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Inoculation of Rice with Different Pathogens: Sheath Blight (Rhizoctonia solani), Damping off Disease (Pythium graminicola) and Barley Powdery Mildew (Blumeria graminis f. sp. hordei)
用不同病原体接种水稻:纹枯病(立枯丝核菌)、猝倒病(禾生腐霉菌)和大麦白粉病(大麦白粉病菌)   

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Abstract

To prevent yield losses in plant cultivation due to plant pathogens, it is an important task to find new disease resistance mechanisms. Recently, Weidenbach et al. (2016) reported about the capacity of the rice gene OsJAC1 to enhance resistance in rice and barley against a broad spectrum of different pathogens. Here, we describe the respective protocols used by Weidenbach and colleagues for inoculation of rice with the basidiomycete Rhizoctonia solani, the oomycete Pythium graminicola and the ascomycete Blumeria graminis f. sp. hordei (Bgh).

Keywords: Rice(水稻), Inoculation protocols(接种方案), Fungal plant pathogens(真菌植物病原体), Oomycete(卵菌纲), Rhizoctonia solani(立枯丝核菌), Pythium graminicola(禾生腐霉菌), Blumeria graminis f. sp. Hordei(大麦白粉病菌), Nonhost resistance(非寄主抗病性)

Background

Following the observation that transcripts of the rice gene OsJAC1 accumulated after pathogen attack or treatment with chemical resistance inducers, transgenic rice plants with constitutive expression or knockout of this gene were investigated in response to inoculation with fungal pathogens. To cover a broad pathogen spectrum, economically important representatives of ascomycete fungi (Magnaporthe oryzae, Blumeria graminis f. sp. hordei), basidiomycete fungi (Rhizoctonia solani) and oomycetes (Pythium graminicola) were chosen. Using protocols for standardized and even inoculation, an enhanced disease resistance phenotype was established for the transgenic plants constitutively expressing OsJAC1 while the respective knockout plants showed enhanced susceptibility (Weidenbach et al., 2016). A detailed bio-protocol for M. oryzae inoculation on rice is already available (Akagi et al., 2015), therefore we focus here on the inoculation protocols for R. solani, P. graminicola and Bgh.

As causal agent of rice sheath blight R. solani is one of the two most important rice diseases (Lee and Rush, 1983). The fungus overwinters as sclerotia or mycelium in the soil and infects rice sheaths by cuticular penetration or through stomata resulting in lesions, necrosis and leaf death (Ou, 1985). Of different methods available for R. solani inoculation, in the present study a time- and space-saving detached leaf assay is described, that was slightly modified from a protocol provided by Monika Höfte (Ghent University, personal communication).

P. graminicola is a causal agent of seedling damping-off and root rot resulting in stunting and yield loss (Hendrix and Campbell, 1973). In this study P. graminicola was inoculated on rice roots growing on agar plates using a protocol adapted from Van Buyten and Höfte (2013).

Fungi of the B. graminis species invade epidermal cells of their host plants with specialized feeding structures called haustoria. All other parts of the fungal mycelium are developed on the leaf surface thereby causing typical powdery mildew disease symptoms. The disease is of permanent importance in cereal agriculture (Dean et al., 2012). Rice plants do not have any powdery mildew pathogens. However, rice can be inoculated with Bgh which allows the investigation of nonhost resistance mechanisms (e.g., Abbruscato et al., 2012; Weidenbach et al., 2016). For an evenly distributed inoculation density, rice leaves have to be fixed and inoculated in a settling tower, as described for barley (Weidenbach et al., 2014).

