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Magnaporthe oryzae Inoculation of Rice Seedlings by Spraying with a Spore Suspension
喷洒孢子悬液对稻秧进行稻瘟病菌接种   

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Abstract

Fungal blast caused by Magnaporthe oryzae (M. oryzae) leads to a serious yield loss of rice. Appropriate assessment of disease occurrence is necessary to investigate the nature of the disease and plant strategies to resist the disease. We describe our assay method of disease severity of M. oryzae on intact rice leaves grown on soil.

Keywords: Rice(水稻), Magnaporthe oryzae(稻瘟病菌), Inoculation(接种)

Materials and Reagents

  1. Fungal blast (Magnaporthe oryzae) stocks (NIAS Genbank, http://www.gene.affrc.go.jp/index_en.php)
  2. Oat meal agar plates (9 cm diameter) (Difco oatmeal agar; Difco)
  3. Rice (Oryza sativa subsp. japonica) seeds
  4. Soil (Bonsol No.2; Sumitomo Chemical Co., Ltd. http://www.sumitomo-chem.co.jp/)
  5. Sterilized water

Equipment

  1. Bacteria spreader
  2. Conical plastic tubes (50 ml) (Greiner)
  3. Eppendorf tubes (1.5 ml) (Eppendorf)
  4. Filter paper (Advantec)
  5. Fungal growth cabinet with black fluorescent lights (Toshiba, model: FL15BLB )
  6. Glass funnel
  7. Hemocytometer (Fuchs-Rosenthal, Hirschmann EM Techcolor)
  8. KimWipe (Nippon Paper Crecia Co., Ltd., model: Wiper S-200 )
  9. Microscope (Leica Microsystems)
  10. Petri dishes (9 cm diameter)
  11. Sprayer (Figure 1C; Tokyo Koyama Plastic, Co., Ltd., model: K380 )
  12. Sterilized painting brush (Figure 1B)
  13. Dew chamber (Ozawa corporation, model: 513A )

Procedure

  1. Preparation of M. oryzae conidia
    1. Grow M. oryzae by placing a stock piece (Figure 2E) on an oatmeal agar plate at 26 °C for 10-12 d.
    2. Transfer the plate to 24 °C and incubate for 3 d under a black fluorescence light to induce conidia formation.
    3. Pour about 20 ml of sterilized water into the plate (Figure 1A) and collect fungal conidia using a painting brush (Figure 1B).
    4. Filter the fungal conidia through KimWipe in a glass funnel set on a 50-ml conical plastic tube (Figure 2A), and determine the concentration of conidia in the filtrate with Hemocytometer under a microscope (Figure 2C).


      Figure 1. A. M. oryzae grown on an oatmeal agar plate. B. Painting brush used for collecting fungal conidia. C. Sprayer used for spraying the conidia.

  2. Rice infection with M. oryzae
    1. Soak rice seeds in water at 30 °C for 1 d in the dark and then sow them in a pot of soil (Bonsal No.2), and leave the pots in a greenhouse at 28 °C in the day and 23 °C at night for 10-14 d. Rice seedlings at four-leaf stage (three true leaves; Figure 2B) with fully expanded top leaves give the best results in this assay and the duration until reaching this stage varies with different seasons. At least ten rice seedlings are required for each infection assay.
    2. Suspend the conidia in 0.02% Silwet L-77 (or 0.05% Tween 20) at a density of 105/ml (in summer) or 1.5 x 105/ml (in winter) and spray them onto rice plants until the uppermost leaf is completely wet (Video 1). After incubation in a dew chamber at 24 °C for 24 h, move the rice plants back to the greenhouse.

      Video 1. Procedure of M. oryzae inoculation

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    3. After 5-7 d of infection, count the number of blast lesion of susceptible-type in unit length (e.g., 10 cm) of the fourth leaves (Figure 3A, 3B and 3C).
    4. In the case of too many blast lesions formed to distinguish and count (Figure 3B), determine the amounts of M. oryzae 28S ribosomal DNA relative to rice ubiquitin 1 (Os06g0681400) by qPCR (Qi and Yang, 2002).


      Figure 2. A. Filtration of conidia. B. Rice seedlings at 4-leaf stage. C. Spores under microscope. D. M. oryzae grown on a filter paper placed on an oatmeal agar plate. E. Filter pieces with M. oryzae grown on in a 1.5 ml-Eppendorf tube.


