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Xanthomonas oryzae pv. oryzae Inoculation and Growth Rate on Rice by Leaf Clipping Method
通过剪叶法接种水稻黄单胞菌水稻致病变种并测定其生长速率   

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Abstract

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases and a major impediment to the increase of rice yield. Appropriate methods for inoculation of Xoo and disease scoring are necessary to investigate the nature of the disease and the mechanism of plant resistance to the pathogen. As the most-widely grown crop in the worldwide, rice yield plays an important role in food security. Uncovering mechanisms of plant-pathogen interaction of rice and Xoo will help develop rice plants that are more resistant to disease caused by Xoo. Here we describe our validated and efficient methods for inoculation of Xoo and disease scoring.

Keywords: Xanthomonas oryzae pv. oryzae(水稻黄单胞菌水稻致病变种), Bacterial blight(白叶枯病), Leaf clipping method(剪叶法), Inoculation(接种), Growth rate(生长速率), Plant-pathogen interactions(植物-病原体相互作用)

Background

Bacterial blight is a vascular disease starting with Xanthomonas oryzae pv. oryzae (Xoo) invasion of rice leaves through wounds, opening and hydathodes at the leaf tip and margin (Niño-Liu et al., 2006). After multiplying in the intercellular spaces of underlying epitheme, Xoo enters and spreads into the rice plant through the xylem, causing long, grey to white, opaque necrotic lesions. The lesion length and bacterial growth rate can be taken as a measure of the progression of blight disease (Mew, 1984; Niño-Liu et al., 2006). Appropriate artificial inoculation methods and assessment of disease occurrence are necessary to investigate the nature of the disease and plant strategies to defend the pathogen. Mew (1984) has briefly summarized several artificial Xoo inoculation methods that include needle-pricking, spraying, and leaf clipping, or dipping of non-leaf parts of rice with bacterial suspension. Leaf clipping method was originally developed by Kauffman et al. (1973), which enables crosscut veins to be exposed to Xoo suspension by cutting off leaf tips with Xoo suspension infected scissors. Our laboratory has found that leaf clipping method is an effective and simple method, and appropriate to the natural infection of Xoo.

Materials and Reagents

  1. Filter paper (Whatman)
  2. Petri dishes (9 cm diameter) (ASONE)
  3. Cultivated land soil
  4. Compound fertilizer (N: 15%; P: 15%; K: 15%)
  5. Kimwipe (Kimberly-Clark)
  6. 1.5 ml Eppendorf tube (Eppendorf)
  7. 10 ml and 50 ml tubes (BD Falcon)
  8. Tips (200 μl and 1,000 μl) (Axygen)
  9. Xanthomonas oryzae pv. oryzae (Xoo) stocks (provided by International Rice Research Institute/IRRI)
    Note: The Xoo strains are maintained as glycerol stock in -80 °C for long term storage. Every strain should be retrieved on a peptone sucrose agar (PSA) plate.
  10. Rice (Oryza sativa subsp. indica) seeds (rice variety IR24, provided by IRRI’s International Rice Genebank)
  11. Sterilized water (Milli-Q)
  12. Ethanol (Sigma-Aldrich, catalog number: 24102 )
    Note: This product has been discontinued.
  13. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  14. Peptone (Sigma-Aldrich, catalog number: P4963 )
  15. L-Glutamic acid (Sigma-Aldrich, catalog number: G5667 )
  16. Sucrose (Sigma-Aldrich, catalog number: V900116 )
  17. Agar (Sigma-Aldrich, catalog number: 17209 )
  18. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
  19. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  20. Peptone sucrose agar (PSA) solid media (see Recipes)
  21. 1 M MgCl2 stock solution (see Recipes)

Equipment

  1. Ruler, scissors, tweezers, mortar and pestle
  2. Pipette (Eppendorf)
  3. Precision balance (Mettler-Toledo, model: ME303TE )
  4. Growth chamber for rice seed germination (Conviron, model: ACT 26 )
  5. Incubation chamber (28 °C) for Xoo growth (Biolab Scientific, model: BIFG-101 )
  6. Spectrophotometer (Beckman Coulter, model: DU-640 )
  7. Greenhouse capable of temperature and humidity control for growing rice plants