Materials and Reagents

  1. Square Petri dishes, (120 x 120) x 17 with vents (Greiner Bio One, catalog number: 688102 )
  2. Round Petri dishes, 94 x 16 with vents (Greiner Bio One, catalog number: 633180 )
  3. Filter paper, e.g., Rotilabo-round filters, type 113A, 125 mm (Carl Roth, Germany)
  4. 50 ml tubes (Cellstar tubes) (Greiner Bio One, Germany)
  5. Surgical 3M Micropore tape (3M Deutschland, Germany)
  6. Aluminum foil
  7. Cover glass
  8. Removable adhesive labels, e.g., multipurpose labels (Avery Zweckform, Germany)
  9. Rice (Oryza sativa L. japonica), transgenic plants and respective wild type
    Note: Cultivar Nipponbare used in this study was kindly provided by the Center de cooperation internationale en recherche agronomique pour le developpement (CIRAD, Montpellier, France).
  10. Barley (Hordeum vulgare) susceptible to Bgh.
    Note: Cultivar Ingrid used in this study was kindly provided by Paul Schulze Lefert (Max-Planck Institute for Plant Breeding Research, Cologne, Germany).
  11. Rhizoctonia solani.
    Note: The isolate NL84 used in this study was kindly provided by Monica Höfte (Ghent University, Gent, Belgium).
  12. Pythium graminicola.
    Note: The isolate 132 used in this study was kindly provided by Monica Höfte (Ghent University, Gent, Belgium).
  13. Blumeria graminis f. sp. hordei.
    Note: In this study race K1 (Hinze et al., 1991) was used, which was kindly provided by Paul Schulze-Lefert (Max Planck Institute for Plant Breeding Research, Cologne, Germany).
  14. Potato extract glucose agar (PDA) (Carl Roth, Germany)
  15. Distilled water
  16. Gamborg B5 medium including vitamins (Duchefa Biochemie, catalog number: G0210.0025 )
  17. Agar-Agar, Kobe I (Carl Roth, Germany)
  18. Sodium hypochlorite (or commercially available bleach water, ‘Eau de Javel’)

Equipment

  1. Scissor, scalpel
  2. Plant growth chamber for rice cultivation (settings: 15 h light/9 h dark period, 24 °C, 75-80% humidity)
  3. Plant growth cabinet for constant Bgh propagation on barley (settings:16 h light/8 h dark period, 18 °C, 65% humidity)
  4. Incubator for maintenance of fungal cultures (22 °C, constant darkness)
  5. Horizontal shaker
  6. Thoma cell counting chamber (Marienfeld, Germany)
  7. Light microscope (ca. 150x magnification)
  8. Trays for Bgh inoculation (min. 30 x 15 cm2)
    Note: Area should at least correspond to the magnitude of two week old rice plants.
  9. Spore settling tower (e.g., a plastic tent of ca. 1 m height with an inoculation opening at the top, see example in Figure 3C)

Procedure

  1. Inoculation protocol for Rhizoctonia solani


    Figure 1. Images of R. solani inoculation. A. Typical R. solani culture on PDA seven days after sub-cultivation. B. Four week old rice plants with fully expanded fourth leaves. C. Four week old rice plant with fourth leaf fully expanded (leaf numbers indicated). D. Rice leaves placed on filter paper and inoculated with agar blocks overgrown with R. solani. E. Example of necrotic lesions developing on an inoculated leaf.

    1. Cultivate R. solani on potato extract glucose agar (PDA) at 22 °C in the dark. For maintenance, transfer agar blocks overgrown with fungal mycelium to fresh PDA plates every two weeks. For inoculation, use a R. solani culture seven days after sub-cultivation (Figure 1A).
    2. Grow rice plants for approx. 4 weeks until full expansion of the fourth leaf (Figures 1B and 1C).
    3. Prepare square Petri dishes (12 x 12 cm) lined with filter paper. Moisten the filter paper with distilled water.
    4. Cut off the upper 10 cm of the fourth leaves of different rice plants and immediately place them on the moist filter paper.
    5. Cut small agar blocks (ca. 0.5 x 0.5 cm) overgrown with fungal mycelium from a 7 day old R. solani culture. For inoculation, place one agar block in the middle of each rice leaf (Figure 1D).
    6. Incubate the Petri dishes with the inoculated leaves in the plant chamber. Avoid desiccation of the leaves by daily moistening the filter paper with distilled water (Note 1).
    7. Observe the development of necrotic lesions (Figure 1E) within four days after inoculation.

