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In vitro Nematode Infection on Potato Plant
体外线虫侵染马铃薯实验   

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Abstract

Potato cyst nematodes (PCNs; Globodera rostochiensis and G. pallida) are devastating pests that infect potato root. We describe an in vitro assay for PCN infection on potato plantlet in tissue culture. This method is useful for studying nematode parasitism on potato and for investigating responses of potato clones/lines to PCN infection.

Keywords: Potato cyst nematode(线虫), Globodera rostochiensis(马铃薯金线虫), Nematode infection(线虫感染), Potato plantlet(马铃薯试管苗)

Materials and Reagents

  1. Potato plant (Solanum tuberosum cv. Désirée)
  2. Potato cyst nematode (G. rostochiensis) cysts
  3. Sodium azide (RICCA Chemical, catalog number: 7144-16 )
  4. Mercuric chloride (RICCA Chemical, catalog number: 4650-16 )
  5. Sterile distilled water
  6. Gentamycin (Fisher Scientific, catalog number: 61398-0010 )
  7. Nystatin (Sigma-Aldrich, catalog number: N-3503 )
  8. Agarose (Fisher Scientific, catalog number: BP160-100 )
  9. Timentin (PhytoTechnology Laboratories®, catalog number: T869 )
  10. Micropore tape (Fisher Scientific, catalog number: 19-027-761 )
  11. Sterilization solution (0.004% mercuric chloride/0.004% sodium azide)
  12. Hoagland's solution (Sigma-Aldrich, catalog number: H2395 )
  13. Amberlite XAD-4 resin (Fisher Scientific, catalog number: AC20223 )
  14. MS salt (Caisson Laboratories, catalog number: MSP01-50LT )
  15. Inositol (Fisher Scientific, catalog number: AC122261000 )
  16. Thiamine HCl (Sigma-Aldrich, catalog number: T1270 )
  17. Gelrite (Fisher Scientific, catalog number: CAS 71010-52-1 )
  18. Potato root diffusate (PRD) (see Recipes)
  19. Propagation medium (see Recipes)
  20. 0.1% agarose (see Recipes)

Equipment

  1. Sieves No. 60 (250 μm), No. 200 (75 μm) and No. 500 (25 μm) (Endecotts, catalog number: 683927 , 681837 , 691131 )
  2. Sterile nematode egg hatching chamber (stainless steel metal pan cover and collection pan may be purchased from Vollrath Products, size six inches; the metal screened pan cover with wire-screen in the central area as shown in Figure 3 was custom made)
  3. Sterile 6-well plates (Greiner Bio-One GmbH, catalog number: 657185 )
  4. Stirplate
  5. Sterile forceps and scalpel
  6. Growth incubator
  7. 30-μm mesh (Small Parts, catalog number: 7050-1220-000-12 )
  8. Tissue culture biosafety cabinet
  9. Centrifuge
  10. Incubator shaker (New Brunswick Scientific, model: C24KC )
  11. S. tuberosum growth chambers (we use several types of growth chambers such as I-66LLVL from Percival)
  12. Rubber stopper (size no. 1) (Fisher Scientific, catalog number: 14-130C )
  13. Beaker
  14. Aluminum foil
  15. Dissecting microscope

Procedure

  1. Plant growth
    1. In a sterile environment, cut nodal stem segments (approximately 1 cm long) (Figure 1), and push the stem piece firmly into the 6-well plate containing 6 to 7 ml of propagation medium per well with timentin (100 μg/ml), leaving the top part of the stem exposed (Figure 2).
    2. Label plates and seal them with micropore tape.
    3. Cultivate in a growth incubator at 24 °C under a 16-h/8-h light/dark cycle for two weeks until roots have emerged for several days.


      Figure 1. Potato plantlet growing in the propagation medium. Nodal section is indicated as shown.


