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Visual Assessment of the Severity of Fusarium Seedling Blight (FSB) and Fusarium Head Blight (FHB) Disease in Barley
大麦中镰刀菌属苗枯病(FSB)和镰刀菌属赤霉病(FHB)严重性的目测评估   

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Abstract

Fusarium pathogens are among the most damaging pathogens of cereals. These pathogens have the ability to attack the roots, seedlings, and flowering heads of barley and wheat plants (Simpson et al., 2004). Resulting in yield loss and head blight disease and also resulting in the contamination of grain with mycotoxins harmful to human and animal health (McMulen et al., 1997; Walter et al., 2010; Agostinelli et al., 2012). The study of Fusarium diseases, including host disease resistance and the effect of exogenous agents (chemicals, biocontrol agents, etc.), requires robust and effective methods for the assessment and quantification of visual disease symptoms. Here we describe the methods commonly used for the assessment and quantification of the severity of Fusarium seedling blight and Fusarium head blight disease.

Materials and Reagents

  1. Fusarium culmorum strain FCF 200 (Courtesy of Paul Nicholson, https://www.jic.ac.uk/directory/paul-nicholson/)
  2. Potato dextrose agar (Difco, catalog number: 213400 )
  3. Mung bean (any grocery store)
  4. John Innes compost No 2 (Westland Horticulture)
  5. Tween 20 (Sigma-Aldrich, catalog number: P2287 )
  6. Fertiliser NPK 10-10-20 (Agrifert)
  7. Agar (OXOID, catalog number: LP0013 )
  8. Filter paper (Whatman, catalog number: 1001-090 )
  9. Mung bean broth (see Recipes)

Equipment

  1. KOVA Glasstic Slide 10 (KOVA International Inc., catalog number: 87144 )
  2. Silicon tubing (0.5 cm diameter)
  3. Plant growth room
  4. -70 °C freezer (Thermo Scientific Revco PLUS)
  5. Shaking incubator (New Brunswick Scientific, model: I26 )
  6. Light microscope (Leica Microsystems, model: DM3000 )
  7. Glasshouse (CambridgeHOK containment glasshouse) [22 °C; maximum temperature of 27 °C and minimum light intensity of 700 μmol/m2/s under a 16 h/8 h day (700 μmol/m/s)/night regime or a contained environment room with an optimal 16 h/8 h day (700 μmol/m/s)/night at 20/12 °C]

Procedure

  1. Preparation of fungal conidial inoculum
    1. Store F. culmorum mycelia at -70 °C in 10% (vv-1) glycerol. Prior to use, culture the Fusarium isolate on potato dextrose agar (PDA) plates and incubated at 25 °C for 7 days.
    2. For conidial production, inoculate 3-4 PDA-agar plugs in 100 ml conical flask containing 25 ml Mung bean broth media (see Recipes) and incubate cultures at 200 rpm, and at 25 °C for 5 days without light.
    3. Pass the conidial suspension through two layers of sterile cheese cloth and wash three times with sterile distilled water. Resuspend conidia in 0.2% Tween 20 and determine the concentration using disposable KOVA Glasstic Slides 10. Adjust the concentration to 2 x 106 spores/ml 0.2% Tween 20.
      Note: For spore counting using KOVA Glasstic slides, determine the average number of spores per small grid and multiply with 90,000 to get number spores/ml.