  2. Inoculation protocol for Pythium graminicola


    Figure 2. Images of P. graminicola inoculation. A. Typical P. graminicola culture on PDA seven days after sub-cultivation. B. Rice seedlings on Gamborg B5 medium inoculated with agar blocks overgrown with P. graminicola.

    1. Cultivate P. graminicola on potato extract glucose agar (PDA) at 22 °C in the dark. For maintenance, transfer agar blocks (ca. 0.5 x 0.5 cm) overgrown with fungal mycelium to fresh PDA plates every two weeks. For inoculation, use P. graminicola cultures seven days after sub-cultivation (Figure 2A).
    2. Prepare square Petri dishes (12 x 12 cm) with 1.5 % agar in Gamborg B5 medium.
    3. Fill rice seeds in a 50 ml tube and add 4 % sodium hypochlorite for surface-sterilization, shake for 10 min. Subsequently, wash three times with sterile distilled water.
    4. Place 3-6 seeds in a line on one Gamborg B5 agar-plate.
    5. Seal the plates with 3M micropore tape and protect root growth from light by covering with aluminum foil.
    6. Incubate the plates upright in a plant growth chamber (conditions see above) until roots have grown to a length of ca. 1 cm (after approx. 5 days).
    7. For inoculation, cut small agar blocks (ca. 0.5 x 0.5 cm) from the marginal zone of a 7 days old P. graminicola culture. Place one agar block between the roots of adjacent plants (Figure 2B). Incubate the plates in a growth chamber as before.
    8. Monitor the disease development for 1 to 2 weeks after inoculation.

  3. Inoculation protocol for Blumeria graminis f. sp. hordei (Bgh)


    Figure 3. Images of Bgh inoculation. A. Two week old rice plants with second leaves fixed on a tray. B. Barley plants heavily infected with Bgh (with white powdery mildew pustules on primary leaves). C. Example for a settling tower that was used for Bgh inoculation (plants were fixed on a tray which was placed at the bottom of the tower). At the top an opening is visible through which infected barley plants can be introduced into the tower.

    1. As the fungus is an obligate biotroph, maintainance has to be done on barley plants in a growth cabinet separated from the test plants. Weekly transfer of Bgh onto fresh, one week old plants is done by shaking off conidiospores from heavily infected barley plants (Figure 3B).
    2. Grow rice plants in soil for approx. 2 weeks until full expansion of the second leaf. Use squared plant pots in order to facilitate a stable fixation on the inoculation trays (see step C4).
    3. One day before inoculation, remove old Bgh conidiospores from barley plants by shaking (Note 2).
    4. For the inoculation of rice, place the plant pots laterally on a tray and fix the second leaf of each plant, adaxial surface up, on the tray by placing 2-3 mm stripes of removable adhesive labels at the tip and the base of each leaf (Note 3, Figure 3A).
    5. Place a Thoma cell counting chamber (without cover glass) beside or between the fixed leaves to monitor the inoculation density.
    6. Cover the trays with a settling tower (Figure 3C).
    7. For inoculation, gently transfer leaves or whole plant pots of Bgh-infected barley plants to the opening of the settling tower and release freshly emerged conidiospores into the tower by shaking. Subsequently, wave with a small tray in the air space of the settling tower to cause a turbulent flow and evenly dispense the conidia.
    8. Let the conidiospores settle for 30-60 min.
    9. Apply a cover glass to the Thoma counting cell and determine the spore density by microscopy. Therefore count the conidiospores in the large central square of each of the two chambers (1 mm2 in a standard Thoma chamber) and take the average. Under our laboratory conditions, 1-5 spores mm-2 may result in distinct, macroscopically observable mildew pustules, while approx. 15-20 spores mm-2 are recommended for microscopic evaluation. If spore density is too low, return to step C5.
    10. Gently remove the adhesive labels from the rice leaves, raise the pots and transfer them to the plant growth chamber.
    11. On host plants, first powdery mildew pustules (as shown in Figure 3B) can be observed within the first week after inoculation. Since rice is a nonhost plant for Bgh, evaluation of the infection process has to be done by microscopy.