      Figure 2. 6-well plate with potato nodal segments embedded in propagation medium

  2. Nematode egg hatching and sterilization (all the following steps are performed at room temperature)
    1. One week before inoculation, soak nematode cysts at room temperature in a small (20-30 ml) beaker covered loosely with aluminum foil and check daily to ensure that cysts are fully soaked in water during the 7-day hydration period.
    2. Crush the cysts using the following method:
      1. Nest the sieves with the No. 60 sieve on top, No. 200 sieve in the middle and the No. 500 sieve on the bottom.
      2. Pour the cysts from their beaker to the sieve stack and rinse out the beaker with water to collect the remaining cysts.
      3. With a rubber stopper, grind the cysts through the No. 60 sieve using a circular motion, washing frequently with tap water to keep cysts off the sides. Grind until no more cyst pieces will go through the screen.
      4. Wash the No. 60 sieve thoroughly with water.
      5. Remove the top sieve and discard the contents. Wash the No. 200 sieve thoroughly with water and remove it. Nematode eggs are collected on the No. 500 sieve.
      6. Use a water bottle to rinse eggs from the No. 500 sieve into a clean 250 ml glass beaker.
    3. Add sodium azide solution (caution: TOXIC! Wear nitrile gloves) to beaker containing nematode eggs and make up to 100 ml with water. The egg suspension should have a final concentration of 0.02% sodium azide.
    4. Stir the egg suspension for 20 min on a stirplate.
    5. Pour the egg suspension back onto the previously used No. 500 sieve and rinse the eggs well with constant flow of tap water for about two min to remove all traces of sodium azide.
    6. Set up the hatching chamber (Figure 3). Nest a large mesh screen pan on a solid pan and place a 30-μm mesh on the screen pan. Rinse the nematode eggs from the No. 500 sieve onto the 30-μm mesh carefully using PRD containing gentamycin (1.5 mg/ml) and nystatin (0.1 mg/ml) antibiotics. Make sure to add enough PRD with antibiotics into the hatching chamber to cover the 30-μm mesh. Cover with an inverted solid pan and place in a tray. All the pans and the 30-μm mesh must be autoclaved before use.


      Figure 3. Nematode egg hatching chamber assembly

    7. Allow eggs to hatch at room temperature for 3-4 days, making sure there is enough PRD (with appropriate antibiotics).
    8. Pour hatched juveniles (J2s) (Figure 4) into a small glass beaker several hours before using to let the worms settle down at the bottom of the beaker.


      Figure 4. Hatched nematode juvenile (J2)

    9. Check the J2 suspension under the dissecting scope for signs of heavy bacterial or fungal contamination. If heavily contaminated, do not use for inoculation work.
    10. Take out PRD as much as possible (without disturbing the settled J2s on the bottom) using a glass pipette and spin down the J2s at 13,400 x g for 2 min in 1.5 ml low retention centrifuge tubes. Resuspend J2s in 1 ml sterile water.
    11. Take counts of J2s by making 1:10 dilution and count J2s in five 10-μl drops. Calculate the total number of J2s in 1 ml according to the average number of J2s in a 10-μl drop, and spin down the amount of J2s needed for inoculation at 13,400 x g for 2 min (spin down more J2s than needed for infection, since some nematodes will die after sterilization). Proceed with sterilization.
    12. Sterilization
      1. Add 1 ml of diluted sterilization solution (0.004% mercuric chloride/0.004% sodium azide; Caution: TOXIC! Wear nitrile gloves) to each tube containing J2s. Close tubes and incubate on a rocking shaker for 10 min.
      2. Spin down the J2s at 13,400 x g for 2 min. Remove sterilization solution and discard in a special waste container (solutions containing mercury must not be discarded in a sink).
      3. Wash J2s with 1 ml sterile distilled water, incubate on the rocking shaker for 5 min and then centrifuge to remove water. Repeat washing steps for three more times.
      4. Resuspend J2s in 1 ml of sterile distilled water and take out 10 μl suspension to count. Calculate the volume needed to obtain the desired number of J2s for infection.
    13. Centrifuge to spin down the J2s and remove water as much as possible. Add calculated volume of 0.1% agarose to get the desired density of J2s for infection (recommend to use 1 - 3 J2 per μl).

