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Rapid Screening and Evaluation of Maize Seedling Resistance to Stalk Rot Caused by Fusarium spp.

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Molecular Plant Microbe Interactions
Aug 2007



Corn stalk rot caused by Fusarium spp., a genus of soil-borne fungal pathogens, has become a major concern of maize production. This disease normally causes significant reduction of maize yield and quality worldwide. The field assay for identifying stalk rot resistance using adult plants is largely relying on large population, yet time-consuming, labor costs, and often influenced by environmental conditions. Therefore, a rapid and reliable assay for investigating maize stalk rot caused by Fusarium spp. is required for screening the resistant lines and functional study of maize resistance to this pathogen. We have developed a seedling assay to rapidly screen the resistant lines using 12-day to 2-week-old seedlings. The entire assay can be completed within approximately 16-18 days post seed germination, with inexpensive labor cost and high repeatability. This simple, rapid and reliable assay can be widely used for identifying the maize resistance to stalk rot caused by Fusarium spp. and other similar fungal pathogens.

Keywords: Corn stalk rot (玉米茎腐病), Disease resistance (抗病性), Fusarium spp. (镰刀菌属), Seedling assay (幼苗分析)


Maize is one of the most important staple food crops and energy plants worldwide. It has been becoming the No.1 crop species in China and worldwide regarding both yield and planting areas since 2012. However, corn stalk rot has become one of the most destructive diseases, which normally lead to significant yield loss and quality reduction of maize extensively (Oerke, 2006). The pathogens causing corn stalk rot mainly include the soil-borne fungal species, such as Fusarium graminearum, F. verticillioides, Colletotrichum graminicola, Pythium aphanidermatum, and Pectobacterium chrysanthemi. Infection of maize by F. graminearum (teleomorph Gibberella zeae Schw. Petch) normally causes Gibberella stalk rot with reddish-pink discoloration inside the stalk, and Gibberella ear rot with carcinogenic mycotoxins deoxynivalenol (DON) and zearalenone produced in mature corn kernels that are harmful to human and animals (Mesterhazy et al., 2012). Moreover, F. graminearum is also the major causal agent infecting other small grain cereal crops, such as wheat, leading to the well-known Fusarium head blight (FHB), which is one of the most serious diseases impacting wheat production in China and is endemic in many wheat-producing countries (Walter et al., 2010; Ma et al., 2017). F. verticillioides [=F. moniliforme J. Sheld.(sexual stage: G. moniliformis Wineland)], on the other hand, predominantly infect maize, resulting in Fusarium stalk rot and Fusarium ear rot, and producing Fumonisins, another type of mycotoxins in maize kernels (Presello et al., 2008). Thus, the increasing prevalence of mycotoxins produced by Fusarium spp., along with their direct impact on yield losses in many cereal-growing regions worldwide, has become one of the major concerns of much research.

In recent years, due to the rapid development and application of mechanization, the harvesting technology of corn grain by machines puts forward a high requirement of disease resistance to corn stalk rot. Currently, the field assay for identifying stalk rot resistance using adult plants is largely relying on large population, yet time-consuming, labor costs, and often influenced by environmental conditions. Therefore, a rapid, reliable and large-scale method for screening disease resistant lines and identifying the resistance to corn stalk rot is extremely in urgent, which can provide a great convenience for the researchers to rapidly excavate the elite genetic resources of maize. We have previously deployed a seedling assay to identify the resistance of maize lines to stalk rot caused by F. verticillioides (Gao et al., 2007). Here, we describe the detailed procedures of modified protocol of a seedling assay, which can be applied to evaluate the stalk rot not only caused by Fusarium spp., but also other fungal pathogens, such as C. graminicola.

