2 users have reported that they have successfully carried out the experiment using this protocol.
Root-knot Nematode Penetration and Sclareol Nematicidal Activity Assays

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Molecular Plant Microbe Interactions
Apr 2015



Plant parasitic nematodes parasitize roots and/or stems of various plants thereby inhibiting absorption of nutrients and moisture. In particular, root-knot nematodes (RKN) are a group of the most devastating pests. Various techniques, such as soil sterilization, cultivation of resistant crops, and chemical application, have been developed to control damage caused by RKN. Among these techniques, diminish by chemicals that induce or activate host defense to RKN is an attractive method because of its potential to reduce the environmental burden caused by crop protection. Sclareol, a diterpene, was identified as a chemical that induces resistance to RKN (Fujimoto et al., 2015). Here we provide a protocol for assessing the impact of sclareol on the penetration of RKNs into tomato and Arabidopsis roots and the direct nematicidal impact of the chemical on nematodes. This protocol can be used for other nematode resistance-inducing chemicals.

Keywords: Root-knot nematode (根结线虫病), Nematicidal activity (杀线虫活性), Nematode penetration (线虫的渗透), Stain method (染色法)

Materials and Reagents

  1. 220 ml bottom opened plastic pot
  2. Glass petri dish, 9 cm in diameter
    Note: not necessary sterilized
  3. Sterilized 50 ml glass culture bottle with cap
  4. 1 ml glass pipette
    Note: Polypropylene pipette tips tend to retain Root-knot nematode (RKN), hence use of such tips should be avoided.
  5. Sterilized 24-well tissue culture plate (e.g., IWAKI, catalog number: 3820-024 )
  6. RKN Meloidogyne incognita (NIAS Genebank, MAFF number: 108258 )
    Note: We obtained a strain 108258 of RKN from the MAFF gene bank of the National Institute of Agrobiological Sciences of Japan (http://www.gene.affrc.go.jp/about_en.php). This strain can survive and propagate on plants carrying Mi-genes for RKN resistance (resistance breaking strain).
  7. Tomato (Solanum lycopersicum), cultivar Momotaro
    Note: The cultivar Momotaro carries Mi-genes for RKN resistance. Seeds of tomato cultivars possessing Mi-genes (e.g., Momotaro) can be obtained from a seed company (Takii & Co. Ltd., Kyoto, Japan).
  8. Arabidopsis thaliana (Columbia)
  9. Water (Milli-Q grade)
  10. Sodium hypochlorite solution (Nacalai Tesque, catalog number: 31518-35 )
  11. Ethanol (99.5%) (Nacalai Tesque, catalog number: 09666-85 )
  12. Sea sand with an average size of 0.3 mm
    Note: Shift and wash sea sand to remove the pebbles and the salt with a large amount of tap water in a metal tub and autoclave the washed sand.
  13. Liquid fertilizer (Hyponex)
  14. Methanol (99.8%) (Nacalai Tesque, catalog number: 21915-93 )
  15. Acid fuchsin (e.g., Sigma-Aldrich, catalog number: F8129-25G , or Wako, catalog number: 061-01332 )
  16. Lactic acid (85~92%) (Nacalai Tesque, catalog number: 20006-75 )
  17. Glycerol (99%) (Nacalai Tesque, catalog number: 17018-83 )
  18. Sclareol (Sigma-Aldrich, catalog number: 357995-1G )
  19. Sodium hydroxide (Nacalai Tesque, catalog number: 31511-05 )
  20. Acetic acid (99%) (Nacalai Tesque, catalog number: 00211-95 )
  21. Acid fuchsin solution (see Recipes)
  22. 100 mM sclareol solution (see Recipes)


  1. Greenhouse or chamber for plant growth
  2. Stainless tweezer
  3. Incubator (e.g., Sibata, model: SMU-0541 )
  4. Light microscope (e.g., OLYMPUS, model: BX53M )
  5. Refrigerator (e.g., PANASONIC, model: MPR-162DCN )
  6. Stainless steel tray (9.2 cm in height x 18 cm depth x 32 cm width)
  7. Scissors (e.g., V. Mueller, catalog number: 25601-164 )
  8. Water bath (e.g., Taitec, model: SH-10N )
    Note: You can also use a commercially available microwave oven for cooking.
  9. Microwave oven (e.g., HITACHI, model: MRO-NT5 )
  10. Stereoscopic microscope (e.g., Nikon, model: SMZ-U )


