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Soybean (Glycine max) is one of the most important crops in the world. Phakopsora pachyrhizi is a plant pathogenic basidiomycete fungus that infects soybean, causing Asian Soybean Rust (ASR) disease and affecting production. Here, we describe how to prepare the plant material and the uredospore suspension (from spores harvested from leaves exhibiting sporulating uredinia) for in vitro leaf infection. Plant material is sprayed with the uredospore suspension and incubated for 12 days. During the incubation period, the presence of lesions and pustules is visually verified. After this incubation period, the leaves are classified according to the lesion type. The number of uredospores per CM2 of leaf was also estimated. The detached-leaf assay is routinely used to test fungicide efficiency (Scherb and Mehl, 2006). Detached-leaf, greenhouse and field results have been shown to be significantly correlated (Twizeyimana et al., 2007). The present protocol was adapted from the two publications cited above. The usefulness of this approach for studying P. pachyrhyzi infection on transgenic soybean was previously demonstrated by our research team (Wiebke-Strohm et al., 2012; Bencke-Malato et al., 2014).

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Phakopsora pachyrhizi Infection Bioassay in Detached Soybean Transgenic Leaves for Candidate Gene Validation
大豆锈菌感染分离的大豆转基因叶片的生物测定和候选基因确认

植物科学 > 植物免疫 > 病害生物测定
作者: Beatriz Wiebke-Strohm
Beatriz Wiebke-StrohmAffiliation: Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
Bio-protocol author page: a2398
Ciliana Rechenmacher
Ciliana RechenmacherAffiliation: Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
Bio-protocol author page: a2399
Luisa Abruzzi de Oliveira
Luisa Abruzzi de OliveiraAffiliation: Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
Bio-protocol author page: a2400
Cláudia Vieira Godoy
Cláudia Vieira GodoyAffiliation: Empresa Brasileira de Pesquisa Agropecuária, Londrina, Brazil
Bio-protocol author page: a2401
 and Maria Helena Bodanese Zanettini
Maria Helena Bodanese ZanettiniAffiliation: Departamento de Genética, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
For correspondence: mhbzanettini@yahoo.com.br
Bio-protocol author page: a2402
Vol 5, Iss 14, 7/20/2015, 2095 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1540

[Abstract] Soybean (Glycine max) is one of the most important crops in the world. Phakopsora pachyrhizi is a plant pathogenic basidiomycete fungus that infects soybean, causing Asian Soybean Rust (ASR) disease and affecting production. Here, we describe how to prepare the plant material and the uredospore suspension (from spores harvested from leaves exhibiting sporulating uredinia) for in vitro leaf infection. Plant material is sprayed with the uredospore suspension and incubated for 12 days. During the incubation period, the presence of lesions and pustules is visually verified. After this incubation period, the leaves are classified according to the lesion type. The number of uredospores per CM2 of leaf was also estimated. The detached-leaf assay is routinely used to test fungicide efficiency (Scherb and Mehl, 2006). Detached-leaf, greenhouse and field results have been shown to be significantly correlated (Twizeyimana et al., 2007). The present protocol was adapted from the two publications cited above. The usefulness of this approach for studying P. pachyrhyzi infection on transgenic soybean was previously demonstrated by our research team (Wiebke-Strohm et al., 2012; Bencke-Malato et al., 2014).

[Abstract]

Materials and Reagents

  1. Soybean transgenic and non-transgenic plants of similar age
  2. Leaves infected with Phakopsora pachyrhizi uredospores (Figure 1A)
  3. Tween 20 (Labsynth)
  4. Uredospore suspension (see Recipes)


    Figure 1. Soybean response to rust infection on the abaxial leaf surface. A. Tan, B. reddish brown and (C) immune.

