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Infection Assay of Cyst Nematodes on Arabidopsis Roots
拟南芥根部的胞囊线虫感染实验   

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Molecular Plant Microbe Interactions
Sep 2014

Abstract

Plant parasitic nematodes are devastating pests on many crops. Juveniles (J2) of cyst nematodes invade the roots to induce a syncytium. This feeding site is their only source of nutrients. Male nematodes leave the roots after the fourth molt to mate with females. The females stay attached to their syncytia throughout their life and produce hundreds of eggs, which are contained in their bodies. When the females die their bodies form the cysts, which protect the eggs. Cysts can survive for many years in the soil until favorable conditions induce hatching of the juveniles.

The beet cyst nematode Heterodera schachtii is a pathogen of sugar beet (Beta vulgaris) but can also complete its life cycle on Arabidopsis roots growing on agar plates under sterile conditions. We present here protocols for a stock culture of H. schachtii and an infection assay on agar plates.

Keywords: Nematode infection assay (线虫感染的检测), Cyst nematode (囊肿线虫), Heterodera schachtii (大豆胞囊线虫schachtii), Arabidopsis (拟南芥)

Materials and Reagents

  1. Heterodera schachtii
  2. Seeds of Sinapis alba cv Albatros
  3. Seeds of Arabidopsis thaliana
  4. Calcium hypochlorite
  5. HgCl2
  6. Tween 20
  7. 70% ethanol
  8. GAMBORG B5 VITAMIN MIXTURE (Duchefa Biochemie, catalog number: G0415 )
  9. Daichin agar (Duchefa Biochemie, catalog number: D1004 )
  10. Saccharose
  11. KNO3
  12. MgSO4.7H2O
  13. Ca(NO3)2.4H2O
  14. KH2PO4
  15. FeNaEDTA
  16. H3BO3
  17. MnCl2 or MnCl2.4H2O
  18. CuSO4.5H2O or CuSO4.2H2O
  19. ZnSO4.7H2O
  20. CoCl2.6H2O
  21. H2MoO4 or NaMoO4.2H2O
  22. NaCl
  23. ZnCl2
  24. GELRITETM (Duchefa Biochemie, catalog number: G1101 )
  25. Plastic Petri dishes 9 cm
  26. Plastic Petri dishes 14.5 cm
  27. Aluminum foil
  28. PVC membrane 100 µm mesh (Buddeberg GmbH, catalog number: 9068289 )
  29. PVC membrane 15 µm mesh (Buddeberg GmbH, catalog number: 9068280 )
  30. 50 ml syringes
  31. Pipettes
  32. Preparation of water agar plates (see Recipes)
  33. Preparation of Knop medium (see Recipes)

Equipment

  1. Nescofilm (or Parafilm)
  2. Growth chamber at 25 °C with 16 h light/8 h dark
  3. Laboratory glassware
  4. Balance
  5. Stereo microscope
  6. Inverse microscope with camera
  7. Clean bench
  8. Autoclave capable of reaching 120 °C
  9. Axiovert 200M inverse microscope (ZEISS) with an integrated camera (ZEISS, model: AxioCam MRc5 )
  10. Funnel for hatching (Figure 1)

Software

  1. Contour tool of the AxioVision software (ZEISS)
  2. SPSS 12.0 (SPSS Inc.)

Procedure

  1. Arabidopsis and mustard seed sterilization:
    1. Immerse seeds for 10 min in 5% calcium hypochlorite with 0.1% Tween 20, shake a few times.
    2. Pour off the solution and add 70% ethanol, incubate for 5 min.
    3. Wash 3 times in sterile water.
    4. Dry the seeds in a clean bench in a sterile Petri dish.
  2. Stock culture of H. schachtii:
    1. Prepare 14.5 cm 0.7% water agar plates.
    2. Prepare 14.5 cm Knop agar plates.
    3. Place 20 sterile mustard seeds on 0.7% water agar plate.
    4. Seal the plates with Nescofilm.
    5. Store in a dark place at RT for 3-4 days.
    6. Transfer 3 seedlings onto a 14.5 cm Knop agar plate and seal the plates with Nescofilm.
    7. Store in a dark place at RT for about 10 days.
    8. Infect the roots with nematodes (3-4 drops per root); it is convenient to use J2 that are left over from an infection test.
    9. Keep the plates in a dark place at room temperature until the cysts are needed. It takes approximately 2 months until the cysts can be harvested. It is possible to store the plates for up to 1 year. However, if the agar in the plates becomes too dry it may be more difficult to pick out the cysts.
  3. Preparation of H. schachtii inoculum:
    1. Prepare a funnel:
    2. Assemble the funnel as shown in Figure 1 with a 100 µm sieve.