  1. Propagation of RKN in tomato plants
    1. Suspend RKNs, which consist of a clonal population of RKN established from a single female, in water to a final concentration of at least 500 RKNs/ml.
      Note: We use stock cultured RKNs obtained from field.
    2. Inoculate 1 ml of RKNs suspension to 4-week-old tomato plants grown in a pot containing soil by injecting the solution on the soil surface surrounding the roots of each plant (Figure 1).

      Figure 1. Inoculation of RKNs suspension on the soil surface using a 1 ml glass pipette

    3. Grow the plants in a greenhouse maintained at 23-27 °C under natural sunlight for approximately 2 months.
    4. Carefully pull the roots out of the pots and wash them in tap water to remove the soil from the roots.
    5. Pick up the egg masses with tweezers from the roots.
    6. Suspend the collected egg masses in a small volume of water in a petri dish and incubate at 25 °C in the dark (Figure 2).

      Figure 2. Petri dishes containing collected RKN egg-masses

    7. After most juveniles hatched (about 10-14 days), vacuum the incubated suspension of nematodes using a glass pipette and release on a glass bottle (Figure 3).

      Figure 3. Transfer of hatched RKN juveniles to a glass bottle

    8. Suspend the collected RKNs in 5-10 ml water.
    9. Count the numbers of RKNs in the suspension under a light microscope (Figure 4).

      Figure 4. Suspension of RKNs under a light microscope prior to the juveniles counting. Scale bar, 5 mm.

    10. Prepare aliquots with an adequate concentration of RKNs in water.
      Note: We adjust 500 RKNs/ml. You can store RKN suspensions without loss of activity in a refrigerator maintained at 10 °C for 2-3 weeks until use.
    11. Use appropriate aliquots for each analysis.

  2. Plant growth 
    1. Tomato plants
      1. Sterilize seeds for 3 min in 1.0% sodium hypochlorite solution.
      2. Wash the sterilized seeds in 70% ethanol.
      3. Wash the sterilized seeds three times in sterilized distilled water.
      4. Germinate the seeds on the sterilized wet paper at 23-27 °C with a photoperiod of 16 h of light and 8 h of dark.
      5. Transfer 7- to 10-day-old seedlings to autoclaved sand packed in 220 ml plastic pot and grow in a chamber maintained at 23-27 °C with a photoperiod of 16 h of light and 8 h of dark. Apply a liquid fertilizer diluted 3,000x with water to the pots every 3 days.
      6. Use tomato seedlings with two fully developed true leaves for each assay (Figure 5).

        Figure 5. Tomato plant grown in soil

    2. Arabidopsis plants
      1. Sterilized seeds for 3 min in 1.0% sodium hypochlorite solution.
      2. Wash the sterilized seeds in 70% ethanol.
      3. Wash the sterilized seeds three times in sterilized distilled water.
      4. Germinate the seeds on the sterilized 1.5% agar plate at 22 °C with a photoperiod of 16 h of light and 8 h of dark.
      5. Transfer 3-week-old seedlings to autoclaved sand packed in 220 ml plastic pot and grow in a chamber maintained at 22 °C with a photoperiod of 16 h of light and 8 h of dark.
      6. Apply a liquid fertilizer diluted 3,000x with water to the pots every 5 to 7 days.
      7. Use 10-week-old plants for each assay (Figure 6).
        Note: We noted that Arabidopsis planted in sand grew slower than in soil. Thus, the size of 10-week-old plants grown in sand are almost the same as that of 6-week-old plants grown in soils.

        Figure 6. Arabidopsis plants grown in vermiculite

  3. Chemical treatments and RKN penetration assay
    1. Use 10 plants for each treatment.
    2. Place five plants per tray.
    3. Pour 2,000 ml of a solution containing adequate concentrations of sclareol or 0.1% methanol alone in the tray, as plant roots are submerged in the solution (Figure 7).
      Note: We present here a protocol for sclareol, however, it can be also used for other chemicals. We used four concentrations; 50, 80, 100 and 200 μM.