Data analysis

Analyses of the inoculated plants can be performed by macroscopic or microscopic evaluation (as described in Weidenbach et al., 2016).

Notes

  1. Carefully avoid ‘flooding’ of the leaves and fungal inoculum.
  2. This step insures fresh inoculum, since the old conidiospores, which differ in virulence, are depleted.
  3. By fixing the position of the leaves on the trays, an even inoculation becomes possible.

Acknowledgments

We thank Monika Höfte for providing R. solani and P. graminicola isolates and for her kind advise in respective inoculation methods. All the protocols presented here were used and described in Weidenbach et al. (2016), whose first author was funded in the framework of the BMBF funding activity ‘Plant Biotechnology for the future, PLANT 2030’ within the project ‘BarleyFortress’.

References

  1. Abbruscato, P., Nepusz, T., Mizzi, L., Del Corvo, M., Morandini, P., Fumasoni, I., Michel, C., Paccanaro, A., Guiderdoni, E., Schaffrath, U., Morel, J. B., Piffanelli, P. and Faivre-Rampant, O. (2012). OsWRKY22, a monocot WRKY gene, plays a role in the resistance response to blast. Mol Plant Pathol 13(8): 828-841.
  2. Akagi, A., Jiang, C. and Takatsuji, H. (2015). Magnaporthe oryzae inoculation of rice seedlings by spraying with a spore suspension. Bio-protocol 5(11): e1486.
  3. Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J. and Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13(4): 414-430.
  4. Hendrix, F. F. and Campbell, W. A. (1973). Pythiums as plant pathogens. Annu Rev Phytopathol 11: 77-98.
  5. Hinze, K., Thompson, R. D., Ritter, E., Salamini, F. and Schulze-Lefert, P. (1991). Restriction fragment length polymorphism-mediated targeting of the ml-o resistance locus in barley (Hordeum vulgare). Proc Natl Acad Sci U S A 88(9): 3691-3695.
  6. Lee, F. N., and Rush, M. C. (1983). Rice sheath blight: A major rice disease. Plant Dis 67(7): 829-833.
  7. Ou, S. H. (1985). Rice diseases. Second edition. Commonwealth Mycological Institute.
  8. Van Buyten, E. and Höfte, M. (2013). Pythium species from rice roots differ in virulence, host colonization and nutritional profile. BMC Plant Biol 13: 203.
  9. Weidenbach, D., Esch, L., Möller, C., Hensel, G., Kumlehn, J., Höfle, C., Hückelhoven, R. and Schaffrath, U. (2016). Polarized defense against fungal pathogens is mediated by the jacalin-related lectin domain of modular Poaceae-specific proteins. Mol Plant 9: 514-527.
  10. Weidenbach, D., Jansen, M., Franke, R. B., Hensel, G., Weissgerber, W., Ulferts, S., Jansen, I., Schreiber, L., Korzun, V., Pontzen, R., Kumlehn, J., Pillen, K. and Schaffrath, U. (2014). Evolutionary conserved function of barley and Arabidopsis 3-ketoacyl-CoA synthases in providing wax signals for germination of powdery mildew fungi. Plant Physiol 166(3): 1621-1633.