  2. Fusarium seedling blight
    1. Place barley seeds in a 9 cm petri plate containing 2 pieces of filter paper (90 mm diameter) soaked with 6ml of sterile distilled water.
    2. Cover the petri plates with aluminum foil and stratify in the dark for 2 days at 4 °C.
    3. Transfer plates to 21 °C in the dark for 2 days to facilitate germination.
    4. Transfer germlings to a 6 cm-diameter pot containing John Innes compost No 2 and place a 2-cm length of sterile silicon tubing (0.5-cm-diam.) as a collar around the stem base.
    5. Place plants in a climate-controlled growth room at 22 °C under long day conditions (16 h/8 h), bottom watering every second day. Relative humidity was maintained at 70%.
    6. Inoculate the stem bases of 10-day-old seedlings with a 400 μl F. culmorum conidial suspension [1 x 106 spores/ml 0.2% Tween 20 in 1% (wv-1) agar]. Treat the mock control plants with 1% agar containing 0.2% Tween 20.
      Note: Prepare 2% (wv-1) sterile agar solution and bring it to 50 °C. Mix the spore suspension (2 x 106 spores/ml) and the agar solution at a 1:1 ratio.
    7. Score visible disease symptoms (Figure 1) on the stems at 12 days post-inoculation using the following disease scoring system: FSB disease scores were the product of lesion length (cm) by lesion colour (lesion colour scale: 0, no disease; 1, very slight brown necrosis; 2, slight/moderate brown necrosis; 3, extensive brown necrosis; 4, extensive black necrosis) (Nicholson et al., 1998).
    8. Repeat the experiment three times, each time including three replicate pots (each containing two plants) per treatment combination in a randomised layout.


      Figure 1. Barley seedlings displaying symptoms of Fusarium (cultivar Lux). Ten-day-old seedlings were treated with: A 0.2% Tween 20 and 1% (wv-1) agar, or B conidia of Fusarium culmorum (strain FCF 200) in 0.2% Tween 20 and 1% agar. Disease symptoms (browning of the stem base) were visualised 12 days post-treatment.

  3. Fusarium head blight
    1. Germinate the barley seeds as described above for the Fusarium seedling blight experiments.
    2. Transfer germinated seed to a 15 cm-diameter pot containing John Innes compost No 2. Cultivate plants in the glasshouse. Add 2 g of NPK 10-10-20 fertiliser to each pot at growth stages (GS; Zadoks et al., 1974) 20 and 30.
      Note: For more information on different Zadoks growth stages go to http://www.cerealcentral.ca/crop-management_cereal-staging.aspx.
    3.  Treat the heads at mid-anthesis (GS 65) by injecting 4 µl of conidial suspension (5x 104 spores/ml 0.2% Tween 20) or mock 0.2% Tween 20 treatment into each of the four middle spikelets of a head.
    4. Immediately cover treated heads with a polythene bag for 48 h in order to promote Fusarium infection.
    5. Score the FHB disease symptoms (Figure 2) (percentages bleached spikelets per head) at GS 70, 80 and 90 and calculate the Area Under the Disease Progress Curve (AUDPC) [as described by Shaner and Finney (1977)].

      Where, Yi is an assessment of a disease progression (percentages bleached spikelets per head) at the ith observation, ti is time (day) at the ith observation, and n is the total number of observations (Shaner and Finney, 1977).
    6. When ripe (GS 91), harvest the cereal heads, freeze-dry for 48 h and determine the number and weight of grains in each head. Use this data to calculate the 1,000 grain weight.
    7. The glasshouse FHB disease trials should be conducted at least twice and each trial should include at least 5 replicate plants (2 heads per plant) per treatment combination arranged in a randomised layout.


      Figure 2. Symptoms of Fusarium head blight in barley (cultivar Akashinriki). Four middle spikelets were injected with 4 µl of A conidia suspension of Fusarium culmorum (strain FCF 200) in 0.2% Tween 20, or B 0.2% Tween 20. Disease symptoms (percentages of bleached spikelets per head) and AUDPC were scored at GS 80. White arrow indicates the point of inoculation.

Recipes

  1. Mung bean broth
    Boil 20 g/L mung bean in distilled water for 20 min
    Pass the broth through two layers of cheese cloths and make up the volume up to 1 L
    Autoclave for 15-20 min at 121 °C

Acknowledgments

This work was supported by the Science Foundation Ireland research fund (IN10/IN.1/B3028) and Department of Agriculture Research Stimulus Grant RSF 07 513.