      Figure 3. Blast lesions on rice leaves. A. Lesion number appropriate to count. B. Lesions too many to count. C. Susceptible-type lesions. D. Resistant-type lesions.

  3. Making M. oryzae stocks
    1. Place a piece of sterile filter paper on an oatmeal agar plate.
    2. Transfer a small piece of M. oryzae mycelium grown on another oatmeal agar plate onto the filter paper.
    3. Grow M. oryzae on the filter paper at 26 °C for 10-12 d until fungal hyphae covers all over the filter paper (Figure 2D).
    4. Take out the filter paper with grown M. oryzae hyphae (Figure 2D), and dry it for 1-2 d in a petri dish with silica gel (Figure 2E).
    5. Cut the filter paper into 5 mm square pieces and dispense 2-3 of them together with 2-3 silica gel into a 1.5 ml-Eppendorf tube, then keep the tube at -20 °C until use.

Notes

  1. It seems that the incidence of blast disease varies seasonally; higher in the summer and lower in the winter. So it is necessary to adjust the conidia density accordingly, e.g., 105/ml in summer and 1.5 x 105/ml in winter respectively, in order to keep the reproducibility of the assay (Akagi et al., 2014).
  2. The induced conidia on the oatmeal agar plate can be stored in a refrigerator (4 °C) for at least 2 weeks without appreciable loss of pathogenicity.
  3. The M. oryzae stocks can be stored at -20 °C for several years.

Acknowledgements

This work was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement, GMO1006b).

References

  1. Akagi, A., Fukushima, S., Okada, K., Jiang, C. J., Yoshida, R., Nakayama, A., Shimono, M., Sugano, S., Yamane, H. and Takatsuji, H. (2014). WRKY45-dependent priming of diterpenoid phytoalexin biosynthesis in rice and the role of cytokinin in triggering the reaction. Plant Mol Biol 86(1-2): 171-183.
  2. Qi, M. and Yang, Y. (2002). Quantification of magnaporthe grisea during infection of rice plants using real-time polymerase chain reaction and Northern Blot/Phosphoimaging analyses. Phytopathology 92(8): 870-876.

简介

由稻瘟病菌(Magnaporthe oryzae)( oryzae )引起的真菌爆炸导致大米的严重产量损失。 对疾病发生的适当评估是必要的,以调查疾病的性质和植物抵抗疾病的策略。 我们描述我们的疾病严重性的测定方法。 在土壤上生长的完整水稻叶上的玉米。

关键字:水稻, 稻瘟病菌, 接种

材料和试剂

  1. 真菌病毒( Magnaporthe oryzae )(NIAS Genbank, http://www.gene.affrc.go.jp/index_en.php
  2. 燕麦粉琼脂板(直径9cm)(Difco燕麦琼脂; Difco)
  3. 水稻( Oryza sativa subsp。 japonica )种子
  4. 土壤(Bonsol No.2;住友化学有限公司 http://www.sumitomo-chem。 co.jp/
  5. 灭菌水

设备

  1. 细菌扩散器
  2. 圆锥形塑料管(50ml)(Greiner)
  3. Eppendorf管(1.5ml)(Eppendorf)
  4. 滤纸(Advantec)
  5. 带黑色荧光灯的真菌生长柜(东芝,型号:FL15BLB)
  6. 玻璃漏斗
  7. 血细胞计数器(Fuchs-Rosenthal,Hirschmann EM Techcolor)
  8. KimWipe(Nippon Paper Crecia Co.,Ltd。,型号:Wiper S-200)
  9. 显微镜(Leica Microsystems)
  10. 培养皿(直径9cm)
  11. 喷雾器(图1C; Tokyo Koyama Plastic,Co.,Ltd。,型号:K380)
  12. 灭菌的刷子(图1B)
  13. Dew chamber(Ozawa corporation,型号:513A)