Equipment

  1. Petri dish (Ø 9 cm)
  2. Hand sprayer that can be adjusted for spraying 1 ml volume (Figure 2A and 2B, Video 1)
  3. Plant culture room
  4. Growth chamber
  5. Stereomicroscope
  6. Laminar flow sterile cabinet
  7. Neubauer chamber


Figure 2. Hand sprayer

Video 1. Hand sprayer adjusted for spraying 1 ml volume

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Procedure

  1. Leaves infected with Phakopsora pachyrhizi uredospores were randomly sampled in the field. Alternatively, uredospores of a determined isolate were propagated on plants under greenhouse conditions. Immediately after sampling, leaves were stored in a dry plastic or paper bag at room temperature up to four days. However, better results are expected from material recently collected or stored under refrigeration.
    In the laboratory, uredospores were tapped off of the leaves into a suitable dry tube using a paint-brush, as shown in Video 2. This step should be done a short time before inoculation, that means, the time necessary to carry out 3-5 steps. The amount of uredospores depends on the amount of leaves that will be included in the experiment. To have an estimate please see steps 3 and 6.

    Video 2. Uredospores tapped off of the leaves using a paint-brush

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  2. Fifty ml uredospore suspension was prepared using sterile distilled water, 1 drop of Tween 20 and uredospores to a final concentration of 1 x 105 spores/ml. The concentration of uredospores was estimated using a Neubauer chamber. The Tween 20 was added using a pipette, as shown in Video 3. Fifty ml solution can be used to infect approximately 50 leaves.

    Video 3. One drop of Tween 20 using a 1 ml pipette

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  3. Soybean transgenic and non-transgenic plants were cultivated in a plant culture room at 26 ± 1 °C with a 16/8 h light/dark cycle at a light intensity of 22.5 µE/m2/s. Fully expanded trifoliate leaves with similar size and position were collected from plants of the same age. The newest fully expanded leaf of 2-month-old plants (V5 or V6 stages) was preferentially used, but plants in other growth stages may be also used.
  4. From this step forward, the material was aseptically manipulated in a laminar flow cabinet. The leaves were rinsed (quick submersion) in sterile distilled water and rapidly dried on sterile filter paper. Each leaflet was cut into 5 cm x 5 cm pieces and individually placed with the abaxial side upwards in a sterile Petri dish (Ø 9 cm) containing filter paper saturated with distilled water to maintain high humidity (Figure 3).


    Figure 3. Soybean leaf piece with the abaxial surface upwards in a Petri dish containing filter paper saturated with water

  5. Each leaf piece was inoculated by spraying 1 ml of the uredospore suspension (105 spores/ml). The uredospore suspension was mixed immediately before each inoculation. A distance of 10 cm was maintained between the hand sprayer and the leaf piece.
  6. The Petri dishes were incubated in a growth chamber at 20 °C with a 12/12 h light/dark cycle for 12 days.
  7. The material was visually evaluated for the presence of lesions and pustules every 2 days during the incubation period.
  8. After 12 days of incubation, leaves showing signals of other diseases or rotting were eliminated (Figure 4). The remaining material was visually classified according to the lesion type (immune, reddish brown or tan) present on the abaxial side of the leaf (Figure 1). Additionally, the number of lesions, the number of lesions with pustules, the number of pustules per lesion, the total number of pustules and the total number of opened pustules present on each leaf piece was recorded under a stereomicroscope (see Note 4; Figure 5). Finally, two infected areas of 1 cm x 1 cm were determined in each leaf piece and cut off. Each 1 cm2 piece was placed in a microtube containing 500 µl water and vortexed vigorously for 1 min. The mean number of uredospores per cm2 was estimated under a stereomicroscope using a Neubauer chamber.


    Figure 4. Examples of leaves showing signals of other diseases or rotting


    Figure 5. Parameters used to compare genotype response to rust infection. A. Lesions without pustule; B. lesion with one pustule (indicated by an arrow); C. lesion with multiple pustules (indicated by arrows); D. a cone-shaped opened pustule (left) and a cone-shaped unopened pustule (right); E. a cone-shaped opened pustule with a pore on the top and spores inside (left) and an opened pustule with spores on top of the cone (right).