References

  1. Agostinelli, A. M., Clark, A. J., Brown-Guedira, G. and Van Sanford, D. A. (2012). Optimizing phenotypic and genotypic selection for Fusarium head blight resistance in wheat. Euphytica 186(1): 115-126.
  2. Ali, S. S., Gunupuru, L. R., Kumar, G. B., Khan, M., Scofield, S., Nicholson, P. and Doohan, F. M. (2014). Plant disease resistance is augmented in uzu barley lines modified in the brassinosteroid receptor BRI1. BMC Plant Biol 14: 227.
  3. Ali, S. S., Kumar, G. B., Khan, M. and Doohan, F. M. (2013). Brassinosteroid enhances resistance to fusarium diseases of barley. Phytopathology 103(12): 1260-1267.
  4. McMullen, M., Jones, R. and Gallenberg, D. (1997). Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Dis 81(12): 1340-1348.
  5. Nicholson, P., Simpson, D., Weston, G., Rezanoor, H., Lees, A., Parry, D. and Joyce, D. (1998). Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol Mol Plant Pathol 53(1): 17-37.
  6. Shaner, G. and Finney, R. (1977). The effect of nitrogen fertilization on the expression of slow-mildewing resistance in Knox wheat. Phytopathology 67(8): 1051-1056.
  7. Simpson, D. R., Thomsett, M. A. and Nicholson, P. (2004). Competitive interactions between Microdochium nivale var. majus, M. nivale var. nivale and Fusarium culmorum in planta and in vitro. Environ Microbiol 6(1): 79-87.
  8. Walter, S., Nicholson, P. and Doohan, F. M. (2010). Action and reaction of host and pathogen during Fusarium head blight disease. New Phytol 185(1): 54-66.
  9. Zadoks J. C., Chang T. T. and Koznak C .F. (1974). A decimal code for the growth stages of cereals. Weed Res 14(6): 415-421.

简介

镰孢属病原体是谷类中最具破坏性的病原体之一。 这些病原体具有攻击大麦和小麦植物的根,幼苗和开花头的能力(Simpson等人,2004)。 导致产量损失和赤霉病,并且还导致谷粒被对人类和动物健康有害的霉菌毒素的污染(McMulen等人,1997; Walter等人, ,2010; Agostinelli et al。,2012)。 镰刀菌属疾病的研究,包括宿主疾病抗性和外源性试剂(化学品,生物控制剂等)的影响,需要用于评估和定量视觉疾病症状的强大且有效的方法。 在这里我们描述了通常用于评估和量化镰刀菌枯萎病和镰孢属赤霉病的严重性的方法。

材料和试剂

  1. Fusarium culmorum 菌株FCF 200(由Paul Nicholson提供, https: //www.jic.ac.uk/directory/paul-nicholson/
  2. 马铃薯葡萄糖琼脂(Difco,目录号:213400)
  3. 绿豆(任何杂货店)
  4. John Innes堆肥No 2(Westland园艺)
  5. 吐温20(Sigma-Aldrich,目录号:P2287)
  6. 肥料NPK 10-10-20(Agrifert)
  7. 琼脂(OXOID,目录号:LP0013)
  8. 滤纸(Whatman,目录号:1001-090)
  9. 绿豆汤(见配方)

设备

  1. KOVA Glasstic Slide 10(KOVA International Inc.,目录号:87144)
  2. 硅管(直径0.5cm)
  3. 植物生长室
  4. -70℃冷冻器(Thermo Scientific Revco PLUS)
  5. 摇动培养箱(New Brunswick Scientific,型号:I26)
  6. 光学显微镜(Leica Microsystems,型号:DM3000)
  7. 玻璃屋(CambridgeHOK containment glasshouse)[22°C; 最大温度为27℃,在16小时/8小时(700μmol/m/s)/夜间条件下的最小光强度为700μmol/m 2/s, 在20/12℃下最佳的16小时/8小时(700μmol/m/s)/夜)