      Figure 1. Funnel for hatching. Glass beakers, funnels, silicone tubes, and clips can be bought from local suppliers.

    3. Cover the funnel with aluminum foil.
    4. Sterilize by autoclaving for 20 min at 120 °C.
    5. Prepare 15 µm and 100 µm sieves (Figure 2).


      Figure 2. 15 µm and 100 µm sieves are needed for sterilization of J2s. Only the 100 µm sieves are shown on this picture.

    6. Cut off 1 cm high rings from a 50 ml syringe.
    7. Heat one side of the ring and glue it to a piece of PVC membrane (15 µm or 100 µm mesh).
    8. Sterilize by autoclaving for 20 min at 120 °C.
    9. Prepare 0.7% gelrite: Add 1.4 g gelrite to 200 ml distilled water, cook until the solution becomes clear.
    10. Autoclave for 20 min at 120 °C.
    11. Pour 5 ml into 15 ml tubes (becomes solid at RT).
    12. Pick cysts from the stock culture on mustard.
    13. Place the cysts into the 100 µm sieve sitting in a 50 ml glass beaker.
    14. Soak the cysts in 5% calcium hypochlorite with 0.1% Tween 20 for 10 min.
    15. Wash 4 times with sterile distilled water.
    16. Put the sieve with the cysts into the hatching funnel (Figure 1).
    17. Fill the funnel with 3 mM ZnCl2 (stimulates hatching of the J2).
    18. Keep the hatching funnel in darkness at RT.
    19. The hatched larvae move through the 100 µm sieve into the silicone tube and can be collected from the silicone tube (Figure 1) by briefly opening the clip after 3-5 days into a 15 µm sieve.
    20. Sterilize the J2 for 2 min by putting the 15 µm sieve with the J2 into a beaker containing 0.05% HgCl2.
    21. Wash four times by transferring the 15 µm sieve with the J2 into a beaker with sterile distilled water.
    22. Resuspend the J2 in 0.7% gelrite prior to inoculation.
    23. Adjust the concentration of the inoculum to 20 J2 per drop of gelrite using a pipette by counting the J2 in the drops under a stereo microscope.
    24. The J2 can be stored for 2-3 days in a fridge at 4 °C without loosing their infectivity.
  4. Arabidopsis cultivation:
    1. Prepare 9 cm Knop agar plates.
    2. Grow the Arabidopsis plants:
    3. Place sterile seeds on Knop agar plate (Figure 3). Use 10 seeds per plate.


      Figure 3. Template for Arabidopsis seeds

    4. Incubate the plates for 4 days at 4 °C to ensure uniform germination.
    5. Place the plates horizontally in the growth chamber for 2 days.
    6. Tilt the plates to approximately 20 degrees so that the roots will grow to the bottom of the plates between the agar and the Petri dish.
  5. Inoculation of Arabidopsis plants:
    1. Inoculate 12-d-old Arabidopsis roots with 60 J2 per plant by putting 3 drops of gelrite containing the J2 on different parts of the root.
    2. Incubate horizontally in the dark over night at RT.
    3. Put the plates back into the light.
  6. Analysis of the nematode infection:
    Take pictures of syncytia with female nematodes at 13 dpi.
    (We use an Axiovert 200M inverse microscope with an integrated camera) at 14 dpi count the numbers of male and female nematodes (Figure 4).


    Figure 4. Male (A), female (B) nematode and the cyst (C)

  7. Size measurement of syncytia and cysts (females):
    1. Select 10 syncytia/cysts at random.
    2. Outline the cysts and the syncytia [we use the Contour tool of the AxioVision software and the software will calculate the size (Figure 5)].