Procedure

  1. Preparation of rice materials
    1. Sow rice seeds (at least 20 seeds per genotype) on a piece of filter paper in each Petri dish with appropriate volume of sterile water and place all the Petri dishes in an incubation chamber (14 h light and 10 h dark photoperiod, at 28-36 °C) for one week.
    2. Transplant the one-week-old seedlings into pots (20 cm in diameter and 30 cm in height) containing 80% volume of cultivated land soil (two seedlings per pot). Water the plants every two days and fertilize the plants every two weeks.

  2. Preparation of Xoo
    1. Grow Xoo by transferring 50 μl of Xoo stock from -80 °C (Figure 1A) to a solid PSA media (see Recipes) (Figure 1B) and incubate at 28 °C for three days until a biofilm is formed (Figure 1C) for Xoo activation. The activated Xoo strain can be stored at 4 °C for up to four weeks.
    2. Two days before inoculating rice plants, subculture of Xoo by transferring activated Xoo from the original solid PSA media to a new solid PSA media and incubating at 28 °C for an additional two days (Figure 1D).
    3. At the day of inoculation, suspend Xoo from the most recent solid PSA media in 10 mM sterilized MgCl2 solution (see Recipes) (pH = 7.0) to OD600 = 0.5 (Figure 1E).


      Figure 1. Xoo preparation. Spread 50 μl Xoo stock solution (A) to a solid PSA media (B) and incubate at 28 °C for three days until a biofilm is formed (C). Two days before inoculation, transfer Xoo from (B) to a new solid PSA media and incubate at 28 °C for two days (D). At the day of inoculation, make Xoo suspension by diluting Xoo to an OD600 of 0.5 from (D) with 10 mM MgCl2 (E).

  3. Inoculation of rice with Xoo and disease scoring
    1. Inoculation of rice leaves by leaf clipping method. Dip scissor tips into the Xoo suspension (Figure 2B) and cut the leaf tip (approximately 2-3 cm for seedling plants and 4-5 cm for adult plants) away from the leaf (Figures 2C and 2D). Plants were grown at 28-32 °C (light, 12 h), 28-32 °C (dark, 12 h), 90% relative humidity.
      Notes:
      1. Clip one to two leaves with one pair of dipped scissor tips into the Xoo suspension for one time.
      2. For statistical analyses of infectious disease, inoculate more than ten leaves per plant and obtain at least five data points for the lesion length.
      3. Rice plants can be transferred into a growth chamber for inoculation.
      4. Cut the tip of the fully extended 1st and 2nd leaves, or just 2nd leaf of seedling plants (before tillering stage). Cut the tip of the fully extended 1st and 2nd leaves, or just 1st leaf per tiller for adult plants (tillering stage or heading stage).


        Figure 2. Xoo inoculation. A. Rice plants grown in green house; B. Dipping scissor tips into the Xoo suspension; C and D. Cutting the leaf tip away from the leaf.

