. 肥料YaraKristalon特殊NPK 18-18-18 + ME [ME - 微量元素:B - 0.025%; Cu * - 0.01%; Fe * - 0.07%; Mn * -0.04%; Mo - 0.004%; Zn * -0.025%; * - 螯合基地(Yara International ASA)
  7. 生长媒介
    1. 对于真菌接种物制剂:液体GFM-通用真菌培养基(Kayser,1992)(参见食谱)
    2. 对于植物:土壤底物(生长植物的土壤底物)
    3. 对于真菌重新分离:马铃薯葡萄糖琼脂+抗生素(硫酸链霉素,新霉素,氯霉素;每100毫克/毫升)= PDA + A板(参见食谱)
  8. 无菌蒸馏水(IDT,目录号:231-791-5)
  9. 96%乙醇
  10. 蛋白胨(Sigma-Aldrich,目录号:73049-73-7)
  11. 酵母提取物(AMRESCO,目录号:J850)
  12. 葡萄糖(Kemika,目录号:07051)
  13. 硝酸钾(KNO 3)(EMD Millipore,目录号:105063)
  14. 马铃薯葡萄糖琼脂(Biolife,目录号:4019352)
  15. 硫酸链霉素(Duchefa Biochemie,目录号:S0148)
  16. 新霉素(Duchefa Biochemie,目录号:M0135)
  17. 氯霉素(Sigma-Aldrich,目录号:C0378)

设备

  1. 500毫升锥形瓶(BRAND,目录号:92824)
  2. 2升塑料杯(BRAND,目录号:87822)
  3. 塑料壶0.5升
  4. 旋转振动筛(Infrost,目录号:29313)
  5. 生长室(Kambič实验室设备,型号:RK-13300)
  6. 荧光灯(Idealo,型号:Osram Fluora L 58 W/77)
  7. Thoma计数室(BRAND,Wertheim,Germany)
  8. 木棍用于跳跳的支持
  9. 手镯和镊子
  10. 光学显微镜(Nikon Instruments)

程序

  1. 宿主植物的制备
    1. 对于接种,使用两个月大的,根植于0.5L塑料盆栽植物的植物,并在温室中从啤酒花母植物中生长为软木屑。植物应由专业种植者直接生产。
    2. 在温室里种植植物在雾台上。每2天浇灌一次植物,每周施肥一次,使用含有微量和微量元素(例如,0.1%YaraKristalon黄NPK 13-40-13 + ME的溶液)的叶面肥料。
    3. 为了保持适当的健康状况,每周喷洒一次植物以防止害虫和叶面病害使用防护植物保护产品。
    4. 使用敏感品种'Celeia'。

  2. 接种物准备
    1. 在装有250ml液体GFM的四个500ml锥形瓶中的每一个中,将一小片真菌菌丝体(尺寸为2mm的球)从储备的PDA板(其应该保持在冰箱中)放置在补充有100mg/ml硫酸链霉素和100mg/ml氯霉素。
    2. 在室温下和黑暗中将振荡器置于旋转振荡器上,在100rpm下孵育约5天(将振荡器置于暗室中)(图1)。


      图1:5天后生长的真菌培养物的图片。可以看到白色真菌菌丝体。具有产生大量孢子的真菌分离物的培养基变钝。

    3. 通过过滤菌丝体和孢子通过miracloth(典型孔径为22-25μm)准备分生孢子接种物(孢子大小5至7μm):
      1. 将不经高压灭菌的miracloth(20 x 20厘米)片放在2升塑料杯的上面,手持,并逐个过滤每个烧瓶。
      2. 清洗孢子并将其悬浮在无菌蒸馏水中。清洗后不需要旋下孢子。
      3. 如果浓度低,则需要所有的过滤材料。
    4. 使用Thoma计数室确定孢子浓度。确定一个过滤悬浮液的四次孢子浓度,并计算平均值
    5. 用无菌蒸馏水将孢子浓度调至5×10 6 /分钟/ml。
    6. 用于人造感染的浓度为5×10 -6 /分钟/ml的接种物的最终体积应为1μL。每一次接种量最多使用12株植物。

  3. 人造感染
    1. 在0.5L塑料罐中种植的根系播种植物。用手尽可能多地使用土壤基底。
    2. 用无菌水冲洗根部(只需轻轻浸泡几秒钟)。
    3. 然后将根浸入1升接种物中10分钟,倒入2升塑料杯中。
    4. 每次治疗至少接种10株植物。
    5. 高压灭菌后处理剩余的真菌接种物。
    6. 对照对照植物相似,但将根浸入无菌水中10分钟
    7. 将植物放在大小为0.5升的新盆中。
    8. 在生长室(RK-13300,Kambič)中,在温度为22℃,相对湿度为65℃的12小时荧光光照(L 58 W/77; Fluora,Osram)下生长植物作为单一生物光照期间为20%,暗期为70%。在室内生长期间,每周两次水(每周一和周五),每周施肥一次,使用含较高氮含量的叶面肥(例如,0.2%的YaraKristalon特殊NPK 18- 18-18 + ME)。

