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Large-scale Maize Seedling Infection with Exserohilum turcicum in the Greenhouse
温室中大规模玉米幼苗感染玉米大斑病菌   

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Abstract

Northern corn leaf blight (NCLB) is a serious foliar disease of maize (Zea mays) worldwide and breeding for resistance is of primary importance for maize crop protection. Phenotyping for NCLB resistance is well established in the field, but such experiments depend on suitable environmental conditions and are seasonal. Here we describe a greenhouse seedling approach that is suitable for testing thousands of seedling plants in a single experiment with a duration of 37 days. Three scoring methods were used to quantify the disease severity: the area under the disease progress curve (AUDPC), the primary diseased leaf area of the inoculated leaves at 16 days post inoculation (PrimDLA at 16 dpi) and the incubation period (IP) that was determined as days from inoculation to symptom appearance. By testing a diverse panel of maize genotypes, a high correlation between the three different methods was observed (81.9% to 94.1%), indicating that each of scoring methods can be applied for disease quantification. Thus, the seedling assay developed served as a relatively simple and high-throughput method for phenotyping NCLB disease resistance under greenhouse condition.

Keywords: Northern corn leaf blight(玉米大斑病), Seedling assay(幼苗实验), High-throughput(高通量), Disease quantification(疾病定量)

Background

Northern corn leaf blight (NCLB) is a ubiquitous foliar wilt disease that threatens maize production worldwide (Welz and Geiger, 2000). The disease is caused by the hemibiotrophic fungus Exserohilum turcicum (anamorph of Setosphaeria turcica), which favors a high-humidity and cool temperature environment. Under favorable conditions, fungal infection manifests itself as large and irregularly emerging lesions that destroy the entire foliage. Therefore, this disease decreases the active leaf area and the accumulation of photosynthesized products. Up to 50% grain yield loss was reported but the reduction largely depended on environmental parameters (e.g., temperature, humidity), phases of maize development and hybrid susceptibility (Ullstrup, 1970; Pataky et al., 1998).

Precision phenotyping for NCLB disease resistance is critical for the determination of host resistance against E. turcicum. Testing for disease resistance in the field is well established, e.g., by placing or distributing inoculums in the leaf whorl at the 4 to 6 leaf stage (or even older) plants (Dingerdissen et al., 1996; Lipps et al., 1997; Brown et al., 2001; Asea et al., 2009; Chung et al., 2010; Chung et al., 2011). Scoring for resistance can be conducted by determining the levels of susceptibility (1 to 9; 1, complete resistance, no symptoms; 9, 90-100% of leaf area infected), the primary diseased leaf area (PrimDLA) that was defined as the percentage of infected leaf area of the inoculated leaf, the diseased leaf area of the entire plant (DLA), the incubation period (IP) rated as the number of days post inoculation until first observing the wilting/lesion, the lesion number (LN) at 14 to 21 days post inoculation and finally the area under the disease progress curve (AUDPC). However, tests for resistance in the field are environmentally-dependent and time-consuming. Here we describe a simple greenhouse seedling assay by testing only the second leaf, thus being suitable for quantifying thousands of seedlings in a single experiment within 37 days.

Materials and Reagents

  1. Pipette tips
  2. General lab materials, including:
    Mesh (0.5 mm)
    Round Petri dish (9 cm)
    Inoculation needle
    Microspore glass
    Vessel
    Funnel
    50 ml Falcon tube, etc.
  3. E. turcicum isolate Passau-1
  4. Potato dextrose agar (PDA) (BD, DifcoTM, catalog number: 213400 )
  5. Tween 20 (Sigma-Aldrich, catalog number: V900548 )
  6. PDA medium (see Recipes)
  7. Tween 20 solution (see Recipes)

Equipment

  1. Pipettes
  2. General greenhouse equipment, including jiffy pots (ø8 cm), tray and sieve tray (L/W: 50 cm/30 cm), etc.
  3. Home-made iron frame cover with non-permeable plastic (L/W/H: 50/30/35 cm)
  4. Sprayer (Semadeni, ø28 mm)
  5. Autoclave
  6. A home-made box (L/W/H: 54/30/25 cm, open at the bottom, 3 cm notches on each side) to shield the Blacklight Blue fluorescent tubes (Philips TL-D BLB, 15 W, peak at λ 356 nm) or any incubators that can fit the fluorescent tubes can be used alternatively
  7. Sterile bench with UV light
  8. Neubauer counting chamber (BRAND, catalog number: 717805 )
  9. Microscope (ZEISS, model: Axio Imager 2 ) or other light microscopes
  10. Centrifuge (Eppendorf, model: 5810 R )