程序

  1. 真菌分生孢子接种物的制备
    1. 存储 F。 大肠杆菌在-70℃在10%(vv <-1)甘油中的菌丝体。 之前 使用,将马铃薯葡萄糖琼脂(PDA)平板上的镰孢分离物培养 并在25℃下培养7天
    2. 对于分生孢子, 接种3-4个PDA琼脂塞在100ml锥形瓶中,含有25ml 绿豆肉汤培养基(见Recipes)并以200rpm培养培养物, 并在25℃,无光照下5天。
    3. 通过分生孢子 悬浮液通过两层无菌奶酪布洗涤三次 次,用无菌蒸馏水。 重悬在0.2%Tween 20中的分生孢子 并使用一次性KOVA Glasstic载玻片测定浓度 10.将浓度调节至2×10 6孢子/ml 0.2%吐温20 注意:对于使用KOVA Glasstic幻灯片的孢子计数,确定 每个小网格的平均孢子数,并乘以90,000得到 数孢子/ml。

  2. 镰刀菌苗枯病
    1. 将大麦种子放在含有2片过滤器的9cm培养皿中 纸(90mm直径),用6ml无菌蒸馏水浸泡
    2. 用铝箔覆盖培养皿,在4℃下在黑暗中分层2天
    3. 转移板21°C在黑暗中2天,以促进发芽
    4. 将胚芽转移到包含John Innes的6厘米直径的锅中 堆肥No 2并放置2cm长的无菌硅管 (0.5-cm直径)作为围绕茎基部的颈圈
    5. 将植物放在   气候控制的增长室在22℃在长日照条件下(16 h/8 h),每隔一天底部浇水。 相对湿度 维持在70%。
    6. 接种10日龄的茎基 幼苗用400μlF。 大肠杆菌悬浮液[1×10 6个孢子/ml 0.2%吐温20的1%(wv < - sup>)琼脂]中。 处理模拟控制 植物用含有0.2%吐温20的1%琼脂 注意:准备2% (wv-sup)无菌琼脂溶液中并使其达到50℃。 混合孢子 悬浮液(2×10 6个孢子/ml)和琼脂溶液以1:1的比例混合。
    7. 在12天时对茎上可见的疾病症状(图1)进行评分 使用以下疾病评分系统进行后接种:FSB疾病   分数是病变长度(cm)与病变颜色(病变)的乘积 色标:0,无病; 1,非常轻微的褐色坏死; 如图2所示, 轻微/中度褐色坏死; 3,广泛的褐色坏死; 如图4所示, 广泛的黑色坏死)(Nicholson等人,1998)。
    8. 重复 实验三次,每次包括三个重复盆(每个 含有两株植物)。


      图1.显示镰孢属(栽培品种Lux)的症状的大麦幼苗。将10天龄的幼苗用以下处理:将0.2%吐温20   和1%(wv -1)琼脂,或大刀镰孢(Fusarium culmorum)(菌株FCF 200)的B分生孢子 在0.2%Tween 20和1%琼脂中。 疾病症状(茎的褐变 碱基)在处理后12天可视化。

  3. 镰孢属枯萎病
    1. 如上所述对镰孢幼芽枯萎实验发芽大麦种子。
    2. 转移发芽的种子到包含约翰的15厘米直径的锅 Innes堆肥No 2.在温室里种植植物。加入2g NPK 10-10-20肥料施用于生长阶段的每个罐(GS; Zadoks等人, 1974)20和30.
      注意:有关不同Zadoks的详细信息 成长阶段请前往 http://www.cerealcentral.ca/crop-management_cereal-staging.aspx
    3.  通过注射4μl分生孢子来处理头部在中期(GS 65) 悬浮液(5×10 4孢子/ml 0.2%吐温20)或模拟0.2%吐温20 处理成一个头的四个中小穗的每一个。
    4. 立即用聚乙烯袋覆盖治疗头48小时,以促进镰刀菌感染。
    5. 评分FHB疾病症状(图2)(漂白的百分比 小穗/头),GS 70,80和90,计算下面的面积  疾病进展曲线(AUDPC)[如Shaner和Finney所述 (1977)]。