    2. Xoo growth rate (Figure 3)
      1. Sample 6 cm-length leaf fragment every two days until to 14 days (Figure 3A).
        Note: At day 0, harvest samples at 30 min after inoculation. For later time points, discard the thoroughly blight part close to the inoculated site (Xoo is a biotrophic bacterium and does not exist in the thoroughly blight part where is dead leaf tissue; the discarded part should not contain any green tissue) and harvest 6 cm-length leaf fragment next to the inoculated site.
      2. The surface of the leaf fragment are sterilized by immersing in 75% ethanol for 1 min (Figure 3C). Dry the leaf fragment with sterilized Kimwipe.
        Note: The scissors and tweezers are sterilized by immersing in 75% ethanol before use (Figure 3B).
      3. Cut the leaf fragment into a mortar and grind the sample to homogenize with 1 ml sterilized water.
      4. Take 100 μl homogenate from the mortar to a 1.5 ml tube (tube 1) containing 900 μl sterilized water and mix thoroughly.
      5. Take 100 μl diluted homogenate from the tube 1 to a new 1.5 ml tube (tube 2) containing 900 μl sterilized water and mix thoroughly. Repeat this step for several times until to tube n with appropriate dilution.
      6. Take 100 μl diluted homogenate from each of the diluted tubes and spread on PSA solid media. Incubated at 28 °C for 2-3 days until obvious colonies formed (Figure 3D).
      7. Calculate the number of bacteria on three serial dilution plates which have measurable colonies formation individually. For statistical analysis of bacteria, take tube n as an example. N = (Nn-2 x 10n-1 + Nn-1 x 10n + Nn x 10n+1)/3 (N, colony forming unit on the selected 6 cm-length leaf fragment; Nn-2 to Nn represent bacteria colonies from three serial dilution plates corresponding to tube n-2 to tube n, respectively; 10n-1 to 10n+1 represent dilution times corresponding to tube n-2 to tube n).
        Notes:
        1. At least calculate three plates and take an average for the number of bacteria of a selected 6 cm-length leaf fragment from one leaf.
        2. For each time point, at least three leaves from three different plants were collected for biological replicates.


          Figure 3. Procedure of Xoo dilution and growth. A and B. Take a 6 cm-length leaf fragment next to the inoculated site with pretreated scissors and tweezers. C. Immerse the leaf fragment into 75% ethanol for 1 min for surface sterilization. D. After being dried, cut the leaf fragment and grind the sample to homogenize with 1 ml sterilized water, and take 100 μl homogenate to a new 1.5 ml tube containing 900 μl sterilized water and mix thoroughly. Repeat this step for several times until to tube n with appropriate dilution. Take 100 μl diluted homogenate from each of the diluted tubes and spread on PSA solid media. Incubated at 28 °C for 2-3 days until obvious colonies formed.

    3. Score the disease by measuring the lesion length at one week (for seedling stage) or two weeks (for adult stage) after inoculation (Figure 4).


      Figure 4. Disease scoring. Two weeks after inoculation lesions developed on inoculated leaves. The disease was scored by measuring the lesion length. The lesion length is the length from inoculation site to the edge of thoroughly blight leaf middle veins.

    4. Preparation of Xoo stocks
      1. Pick several colonies from PSA solid media streaked with diluted homogenate to 10 ml tubes containing 3 ml PSA liquid medium (one colony per tube) and incubate at 28 °C for two days.
      2. Transfer 100 μl liquid culture into a new 1.5 ml tube and add the same volume of 50% sterilized glycerol, then mix and store at -80 °C.

Data analysis

Data analysis see Yuan et al. (2016).

Recipes

  1. Peptone sucrose agar (PSA) solid media (1 L)
    10 g peptone
    1 g L-glutamic acid
    10 g sucrose
    Dissolve the above gradients in 900 ml sterile distilled H2O and adjust the pH of the medium to 7.0 using 1 N KOH and bring volume up to 1 L. Add 20 g agar and autoclave at 121 °C for 30 min
  2. 1 M MgCl2 stock solution
    Dissolve 203.3 g MgCl2·6H2O in 800 ml sterile H2O and adjust the volume to 1 L and sterilize by autoclaving for 30 min at 121 °C

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (31371926, 31501618). The protocol was adapted from Yuan et al. (2016).

References

  1. Kauffman, H. E., Reddy, A. P. K., Hsieh, S. P. Y and Merca, S. D. (1973). An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Dis Rep 57(6): 537-541.
  2. Mew, T. W. (1984). Scanning electron microscopy of virulent and avirulent strains of Xanthomonas campestris pv. oryzae on rice leaves. Phytopathology 74(6):635-641.
  3. Niño-Liu, D. O., Ronald, P. C. and Bogdanove, A. J. (2006). Xanthomonas oryzae pathovars: model pathogens of a model crop. Mol Plant Pathol 7(5): 303-324.
  4. Yuan, M., Ke, Y., Huang, R., Ma, L., Yang, Z., Chu, Z., Xiao, J., Li, X. and Wang, S. (2016). A host basal transcription factor is a key component for infection of rice by TALE-carrying bacteria. Elife 5.