Procedure

  1. Preparation of seedling plants for infection
    This seedling assay was conducted under greenhouse conditions (16 h day, 20 °C/8 h night, 18 °C; light intensity, 160 μmol/m2 sec-1 (400 W, MHL); relative humidity, ca. 60%) during the whole experiment. Three to four maize seeds were sown in each jiffy pot, and fifteen pots were placed in one tray (5 per row). After germination, up to 3 seedlings per pot (ca. 45 seedlings per tray) were kept. In general, the second leaves had fully emerged two weeks after sowing. Newly emerged leaves were completely removed by cutting every 2-3 days until the end of each experiment.
    Note: Removing the newly emerged leaves can keep seedling plants small and delay leaf senescence, which gives nice disease symptoms and the seedlings can be easily kept under space-limited high-humidity micro-conditions.
  2. Propagation of E. turcicum isolate
    1. Inoculation with mycelia and culture on PDA medium plate (35 ml per plate, see Recipes) was carried out at the same day seed sowing (Figure 1A).
    2. Add 500 ml ddH2O to 19.5 g of PDA powder and autoclave at 121 °C for 20 min. Pour approximate 35 ml of liquid media into round Petri dish plates in a sterile bench. Sterilize the PDA plates under UV light (30 min).
      Note: Less media will inhibit fungal growth after some time.
    3. Inoculation of PDA plates was conducted using an inoculation needle to pick up a small piece of E. turcicum mycelia that is generally kept at room temperature for long-term storage (up to one year). Fix the PDA plate using the permeable surgical tape.
      Note: The dehydration and contamination of conidia PDA plates were often observed several months later. Inoculation of PDA plates needs uncontaminated mycelia.
    4. Place the plates upside down and incubate at room temperature in the dark. When the conidia cover the complete medium plate (after about two weeks), incubate the plates under BLB UV light until harvest (10 h per day, 7 days). This may induce the fungal sporulation to produce more spores.
  3. Harvest and preparation of spore suspension
    1. Freshly prepare 0.1% Tween 20 (v/v) (see Recipes) in sterile water. Pour 10 ml of 0.1% Tween 20 on the plate and scrape the surface with a glass slide to dislodge the spores from the hyphae.
    2. Collect and pour the spore suspension by filtering through one layer of a fine mesh (ca. 0.5 mm), which is placed in a funnel to remove most of the mycelia.
    3. Before counting, mix spore suspension thoroughly by inverting 2-3 times, since the spores settled at the bottom of the collection vessels.
    4. Determine the spore density by using a 0.1-mm thick Neubauer counting chamber. Adjust the concentration to 4.5 x 104 spores/ml for use. If the spore concentration of the suspension is lower than the concentration needed, concentrate the spores by centrifuging for 5 min (room temperature, 1,811 x g) and removing the extensive supernatant. In general, the spores from one PDA plate after 21 days culture would be enough for infecting 2-4 trays. The conidia PDA plates can be stored up to one year at room temperature, but the spore suspension should be freshly prepared for each infection experiment.
  4. Infection of maize seedlings by spray
    1. Inoculate E. turcicum to maize seedlings at 21 days after sowing when the second leaf fully emerges and becomes deep green. This date can be adjusted according to the growth status of seedling plants. Spray four trays using a total of 4 ml of spore suspension.
    2. After infection, water the tray extensively from the bottom to promote transpiration. A very high humidity micro-environment is achieved by placing non-permeable plastic hoods on top of each tray (Figure 1A).
      Note: The high humidity micro-environment can promote E. turcicum infection.


      Figure 1. The seedling assay for testing NCLB disease resistance in the greenhouse. A. The pipeline for making inoculation. Parallel sowing and culture start of E. turcicum on a PDA medium plate performed 21 days before inoculation. The first and second leaves were subjected for inoculation by spraying spore suspension (4.5 x 104 spores/ml, 4 ml for 4 trays). The micro-environment with higher humidity was achieved by watering the tray extensively to promote transpiration and by covering the trays with a non-permeable cover until the end of each experiment. B. Symptoms of E. turcicum infected seedling. dpi, days post inoculation.