      其中,Yi 是疾病进展的评估 ( 观察时, ti 的百分比漂白小穗数) 在 观察时的时间(天),以及 n 观察(Shaner和Finney,1977)。
    6. 成熟时(GS 91), 收获谷物头,冷冻干燥48小时,并确定数量 和每个头中的颗粒重量。 使用此数据计算1,000 颗粒重量。
    7. 温室FHB疾病试验应该 进行至少两次,每次试验至少应包括5次 每个处理组合安排的复制植物(每株植物2头) 在随机布局中

      图2.镰孢属枯萎病的症状 在大麦(栽培品种Akashinriki)中。注射四个中部小穗 与4μl的大刀镰孢(Fusarium culmorum)(菌株FCF 200)的分生孢子悬浮液 在0.2%吐温20或B 0.2%吐温20中 漂白的小穗/头)和AUDPC得分在GS80。白色箭头   表示接种点。

食谱

  1. 绿豆汤
    煮20克/升绿豆在蒸馏水中20分钟
    将肉汤通过两层奶酪布,并使体积达到1 L
    在121℃下高压灭菌15-20分钟

致谢

这项工作得到了科学基金会爱尔兰研究基金(IN10/IN.1/B3028)和农业研究刺激捐赠基金RSF 07 513的支持。

参考

  1. Agostinelli,A.M.,Clark,A.J.,Brown-Guedira,G。和Van Sanford,D.A。(2012)。 优化镰刀菌抗虫抗性的表型和基因型选择 在小麦中。 Euphytica 186(1):115-126。
  2. Ali,S.S.,Gunupuru,L.R.,Kumar,G.B.,Khan,M.,Scofield,S.,Nicholson,P.and Doohan,F.M。 在油菜素类固醇受体BRI1中修饰的uzu大麦品系中植物病害抗性增强。 em> BMC Plant Biol 14:227.
  3. Ali,S.S.,Kumar,G.B.,Khan,M。和Doohan,F.M。(2013)。 油菜素类固醇增强抗大麦镰刀菌病的能力。植物病理 103(12):1260-1267。
  4. McMullen,M.,Jones,R。和Gallenberg,D。(1997)。 小麦和大麦的疤痕:一种重新出现的破坏性影响疾病。 a> Plant Dis 81(12):1340-1348。
  5. Nicholson,P.,Simpson,D.,Weston,G.,Rezanoor,H.,Lees,A.,Parry,D。和Joyce,D。(1998)。 大镰刀菌和禾本科镰孢菌的检测和定量使用PCR测定法在谷类中进行。 Physiol Mol Plant Pathol 53(1):17-37。
  6. Shaner,G.and Finney,R。(1977)。 氮施肥对诺克斯慢霉病抗性表达的影响小麦。 Phytopathology 67(8):1051-1056。
  7. Simpson,D.R.,Thomsett,M.A。和Nicholson,P。(2004)。 Microdochium nivale var。 majus , nivale var。 1):79-87。
  8. Walter,S.,Nicholson,P.and Doohan,F.M。(2010)。 镰刀菌赤霉病病毒中宿主和病原体的作用和反应。/a> New Phytol 185(1):54-66。
  9. Zadoks J.C.,Chang T.T.和Koznak C.F. (1974)。 谷物生长阶段的十进制代码。 Weed Res 14(6):415-421。
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引用:Ali, S. S., Gunupuru, L. R. and Doohan, F. M. (2015). Visual Assessment of the Severity of Fusarium Seedling Blight (FSB) and Fusarium Head Blight (FHB) Disease in Barley. Bio-protocol 5(12): e1507. DOI: 10.21769/BioProtoc.1507.
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