      Figure 5. Size measurement of female nematodes and syncytia. Numbers are the sizes calculated by the contour tool of the AxioVision software for nematodes (blue) and syncytia (red). Shown are syncytia induced in transgenic lines overexpressing a WRKY gene and the wild type (Col) [from Ali et al. (2014)]

    3. Analyze the significance of the results using analysis of variance (ANOVA) (P < 0.05) (we use SPSS 12.0).
  8. Counting the nematodes:
    1. Use a stereo microscope.
    2. Invert the plates and use waterproof markers with different colors to mark males and females by placing a dot besides the nematodes.
    3. Count the dots for males and females.
    4. Calculate the total number of males and females for each line per plant or root length. From these data calculate the male/female ratio.
    5. If there is a difference in the growth (size) of control and test (mutants, transgenic lines) lines at the time of inoculation classify the lines on the Petri dishes according to Figure 6.
    6. Analyze the significance of the results using analysis of variance (ANOVA) (P < 0.05) (we use SPSS 12.0).


      Figure 6. Growth classes of Arabidopsis plants. This figure can be used to classify the Arabidopsis lines if there are differences in plant growth to adjust the number of nematodes [from Jürgensen (2001)].

Notes

  1. It is known that in comparison to male nematodes more female nematodes can develop under optimal nutritional and environmental conditions. Therefore the female/male ratio (F/M) is a meaningful indicator for the developmental conditions provided by the host plant (Triantaphyllou, 1973).
  2. It was found that growth of Arabidopsis plants under sterile conditions for infection with H. schachtii was best on 0.2x concentrated Knop medium (Sijmons et al., 1992) prepared with Daichin agar. Other agar types may also work but have to be tested.
  3. There is seasonal variation in growth and hatching of the nematodes. During the summer months the hatching and infection rate is significantly higher than during the wintertime.
  4. Each test should include a control to adjust for differences in inoculum.
  5. We do three replicates with 40 plants per line (4 Petri dishes).
  6. Growth of plants and nematodes is under sterile conditions. Also the preparation of the inoculum has to be done under sterile conditions.

Recipes

  1. Preparation of water agar plates
    1. Combine 7 g Daichin agar and 1 L distilled water and autoclave for 20 min at 120 °C
    2. Pour into 14.5 cm Petri dishes
  2. Preparation of Knop medium
    1. Prepare the following stock solutions for Knop medium (per liter)
      Stock solution
      Chemical
      g per liter
      Alternative chem.
      g per liter
      Stock solution I
      KNO3
      121.32 g


      MgSO4.7H2O
      19.71 g


      Stock solution II
      Ca(NO3)2.4H2O
      120 g


      Stock solution III
      KH2PO4
      27.22 g


      Stock solution IV
      FeNaEDTA
      7.34 g


      Stock solution V
      H3BO3
      2,86 g


      MnCl2
      1.81 g
      MnCl2.4H2O
      2.85 g
      CuSO4.5H2O
      0.073 g
      CuSO4.2H2O
      0.05 g
      ZnSO4.7H2O
      0.36 g


      CoCl2.6H2O
      0.03 g


      H2MoO4
      0.052 g
      NaMoO4.2H2O
      0.0775 g
      NaCl
      2 g



    2. Prepare 1 L, 0.2x Knop agar
      Saccharose
      20 g
      Daichin agar
      8 g
      B5 Vitamins
      1 ml
      Stock solution I
      2 ml
      Stock solution II
      2 ml
      Stock solution III
      2 ml
      Stock solution IV
      0.4 ml
      Stock solution V
      0.2 ml

    3. Add deionized water to 1 L
    4. Adjust the pH to 6.4 with KOH solution
    5. Autoclave for 20 min at 120 °C and pour plates the same day

Acknowledgments

This work was supported by FWF Austrian Science Fund grants P16296-B06, P16897-B06 and P21067-B12.

References

  1. Ali, M. A., Wieczorek, K., Kreil, D. P. and Bohlmann, H. (2014). The beet cyst nematode Heterodera schachtii modulates the expression of WRKY transcription factors in syncytia to favour its development in Arabidopsis roots. PLoS One 9(7): e102360.
  2. Jürgensen, K (2001). Untersuchungen zum Assimilat- und Wassertransfer in der Interaktion zwischen Arabidopsis thaliana und Heterodera schachtii. Dissertation Kiel, Germany: Christian-Albrechts Universität.
  3. Sijmons, P. C., Grundler, F. M. W., von Mende, N., Burrows, P. R. and Wyss, U. (1991). Arabidopsis thaliana as a new model host for plant-parasitic nematodes. Plant J 1: 245-254.
  4. Triantaphyllou, A. C. (1973). Gametogenesis and reproduction of Meloidogyne graminis and M. ottersoni (Nematoda: Heteroderidae). J Nematol 5(2): 84-87.

