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Fusarium Virulence Assay on Wheat and Barley Seedlings
小麦和大麦幼苗的抗镰刀菌毒性试验   

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Abstract

Fusarium root and crown rot is a very important complex disease of small grain cereals worldwide which may lead to very high yield losses. Traditional virulence assays are time consuming and often require plants to be grown in greenhouses or climatic chambers in soil. We describe a rapid laboratory assay for assessing such a disease in wheat and barley seedlings. The method could be successfully used for testing fungal virulence as well as to assess plant resistance.

Materials and Reagents

  1. Fungal strains
  2. 25% Campbell`s V8 juice (pH 6.5 with NaOH)
  3. 1.2% agar plates (9 cm diameter)
  4. Wheat and barley seeds
  5. Sterilizing solution (0.64% sodium hypochlorite - 10% ethanol)
  6. Sterile deionized water

Equipment

  1. Petri dishes (9 and 14 cm diameter)
  2. Filter paper (Whatman No.3, 8 and 12.5 cm diameter)
  3. Pipette tip or cork borer
  4. Sealing film (Phyto Technology Laboratories) or parafilm
  5. Ruler
  6. Razor blades
  7. Laminar flowhood
  8. Illuminated incubator at 21 ± 1 °C, light intensity > 90 μmols
  9. Fungal growth cabinet with white (one 18 W lamp) and black fluorescent (one 18 W lamp) lights on 12/12 h day/night cycle at 20-22 °C
  10. Incubator

Procedure

  1. Day 1
    Grow fungal colonies on V8 agar plates for 7 days at 20-22 °C under white and black fluorescent light on a 12/12 h day/night cycle, starting from stored fungal cultures or colonies from plates.
  2. Day 3 (wheat) or 4 (barley)
    For each fungal isolate or plant genotype 16 germinated seeds are required. If testing fungal isolate virulence, a mock treatment is also required. Barley is more vigorous in the early stages of growth and seeds are allowed to germinate for 2 days whereas wheat is allowed to germinate for 3 days prior to inoculation.
    1. Disinfect wheat or barley seeds in sterilizing solution for 5 min and wash 3 times with sterile deionized water.
    2. Imbibe disinfected seeds in 9 cm Petri dishes containing one 8 cm filter paper soaked with sterile deionized water at 4 °C for 1 day. Keep in dark by wrapping plates in aluminium foil.
  3. Day 4 (wheat) or 5 (barley)
    Transfer plates to plant growth chamber but maintain in darkness (wrapped in foil).
  4. Day 6
    1. Place three 12.5 cm diameter filter paper layers into 14 cm Petri dishes.
    2. Add sterile deionized water to filter paper until completely soaked.
    3. Remove excess water from plates.
    4. Place 16 pre-germinated wheat or barley seeds on filter paper in rows of 3-5-5-3 seeds.
    5. Seal plates with film and place plates to plant growth chamber in dark overnight.
  5. Day 7
    1. Make mycelial plugs of approximately 5-6 mm diameter by using the reverse of a pipette tip or a cork borer from the edge of the growing colony (Figure 1).


      Figure 1. Mycelial plugs obtained from the edge of a growing Fusarium colony.
       
    2. Place mycelial plugs upside down on the main root of each germinated seed when about 3 cm long, at approximately 1 cm from the seed, with the mycelium in direct contact with the root (Figure 2).


      Figure 2. Mycelial plugs on a wheat rootle.

    3. Seal plates with sealing film or Parafilm.
    4. Put plates in incubator at 21 ± 1 °C for 4 to 6 days with 12 h of light.
  6. Day 11-13 as required
    1. Determination of root-rot: observation of root necrosis at 4 days post-inoculation by measuring symptom extension (SE) (cm) and visually observing the browning index (BI, 0 = symptomless; 1 = slightly necrotic; 2 moderately necrotic; 3 = severely necrotic; 4 = completely necrotic).
    2. Determination of crown-rot: Visual observation of crown necrosis at 6 days post-inoculation by a 0-4 scale (0 = symptomless; 1 = slightly necrotic; 2 moderately necrotic; 3 = severely necrotic; 4 = completely necrotic), measuring of shoot length with a ruler (cm) and weighing the shoots after their dissection with a razor blade.

      Please see Figure 1 of Beccari et al. (2011) and Figures 6 and 8 of Gardiner et al. (2012).

Acknowledgments

The present protocol here described in detail was adopted in experiments reported in the following publications: Beccari et al. (2011) and Gardiner et al. (2012). Work was supported by the Australian Grains Research and Development Corporation, an Australian statutory authority.

References

  1. Beccari, G., Covarelli, L. and Nicholson, P. (2011). Infection processes and soft wheat response to root rot and crown rot caused by Fusarium culmorum. Plant Pathol 60(4): 671-684.
  2. Gardiner, D. M., McDonald, M. C., Covarelli, L., Solomon, P. S., Rusu, A. G., Marshall, M., Kazan, K., Chakraborty, S., McDonald, B. A. and Manners, J. M. (2012). Comparative pathogenomics reveals horizontally acquired novel virulence genes in fungi infecting cereal hosts. PLoS Pathog 8(9): e1002952.