简介

由黄单胞菌(Xanthomonas oryzae)引起的细菌性病害pv。 Xoo )是最严重的细菌性疾病之一,也是水稻产量增加的主要障碍。 用于接种“Xoo”和疾病评分的适当方法对于调查疾病的性质和植物对病原体的抗性的机制是必要的。 作为世界上种植最广泛的作物,水稻产量在粮食安全方面发挥重要作用。 水稻和Xoo的植物 - 病原体相互作用的揭示机制将有助于开发更易抵抗Xoo 引起的疾病的水稻植物。 在这里,我们描述了我们验证和有效的接种Xoo 和疾病评分的方法。
【背景】细菌性疾病是以黄单胞菌(Xanthomonas oryzae)/ pv。 (Xoo )侵入稻叶通过伤口,叶尖和边缘的开放和水平方向(Niño-Liu等人,2006) )。在下层表皮的细胞间隙中增殖后,Xoo 通过木质部进入并传播到水稻植物中,导致长而灰白色的不透明坏死病变。病变长度和细菌生长率可以作为疾病进展的量度(Mew,1984;Niño-Liu等,2006)。适当的人工接种方法和疾病发生的评估对于调查疾病的性质和防治病原体的植物策略是必要的。 Mew(1984)简要总结了几种人造Xoo 接种方法,包括针刺,喷洒和叶片剪切,或用细菌悬浮液浸渍水稻的非叶片部分。叶片剪切方法最初是由Kauffman等人(1973)开发的,其使横切静脉暴露于Xoo 悬浮液,通过用Xoo切断叶片尖端, 悬浮感染的剪刀。我们的实验室发现叶片剪切方法是一种有效和简单的方法,适合于Xoo 的自然感染。

关键字:水稻黄单胞菌水稻致病变种, 白叶枯病, 剪叶法, 接种, 生长速率, 植物-病原体相互作用

材料和试剂

  1. 滤纸(Whatman)
  2. 培养皿(直径9厘米)(ASONE)
  3. 耕地土壤
  4. 复合肥(N:15%; P:15%; K:15%)
  5. Kimwipe(Kimberly-Clark)
  6. 1.5 ml Eppendorf管(Eppendorf)
  7. 10ml和50ml管(BD Falcon)
  8. 提示(200μl和1,000μl)(Axygen)
  9. xanthomonas oryzae pv。 ( Xoo )股票(由国际水稻研究所/ IRRI提供)
    注意:Xoo菌株在-80℃下保存为甘油储备,以进行长期储存。应在蛋白胨蔗糖琼脂(PSA)板上取出每个菌株。
  10. 种子(IRRI国际水稻基因库提供的水稻品种IR24)
    水稻(Oryza sativa subsp。
  11. 灭菌水(Milli-Q)
  12. 乙醇(Sigma-Aldrich,目录号:24102)
    注意:本产品已停产。
  13. 甘油(Sigma-Aldrich,目录号:G5516)
  14. 蛋白胨(Sigma-Aldrich,目录号:P4963)
  15. L-谷氨酸(Sigma-Aldrich,目录号:G5667)
  16. 蔗糖(Sigma-Aldrich,目录号:V900116)
  17. 琼脂(Sigma-Aldrich,目录号:17209)
  18. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P5958)
  19. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  20. 胨蔗糖琼脂(PSA)固体培养基(见食谱)
  21. 1 M MgCl 2 2储备溶液(参见食谱)

设备

  1. 标尺,剪刀,镊子,迫击炮和杵
  2. 移液器(Eppendorf)
  3. 精密平衡(Mettler-Toledo,型号:ME303TE)
  4. 水稻种子萌发生长室(环境,型号:ACT 26)
  5. 孵化室(28°C),用于Xoo生长(Biolab Scientific,型号:BIFG-101)
  6. 分光光度计(Beckman Coulter,型号:DU-640)
  7. 温室能够对种植水稻进行温湿度控制