  9. Results obtained from each leaf piece were considered replicates. Tree leaf pieces from the same plant and at least two plants from each transgenic event were used.
  10. Non-transformed plants of the same genetic background were used as a control. Ideally, a susceptible and a partially resistant variety should be used as positive and negative controls of the experimental conditions.

Notes

  1. Whenever possible the material should be aseptically manipulated to avoid cross contamination.
  2. Twizeyimana et al. (2007) placed the leaves on medium. We were not able to establish the protocol using the same medium due to cross contamination and rotting.
  3. The use of intact leaves will help to prevent cross contamination and rotting. However, bigger Petri dishes and more space in growth chambers will be necessary to carry out the experiment.
  4. Rust symptoms begin as small lesions on the abaxial leaf surface that gradually increase in size and change from gray to tan or reddish brown. Soybean rust produces two types of lesions: tan (Figure 1A) and reddish brown (Figure 1B). When mature, tan lesions consist of small pustules surrounded by slightly discolored necrotic area with masses of tan spores (Figure 5B-E). Reddish brown lesions have a larger reddish brown necrotic area, with a limited number of pustules and few visible spores. The mature soybean rust pustules are cone-shaped with a pore on the top and spores inside or on the cone (Figure 5D-E). (http://www.aphis.usda.gov/publications/plant_health/content/printable_version/Soybean_Rust_22.pdf).

Recipes

  1. Uredospore suspension
    P. pachyrhizi
    uredospores, 1 drop of Tween 20 (Video 2) and sterile distilled water to a final concentration of 1 x 105 spores/ml.

Acknowledgments

We thank Dr. Emerson M. Del Ponte, Dr. Juliano dos Santos and Larissa Bittecourt for their technical assistance. This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS). The present protocol was adapted from Scherb and Mehl (2006) and Twizeyimana et al. (2007).

References

  1. Bencke-Malato, M., Cabreira, C., Wiebke-Strohm, B., Bucker-Neto, L., Mancini, E., Osorio, M. B., Homrich, M. S., Turchetto-Zolet, A., De Carvalho, M., Stolf, R., Weber, R., Westergaard, G., Castagnaro, A. P., Abdelnoor, R. V., Marcelino-Guimaraes, F. C., Margis-Pinheiro, M. and Bodanese-Zanettini, M. (2014). Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection. BMC Plant Biol 14(1): 236.
  2. Scherb, C. T. and Mehl, A. (2006). SBI fungicides, also suited for other fungicide classes - detached leaf test. FRAC (Fungicide Resistance Action Committee)
  3. Twizeyimana, M., Ojiambo, P., Ikotun, T., Paul, C., Hartman, G. and Bandyopadhyay, R. (2007). Comparison of field, greenhouse, and detached-leaf evaluations of soybean germplasm for resistance to Phakopsora pachyrhizi. Plant Dis 91(9): 1161-1169.
  4. Wiebke-Strohm, B., Pasquali, G., Margis-Pinheiro, M., Bencke, M., Bucker-Neto, L., Becker-Ritt, A. B., Martinelli, A. H., Rechenmacher, C., Polacco, J. C., Stolf, R., Marcelino, F. C., Abdelnoor, R. V., Homrich, M. S., Del Ponte, E. M., Carlini, C. R., De Carvalho, M. C. and Bodanese-Zanettini, M. H. (2012). Ubiquitous urease affects soybean susceptibility to fungi. Plant Mol Biol 79(1-2): 75-87.