  4. 症状监测
    1. 第一种疾病症状发生在接种后约20天(dpi)。第一个症状可以看作是一些叶子的淡黄色(图2 - 第1节)。当时没有观察到萎靡。


      图2.用于评估跳叶枯萎病叶片症状的0-5标度 0表示无叶症状,1 = 1〜20%叶面积枯萎,2 = 21〜40%的叶面积枯萎,3 = 41〜60%叶面积枯萎,4 = 61〜80%叶面积枯萎,5 = 81〜100%叶面枯萎。
    2. 随后,以7天的时间间隔使用0-5量表(Radisek等人,2003)(图2)视觉评估植物的叶面症状。
    3. 有3到5个评估时间点。在最后一个时间点拍摄植物,当时疾病症状最严重。在较早的时间点拍摄并不表明真菌接种和模拟接种植物之间的最大差异
    4. 在视觉疾病评估结束时,进行病原体的真菌学重新分离,以确认接种植物中病原体的存在。
      1. 在地面切割植物并将其转移到层流。用96%乙醇喷洒切割的茎,并将其放在明火上,以便对表面进行灭菌。
      2. 通过使用从接种植物的茎无菌收获的木质部切片进行重新分离,并放置在PDA + A板上。
      3. 木质部实际上是茎的整个内部,它应该被切成1-2厘米长的碎片。在PDA + A板上放置三至六块植物。
      4. 在板在24℃的黑暗中孵育3-5天后,可以检测到真菌生长。检查出现的菌丝体(如果有)通过光学显微镜检查
    5. 只有受感染的植物才能被考虑并用于进一步分析。
    6. 计算每个植物每个时间点的疾病严重度指数(DSI),并确定平均疾病严重程度指数(方程式1)。也可以图形地显示DSI结果(图3) 公式1:
      平均疾病严重程度指数=感染植物对最近评估时间点/受感染植物数量的评估总和(Jakse等人,2013)。


      图3. DSI指数的图形呈现示例感染野生型和两种V突变体的啤酒花植株的DSI曲线。恶性疟原虫,其毒力有所减弱,表现在五个不同时间点受感染植物的症状评估。

    7. 疾病进展曲线(AUDPC)下的面积按照Campbell和Madden(1990)(等式2)所示计算,并以相对的AUDPC-rAUDPC(Simko和Piepho,2012)表示(公式3)。
      公式2:

      哪里,
      n =观察总数,
      y =每个植物的DSI指数,
      t =接种天数。

      公式3:
      rAUDPC =实际AUDPC值/最大潜在AUDPC值

数据分析

图4显示了致病性测定结果的代表性实例

图4.致病性测定结果 A.啤酒花植物的疾病症状左侧是模拟接种的无症状植物,正确的是用野生型感染的啤酒花植物的疾病症状。植物在接种31天后成像。 B.一个感染啤酒花植物的真菌学重新分离。在室温下在PDA + A板上铺板木质部切片后3天显示真菌生长。白色,蓬松的菌丝体是典型的V。 nonalfalfae 。然而,一些其它内生真菌的生长也可以出现,正如在第一行中的第一木质部部分清楚地看到的,并且在较小程度上与第二行中的第一和第二木质部部分相似。其他微生物也可能由木质部产生(见注释)。 V。非苜蓿科菌株优选在植物材料上生长(木质部分被菌丝体完全长满),而不是在PDA培养基上生长。

统计分析
按照以下顺序对rAUDPC值进行致病性测定结果的统计分析:

  1. 首先,使用Levene的测试者来评估方差值的差异。
  2. 其次,如果治疗rAUDPC值的差异没有显着性差异,则将每个治疗计算的受试者rAUDPC值与标准单因素方差分析随后进行Dunnett检验,其将突变感染植物的平均rAUDPC值与平均rAUDPC野生型感染植物的价值。
  3. 然而,如果处理rAUDPC值的差异存在显着差异,则应使用非参数统计分析的Kruskal-Wallis检验,比较rAUDPC值的等级。
  4. 如果存在差异,请执行Dunn的测试,以便将突变体感染植物的平均等级值与野生型感染植物的平均等级值进行比较。
  5. 结果以点图形式显示(图5)

    图5. rAUDPC值的图形显示示例。提供了具有受损的毒力的野生型和两种突变体的平均rAUDPC值。星号显示出显着差异(*** P <0.001; ** <0.01)。误差条表示平均值的标准误差。