  5. Scoring symptoms
    1. Scoring individual seedling for disease symptoms was conducted between 11 and 25 days post inoculation in an interval of 1 to 2 days (Figure 1B). The period for scoring can be adjusted according to the levels of susceptibility/resistance.
    2. Perform scoring for disease symptoms manually by visualization. Three disease parameters AUDPC, PrimDLA at 16 dpi and IP are used for disease quantification. IP and PrimDLA are rated individually, while AUDPC is calculated using the mean of all test plants in each genotype. In case of plants that were uninfected at 25 dpi, the respective IP was rated as 25 days. In general, 10 to 15 plants of each genotype are tested in each experiment. The IP, PrimDLA and AUDPC are calculated as follows:



      where, d1 = days of symptom appearance post inoculation in plant d1, n = number of total inoculated plants.



      where, p1 = percentage of diseased leaf area of the second leaf in plant p1, n = number of total inoculated plants.



      where, yi = PrimDLA at day i, ti+1 - ti = day interval between two ratings, n = number of ratings (Chung et al., 2011).
      Note: The use of less plants may lead to large variation and less reliability because of the quantitative nature of NCLB resistance.

Data analysis

Our previous work demonstrated that a seedling assay can be used to determine the presence of the quantitative NCLB resistance gene Htn1 in maize, as well as the wheat broad-spectrum fungal disease resistance gene Lr34 in transgenic maize lines (Hurni et al., 2015; Sucher et al., 2017). To test if the seedling assay can be used in diverse maize germplasm, we tested this method in a panel of maize lines (Table S1). This panel included six maize breeding lines that were kindly provided by KWS (Einbeck, Germany), and 127 exotic and historic maize lines that were collected before the 1990s from dozens of countries (IPK Genebank, Gatersleben, Germany). Interestingly, a continuous range of NCLB disease resistance/susceptibility was observed (Figures 2A-2C). The susceptible recurrent parental line RP1 was strongly infected, while near-isogenic line containing the introgressed resistance gene Htn1 was highly resistant (Figures 2A-2C). While no visible disease symptoms were detected in ten genotypes, most accessions were infected with visible disease symptoms (AUDPC > 0) and 59% of genotypes were highly susceptible (PrimDLA_16 dpi ≥ 40%). Very importantly, three disease parameters AUDPC, PrimDLA_16 dpi and IP were highly correlated (R2, 81.9 to 94.1) (Figures 2D-2F). Thus, each of the three parameters can be used for quantifying NCLB disease. For example, if PrimDLA is determined at 16 dpi for disease quantification, the data can be obtained after only 37 days.


Figure 2. NCLB disease severity and correlation among disease parameters. Six genotypes from KWS Einbeck and 127 exotic maize genotypes from the IPK Genebank were tested in the seedling assay (Table S1). A. The area under the disease progress curve between 11 and 25 dpi; B. The primary diseased leaf (PrimDLA) area at 16 dpi; C. The incubation period that was rated as days from inoculation until appearance of disease symptoms; D. Correlation between AUDPC and PrimDLA at 16 dpi; E. Correlation between AUDPC and IP; F. Correlation between IP and PrimDLA at 16 dpi. Error bars indicate ± standard error (SE). dpi, days after inoculation.

Recipes

  1. PDA medium
    Add 500 ml ddH2O to 19.5 g of PDA powder and autoclave at 121 °C for 20 min
  2. Tween 20 solution
    Add 200 µl Tween 20 to 100 ml of sterile water

Acknowledgments

The authors would like to thank IPK Genebank (Gatersleben, Germany) and KWS (Einbeck, Germany) for kindly providing maize lines, Dr. Severine Hurni for discussion, and Mr. Alessandro Artemisio, Mr. Karl Huwiler for technical support. This work was supported by Swiss National Science Foundation Grant 310030_163260. The protocol was adapted and updated from methods reported in Hurni et al. (2015).