简介

镰刀根和冠腐病是全世界小谷物谷物非常重要的复杂疾病,可能导致非常高的产量损失。 传统的毒力测定是耗时的,并且通常需要植物在土壤中的温室或气候室中生长。 我们描述了用于在小麦和大麦幼苗中评估这种疾病的快速实验室测定法。 该方法可以成功地用于测试真菌毒力以及评估植物抗性。

材料和试剂

  1. 真菌菌株
  2. 25%Campbell V8汁(pH6.5,NaOH)
  3. 1.2%琼脂板(直径9cm)
  4. 小麦和大麦种子
  5. 灭菌溶液(0.64%次氯酸钠-10%乙醇)
  6. 无菌去离子水

设备

  1. 培养皿(9和14cm直径)
  2. 滤纸(Whatman No.3,8和12.5cm直径)
  3. 移液器吸头或软木钻孔器
  4. 密封膜(Phyto Technology Laboratories)或parafilm
  5. 标尺
  6. 剃刀刀片
  7. 层流模式
  8. 在21±1℃的发光培养箱中,光强度> 90μmols
  9. 具有白色(一个18W灯)和黑色荧光灯(一个18W灯)的真菌生长柜在12-22小时日/夜循环在20-22℃照明
  10. 孵化器

程序

  1. 第1天
    在储存的真菌培养物或来自平板的菌落开始的12-12小时白天/黑夜循环,在白色和黑色荧光灯下,在20-22℃下,在V8琼脂平板上生长真菌菌落7天。
  2. 第3天(小麦)或4(大麦)
    对于每种真菌分离物或植物基因型16,需要发芽的种子。如果测试真菌分离株的毒力,还需要模拟处理。大麦在生长的早期阶段更有活力,种子允许发芽2天,而小麦在接种前允许发芽3天。
    1. 在灭菌溶液中消毒小麦或大麦种子5分钟,用无菌去离子水洗涤3次
    2. Imbibe消毒的种子在9cm培养皿中,所述培养皿含有在4℃下用无菌去离子水浸泡的1cm 8cm滤纸1天。通过在铝箔中包装板保持在黑暗中。
  3. 第4天(小麦)或5(大麦)
    转移板到植物生长室,但保持在黑暗中(包裹在箔)。
  4. 第6天
    1. 将三个12.5厘米直径的滤纸层放入14厘米培养皿
    2. 向滤纸中加入无菌去离子水,直到完全浸泡
    3. 清除板上多余的水。
    4. 将16颗预发芽的小麦或大麦种子放在滤纸上,每排3-5-5-3粒种子
    5. 用薄膜密封板,并在黑暗中过夜培养植物生长室
  5. 第7天
    1. 通过使用移液管尖端或软木钻孔从生长的殖民地的边缘(图1)的直径大约5-6毫米的菌丝体插头。


      图1.从生长的Fusarium菌落边缘获得的菌丝体 。
       
    2. 将菌丝体倒置放置在每个发芽种子的主根上,长约3厘米,距离种子约1厘米,菌丝体与根直接接触(图2)。


      图2.小麦根上的菌丝插头。

    3. 密封板带密封膜或石蜡膜
    4. 将培养皿放在培养箱中,在21±1℃下培养4至6天,12小时光照
  6. 第11-13天
    1. 根腐病的测定:通过测量症状延伸(SE)(cm)和肉眼观察褐变指数(BI,0 =无症状; 1 =轻微 坏死 2中度坏死; 3 =严重坏死; 4 =完全坏死)。
    2. 冠腐病的测定:通过0-4量表(0 =无症状; 1 =轻微坏死; 2中度坏死; 3 =严重坏死; 4 =完全坏死)目测观察冠状坏死在6天后的测量, 用直尺(cm)拍摄长度,并用刀片称重切片后称重
      请参阅Beccari等人(2011)的图1和Gardiner等人(2012年)的图6和8。

致谢

这里详细描述的本方案在以下出版物中报道的实验中采用:Beccari等人(2011)和Gardiner等人(2012)。 澳大利亚谷物研究与开发公司(Australian Grains Research and Development Corporation)是澳大利亚法定机构的支持者。

参考文献

  1. Beccari,G.,Covarelli,L.和Nicholson,P。(2011)。 感染过程和软小麦对根腐病和冠腐病的反应 。 植物病理学 60(4):671-684
  2. Gardiner,D.M.,McDonald,M.C.,Covarelli,L.,Solomon,P.S.,Rusu,A.G.,Marshall,M.,Kazan,K.,Chakraborty,S.,McDonald,B.A.and Manners, 比较病原体显示在感染谷类宿主的真菌中水平获得的新型毒力基因。 PLoS Pathog 8(9):e1002952。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Covarelli, L., Gardiner, D., Beccari, G. and Nicholson, P. (2013). Fusarium Virulence Assay on Wheat and Barley Seedlings. Bio-protocol 3(7): e446. DOI: 10.21769/BioProtoc.446.
  2. Gardiner, D. M., McDonald, M. C., Covarelli, L., Solomon, P. S., Rusu, A. G., Marshall, M., Kazan, K., Chakraborty, S., McDonald, B. A. and Manners, J. M. (2012). Comparative pathogenomics reveals horizontally acquired novel virulence genes in fungi infecting cereal hosts. PLoS Pathog 8(9): e1002952.
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