Materials and Reagents

  1. 1.5 ml centrifuge tubes (Nanjing Qingke Biological Company)
  2. 50 ml centrifuge tubes (Nanjing Qingke Biological Company)
  3. Long pot (20 cm tall x 5 cm diameter), hand-made with PVC tubes with a holed bottom cover
  4. Parafilm (Bemis, catalog number: PM996 )
  5. Press-in Saran Wrap (GLADR, Pressin Seal, 21.6 m x 30 cm), a type of sealing saran wrap with stickiness that can be pressed on the surface to seal tightly the object
  6. Conical bottle (200 ml)
  7. Paper towel (230 x 225 mm, Yong Li Yu Investment Company Limited, May Flower, catalog number: A18250S )
  8. Cheese cloth (40S Warp x 40S Weft; 5 m x 1.2 m), made by combed cotton
  9. Soil (Nanjing Shoude Company, nutrient soil: vermiculite = 2:1)
  10. Syringe needle (Luer-Lok 20G, BD, catalog number: 309634 )
  11. Plastic square trays (Purchased from market) (100 cm length x 80 cm width x 10 cm height)
  12. Maize seeds (Inbred line: B73; select the similar size seeds with germinating rate > 90%)
  13. Fungal strains: Fusarium graminearum (isolate: F0609); F. verticillioides (isolate: 7600)
  14. Sterile ddH2O
  15. Glycerin
  16. PDA (Potato dextrose agar) (Qingdao Hope Bio-Technology, catalog number: HB0233 )
  17. Tween-20 (SunShine Bio, catalog number: T0014 )
  18. Mung Bean (Purchased from market)
  19. PDA media (see Recipes)
  20. Mung bean soup (see Recipes)


  1. Pipettes (Eppendorf, Research Plus, catalog numbers: 3120000020 , 3120000046 , 3120000062 )
  2. Hemacytometer (0.100 mm, Hausser Scientific, catalog number: 3110 )
  3. Water bath (Changzhou Nuoji Instrument, catalog number: HHS-11-1 )
  4. Clean bench (AIRTECH, model: SW-CJ-1FD , catalog number: A16116317)
  5. Constant temperature incubator (Ningbo Jiangnan Instrument Factory, catalog number: DNP-9162 )
  6. Growth chamber (Ningbo Jiangnan Instrument Factory, model: RXM-508C-3 , catalog number: 17091559)
  7. Centrifuge (Eppendorf, model: 5424 , catalog number: 5424FL570190)
  8. Microscope (Olympus, catalog number: BX41 )
  9. Autoclave


  1. Growing maize Seedlings
    1. Sow the seeds of different maize inbred lines in the soil in long pots made with PVC cylinders (5 cm in diameter and 20 cm in depth), with 4 seeds per cylinder, covered with a layer of soil in 2 cm depth. To prove the stalk rot phenotype, a resistant line 1145 and a susceptible line Y331 to Gibberella stalk rot, described by Yang et al. (2010) can be included as controls.
    2. Place the pots in a controlled growth chamber (light cycle and temperature: 14 light/10 dark at 26 ± 2 °C; light intensity: 600 Lux) to allow the seedling growth.
    3. Select the seedlings in similar size at approximately 12-d to 2-week-old stage and transfer to a culture room for infection assay.

  2. Preparation of fungal spore suspension
    1. Fungal strain culture
      Fungal isolates (F. graminearum or F. verticillioides) are initially stored in 20% glycerin at -80 °C. Inoculate the fungal isolate on a PDA plate and incubate it in an incubator (25 °C) in the dark for at least 10 days.
    2. Stimulating sporulation
      Cut several agar blocks (5 x 5 mm) aseptically and place them in sterilized mung bean soup, and incubate in an incubator at 200 rpm, 28 °C for 2-3 d (Figure 1). 

      Figure 1. Mung bean soup with F. graminearum cultured for 2 days

    3. Spore enumeration and preparation of inoculum
      Filter the spore suspension in mung bean soup through two layers of cheese cloth, followed by centrifugation at 3,800 x g for 5 min. Discard the supernatant, re-suspend and wash the concentrated conidia twice with sterile water before diluting it to a concentration of 1.0 x 106/ml in 0.001% Tween-20.