References

  1. Asea, G., Vivek, B. S., Bigirwa, G., Lipps, P. E. and Pratt, R. C. (2009). Validation of consensus quantitative trait loci associated with resistance to multiple foliar pathogens of maize. Phytopathology 99(5): 540-547.
  2. Brown, A. F., Juvik, J. A. and Pataky, J. K. (2001). Quantitative trait loci in sweet corn associated with partial resistance to Stewart's wilt, northern corn leaf blight, and common rust. Phytopathology 91(3): 293-300.
  3. Chung, C. L., Jamann, T., Longfellow, J. and Nelson, R. (2010). Characterization and fine-mapping of a resistance locus for northern leaf blight in maize bin 8.06. Theor Appl Genet 121(2): 205-227.
  4. Chung, C. L., Poland, J., Kump, K., Benson, J., Longfellow, J., Walsh, E., Balint-Kurti, P. and Nelson, R. (2011). Targeted discovery of quantitative trait loci for resistance to northern leaf blight and other diseases of maize. Theor Appl Genet 123(2): 307-326.
  5. Dingerdissen, A. L., Geiger, H. H., Lee, M., Schechert, A., Welz, H. G. (1996). Interval mapping of genes for quantitative resistance of maize to Setosphaeria turcica, cause of northern leaf blight, in a tropical environment. Mol Breed 2(2): 143-156.
  6. Hurni, S., Scheuermann, D., Krattinger, S. G., Kessel, B., Wicker, T., Herren, G., Fitze, M. N., Breen, J., Presterl, T., Ouzunova, M. and Keller, B. (2015). The maize disease resistance gene Htn1 against northern corn leaf blight encodes a wall-associated receptor-like kinase. Proc Natl Acad Sci U S A 112(28): 8780-8785.
  7. Lipps, P. E., Pratt, R. C., Hakiza, J. J. (1997). Interaction of Ht and partial resistance to Exserohilum turcicum in maize. Plant Dis 81(3): 277-282.
  8. Pataky, J. K., Raid, R. N., Du, T. L. and Schueneman, T. J. (1998). Disease severity and yield of sweet corn hybrids with resistance to northern leaf blight. Plant Dis 82(1): 57-63.
  9. Sucher, J., Boni, R., Yang, P., Rogowsky, P., Buchner, H., Kastner, C., Kumlehn, J., Krattinger, S. G. and Keller, B. (2017). The durable wheat disease resistance gene Lr34 confers common rust and northern corn leaf blight resistance in maize. Plant Biotechnol J 15(4): 489-496.
  10. Ullstrup, A. J. (1970). A comparison of monogenic and polygenic resistance to Helminthosporium turcicum in corn. Phytopathology 60(11): 1597-1599.
  11. Welz, H. G. and Geiger, H. H. (2000). Genes for resistance to northern corn leaf blight in diverse maize populations. Plant Breeding 119: 1-14.

简介

北方玉米叶枯病(NCLB)是世界范围内严重的玉米叶片病(Zea mays),对抗玉米作物保护最为重要。 NCLB抗性的表型在该领域已经确立,但是这种实验取决于合适的环境条件并且是季节性的。在这里,我们描述了一种温室幼苗方法,适用于在单次实验中测试数千株幼苗,持续时间为37天。使用三种评分方法来定量疾病严重程度:疾病进展曲线(AUDPC)下的面积,接种后16天接种的叶的主要病叶面积(16dpi的PrimDLA)和潜伏期(IP),被确定为从接种到症状外观的天数。通过测试多样化的玉米基因型,观察到三种不同方法之间的高度相关性(81.9%至94.1%),表明每种评分方法可用于疾病定量。因此,开发的幼苗测定作为在温室条件下表型NCLB抗病性的相对简单且高通量的方法。
【背景】北方玉米叶枯病(NCLB)是全球威胁到玉米生产的无处不在的叶枯病(Welz和Geiger,2000)。该疾病是由有利于高湿度和凉爽的温度环境的半抗生物真菌(Exserohilum turcicum)((Setosphaeria turcica )的变态)引起的。在有利的条件下,真菌感染本身就表现为破坏整个叶子的大型和不规则出现的病变。因此,这种疾病减少了活性叶面积和光合产物的积累。报道了高达50%的谷物产量损失,但减少主要取决于环境参数(例如,温度,湿度),玉米发育阶段和杂交易感性(Ullstrup,1970; Pataky et al。 ,1998)。
NCLB抗病性的精确表型对于确定宿主对E的抵抗力至关重要。大斑病菌。通过将接种物置于或分配在4至6叶期(甚至更老的)植物的叶子中,测定该领域的抗病性是确定的(例如,Dingerdissen等人)。 ,1996; Lipps等人,1997; Brown等人,2001; Asea等人,2009; Chung 等人,2010; Chung等人,2011)。耐药性评分可以通过确定易感性水平(1〜9; 1,完全抗性,无症状;感染叶面积的9,90-100%),将主要病叶面积(PrimDLA)定义为病虫数(LN),接种叶片受感染叶面积百分比,全株病害面积(DLA),潜伏期(IP)等级为接种后天数,直到首先观察萎ting /病变,接种后14〜21天,最终达到疾病进展曲线下面积(AUDPC)。然而,该领域的电阻测试是环境依赖和耗时的。在这里,我们通过仅测试第二叶来描述简单的温室幼苗测定,因此适合于在37天内在单个实验中量化数千只幼苗。