程序

  1. M的制备。 oryzae 分生孢子
    1. 成长M。 (图2E)放置在燕麦粥琼脂板上,在26℃下10-12天。
    2. 将板转移到24℃,并在黑色荧光灯下孵育3天以诱导分生孢子形成
    3. 将约20ml的无菌水倒入平板(图1A)和 使用画刷收集真菌分生孢子(图1B)。
    4. 通过设置在玻璃漏斗中的KimWipe过滤真菌分生孢子 50 ml锥形塑料管(图2A),并测定浓度 在显微镜下用血细胞计数器测定滤液中的分生孢子   2C)。


      图1。 A. M。 在燕麦琼脂平板上生长的米曲霉。   B.用于收集真菌分生孢子的涂刷。 C.使用喷雾器 用于喷洒分生孢子。

  2. 用 M的水稻感染。 oryzae
    1. 将水稻种子在30℃水中浸泡1天,然后播种 在一盆土壤(Bonsal No.2),并在28年的温室离开锅   ℃,夜间为23℃,10-14天。 水稻幼苗 四叶期(三个真叶;图2B)具有完全展开的顶部 叶在这个测定和持续时间中给出最好的结果 达到这个阶段随不同季节而变化。 至少10米 每次感染测定需要幼苗
    2. 暂停 分生孢子在0.02%Silwet L-77(或0.05%Tween 20)中以10 5 / (夏季)或1.5×10 5 s/ml(冬季),并将其喷雾到水稻植物上 直到最上面的叶片完全变湿(视频1)。 孵育后 在24℃的露水室中24小时,将稻植物移回 温室。

      视频1. M的过程。 oryzae 接种
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    3. 感染后5-7天,计数 单位长度中易受影响类型的爆炸病变的数目(例如 cm)(图3A,3B和3C)
    4. 如果是 太多的胚细胞损伤形成以区分和计数(图3B), 通过qPCR确定米曲霉28S核糖体DNA相对于水稻泛素1(Os06g0681400)的量(Qi和Yang,2002)。


      图2。 A.  分生孢子过滤。 B. 4叶期的水稻幼苗。 C.孢子 在显微镜下。 D. M。 oryzae 生长在滤纸上 燕麦片琼脂平板。 E.用 M过滤器。 oryzae 生长在1.5 ml-Eppendorf管

      图3.水稻叶子上的病斑。 A.病变  数量适合计数。 B.损伤太多无法计数。 C。 易感型病变。 D.抗性型病变。

  3. 制作 M。 oryzae 股票
    1. 将一块无菌滤纸放在燕麦片琼脂板上。
    2. 将在另一个燕麦片琼脂平板上生长的一小块米曲霉菌丝体转移到滤纸上。
    3. 成长M。 oryzae 在滤纸上在26℃下10-12天直到真菌菌丝覆盖整个滤纸(图2D)。
    4. 取出生长的M的滤纸。 oryzae 菌丝(图2D), 并在具有硅胶的培养皿中干燥1-2天(图2E)。
    5. 将滤纸切成5平方毫米,并分配2-3份   与2-3个硅胶一起放入1.5ml的Eppendorf管中,然后保存 将管在-20℃下使用。

笔记

  1. 似乎爆发疾病的发病率季节不同; 在夏天更高,在冬天更低。 因此,有必要相应地调整分生孢子密度,例如在夏天分别调整10/sup/ml和1.5×10 5/sup/ml ,以保持测定的再现性(Akagi等人,2014)。
  2. 在燕麦片琼脂平板上诱导的分生孢子可以在冰箱(4℃)中储存至少2周,而没有明显的致病性损失。
  3. M。 oryzae 库存可以在-20°C储存几年

致谢

这项工作得到了日本农业,林业和渔业部的一项赠款(基于基因组的农业改进技术,GMO1006b)的支持。

参考文献

  1. Akagi,A.,Fukushima,S.,Okada,K.,Jiang,CJ,Yoshida,R.,Nakayama,A.,Shimono,M.,Sugano,S.,Yamane,H.and Takatsuji, )。 水稻中二萜植物抗毒素生物合成的WRKY45依赖性引发和细胞分裂素在触发反应中的作用。/a> Plant Mol Biol 86(1-2):171-183。
  2. Qi,M。和Yang,Y。(2002)。 使用实时聚合酶链反应和Northern印迹/磷光成像技术在水稻植物感染期间量子稻瘟病菌 分析。 Phytopathology 92(8):870-876。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: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. DOI: 10.21769/BioProtoc.1486.
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