简介

为防止植物病原体引起的植物栽培产量损失,寻找新的抗病机制是重中之重。最近,Weidenbach等人。 (2016)报道了水稻基因OsJAC1的能力,以增强水稻和大麦对广谱不同病原体的抵抗力。在这里,我们描述了Weidenbach及其同事使用的各种协议,用于接种担子菌丝核菌(Rhizoctonia solani),卵菌(Pseudium graminicola)和子囊菌(Blumeria graminis) em> f。 sp。 hordei ( Bgh )。

背景 在观察到病原体攻击或用化学抗性诱导剂处理之后积累的水稻基因OsJAC1的转录物观察到,具有组成型表达或敲除该基因的转基因水稻植物对接种真菌病原体进行了研究。为了覆盖广泛的病原体谱,在子囊菌真菌(稻瘟病菌选择立枯丝核菌(Rhizoctonia solani)和oomycetes(镰刀菌腐霉菌)。使用用于标准化和均匀接种的方案,建立了组成型表达OsJAC1的转基因植物的增强的抗病性表型,而相应的敲除植物显示增强的易感性(Weidenbach等人, 2016)。详细的生物学协议。稻米接种米饭已经可以使用(Akagi等人,2015),因此我们重点介绍了这种接种方案。 solani , P。 graminicola Bgh
  作为水稻纹枯病的原因。 solani 是两个最重要的水稻病之一(Lee和Rush,1983)。真菌在土壤中作为菌核或菌丝体越冬,通过角质渗透或通过气孔感染水稻鞘,导致病变,坏死和叶死亡(Ou,1985)。对于可用于 R的不同方法。 Solani 接种,在本研究中描述了一种时间和空间节约的分离叶测定法,该方法从MonikaHöfte(根特大学个人通信)提供的方案进行了稍微修改。
   P。 graminicola 是幼苗阻尼和根腐病的致病因子,导致发育迟缓和产量损失(Hendrix和Campbell,1973)。在这项研究中使用Van Buyten和Höfte(2013)的方案,在生长在琼脂平板上的稻根上接种graminicola 。
   B的真菌。禾本科植物物种入侵其宿主植物的表皮细胞,其特殊的饲养结构称为ust ust。真菌菌丝体的所有其他部分在叶面上发育,从而引起典型的白粉病症状。该疾病在谷物农业中是永久性的(Dean等人,2012)。水稻植物没有任何白粉病菌。然而,可以使用允许调查非宿主抗性机制(例如,,Abbruscato et al。,2012; Weidenbach > et al。,2016)。对于均匀分布的接种密度,如对于大麦(Weidenbach等人,2014)所述,必须将稻叶固定并接种在沉降塔中。

关键字:水稻, 接种方案, 真菌植物病原体, 卵菌纲, 立枯丝核菌, 禾生腐霉菌, 大麦白粉病菌, 非寄主抗病性

材料和试剂

  1. 方格培养皿(120 x 120)x 17带通风口(Greiner Bio One,目录号:688102)
  2. 圆形培养皿,94 x 16通风口(Greiner Bio One,目录号:633180)
  3. 滤纸,例如,Rotilabo圆形过滤器,113A型,125mm(Carl Roth,德国)
  4. 50ml管(Cellstar管)(Greiner Bio One,德国)
  5. 手术3M微孔胶带(3M德国,德国)
  6. 铝箔
  7. 盖玻璃
  8. 可拆卸的粘合标签,例如,多功能标签(Avery Zweckform,德国)
  9. 水稻(za iva>> j ica ica ica ica ica ica ica ica ica ica>>>>>>>>>>>>>>>>>>>> 注意:本研究中使用的栽培品种Nipponbare由国际农业研究中心(CIRAD,Montpellier,France)提供。
  10. 大麦(Hordeum vulgare )易受 的影响。
    注意:本研究中使用的Cultivar Ingrid由Paul Schulze Lefert(德国科隆Max-Planck植物育种研究所)提供。
  11. 丝核菌(Rhizoctonia solani)
    注意:本研究中使用的分离物NL84由MonicaHöfte(根特大学,根特,比利时)提供。
  12. 腐霉腐霉
    注意:本研究中使用的隔离物132由MonicaHöfte(根特大学,根特,比利时)提供。
  13. Blumeria graminis f。 sp。 hordei。
    注意:在本研究中,使用了K1(Hinze等,1991),由Paul Schulze-Lefert(马克斯·普朗克植物育种研究所,德国科隆)提供。
  14. 土豆提取物葡萄糖琼脂(PDA)(Carl Roth,德国)
  15. 蒸馏水
  16. 含有维生素的Gamborg B5培养基(Duchefa Biochemie,目录号:G0210.0025)
  17. 琼脂,神户一(Carl Roth,德国)
  18. 次氯酸钠(或市售的漂白水,"Eau de Javel")