关键字:玉米大斑病, 幼苗实验, 高通量, 疾病定量

材料和试剂

  1. 移液器提示
  2. 一般实验室材料,包括:
    网格(0.5毫米)
    圆形培养皿(9厘米)
    接种针头
    微孢子玻璃
    船舶
    漏斗
    50ml猎鹰管,
  3. 电子。 turcicum 隔离Passau-1
  4. 马铃薯葡萄糖琼脂(PDA)(BD,Difco TM,目录号:213400)
  5. 吐温20(Sigma-Aldrich,目录号:V900548)
  6. PDA媒体(请参阅食谱)
  7. 吐温20溶液(参见食谱)

设备

  1. 移液器
  2. 一般温室设备,包括y锅(ø8厘米),托盘和筛板(长/宽:50厘米/ 30厘米),等。
  3. 自制铁框架盖,不透水塑料(长/宽:50/30/35厘米)
  4. 喷雾器(Semadeni,ø28mm)
  5. 高压灭菌器
  6. 自制盒(L / W / H:54/30/25厘米,底部开放,每边3厘米凹口),以屏蔽Blacklight蓝色荧光灯管(Philips TL-D BLB,15 W, λ356 nm)或任何可以安装荧光管的孵化器可以交替使用
  7. 紫外线无菌台灯
  8. Neubauer计数室(BRAND,目录号:717805)
  9. 显微镜(ZEISS,型号:Axio Imager 2)或其他光学显微镜
  10. 离心机(Eppendorf,型号:5810 R)

程序

  1. 用于感染的幼苗的制备
    这种幼苗测定在温室条件下(16小时日,20℃/ 8小时夜晚,18℃;光强度,160微摩尔/平方米秒 (400W,MHL);相对湿度,大约60%)。将三至四个玉米种子播种在每个y锅中,将十五盆置于一个盘中(每行5个)。在萌发后,保存每盆多达3个幼苗(大约每个托盘45个幼苗)。一般来说,播种后两周完全出现了第二片叶子。通过每2-3天直到每次实验结束切割完全除去新出现的叶子。
    注意:去除新出现的叶子可以使幼苗植物保持较小并延缓叶片衰老,这样可以很好地发挥疾病症状,幼苗可以很容易地保持在空间有限的高湿度微条件下。 >
  2. E的传播。 turcicum 分离株
    1. 在同一天种子播种时,在PDA培养板上接种菌丝体和培养物(每片35毫升,参见食谱)进行(图1A)。
    2. 向19.5g PDA粉末中加入500ml ddH 2 O,并在121℃下高压灭菌20分钟。将约35ml液体培养基倒入无菌台中的圆形培养皿中。在紫外光下灭菌PDA板(30分钟) 注意:一段时间后,较少的培养基会抑制真菌生长。
    3. 使用接种针进行PDA板的接种以拾取一小块E。 turcicum 菌丝体,通常保存在室温下长期储存(长达一年)。使用可渗透手术胶带固定PDA板。
      注意:几个月后经常会观察到分生孢子PDA板的脱水和污染。 PDA板的接种需要未受污染的菌丝体。
    4. 将板倒置并在室温下在黑暗中孵育。当分生孢子覆盖完整的培养基(大约两周)后,在BLB紫外线下培养板,直到收获(每天10小时,7天)。这可能导致真菌孢子生成更多的孢子
  3. 收获和准备孢子悬浮液
    1. 在无菌水中新鲜制备0.1%Tween 20(v / v)(见食谱)。将10毫升0.1%吐温20倒入板上,用玻片刮去表面,以从菌丝上除去孢子。
    2. 收集并倒出孢子悬浮液,过滤通过一层精细网格( ca。 0.5 mm),将其放置在漏斗中以去除大部分菌丝体。
    3. 在计数之前,通过反复2-3次彻底混合孢子悬浮液,因为孢子沉淀在收集容器的底部。
    4. 通过使用0.1毫米厚的Neubauer计数室确定孢子密度。将浓度调整至4.5×10 4个/ ml以上孢子/ ml。如果悬浮液的孢子浓度低于所需浓度,则通过离心浓缩孢子5分钟(室温,1,811×g)并除去广泛的上清液。一般来说,培养21天后一个PDA板的孢子足以感染2-4个盘。分生孢子PDA板可以在室温下储存长达一年,但应对每次感染实验新鲜准备孢子悬浮液。
  4. 通过喷雾感染玉米幼苗
    1. 接种E。 turcicum 在播种后21天对玉米幼苗进行玉米育种,当第二片叶子完全出现并变成深绿色时。这个日期可以根据苗木的生长状况进行调整。使用总共4毫升孢子悬浮液喷洒四个托盘。
    2. 感染后,从底部大量吸收托盘以促进蒸腾。通过将不可渗透的塑料罩放置在每个托盘的顶部(图1A)上实现非常高湿度的微环境。
      注意:高湿度微环境可以促进土耳其感染。