      Figure 7. The pots and tray used in our protocol

    4. After submerging for 48 h, inoculate second-stage juveniles of RKNs to plants by injecting 1 ml of the RKN suspension (200 RKNs for tomato and 500 RKNs for Arabidopsis) on the sand surrounding the roots with 1 ml glass pipette.
      Note: Because the efficiency of penetration of RKN to Arabidopsis roots is lower than that to tomato roots, we always use higher number of RKNs for Arabidopsis.
    5. Grow the inoculated plants in a chamber maintained at 25 °C for tomato and 22 °C for Arabidopsis with a photoperiod of 16 h of light and 8 h of dark.

  4. Staining with acid fuchsin
    Note: Acid fuchsin is a dye that stains nematodes. The procedure described below is a modified version of the staining method reported by McBeth et al. (1941).
    1. Seven days after inoculation, pull the roots out of the pots and wash them with tap water to remove the sea sand.
    2. Separate the roots from plants using scissors and put the roots in a 50 ml glass culture bottle.
    3. To decolorize the roots, pour 40 ml 1.0% sodium hypochlorite in the culture bottle and gently invert the bottle 10 or more times.
    4. Incubate for 5 min.
    5. After discarding the sodium hypochlorite, wash the roots with a large amount of tap water.
      Note: Because sodium hypochlorite blocks the acid fuchsin’s staining effect, sodium hypochlorite should be completely removed at this step.
    6. Pour 30 ml water in the culture bottle and then add 1 ml acid fuchsin. Mix well by inverting the bottle several times.
      Note: The acid fuchsin solution in the bottle turns pale red. If the solution turns pale orange, it means that sodium hypochlorite still remains in the solution.
    7. Boil the culture bottle containing the roots in the acid fuchsin solution in a preheated water bath at 100 °C.
      Note: You can also use a microwave oven instead of water bath. In this case, the microwave oven should be stopped when the solution in the bottle starts to bubble.
    8. Cool down the boiled roots and solution to room temperature.
    9. Discard the acid fuchsin solution and wash the roots with tap water to remove excess of acid fuchsin solution.
    10. Add 30 ml of a mixture of lactic acid and glycerol (1:1, v/v).
    11. Boil the bottle according to the step D7.
    12. Cool down the bottle to room temperature.
    13. After washing the roots with tap water, count the number of stained RKNs in the roots under a stereoscopic microscope. A representative example of stained RKNs is shown in Figure 8 and a representative result of sclareol-treated or non-treated plants for the penetration rate is shown in Figure 9.

      Figure 8. RKN in tomato roots stained with acid fuchsin. Scale bar, 200 μm.

      Figure 9. Effect of sclareol on the penetration of RKNs into tomato roots. Values are the mean ± SD from two independent experiments, each performed in 10 replicates. Asterisks denote statistically significant differences compared with the 0.1% methanol control (Tukey’s HSD test; P < 0.05).

  5. Nematicidal activity assay
    Note: The procedure described below is a modified version of the assay reported by Chen and Dickson (2000).
    1. Dilute RKN suspension to a concentration of 100 RKNs per ml with water.
    2. Add 1 ml RKN suspension to each well of 24-well tissue culture plates.
    3. Add 1 ml solution containing adequate concentration of sclareol or 0.1% methanol to each well.
    4. Incubate the plates in the dark at 25 °C.
    5. Count the numbers of motile and non-motile RKNs 1, 3, 6, 12, 24 and 48 h after the chemical application under a light microscope.
    6. To check motility of RKNs, add a few drops of 1 M sodium hydroxide (NaOH) to each well.
      Note: Sodium hydroxide can stimulate movement of RKN. If RKN moves by the addition of NaOH, we count it as living RKN.
    7. Calculate the ratio of living RKN to dead RKN (Figure 10).