设备

  1. 剪刀,手术刀
  2. 用于水稻种植的植物生长室(设置:15小时灯/9小时黑暗时期,24℃,75-80%湿度)
  3. 植物生长箱用于常规繁殖(设置:16小时灯/8小时黑暗时期,18℃,65%湿度)
  4. 用于维护真菌培养物的培养箱(22°C,恒定黑暗)
  5. 卧式摇床
  6. Thoma细胞计数室(Marienfeld,德国)
  7. 光学显微镜(约150倍放大)
  8. 接种的托盘(最小30 x 15厘米 2)
    注意:面积应至少对应于两周龄水稻植物的大小。
  9. 孢子沉降塔(例如,,高约1米的塑料帐篷,顶部有接种开口,参见图3C中的示例)

程序

  1. 接种立枯丝核菌的方案


    solani 接种。 A.典型的 R。在培育7天后,PDA上的solani 文化。 B.四周龄的水稻植株,四叶完全膨大。 C.四周龄水稻,第四叶充分膨大(叶数)。 D.将大米叶放置在滤纸上,并用长满大块的琼脂块接种。 solani 。 E.在接种的叶上发育的坏死病变的实例。

    1. 培养 R。 solani在土豆提取物葡萄糖琼脂(PDA)上在22℃下在黑暗中。为了维护,每两周将带有真菌菌丝体的琼脂块转移到新鲜的PDA板上。对于接种,使用 R。分蘖后七天(图1A)的solani 文化
    2. 种植水稻约为4周直到第四叶充分膨胀(图1B和1C)
    3. 准备内置滤纸的方格培养皿(12 x 12厘米)。用蒸馏水润湿滤纸。
    4. 切掉不同水稻植物的第四个叶子的上部10厘米,并立即将其放在潮湿的滤纸上
    5. 切割来自7天龄的真菌菌丝体的小琼脂块(约0.5×0.5厘米)。 solani 文化。对于接种,将一个琼脂块放在每个稻叶的中间(图1D)
    6. 孵化培养皿与接种的叶子在植物室。避免用蒸馏水润湿滤纸来干燥叶子(注1)
    7. 接种后4天内观察坏死性病变(图1E)的发展情况
  2. 葛根腐霉的接种方案

    图2. < graminicola 接种。 A.典型的。培养七天后,在PDA上培养禾本科植物。 B.接种有琼脂块的Gamborg B5培养基上的水稻幼苗,其中长满了P. graminicola 。

    1. 培养P。 graminicola 在土豆提取物葡萄糖琼脂(PDA)中在22℃下在黑暗中。为了维护,每两周将含有真菌菌丝体的琼脂块(约0.5 x 0.5厘米)转移到新鲜的PDA板上。对于接种,请使用P.分蘖后7天培养禾本科植物(图2A)
    2. 在Gamborg B5培养基中准备含1.5%琼脂的方形培养皿(12 x 12厘米)。
    3. 将米粒填充在50ml管中,加入4%次氯酸钠进行表面消毒,摇动10分钟。随后用无菌蒸馏水洗三次
    4. 在一个Gamborg B5琼脂平板上放置3-6粒种子。
    5. 用3M微孔带密封板,并用铝箔覆盖保护根生长。
    6. 将板直立放置在植物生长室中(条件见上文),直到根长出长约ca。 1厘米(约5天后)。
    7. 对于接种,从7天龄的边缘区切下小琼脂块(约0.5×0.5厘米)。 graminicola 文化。在相邻植物的根之间放置一个琼脂块(图2B)。如前所述,在生长室中孵育平板。
    8. 接种后1〜2周监测疾病发展情况
  3. f。Blumeria graminis的接种方案f。f。 sp。 hordei ( Bgh