程序

  1. 米料的制备
    1. 在每个陪替氏培养皿上的一张滤纸上播种水稻种子(每个基因型至少20个种子),并将所有培养皿放置在孵育室中(14小时光照,10小时黑暗光周期, 36℃)一周。
    2. 将一周龄的幼苗移植到含有80%体积耕地土壤(每盆两株幼苗)的盆(直径20厘米,高30厘米)中。每两天浇灌植物,每两周施肥一次植物
  2. 准备 Xoo
    1. 通过将50μl的Xoo 库存从-80°C(图1A)转移到固体PSA介质(参见食谱)(见图1B)并在28℃温育,生长Xoo 直到形成生物膜(图1C),以进行激活。活化的Xoo 菌株可以在4℃下储存长达四周。
    2. 在接种水稻植物前两天,通过将活化的Xoo从原始的固体PSA培养基转移到新的固体PSA培养基上并在28℃下再孵育另外两次,从而传代Xoo 天(图1D)。
    3. 在接种当天,将10mM无菌MgCl 2溶液(参见食谱)(pH = 7.0)中最新的固体PSA介质悬浮于Xio, 600 <= 0.5(图1E)

      图1. Xoo 准备。将50μlXoo 储备溶液(A)扩散至固体PSA介质(B),并在28°C温育直到形成生物膜(C)。在接种前两天,将(B)的Xoo转移到新的固体PSA培养基中,并在28℃下孵育两天(D)。在接种当天,通过用10mM MgCl 2从(D)稀释Xoo 至0.5的OD 600,使得Xoo < 2(E)。

  3. 用Xoo接种水稻和疾病评分
    1. 通过叶剪接法接种稻叶。将剪刀尖端浸入Xoo 悬浮液(图2B)中,并将叶片切成叶片(图2-3和图2d),叶片顶端(幼苗植物约2-3厘米,成年植物4-5厘米) )。植物在28-32℃(光,12小时),28-32℃(黑暗,12小时),90%相对湿度下生长。
      注意:
      1. 将一对两个叶子用一对浸渍的剪刀提示夹入Xoo暂停一次。
      2. 为了对传染病进行统计学分析,每个植物接种超过10个叶,并获得至少五个病变长度的数据点。
      3. 水稻植物可以转移到生长室进行接种。
      4. 切割完全扩展的1 st 和2 nd (或分蘖期之前)的叶子,或只有2 nd 。剪切完全扩展的1 st 和2 nd />叶子,或只有成年植物(分蘖阶段或穗阶段)每个耕耘机的1 / sup> st 叶。


        图2. Xoo 接种。 :一种。在温室种植的水稻植物; B.将剪刀技巧浸入Xoo 悬浮液; C和D.将叶尖远离叶子。

    2. Xoo 增长率(图3)
      1. 样品6厘米长叶片每两天直到14天(图3A)。
        注意:在第0天,接种后30分钟收获样品。对于后来的时间点,丢弃靠近接种部位的彻底枯萎部位(Xoo是生物营养细菌,不存在于死叶组织的枯萎部位;丢弃的部分不应含有任何绿色组织),并收获6厘米在接种的站点旁边的长叶片。
      2. 将叶片的表面浸入75%乙醇中灭菌1分钟(图3C)。用灭菌的Kimwipe干燥叶片。
        注意:剪刀和镊子在使用前浸入75%乙醇中进行灭菌(图3B)。
      3. 将叶片切成砂浆,研磨样品,用1ml无菌水均质化
      4. 从研钵中取100μl匀浆至含有900μl灭菌水的1.5ml管(管1)并充分混合。
      5. 将100μl稀释的匀浆从管1中取出至含有900μl灭菌水的新的1.5ml管(管2)并充分混合。重复此步骤多次,直到适当稀释至管n
      6. 从每个稀释的管中取100μl稀释的匀浆并铺展在PSA固体培养基上。在28℃下孵育2-3天,直到形成明显的菌落(图3D)
      7. 计算三个连续稀释板上的细菌数量,这些细菌分别具有可测量的菌落形成。对于细菌的统计分析,以管n为例。 N =(N n-2×10 n-1 + N n-1 10 + N (N,在所选择的6cm长的叶片上的集落形成单元; N n-2 x 10 n + 1 )/至N n分别表示来自对应于管n-2至管n的三个连续稀释板的细菌菌落; 10-n-1至10-n + 1 < / sup>表示与管n-2对应管n)的稀释倍数 注意:
        1. 至少计算三个平板,并从一片叶子中平均选择6厘米长的叶片的细菌数。
        2. 对于每个时间点,收集来自三个不同植物的至少三个叶子用于生物重复。