      Figure 10. Nematicidal activity of 100 μM sclareol. Approximately 100 RKNs in 1 ml of nematode suspension were added to 1 ml of 200 μM sclareol solution or control solution (0.2% methanol) in 24-well tissue culture plates and incubated in the dark at 25 °C. At 1, 3, 6, 12, 24 and 48 h after the onset of incubation, the numbers of motile and non-motile RKNs were counted (data for 1, 3 and 6 h are not shown).


  1. Acid fuchsin solution (for 1,000 ml)
    Dissolve 2.5 g acid fuchsin in 500 ml water and add 250 ml acetic acid to the solution
    Adjusted with water to the final volume of 1,000 ml
    Stored at room temperature in the dark
  2. 100 mM sclareol solution
    Dissolve sclareol in methanol to a concentration of 100 mM
    You can use the solution as a stock solution
    Store the stock solution at -20 °C in the dark
    Dilute the stock solution to each concentration with water


This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, the Japanese Program for the Promotion of Basic and Applied Research for Innovation in Bio-oriented Industry (BRAIN), Japan, and a grant from Cross-ministerial Strategic Innovation Promotion Program, Japan.


  1. Chen, S. Y. and Dickson, D. W. (2000). A technique for determining live second-stage juveniles of Heterodera glycines. J Nematol 32:117-121.
  2. Fujimoto, T., Mizukubo, T., Abe, H. and Seo, S. (2015). Sclareol induces plant resistance to root-knot nematode partially through ethylene-dependent enhancement of lignin accumulation. Mol Plant Microbe Interact 28: 398-407.
  3. McBeth, C. W., Taylor, A. L. and Smith, A. L. (1941). Note on staining nematodes in root tissues. Proc Helminthol Soc Wash 16: 3-6.


植物寄生线虫寄生于各种植物的根和/或茎,从而抑制营养物和水分的吸收。 特别地,根结线虫(RKN)是最具破坏性的害虫的组。 已经开发了各种技术,例如土壤灭菌,抗性作物的栽培和化学施用,以控制由RKN引起的损害。 在这些技术中,诱导或激活RKN的宿主防御的化学物质的减少是有吸引力的方法,因为其具有减少作物保护所引起的环境负担的潜力。 Sclareol,一种二萜,被鉴定为诱导对RKN的抗性的化学物质(Fujimoto等人,2015)。 在这里我们提供了评估香紫苏醇对RKNs对番茄和拟南芥根的渗透以及化学物对线虫的直接杀线虫影响的影响的方案。 该方案可用于其它线虫抗性诱导化学品。

关键字:根结线虫病, 杀线虫活性, 线虫的渗透, 染色法


  1. 220毫升底部打开的塑料盆
  2. 玻璃培养皿,直径9厘米
  3. 灭菌50ml玻璃培养瓶带盖
  4. 1 ml玻璃移液管
  5. 灭菌的24孔组织培养板(例如,IWAKI,目录号:3820-024)
  6. RKN (NIAS Genebank,MAFF编号:108258)
    注意:我们从日本农业生物科学国家研究所的MAFF基因库获得了RKN的菌株108258( http://www.gene.affrc.go.jp/about_en.php )。该菌株可以在携带用于RKN抗性(抗性断裂应变)的Mi基因的植物上存活和繁殖。
  7. 番茄( Solanum lycopersicum ),桃太郎栽培品种
    注意:品种Momotaro携带用于RKN抗性的Mi基因。可以从种子公司(Takii& Co.Ltd。,Kyoto,Japan)获得具有Mi基因的番茄栽培种的种子(例如,Momotaro)。
  8. 拟南芥(哥伦比亚)
  9. 水(Milli-Q级)
  10. 次氯酸钠溶液(Nacalai Tesque,目录号:31518-35)
  11. 乙醇(99.5%)(Nacalai Tesque,目录号:09666-85)
  12. 平均尺寸为0.3毫米的海砂
  13. 液体肥料(Hyponex)
  14. 甲醇(99.8%)(Nacalai Tesque,目录号:21915-93)
  15. 酸性品红(例如,Sigma-Aldrich,目录号:F8129-25G或Wako,目录号:061-01332)
  16. 乳酸(85〜92%)(Nacalai Tesque,目录号:20006-75)
  17. 甘油(99%)(Nacalai Tesque,目录号:17018-83)
  18. Sclareol(Sigma-Aldrich,目录号:357995-1G)
  19. 氢氧化钠(Nacalai Tesque,目录号:31511-05)
  20. 乙酸(99%)(Nacalai Tesque,目录号:00211-95)
  21. 酸性品红溶液(见配方)
  22. 100 mM香紫苏醇溶液(见配方)