      图1.用于测试温室中NCLB抗病性的幼苗测定。 A.接种的管道。平均播种文化开始。在接种前21天在PDA培养基平板上进行草皮。通过喷洒孢子悬浮液(4.5×10 4个/孢子/ ml,4个塔盘4ml)对第一和第二叶进行接种。具有较高湿度的微环境是通过大量浇灌托盘来促进蒸腾和通过用不可渗透的盖子覆盖托盘直到每个实验结束来实现的。 B.症状E。 turcicum 感染幼苗。 dpi,接种后的天数。

  5. 得分症状
    1. 在接种后11至25天以1至2天的间隔进行疾病症状评估个体幼苗(图1B)。评分期可以根据敏感度/抵抗力的水平进行调整
    2. 通过可视化手段对疾病症状进行评分。三种疾病参数AUDPC,16dpi和IP的PrimDLA用于疾病定量。 IP和PrimDLA被单独评估,而AUDPC是使用每种基因型中所有测试植物的平均值计算的。在25dpi未感染的植物的情况下,各自的IP被评为25天。通常,在每个实验中测试每种基因型的10至15株植物。 IP,PrimDLA和AUDPC计算如下:



      其中, d 1 =植物接种后症状出现天数 , n =总接种植物的数量。



      其中, p 1 =植物中第二叶的病叶面积百分比 > 1 , n =总接种植物的数量。



      其中, y i =第一天的PrimDLA , i + 1 - i =两个评级之间的日期间隔, =评级数(Chung et al。,2011)。
      注意:由于NCLB抗性的数量性质,使用较少的植物可能导致较大的变异和较低的可靠性。

数据分析

我们以前的工作表明,可以使用幼苗测定来确定玉米中定量NCLB抗性基因Htn1的存在,以及小麦广谱真菌病抗性基因Lr34,转基因玉米品系(Hurni等人,2015; Sucher等人,2017)。为了测试是否可以在不同的玉米种质中使用幼苗测定,我们在一组玉米品系中测试了该方法(表S1 )。该小组包括由KWS(德国Einbeck)提供的六个玉米育种系,以及在数十个国家(IPK Genebank,Gatersleben,Germany)之前收集的127种外来和历史的玉米品系。有趣的是,观察到NCLB疾病耐药/易感性的连续范围(图2A-2C)。敏感的亲本亲本系RP1被强烈感染,而含有渐渗抗性基因Htn1的近等基因系具有高度抗性(图2A-2C)。虽然在10种基因型中没有发现可见的疾病症状,但大多数种质感染了可见的疾病症状(AUDPC≥0),59%的基因型是高度易感的(PrimDLA_16 dpi≥40%)。非常重要的是,三种疾病参数AUDPC,PrimDLA_16dpi和IP都具有高度相关性(图2D-2F)(图7-10) 。因此,三个参数中的每一个可以用于定量NCLB疾病。例如,如果PrimDLA以16dpi确定疾病定量,则可以在仅37天之后获得数据。