  3. Inoculation of seedling stem with fungal spores
    1. Preparation of maize seedlings
      Keep the plastic square tray (100 x 80 x 10 cm) in a controlled incubation room (light cycle and temperature: 14 light/10 dark at 24 ± 2 °C; light intensity: 200 Lux). To maintain a high humidity (~75%) inside the tray, add 250 ml sterile water to a layer of paper towel on the bottom of the tray, until the towel is wetted completely. Discard the extra water if needed. Line-up the pots with seedlings horizontally in the tray (Figure 2).

      Figure 2. The arrangement of pre-inoculation environment. Maize seeds are planted and grown in a PVC cylinder in a control room for 12-14 days. The pots with seedlings are then lined up horizontally in a tray with a layer of paper towel completely wetted on the bottom of the tray.

    2. Artificial inoculation process
      To facilitate the infection of fungal spores, punch a tiny hole in approximately 1 mm depth in the middle position of first internode of the seedling stem using a syringe needle (Figure 3, Video 1), then drop 20 μl of spore suspensions in 0.001% Tween-20 to the wounded point carefully using a pipette (Figure 4, Video 2). Seal the plastic square tray with Press-in saran wrap to maintain the moisture. To allow the air exchange, make 6-8 uniform strip holes (in approximately 2 cm length) on the wrap. Upon infection, maintain the seedlings under a condition with 14 h light/10 h dark at 24 ± 2 °C for 3 d without moving (Figure 5). Check the humidity daily to ensure a high humidity at approximately 75%. Add the water if needed.

      Figure 3. Punching a hole on the stem of maize seedling

      Video 1. Punch a hole on the stem of maize seedling. Use a syringe needle to carefully make a tiny hole in approximately 1 mm depth on the first internode of maize seedling.

      Figure 4. Inoculation of maize stem with spore suspensions. 20 μl of spore suspensions in 0.001% Tween-20 is dropped to the wounded site on the seedling stem.

      Video 2. Inoculate maize seedling stem with Fusarium spp. spore suspensions. Drop 20 μl of spore suspensions in 0.001% Tween-20 to the wounded point carefully using a pipette.

      Figure 5. Maintainance of seedlings after inoculation. The plastic square tray is sealed with Press-in saran wrap to maintain a moisture > 75%. Several uniform holes are made on the cover surface. The tray is kept steadily in a controlled condition with 14 h light/10 h dark at 24 ± 2 °C.

Data analysis

  1. Rating scales of disease severity
    Score the stalk rot symptoms 3 days after artificial inoculation, with at least three scorings (15-20 seedlings per scoring per maize line). Based on the data collected from three or more scorings, estimate the disease severity for each individual plant using a 5-point classification scale ranging from 1 (highest resistance) to 5 (highest susceptibility) (Figure 6). Rating standards of corn stalk rot symptom are described in Table 1.

    Figure 6. Phenotypic characteristics of seedlings by a 5-point classification scale. CK: non-inoculated control; 1, the most resistant; 5, the most susceptible.

    Table 1. Scales and scoring standards of Gibberella stalk rot of maize seedling

  2. Calculation of disease severity index (DSI)
    Calculate the DSI according to the formula described by Ma et al. (2017) with some modifications:
    DSI (%) = ∑(ranking value x number of plants with that value) x 100/(1 x total number of plants). All of the statistical analyses were performed using SPSS (14.0) software for ANOVA and the Student’s t-test, with the level of significance set at P < 0.05.


  1. Select the healthy, non-stressed and uniform seedlings for infection assay.
  2. If the inoculum drops immediately from the infection site after inoculation, redo the infection.
  3. Maintain the appropriate humidity and temperature is the key for successful development and penetration of fungal pathogens.