  1. 温室或植物生长室
  2. 不锈钢镊子
  3. 孵化器(,例如,Sibata,型号:SMU-0541)
  4. 光显微镜(,例如,OLYMPUS,型号:BX53M)
  5. 冰箱(例如,PANASONIC,型号:MPR-162DCN)
  6. 不锈钢托盘(高9.2厘米×深18厘米×宽32厘米)
  7. 剪刀(例如,V.Mueller,目录号:25601-164)
  8. 水浴(,例如,Taitec,型号:SH-10N)
  9. 微波炉(如,HITACHI,型号:MRO-NT5)
  10. 立体显微镜(例如,Nikon,型号:SMZ-U)


  1. RKN在番茄植物中的繁殖
    1. 悬浮RKNs,其由从单个雌性建立的RKN的克隆群体组成,在水中至终浓度为至少500RKNs/ml。
    2. 通过将溶液注入围绕每株植物根部的土壤表面上,将1ml RKNs悬浮液接种在含有土壤的盆中生长的4周龄番茄植株(图1)。


    3. 在自然阳光下在23-27℃的温室中生长植物约2个月
    4. 小心地将根从花盆里拉出,用自来水冲洗,以除去根部的土壤
    5. 用镊子从根部拿起鸡蛋块。
    6. 将收集的蛋团悬浮在培养皿中的小体积水中,在25℃下在黑暗中孵育(图2)。


    7. 在大多数幼体孵化(约10-14天)后,使用玻璃吸管真空孵育线虫的悬浮液并释放在玻璃瓶上(图3)。


    8. 将收集的RKNs悬浮于5-10ml水中。
    9. 在光学显微镜下计数悬浮液中RKN的数目(图4)

      图4.在幼年计数之前在光学显微镜下悬浮RKN。 比例尺,5毫米。

    10. 准备足够浓度的RKNs在水中的等分试样 注意:我们调整500 RKNs/ml。 您可以在保持在10°C的冰箱中储存RKN悬浮液,而不会失去活动2-3周,直到使用。
    11. 每次分析使用适当的等分试样。

  2. 植物生长
    1. 西红柿
      1. 在1.0%次氯酸钠溶液中灭菌种子3分钟
      2. 在70%乙醇中洗涤灭菌的种子
      3. 在灭菌蒸馏水中洗涤灭菌的种子三次。
      4. 在灭菌的湿纸上,在23-27℃下,以16小时光照和8小时黑暗的光周期发芽种子。
      5. 将7至10天龄的幼苗转移到装在220ml塑料盆中的高压灭菌的沙子中,并在保持在23-27℃的室中生长,光周期为16小时光照和8小时黑暗。 每3天向盆中施加用水稀释3,000倍的液体肥料。
      6. 每个测定使用番茄幼苗两个完全发育的真叶(图5)


    2. 拟南芥植物
      1. 在1.0%次氯酸钠溶液中灭菌种子3分钟
      2. 在70%乙醇中洗涤灭菌的种子
      3. 在灭菌蒸馏水中洗涤灭菌的种子三次。
      4. 在灭菌的1.5%琼脂板上在22℃下发芽种子,光周期为16小时光照和8小时黑暗。
      5. 将3周龄的幼苗转移到装在220ml塑料罐中的高压灭菌的沙子中,并在保持在22℃的室中生长,光周期为16小时光照和8小时黑暗。
      6. 每5至7天,用水稀释3,000倍的液体肥料施用于盆中
      7. 每个测定使用10周龄的植物(图6)注意:我们注意到,种植在沙中的拟南芥生长速度慢于土壤。 因此,生长在沙中的10周龄植物的大小几乎与生长在土壤中的6周龄植物的大小相同。