材料和试剂

  1. 相似年龄的大豆转基因和非转基因植物
  2. 感染了豆薯层锈菌(Phakopsora pachyrhiziz)的叶子(图1A)
  3. Tween 20(Labsynth)
  4. Uredospore悬浮液(参见配方)


    图1.大豆对背叶表面锈病感染的反应 A. Tan,B.红棕色和(C)免疫。

设备

  1. 培养皿(Ø9厘米)
  2. 手动喷雾器,可以调整喷涂1毫升体积(图2A和2B,视频1)
  3. 植物培养室
  4. 生长室
  5. 立体显微镜
  6. 层流无菌室
  7. Neubauer房间


图2.手动喷雾器

视频1.手动喷雾器调整为喷涂1 ml容积
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程序

  1. 在田间随机取样感染了豆薯层锈菌的叶子。或者,将确定的分离株的孢子在温室条件下在植物上繁殖。取样后立即将叶子在室温下储存在干燥的塑料或纸袋中至多4天。然而,预期最近在冷藏下收集或存储的材料的结果更好 在实验室中,使用油漆刷将旋孢子从叶子中分离到合适的干燥管中,如视频2所示。该步骤应在接种之前短时间进行,这意味着,进行3- 5个步骤。子孢子的量取决于将包括在实验中的叶子的量。要获得估算,请参阅步骤3和步骤6.

    视频2. Uredospores使用画笔刷出树叶
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  2. 使用无菌蒸馏水,1滴Tween 20和冰孢子制备50ml冰孢子悬浮液至终浓度为1×10 5孢子/ml。使用Neubauer室估计冰孢子的浓度。使用移液管加入吐温20,如视频3所示.50ml溶液可用于感染大约50片叶子。

    视频3.使用1 ml移液器一滴Tween 20
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  3. 将大豆转基因和非转基因植物在26±1℃的植物培养室中以22.5μE/m 2/s的光强度以16/8小时光/暗循环进行培养。从相同年龄的植物收集具有相似大小和位置的完全扩展的三叶叶。优选使用2个月大的植物的最新完全扩展的叶(V5或V6阶段),但也可以使用其它生长阶段的植物。
  4. 从该步骤向前,材料在层流柜中无菌操作。将叶子在无菌蒸馏水中漂洗(快速浸没)并在无菌滤纸上快速干燥。将每个小叶切成5cm×5cm的片,并将其背侧向上单独放置在含有用蒸馏水饱和的滤纸的无菌培养皿(Ø9cm)中以保持高湿度(图3)。


    图3.在含有饱和水的滤纸的培养皿中,大豆叶片的背面向上。

  5. 通过喷洒1ml冰孢子悬浮液(10μL孢子/ml)接种每片叶片。在每次接种之前立即混合冰孢子悬浮液。手持喷雾器和叶片之间保持10cm的距离。
  6. 将培养皿在20℃的生长室中以12/12小时光/暗循环温育12天。
  7. 在孵育期间每2天目测评价材料的损伤和脓疱的存在。
  8. 在温育12天后,消除显示其它疾病或腐烂的信号的叶子(图4)。根据存在于叶的背侧的损伤类型(免疫,红棕色或棕褐色)将剩余材料视觉分类(图1)。另外,在立体显微镜下记录每片叶片上的损伤数,具有脓疱的损伤数,每个损伤的脓疱数,脓疱总数和开放的脓疱总数,(见注4;图5 )。最后,在每个叶片中测定两个1cm×1cm的感染区域并切下。将每1cm 2片放置在含有500μl水的微量管中并剧烈涡旋1分钟。使用Neubauer室在立体显微镜下估计每cm 2的平均孢子数。


    图4.显示其他疾病或腐烂信号的叶片示例


    图5.用于比较基因型对铁锈感染的反应的参数。 A.无脓疱的病变; B.具有一个脓管的病变(由箭头指示); C.具有多个脓疱的病变(由箭头指示); D.锥形开放性脓疱(左)和锥状未开放性脓疱(右); E.锥形开放的脓疱,在顶部具有孔,在内部(左)具有孢子,在锥体顶部具有孢子的开放脓疱(右)。