笔记

  1. 如"程序"一节(部分"B.接种物制备",步骤6)中所述,每一个体积的接种物(其浓度为5×10 6)的最大12株植物分生孢子/ml)。在这些植物的根浸时,接种物被稀释。如果有更多的植物接种相同的接种物,则必须通过加入新鲜的分生孢子将所用接种物的浓度回收至5×10 6分配孢子/ml。更好地准备浓度为5×10 6 /分钟/ml的新接种物。
  2. 关于疾病症状评估,我们强烈建议同一人总是评估疾病症状。因此评估的任何错误都会较小,或至少相等于所有评估。
  3. 关于真菌学重新分离:虽然重新分离培养基含有300mg/ml的抗生素,并且在无菌环境中进行再分离,但是在培养基上铺板木质部之后会出现许多不同的微生物。室温下培养板应不超过5天。
  4. 必须使用光学显微镜对再隔离板上的新生菌丝进行形态学分析,以确认V的存在。 nonalfalfae 。 V的空气菌丝体非苜蓿通常是丰富的,絮状的,细菌的,菌丝是光滑的,宽1.5-3μm。分生孢子梗是直立或倾斜的,通常是确定的,分枝的或不分枝的,在整个殖民地和透明质素中不连贯地形成。

食谱

  1. GFM
    2克蛋白胨
    2克酵母提取物
    20 g葡萄糖 1 g KNO <3>
    在1000毫升蒸馏水中混合,121℃高压灭菌20分钟
  2. PDA用300毫克/毫升抗生素
    1. 在1000 ml蒸馏水中加入35 g马铃薯葡萄糖琼脂
    2. 摇动并混合粉末,高压灭菌20分钟
    3. 将PDA肉汤冷却至55°C
    4. 加入1 ml的100 mg/ml硫酸链霉素,1 ml的100mg/ml新霉素和1 ml的100mg/ml氯霉素,并混匀
    5. 倒入90 x 15毫米的培养皿

致谢

该协议改编自以前发表的论文(Radisek等人,2006;Jakše等人,2013; Cregeen等人,2015年) ; Mandelc等人,2013; Flajsman等人,2016)。我们承认斯洛文尼亚研究机构P4-0077研究计划的资金。

参考文献

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  2. Cregeen,S.,Radisek,S.,Mandelc,S.,Turk,B.,Stajner,N.,Jakse,J.and Javornik,B。(2015)。  抗性和敏感性啤酒花品种的不同基因表达响应于具有高侵袭性菌株的轮枝孢菌-atrum 。植物分子生物学报告 33(3):689-704。
  3. Ellendorff,U.,Fradin,EF,De Jonge,R.and Thomma,BP(2009)。< a class ="ke-insertfile"href ="http://jxb.oxfordjournals.org/content/60/2/591.full"target ="_ blank">对于
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  4. Flajsman,M.,Mandelc,S.,Radisek,S.,Stajner,N.,Jakse,J.,Kosmelj,K.and Javornik,B。(2016)。  通过在蟑螂定居期间通过轮枝孢属非苜蓿草分泌的木质部的新型毒力相关蛋白的鉴定植物微生物相互作用 29(5):362-373。
  5. Fradin,EF,Zhang,Z.,Juarez Ayala,JC,Castroverde,CD,Nazar,RN,Robb,J.,Liu,CM和Thomma,BP(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/19321708"target ="_ blank">由番茄Ve1介导的黄萎病的遗传解剖。 >植物生理学 150(1):320-332。
  6. Jakše,J.,Cerenak,A.,Radisek,S.,Satovic,Z.,Luthar,Z。和Javornik,B。(2013)。  确定抗轮霉病的数量性状基因座的确定和产量参数(葎草属)/em> L.)。 Theor Appl Genet 126(6):1431-1443。
  7. Kayser,T。(1992)。原生质体融合筛选嗜血杆菌染色体和Genkartierung beifilamentösenPilzen:青霉属青霉素,茜草a a>> und>>。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。论文。
  8. Klosterman,SJ,Subbarao,KV,Kang,S.,Veronese,P.,Gold,SE,Thomma,BP,Chen,Z.,Henrissat,B.,Lee,YH,Park,J.,Garcia-Pedrajas,MD ,Barbara,DJ,Anchieta,A.,de Jonge,R.,Santhanam,P.,Maruthachalam,K.,Atallah,Z.,Amyotte,SG,Paz,Z.,Inderbitzin,P.,Hayes,RJ,Heiman ,DI,Young,S.,Zeng,Q.,Engels,R.,Galagan,J.,Cuomo,CA,Dobinson,KF和Ma,LJ(2011)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/21829347"target ="_ blank">比较基因组学得出植物血管枯萎病原体的利基适应性的见解 PLoS Pathog 7(7):e1002137。
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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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