简介

植物寄生线虫是许多作物上的毁灭性害虫。 孢囊线虫的幼虫(J2)侵入根以诱导合胞体。 这个饲养场是他们唯一的营养来源。 雄性线虫在第四蜕皮后离开根以与雌性交配。 雌性在其整个生命期间保持附着于其合胞体,并产生数百个鸡蛋,其包含在它们的身体中。 当女性死于他们的身体形成囊肿,保护卵。 囊肿可以在土壤中存活多年,直到有利条件诱导幼鱼孵化。
甜菜孢囊线虫 Heterodera schachtii 是甜菜的病原体( vulgaris ),但也可以在拟南芥 根在无菌条件下在琼脂平板上生长。 我们在这里介绍的H的股票文化的协议。 schachtii)和在琼脂平板上的感染测定。

关键字:线虫感染的检测, 囊肿线虫, 大豆胞囊线虫schachtii, 拟南芥

材料和试剂

  1. Heterodera schachtii
  2. Albatros的种子。
  3. 拟南芥种子
  4. 次氯酸钙
  5. HgCl <2>
  6. 吐温20
  7. 70%乙醇
  8. GAMBORG B5维生素混合物(Duchefa Biochemie,目录号:G0415)
  9. Daichin琼脂(Duchefa Biochemie,目录号:D1004)
  10. 蔗糖
  11. KNO 3
  12. MgSO 4。 。 O
  13. Ca(NO 3)2 sub 2 4H 2 O
  14. KH 2 PO 4
  15. FeNaEDTA
  16. H 3 BO 3
  17. MnCl 2或MnCl 2 sub。 4H 2 O
  18. CuSO 4或者CuSO 4或者CuSO 4或者CuSO 4,或者CuSO 4和/ O
  19. ZnSO 4 。 7H O
  20. CoCl <2> 6H <2> O
  21. H sub 2 MoO 4或NaMoO 4 sub 。 2H O
  22. NaCl
  23. ZnCl 2
  24. GELRITE TM (Duchefa Biochemie,目录号:G1101)
  25. 塑料培养皿9厘米
  26. 塑料培养皿14.5厘米
  27. 铝箔
  28. PVC膜100μm筛(Buddeberg GmbH,目录号:9068289)
  29. PVC膜15μm网目(Buddeberg GmbH,目录号:9068280)
  30. 50ml注射器
  31. 移液器
  32. 水琼脂板的制备(参见配方)
  33. Knop培养基的制备(参见配方)

设备

  1. Nescofilm(或Parafilm)
  2. 生长室在25℃下用16小时光/8小时黑暗
  3. 实验室玻璃器皿
  4. 余额
  5. 立体显微镜
  6. 带有相机的反显微镜
  7. 清洁长椅
  8. 高压灭菌器能够达到120°C
  9. 带集成摄像头的Axiovert 200M反相显微镜(ZEISS)(ZEISS,型号:AxioCam MRc5)
  10. 孵化漏斗(图1)

软件

  1. AxioVision软件(ZEISS)的轮廓工具
  2. SPSS 12.0(SPSS Inc.)

程序

  1. 拟南芥和芥菜种子灭菌:
    1. 在5%次氯酸钙和0.1%吐温20中浸泡种子10分钟,摇动几次。
    2. 倒出溶液,加入70%乙醇,孵育5分钟
    3. 在无菌水中洗3次。
    4. 在无菌培养皿中的干净的工作台中干燥种子。
  2. H的储存培养物。 schachtii :
    1. 准备14.5厘米0.7%的琼脂平板
    2. 准备14.5厘米Knop琼脂平板
    3. 将20个无菌芥菜种子放在0.7%的琼脂平板上
    4. 用Nescofilm密封板。
    5. 在室温避光存放3-4天。
    6. 将3株幼苗转移到14.5cm的Knop琼脂平板上,并用Nescofilm密封平板
    7. 储存在暗处,室温下约10天。
    8. 用线虫感染根(每根根3-4滴); 使用从感染测试中遗留下来的J2是很方便的
    9. 保持板在黑暗的地方在室温,直到囊肿 需要。 它需要大约2个月,直到囊肿可以 收获。 它可以存储板最多1年。 然而,   如果板中的琼脂变得太干,则可能更难 挑出囊肿。
  3. 制备H。 schachtii 接种物:
    1. 准备渠道:
    2. 如图1所示,用100μm的筛子组装漏斗。