          图3. Xoo 稀释和生长的步骤 A和B.用预处理的剪刀和镊子在接种部位旁边取6厘米长的叶片。 C.将叶片浸入75%乙醇中1分钟进行表面灭菌。 D.干燥后,切碎叶片并研磨样品,用1ml无菌水均质化,取100μl匀浆至含有900μl灭菌水的新1.5ml试管中,充分混合。重复此步骤多次,直到适当稀释至管n。从每个稀释的管中取100μl稀释的匀浆并铺展在PSA固体培养基上。在28℃下孵育2-3天,直到形成明显的菌落
    3. 通过测量接种后一周(幼苗期)或两周(成人阶段)的病变长度来评定疾病(图4)。


      图4.疾病评分。 接种两周后,在接种的叶上发育病变。通过测量病变长度对疾病进行评分。病变长度为从接种部位到枯叶叶中脉边缘的长度
    4. 准备Xoo 股票
      1. 从PSA固体培养基中挑选几个菌落,稀释后的匀浆至含有3 ml PSA液体培养基(每个菌落一个菌落)的10ml管,并在28℃下孵育两天。
      2. 将100μl液体培养物转移到新的1.5ml管中,并加入相同体积的50%灭菌的甘油,然后混合并储存在-80℃。

数据分析

数据分析见袁>等。(2016)。

食谱

  1. 蛋白胨蔗糖琼脂(PSA)固体培养基(1L)
    10g蛋白胨
    1g L-谷氨酸
    10g蔗糖
    将上述梯度溶解在900ml无菌蒸馏的H 2 O中,并使用1N NaOH将培养基的pH调节至7.0,并使体积达到1L。加入20g琼脂和121℃的高压釜30分钟
  2. 1 M MgCl 2 储备溶液
    将203.3g MgCl 2•6H 2 O溶解在800ml无菌H 2 O中,并将体积调节至1L,并通过高压灭菌30分钟灭菌分钟在121°C

致谢

这项工作得到了中国国家自然科学基金资助(31371926,31501618)的支持。该方案由Yuan等人(2016)进行了改编。

参考

  1. Kauffman,H.E.,Reddy,A.P.K.,Hsieh,S.P.Y和Merca,S.D。(1973)。 评估水稻品种对米色黄单胞菌属的抗性的改进技术 植物遗症 57(6):537-541。
  2. Mew,T.W。(1984)。 PV。 植物病理学 74(6):635-641。
  3. Niño-Liu,D.O。,Ronald,P.C。和Bogdanove,A.J。(2006)。 xanthomonas oryzae 病毒:模型作物的模型病原体。 Mol Plant Pathol 7(5):303-324。
  4. Yuan,M.,Ke,Y.,Huang,R.,Ma,L.,Yang,Z.,Chu,Z.,Xiao,J.,Li,X.and Wang,S.(2016)。 宿主基础转录因子是携带TALE的细菌感染水稻的关键组成部分。 Elife 5.
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Copyright Ke et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ke, Y., Hui, S. and Yuan, M. (2017). Xanthomonas oryzae pv. oryzae Inoculation and Growth Rate on Rice by Leaf Clipping Method. Bio-protocol 7(19): e2568. DOI: 10.21769/BioProtoc.2568.
  2. Yuan, M., Ke, Y., Huang, R., Ma, L., Yang, Z., Chu, Z., Xiao, J., Li, X. and Wang, S. (2016). A host basal transcription factor is a key component for infection of rice by TALE-carrying bacteria. Elife 5.
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