  3. 化学处理和RKN渗透测定
    1. 每次处理使用10株植物。
    2. 每个托盘放五个植物。
    3. 当植物根浸没在溶液中时,将2,000ml含有足够浓度的香紫苏醇或0.1%甲醇的溶液单独倒入托盘中(图7)。
      注意:我们在这里提出了sclareol的方案,但是,它也可以用于其他化学品。我们使用四种浓度; 50,80,100和200μM


    4. 在浸没48小时后,通过在根周围的沙子上注射1ml RKN悬浮液(用于番茄的200RKNs和用于拟南芥的500RKN),用1ml RKN悬浮液接种RKN的第二阶段幼体玻璃移液管 注意:因为RKN对拟南芥根的穿透效率低于对番茄根的穿透效率,所以我们总是使用更多数量的RKN用于拟南芥。
    5. 将接种的植物在维持25℃番茄和22℃的拟南芥中的室中生长,光周期为16小时光照和8小时黑暗。
  4. 酸性品红染色
    注意:酸性品红是染色线虫的染料。下面描述的方法是由McBeth等人报道的染色方法的修改版本。 (1941)。
    1. 接种7天后,将根从花盆中取出,用自来水冲洗以除去海沙。
    2. 用剪刀将植物的根分离,并将根置于50ml玻璃培养瓶中
    3. 要使根脱色,在培养瓶中倒入40 ml 1.0%次氯酸钠,轻轻翻转瓶子10次以上。
    4. 孵育5分钟。
    5. 丢弃次氯酸钠后,用大量自来水冲洗根 注意:因为次氯酸钠阻挡了酸性品红的染色效果,所以次氯酸钠应该在此步骤完全除去。
    6. 在培养瓶中倒入30毫升水,然后加入1毫升酸性品红。通过倒置瓶子几次混合好。
    7. 将含有根的培养瓶在酸性品红溶液中在100℃的预热水浴中煮沸 注意:您也可以使用微波炉代替水浴。在这种情况下,当瓶中的溶液开始起泡时,应停止微波炉。
    8. 将煮熟的根和溶液冷却至室温
    9. 丢弃酸性品红溶液,用自来水冲洗根以除去过量的酸性品红溶液
    10. 加入30ml乳酸和甘油(1:1,v/v)的混合物
    11. 根据步骤D7煮瓶。
    12. 将瓶子冷却至室温。
    13. 用自来水清洗根后,在立体显微镜下计数根中染色的RKN的数目。染色的RKN的代表性实例显示在图8中,并且对于渗透速率的香紫苏醇处理或未处理的植物的代表性结果显示在图9中。

      图8.用酸性品红染色的番茄根中的RKN。 比例尺,200μm。

      图9.香紫苏醇对RKNs侵入番茄根部的影响。 值是来自两次独立实验的平均值±SD,每次实验重复10次。星号表示与0.1%甲醇对照(Tukey's HSD试验; P <0.05)相比的统计学显着差异。

  5. 杀线虫活性测定
    1. 用水稀释RKN悬浮液至浓度为100 RKNs/ml
    2. 向24孔组织培养板的每个孔中加入1ml RKN悬浮液
    3. 向每个孔中加入1ml含有足够浓度的香紫苏醇或0.1%甲醇的溶液
    4. 在25℃的黑暗中孵育平板。
    5. 在光学显微镜下计数施用化学药剂后1小时,3小时,6小时,12小时,24小时和48小时的动力和非动力RKNs的数量。
    6. 为了检查RKN的运动性,向每个孔中加入几滴1M氢氧化钠(NaOH) 注意:氢氧化钠可刺激RKN的运动。如果RKN通过加入NaOH而移动,我们将其视为活的RKN。
    7. 计算活RKN与死RKN的比率(图10)。



  1. 酸性品红溶液(1000ml)
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fujimoto, T., Mizukubo, T., Abe, H. and Seo, S. (2016). Root-knot Nematode Penetration and Sclareol Nematicidal Activity Assays. Bio-protocol 6(12): e1848. DOI: 10.21769/BioProtoc.1848.