      图1.孵化的漏斗。 玻璃烧杯,漏斗,硅胶管和夹子可以从当地供应商处购买
    3. 用铝箔覆盖漏斗。
    4. 在120℃下高压灭菌20分钟灭菌。
    5. 准备15μm和100μm筛(图2)。


      图2.需要15μm和100μm筛网用于J2s的灭菌。 在此图片上仅显示100μm筛。

    6. 从50毫升注射器切下1厘米的高环
    7. 加热环的一侧并将其粘合到一块PVC膜(15μm或100μm网)上
    8. 在120℃下高压灭菌20分钟灭菌。
    9. 制备0.7%gelrite:将1.4g gelrite加入到200ml蒸馏水中,煮至溶液变澄清
    10. 在120℃下高压灭菌20分钟。
    11. 将5ml倒入15ml管中(在RT下变成固体)
    12. 在芥末上从储存培养物中挑选孢囊。
    13. 将囊肿放入放置在50ml玻璃烧杯中的100μm筛中
    14. 用5%次氯酸钙和0.1%吐温20浸泡囊肿10分钟
    15. 用无菌蒸馏水洗涤4次
    16. 将带有孢囊的筛子放入孵化漏斗(图1)。
    17. 向漏斗中加入3mM ZnCl 2(刺激J2的孵化)。
    18. 将孵化漏斗置于室温黑暗中。
    19. 孵出的幼虫通过100μm筛进入硅树脂 管,并且可以通过简单地从硅胶管(图1)收集 3-5天后打开夹子进入15μm筛子
    20. 通过将具有J2的15μm筛子放入含有0.05%HgCl 2的烧杯中消毒J2 2分钟。
    21. 通过将带有J2的15μm筛子转移到装有无菌蒸馏水的烧杯中洗涤四次
    22. 在接种前将J2重悬于0.7%的gelrite中
    23. 调节接种物的浓度为每滴20 J2 通过计数立体声下的液滴中的J2,使用移液管进行凝胶化 显微镜。
    24. J2可以在4℃的冰箱中储存2-3天,而不丧失其感染性
  4. 拟南芥栽培:
    1. 准备9厘米的Knop琼脂平板
    2. 种植拟南芥植物:
    3. 将无菌种子放在Knop琼脂平板上(图3)。 每板使用10粒种子。


      图3. 拟南芥 种子的模板

    4. 在4℃下孵育平板4天,以确保均匀发芽
    5. 将板水平放置在生长室中2天
    6. 倾斜板大约20度,以便根 生长到琼脂和培养皿之间的板的底部。
  5. 拟南芥植物的接种:
    1. 通过将3只植物与每株植物接种60只J2,接种12-d的拟南芥根 在根的不同部分含有J2的凝胶状液滴
    2. 在室温下在黑暗中水平孵育过夜。
    3. 把板放回光。
  6. 线虫感染分析:
    在13 dpi时拍摄合胞体与女性线虫的照片。
    (我们使用具有集成相机的Axiovert 200M反相显微镜)以14dpi计数雄性和雌性线虫的数目(图4)。


    图4.雄性(A),雌性(B)线虫和孢囊(C)

  7. 合胞体和囊肿(雌性)的大小测量:
    1. 随机选择10个合胞体/囊肿。
    2. 概述囊肿和 syncytia [我们使用AxioVision软件的Contour工具和 软件将计算大小(图5)]。


      图5.大小 测量雌性线虫和合胞体 tia 。 通过AxioVision软件的轮廓工具计算线虫 (蓝色)和合胞体(红色)。 显示在转基因中诱导的合胞体 线过表达WRKY基因和野生型(Col)[来自Ali等人 al。(2014)]