  1. PDA media (200 ml)
    9.2 g Potato glucose agar powder dissolved in 200 ml ddH2O
    Sterilize with autoclave for 15 min at 120 °C
  2. Mung bean soup (1 L)
    1. Boil 40 g Mung bean in 250 ml ddH2O in a 85 °C water bath for 1 h
    2. Then filter through a layer of gauze, and dilute in autoclaved ddH2O to 1 L
    3. Sterilize the soup at 120 °C for 15 min


This protocol is partially adapted from our previous work (Gao et al., 2007), with some modifications. This work was supported by The National Key Research and Development Program of China (No. 2016YFD0101002), the NSFC (No. 31471508, No. 31670702), and from the Technology Foundation for Selected Overseas Chinese Scholar, Ministry of Personnel of China (No. G0101500090) and the Innovation Team Program for Jiangsu Universities (2014). The authors declare no conflicts of interest or competing interests.


  1. Gao, X., Shim, W. B., Gobel, C., Kunze, S., Feussner, I., Meeley, R., Balint-Kurti, P. and Kolomiets, M. (2007). Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin. Mol Plant Microbe Interact 20(8): 922-933.
  2. Ma, C., Ma, X., Yao, L., Liu, Y., Du, F., Yang, X. and Xu, M. (2017). qRfg3, a novel quantitative resistance locus against Gibberella stalk rot in maize. Theor Appl Genet 130(8): 1723-1734.
  3. Mesterhazy, A., Lemmens, M. and Reid, L. M. (2012). Breeding for resistance to ear rots caused by Fusarium spp. in maize – a review. Plant Breeding 131: 1-19.
  4. Oerke, E. C. (2006). Crop losses to pests. Journal of Agricultural Science 144: 31-43.
  5. Presello, D. A., Botta, G., Iglesias, J., Eytherabide, G. H. (2008). Effect of disease severity on yield and grain 591 fumonisin concentration of maize hybrids inoculated with Fusarium verticillioides. Crop Protection 27: (3-5): 572-576.
  6. 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.
  7. Yang, Q., Yin, G., Guo, Y., Zhang, D., Chen, S. and Xu, M. (2010). A major QTL for resistance to Gibberella stalk rot in maize. Theor Appl Genet 121: 673-687.



【背景】玉米是全球最重要的主粮作物和能源植物之一。自2012年以来,它已成为中国乃至全世界第一大产量和种植面积的作物品种。然而,玉米秸秆腐烂已成为最具破坏性的疾病之一,其通常导致玉米产量大幅下降和玉米质量下降(Oerke,2006)。引起玉米秸秆腐烂的病原体主要包括土壤真菌物种,例如禾谷镰刀菌F, (Verticillioides),
近年来,由于机械化发展和应用的迅速发展,玉米籽粒机械化采收技术对玉米秸秆腐病的抗病性提出了较高的要求。目前,使用成年植物鉴定茎腐病抗性的田间试验在很大程度上依赖于大量人群,但费时费力,并且经常受环境条件的影响。因此,一种快速,可靠,大规模筛选抗病品系和鉴定玉米茎腐病抗性的方法极为迫切,为研究人员迅速挖掘玉米精英遗传资源提供了极大的便利。我们之前已经部署了一个幼苗分析来鉴定玉米系对由F引起的茎腐病的抗性。 verticillioides (Gao等人,2007年)。在这里,我们描述了苗实验的修改方案的详细程序,其可以用于评估不仅由镰刀菌属引起的茎腐病,而且还可以用于其他真菌病原体如 C。蛛。

关键字:玉米茎腐病, 抗病性, 镰刀菌属, 幼苗分析


  1. 1.5 ml离心管(南京青科生物公司)

  2. 50毫升离心管(南京青科生物公司)
  3. 长盆(20厘米高x 5厘米直径),手工制作,PVC管带有一个带孔的底盖
  4. Parafilm(Bemis,目录号:PM996)
  5. 压入式Saran Wrap(GLAD R ,Pressin Seal,21.6 m x 30 cm),一种可以压在表面上以密封物体的粘性saran包装
  6. 圆锥形瓶(200毫升)
  7. 纸巾(230 x 225毫米,永利郁投资有限公司,五月花,产品编号:A18250S)