图2. NCLB疾病严重程度和疾病参数之间的相关性。来自KWS Einbeck的六种基因型和来自IPK基因库的127种外来玉米基因型在幼苗试验中测试(表S1 )。 A.疾病进展曲线在11和25 dpi之间的区域; B.主要病叶(PrimDLA)面积为16dpi; C.从接种直到出现疾病症状为止的潜伏期; D.PCD和PrimDLA之间的相关性为16dpi; E.澳门币与知识产权相关; F.在16dpi下IP和PrimDLA之间的相关性。误差条表示±标准误差(SE)。 dpi,接种后数天。

食谱

  1. PDA媒体
    向19.5g PDA粉末中加入500ml ddH 2 O,并在121℃下高压灭菌20分钟
  2. 吐温20溶液
    加入200μlTween 20至100ml无菌水

致谢

作者要感谢IPK Genebank(德国Gatersleben)和KWS(德国Einbeck),提供玉米生产线,Severine Hurni博士和Alessandro Artemisio先生,Karl Huwiler先生的技术支持。这项工作得到瑞士国家科学基金会拨款310030_163260的支持。该方案根据Hurni等人报道的方法进行了修改和更新。 (2015)。

参考

  1. Asea,G.,Vivek,B.S.,Bigirwa,G.,Lipps,P.E。和Pratt,R.C。(2009)。 确认与玉米多重叶酸病原体的抗性相关的一致数量性状位点。 Phytopathology 99(5):540-547。
  2. Brown,A.F.,Juvik,J.A。和Pataky,J.K。(2001)。 甜玉米与斯图尔特枯萎部分抵抗相关的定量性状位点,北部玉米叶枯病和常见生锈。 植物病理学 91(3):293-300。
  3. Chung,C.L.,Jamann,T.,Longfellow,J.and Nelson,R。(2010)。 玉米仓8.06中北部叶枯病抗性位点的表征和精细绘图。理论应用基因 121(2):205-227。
  4. Chung,C.L。,波兰,J.,Kump,K.,Benson,J.,Longfellow,J.,Walsh,E.,Balint-Kurti,P。和Nelson,R。(2011)。 有针对性的发现数量性状位点,以抵抗北部叶枯病和其他玉米病。理论应用基因 123(2):307-326。
  5. Dingerdissen,A.L.,Geiger,H.H.,Lee,M.,Schechert,A.,Welz,H.G。(1996)。 将玉米定量抗性的基因与西班牙黑腹果蝇的间隔作图,北方的原因Mol.Breed 2(2):143-156。
  6. Hurni,S.,Scheuermann,D.,Krattinger,SG,Kessel,B.,Wicker,T.,Herren,G.,Fitze,MN,Breen,J.,Presterl,T.,Ouzunova,M.and Keller, B.(2015)。 针对北方玉米叶枯病的玉米抗病基因Htn1编码一个墙相关受体样激酶。
    Proc Natl Acad Sci U S A 112(28):8780-8785。
  7. Lipps,P.E.,Pratt,R.C.,Hakiza,J.J。(1997)。 的互动和的部分抵制玉米中的Exserohilum turcicum 81(3):277-282。
  8. Pataky,J.K.,Raid,R.N.,Du,T.L.and Schueneman,T.J。(1998)。 具有抵抗北部叶枯病的甜玉米杂种的疾病严重性和产量。 a> Plant Dis 82(1):57-63。
  9. Sucher,J.,Boni,R.,Yang,P.,Rogowsky,P.,Buchner,H.,Kastner,C.,Kumlehn,J.,Krattinger,S.G.and Keller,B。(2017)。 耐用小麦抗病基因Lr34 赋予常见的锈病和北方玉米叶玉米中的抗病性。植物生物技术J 15(4):489-496。
  10. Ullstrup,A.J。(1970)。 将单基因和多基因耐药性与 Helminthosporium turcicum 在玉米中。 植物病理学 60(11):1597-1599。
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引用:Yang, P., Herren, G., Krattinger, S. G. and Keller, B. (2017). Large-scale Maize Seedling Infection with Exserohilum turcicum in the Greenhouse. Bio-protocol 7(19): e2567. DOI: 10.21769/BioProtoc.2567.
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