  9. 从每个叶片获得的结果被认为是重复的。使用来自相同植物的树叶片段和来自每个转基因事件的至少两个植物。
  10. 使用相同遗传背景的未转化植物作为对照。理想地,易感和部分抗性品种应当用作实验条件的阳性和阴性对照

笔记

  1. 只要有可能,材料应进行无菌操作,以避免交叉污染
  2. Twizeyimana等人(2007)将叶放在培养基上。由于交叉污染和腐烂,我们不能使用相同的培养基建立协议
  3. 使用完整的叶子将有助于防止交叉污染和腐烂。然而,更大的培养皿和更多的生长室中的空间将是进行实验所必需的
  4. 锈病症状开始于叶轴表面上的小病变,其尺寸逐渐增大,并且从灰色变为褐色或红棕色。大豆锈病产生两种类型的损伤:褐色(图1A)和红棕色(图1B)。当成熟时,棕褐色损伤由被稍微变色的坏死区域和大量褐色孢子包围的小脓疱组成(图5B-E)。红棕色病变具有较大的红棕色坏死区,具有有限数目的脓疱和很少可见的孢子。成熟的大豆锈病脓疱是圆锥形的,在顶部具有孔,在圆锥体内部或圆锥上具有孢子(图5D-E)。 ( http://www.aphis.usda.gov/publications/plant_health/content/printable_version/Soybean_Rust_22.pdf )。

食谱

  1. 悬浮孢子悬浮液
    豆腐孢子,1滴吐温20(视频2)和无菌蒸馏水至终浓度为1×10 5孢子/ml。

致谢

我们感谢Emerson M. Del Ponte博士,Juliano dos Santos博士和Larissa Bittecourt的技术援助。 这项工作得到了来自国家科学技术委员会(CNPq),协调高级人员协会(CAPES),基础设施建设委员会(FAPERGS)的资助。 本方案改编自Scherb和Mehl(2006)和Twizeyimana等人(2007)。

参考文献

  1. Bencke-Malato,M.,Cabreira,C.,Wiebke-Strohm,B.,Bucker-Neto,L.,Mancini,E.,Osorio,MB,Homrich,MS,Turchetto-Zolet,A.,De Carvalho,M 。,Stolf,R.,Weber,R.,Westergaard,G.,Castagnaro,AP,Abdelnoor,RV,Marcelino-Guimaraes,FC,Margis-Pinheiro,M.and Bodanese-Zanettini, 大豆WRKY家族的全基因组注释和参与响应的基因的功能表征 Phakopsora pachyrhiziz 感染。 BMC Plant Biol 14(1):236。
  2. Scherb,C.T。和Mehl,A。(2006)。 SBI杀真菌剂,也适用于其他杀真菌剂类 - 离体叶试验。 FRAC (杀菌剂抗性行动委员会)
  3. Twizeyimana,M.,Ojiambo,P.,Ikotun,T.,Paul,C.,Hartman,G.and Bandyopadhyay,R。(2007)。 大豆种质田间,温室和独立叶评估的比较 Plant Dis 91(9):1161-1169。
  4. Wiebke-Strohm,B.,Pasquali,G.,Margis-Pinheiro,M.,Bencke,M.,Bucker-Neto,L.,Becker-Ritt,AB,Martinelli,AH,Rechenmacher,C.,Polacco, Stolf,R.,Marcelino,FC,Abdelnoor,RV,Homrich,MS,Del Ponte,EM,Carlini,CR,De Carvalho,MC和Bodanese-Zanettini,MH(2012)。 无处不在的尿素酶影响大豆对真菌的易感性。 植物分子生物学> 79(1-2):75-87。
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How to cite this protocol: Wiebke-Strohm, B., Rechenmacher, C., Oliveira, L. A., Godoy, C. V. and Zanettini, M. H. (2015). Phakopsora pachyrhizi Infection Bioassay in Detached Soybean Transgenic Leaves for Candidate Gene Validation. Bio-protocol 5(14): e1540. DOI: 10.21769/BioProtoc.1540; Full Text



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