  8. 用精梳棉制成的奶酪布(40S经纱×40S纬纱; 5米×1.2米)
  9. 土壤(南京首德公司,营养土:蛭石= 2:1)
  10. 注射器针头(Luer-Lok 20G,BD,目录号:309634)
  11. 塑料方盘(购自市场)(100厘米长×80厘米宽×10厘米高)
  12. 玉米种子(自交系:B73;选择发芽率> 90%的相似大小的种子)
  13. 真菌菌株:禾谷镰刀菌(Fusarium graminearum)(分离株:F0609); F。 verticillioides (分离:7600)
  14. 无菌ddH <2>
  15. 甘油
  16. PDA(马铃薯葡萄糖琼脂)(青岛希望生物技术有限公司,产品目录号:HB0233)
  17. Tween-20(SunShine Bio,目录号:T0014)
  18. 绿豆(从市场购买)
  19. PDA媒体(见食谱)
  20. 绿豆汤(见食谱)


  1. 移液器(Eppendorf,Research Plus,产品目录号:3120000020,3120000046,3120000062)
  2. 血细胞计数器(0.100毫米,Hausser科学,目录号:3110)
  3. 水浴(常州诺基仪器,目录号:HHS-11-1)
  4. 洁净工作台(AIRTECH,型号:SW-CJ-1FD,目录号:A16116317)
  5. 恒温培养箱(宁波江南仪器厂,目录号:DNP-9162)
  6. 生长室(宁波江南仪器厂,型号:RXM-508C-3,产品编号:17091559)
  7. 离心机(Eppendorf,型号:5424,目录号:5424FL570190)
  8. 显微镜(奥林巴斯,目录号:BX41)
  9. 高压灭菌器


  1. 越来越多的玉米幼苗
    1. 在用PVC圆柱体(直径5厘米,深20厘米)制成的长盆中的土壤中播种不同玉米自交系的种子,每个圆柱4粒种子,覆盖一层2厘米厚的土壤。为了证明茎腐病表型,可以包括由杨等人(2010)描述的抗性品系1145和对赤霉病茎腐病的敏感品系Y331作为对照。
    2. 将盆放置在受控的生长室(光周期和温度:26±2℃14光/ 10黑暗;光强度:600勒克斯)以允许幼苗生长。
    3. 在大约12天到2周龄阶段选择相似大小的幼苗,并转移到培养室进行感染测定。

  2. 制备真菌孢子悬浮液
    1. 真菌菌株培养
      真菌分离物( F. graminearum 或 verticillioides )最初储存在-80℃的20%甘油中。在PDA平板上接种真菌分离物,并在黑暗中孵育器(25°C)中孵育至少10天。
    2. 刺激孢子形成

      图1.绿豆汤与 F。 graminearum 培养2天

    3. 孢子枚举和接种物的准备
      通过两层奶酪布过滤绿豆汤中的孢子悬浮液,然后在3,800gxg离心5分钟。丢弃上清液,用无菌水重新悬浮并洗涤浓缩的分生孢子两次,然后在0.001%Tween-20中稀释至浓度为1.0×10 6 / ml / ml。

  3. 用真菌孢子接种幼苗茎
    1. 玉米苗的制备
      将塑料方形托盘(100 x 80 x 10厘米)放在受控的孵化室中(光周期和温度:24±2°C时14光/ 10暗;光强度:200勒克斯)。要在托盘内保持高湿度(〜75%),请在托盘底部的一层纸巾中加入250 ml无菌水,直至毛巾完全湿润。如果需要,丢弃额外的水。