    图3. 接种。A.将二周龄的水稻植物与第二叶固定在托盘上。 B.大麦感染的大麦植物(原生叶上有白色白粉病脓疱)。 C.用于接种的沉降塔的实施例(将植物固定在放置在塔底部的托盘上)。在顶部,可以看到可以将感染的大麦植物引入塔中的开口。

    1. 由于真菌是专性生物体,因此必须在与试验植物分离的生长箱中的大麦植物上进行维持。通过从严重感染的大麦植物中摆脱分生孢子,将Bgh 每周转移到新鲜的一周龄植物中(图3B)。
    2. 在土壤中种植大米植物约2周直到第二叶充分膨胀。使用平方的植物盆,以便于在接种盘上稳定地固定(参见步骤C4)。
    3. 在接种前一天,通过摇晃从大麦植物中除去老的分生孢子(注2)。
    4. 对于接种米饭,将植物盆横向放置在托盘上,通过在每个叶片的顶端和底部放置2-3毫米可移除的粘合剂标签条,将每个植物的第二叶子(近轴表面)固定在托盘上(注3,图3A)
    5. 将固定叶片旁边或两侧的Thoma细胞计数室(无盖玻片)放置,以监测接种密度
    6. 用沉淀塔盖住托盘(图3C)。
    7. 对于接种,将感染的大麦植物的叶子或整个植物盆轻轻地转移到沉降塔的开口处,并通过摇动将新鲜出现的分生孢子释放到塔中。随后,在沉降塔的空气空间内用小托盘挥动,引起湍流,均匀分配分生孢子。
    8. 让分生孢子定居30-60分钟。
    9. 在托马计数细胞上涂上盖玻片,用显微镜确定孢子密度。因此,计算两个房间的大中央正方形(在标准Thoma室中为1 mm 2 )的分生孢子,并取平均值。在我们的实验室条件下,1-5个孢子mm -2 可能导致明显的宏观可观察到的霉菌脓疱, 15-20孢子mm -2 推荐用于显微镜评估。如果孢子密度太低,返回步骤C5。
    10. 轻轻地从米叶中取出粘合剂标签,抬起锅并将其转移到植物生长室。
    11. 在宿主植物上,可以在接种后的第一周内观察到第一次白粉病脓疱(如图3B所示)。由于大米是一种不育植物,因此必须通过显微镜来评估感染过程。

数据分析

接种的植物的分析可以通过宏观或微观评估进行(如在Weidenbach等人,2016中所述)。

笔记

  1. 小心避免叶子和真菌接种物的"淹水"。
  2. 这一步确保新鲜的接种物,因为毒力不同的老的分生孢子已经枯竭了。
  3. 通过将叶片的位置固定在托盘上,可以进行均匀的接种。

致谢

我们感谢MonikaHöfte提供 R。 solani P。 graminicola 分离,并在各自的接种方法中为她提供建议。 Weidenbach等人(2016)中使用和描述了这里提供的所有协议,其第一作者是在BMBF资助活动"未来植物生物技术"(Plant Biotechnology for the Future,BASANT 2030)的框架内资助的项目"BarleyFortress"。

参考文献

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  2. Akagi,A.,Jiang,C.和Takatsuji,H。(2015)。 稻瘟病菌通过喷洒孢子接种水稻幼苗悬挂。 生物协议 5(11):e1486。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Delventhal, R., Loehrer, M., Weidenbach, D. and Schaffrath, U. (2016). Inoculation of Rice with Different Pathogens: Sheath Blight (Rhizoctonia solani), Damping off Disease (Pythium graminicola) and Barley Powdery Mildew (Blumeria graminis f. sp. hordei). Bio-protocol 6(24): e2070. DOI: 10.21769/BioProtoc.2070.
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