    3. 使用方差分析(ANOVA)(P <0.05)分析结果的显着性(我们使用SPSS 12.0)。
  8. 计数线虫:
    1. 使用立体显微镜。
    2. 翻转板材并使用防水 标记用不同的颜色标记男性和女性通过放置一个点   除了线虫。
    3. 计数男性和女性的点。
    4. 计算每行的男性和女性的总数 植物或根长度。 从这些数据计算男/女比例
    5. 如果对照和试验的生长(尺寸)存在差异 (突变体,转基因株系)在接种分类时 根据图6的培养皿上的线
    6. 使用方差分析(ANOVA)(P <0.05)(我们使用SPSS 12.0)分析结果的显着性。


      图6. 拟南芥植物的生长类。  如果植物存在差异,则对拟南芥品系进行分类 生长以调节线虫的数量[来自Jürgensen(2001)]。

笔记

  1. 已知与男性线虫相比,更多的雌性线虫可以在最佳营养和环境条件下发育。因此,雌性/雄性比率(F/M)是宿主植物提供的发育条件的有意义的指标(Triantaphyllou,1973)。
  2. 发现拟南芥植物在用H感染的无菌条件下的生长。 schachtii最适用于用Daichin琼脂制备的0.2倍浓缩的Knop培养基(Sijmons等人,1992)。其他琼脂类型也可以工作,但必须进行测试
  3. 线虫的生长和孵化有季节性变化。 在夏季,孵化和感染率明显高于冬季
  4. 每个测试应包括一个控制以调整接种物的差异
  5. 我们做三个重复,每行40株植物(4个培养皿)。
  6. 植物和线虫的生长在无菌条件下。 此外,接种物的制备必须在无菌条件下进行

食谱

  1. 水琼脂平板的制备
    1. 将7g Daichin琼脂和1L蒸馏水混合,并在120℃下高压灭菌20分钟
    2. 倒入14.5厘米培养皿
  2. Knop培养基的制备
    1. 准备Knop培养基(每升)的以下储备溶液
      库存解决方案
      化学品
      g /升
      替代化学。
      g /升
      库存解决方案
      KNO 3
      121.32克


      MgSO 4。 。 O
      19.71克


      库存解决方案II
      Ca(NO 3)2 sub 2 4H 2 O
      120克


      储备解决方案III
      KH 2 PO 4
      27.22克


      库存解决方案IV
      FeNaEDTA
      7.34克


      库存解决方案V
      H 3 BO 3
      2,86克


      MnCl 2
      1.81克
      MnCl 2 4H O
      2.85克
      CuSO 4 5H sub 2 O
      0.073克
      CuSO 4 2H <2> O
      0.05 g
      ZnSO 4 。 7H O
      0.36克


      CoCl <2> 6H <2> O
      0.03 g


      H 2 MoO 4
      0.052克
      NaMoO 4 2H <2> O
      0.0775克
      NaCl
      2克



    2. 准备1 L,0.2x Knop琼脂
      蔗糖
      20克
      大豆琼脂
      8克
      B5维生素
      1 ml
      库存解决方案
      2 ml
      库存解决方案II
      2 ml
      库存解决方案III
      2 ml
      库存解决方案IV
      0.4 ml
      库存解决方案V
      0.2 ml

    3. 加入去离子水至1 L
    4. 用KOH溶液
      调节pH至6.4
    5. 在120°C高压灭菌20分钟,并在同一天倒板

致谢

这项工作得到FWF奥地利科学基金拨款P16296-B06,P16897-B06和P21067-B12的支持。

参考文献

  1. Ali,M.A.,Wieczorek,K.,Kreil,D.P。和Bohlmann,H。(2014)。 甜菜孢囊线虫 Heterodera schachtii 调节WRKY转录因子的表达syncytia以促进其在拟南芥根中的发育。 9(7):e102360。
  2. Jürgensen,K(2001)。 拟南芥作为新模型 植物寄生线虫的宿主。植物J 1:245-254
  3. Triantaphyllou,A.C。(1973)。 禾本科植物禾本科植物的生殖和繁殖 ottersoni (Nematoda:Heteroderidae) J Nematol 5(2):84-87。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bohlmann, H. and Wieczorek, K. (2015). Infection Assay of Cyst Nematodes on Arabidopsis Roots. Bio-protocol 5(18): e1596. DOI: 10.21769/BioProtoc.1596.
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