    2. 人工接种过程
      为了促进真菌孢子的感染,使用注射器针头(图3,视频1)在苗茎的第一节间的中间位置打出约1mm深度的小孔,然后将20μl的孢子悬浮液以0.001% Tween-20小心使用移液管(图4,视频2)到受伤部位。用压入式saran包装密封塑料方形托盘以保持湿度。为了进行换气,在包装上制作6-8个均匀的带孔(约2厘米长)。感染后,将幼苗保持在24±2℃下14小时光照/ 10小时黑暗条件下3天,而不移动(图5)。每天检查湿度以确保大约75%的高湿度。



      图5.接种后幼苗的维护使用压入式saran包装将塑料方形托盘密封以保持湿度&gt; 75%。盖表面上有几个均匀的孔。



  1. 疾病严重程度评分等级

    图6.通过5分类分级量表的幼苗的表型特征CK:未接种的对照; 1,最有抵抗力; 5,最易感染。


  2. 计算疾病严重程度指数(DSI)
    DSI(%)=Σ(排名值x具有该值的植物数量)×100 /(1×植物总数)。所有统计分析均使用SPSS(14.0)软件进行方差分析和Student's检验,其显着性水平设定为 P 0.05。


  1. 选择健康,无压力和均匀的幼苗进行感染测定。

  2. 接种后如果接种物立即从感染部位滴下,则重新感染。

  3. 维持适当的湿度和温度是成功开发和渗透真菌病原体的关键


  1. PDA媒体(200毫升)
  2. 绿豆汤(1升)
    1. 在85℃水浴中将40g绿豆在250ml ddH2O中煮沸1小时
    2. 然后过滤一层纱布,并在高压灭菌的ddH 2 O中稀释至1L。

    3. 在120°C下消毒15分钟


该协议部分适用于我们以前的工作(Gao等人,2007年),并进行了一些修改。这项工作得到了中国国家重点研究与发展计划(编号2016YFD0101002),国家自然科学基金委编号(编号31471508,编号31670702)的支持,并得到了中国人事部选定华侨华人科技基金会的支持(No G0101500090)和江苏省高校创新团队项目(2014)。作者声明不存在利益冲突或利益冲突。


  1. Gao,X。,Shim,W. B.,Gobel,C.,Kunze,S.,Feussner,I.,Meeley,R.,Balint-Kurti,P.和Kolomiets,M.(2007)。 玉米9-脂氧合酶的破坏导致对真菌病原体的抗性增加和霉菌毒素污染水平降低伏马菌素。 Mol Plant Microbe Interact 20(8):922-933。
  2. Ma,C.,Ma,X.,Yao,L.,Liu,Y.,Du,F.,Yang,X.和Xu,M.(2017)。 qRfg3,一种新的抗玉米赤霉病茎锈病的定量抗性基因座。 Theor Appl Genet 130(8):1723-1734。
  3. Mesterhazy,A.,Lemmens,M.和Reid,L.M。(2012)。 抗镰刀菌引起的穗腐病育种 spp。在玉米 - 评论。 植物育种 131:1-19。
  4. Oerke,E.C。(2006)。 作物对害虫的损失。 农业科学杂志 144:31-43。
  5. Presello,D.A.,Botta,G.,Iglesias,J.,Eytherabide,G.H。(2008)。 疾病严重度对产量和谷物591伏马菌素浓度的影响的玉米杂交种接种'Fusarium verticillioides 。“作物保护 27:(3-5):572-576。
  6. Walter,S.,Nicholson,P。和Doohan,F.M。(2010)。 镰刀菌病疫病期间宿主和病原体的作用和反应< / a> New Phytol 185(1):54-66。
  7. Yang,Q.,Yin,G.,Guo,Y.,Zhang,D.,Chen,S.and Xu,M。(2010)。 抗玉米赤霉病茎腐病的主要QTL。 Theor Appl Genet 121:673-687。
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引用:Sun, Y., Ruan, X., Ma, L., Wang, F. and Gao, X. (2018). Rapid Screening and Evaluation of Maize Seedling Resistance to Stalk Rot Caused by Fusarium spp.. Bio-protocol 8(10): e2859. DOI: 10.21769/BioProtoc.2859.