  3. Infection of S. tuberosum with potato cyst nematode G. rostochiensis
    1. Cut off the tops of the potato plantlets in the 6-well plates using a sterile technique.
    2. Inoculate each potato plantlet with 100 - 200 nematode J2s depending on experiments. Pipette out a desired volume of J2 agarose suspension and punch the pipet tip into the medium and release the nematode suspension around the root tips (perform the inoculation within half an hour after J2s are suspended in 0.1% agarose since long suspension in this agarose solution can reduce nematode infectivity).
    3. Seal with micropore tape and return plates in the growth chamber (24 °C, dark condition).
    4. Monitor the development of G. rostochiensis on potato plantlet roots each week for the next four to five weeks. After two weeks of infection nematodes at J3 stage should be visible.
    5. Count nematode females four to five weeks post infection
      1. Take out each plantlet (including medium) and put on the lid of a petri dish (100 mm in diameter). Cut off the top of the plantlet and just keep the roots, add several drops of water on the plate, cover with another half of the petri dish on top. Gently press the top petri dish until the medium and the roots fully expanded on the bottom petri dish (Figure 5).
      2. Count the females on this plate under a dissecting microscope.


        Figure 5. Nematode females formed on a potato root (some were detached from the root due to handling)

Recipes

  1. Potato root diffusate (PRD)
    Potatoes (tuber pieces with sprouts) are planted in 4 L inverted amber glass bottles filled with perlite and fitted at the bottom with tubing, grow within a greenhouse. Plants were fertilized with 1/2 strength Hoagland's solution and kept moist throughout the growing season. At weekly intervals, starting at three to ten weeks post planting, pots were filled with distilled water, let stand for 1 h, and then the water containing root diffusate was drained and collected. This material was passed through a column bed of Amberlite XAD-4 resin, column rinsed with 500 ml water, and then eluted with 500 ml methanol. The methanol elutant was rotor vaporated to a minimal volume (typically 2 ml), transferred to a vial, frozen with liquid nitrogen, and placed into a -80 °C freezer. Use 1:800 dilution with sterile distilled water for nematode egg hatching.
  2. Propagation medium (1 L)
    4.3 g MS salt
    0.17 g NaH2PO4.H2O
    0.10 g inositol
    0.4 mg thiamine HCl
    30 g sucrose
    2.5 g gelrite
    Adjust pH to 6.0 with KOH and autoclave
  3. 0.1% Agarose
    0.1 g agarose in 100 ml distilled water
    Autoclave and store at room temperature

Acknowledgments

This work was supported by funding from USDA-ARS.

References

  1. Baum, T. J., Wubben, M. J., Hardyy, K. A., Su, H. and Rodermel, S. R. (2000). A screen for Arabidopsis thaliana mutants with altered susceptibility to Heterodera schachtii. J Nematol 32(2): 166-173.
  2. Brodie, B. B. (1996). Effect of initial nematode density on managing Globodera rostochiensis with resistant cultivars and nonhosts. J Nematol 28: 510–519.
  3. Brodie, B. B., Plaisted, R. L. and de Scurrah, M. M. (1991). The incorporation of resistance to Globodera pallida into Solanum tuberosum germplasm adapted to North America. Am Potato J 68: 1-11.
  4. Chronis, D., Chen, S., Lu, S., Hewezi, T., Carpenter, S. C., Loria, R., Baum, T. J. and Wang, X. (2013). A ubiquitin carboxyl extension protein secreted from a plant-parasitic nematode Globodera rostochiensis is cleaved in planta to promote plant parasitism. Plant J 74(2): 185-196.
  5. Clarke A. J. and Perry R. N. (1977). Hatching of cyst-nematodes. Nematologica 23: 350-368.
  6. Lu, S. W., Tian, D., Borchardt-Wier, H. B. and Wang, X. (2008). Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis. Mol Biochem Parasitol 162(1): 1-15.
  7. Sijmons, P. C., Grundler, F. M., Mende, N., Burrows, P. R. and Wyss, U. (1991). Arabidopsis thaliana as a new model host for plant‐parasitic nematodes. Plant J 1(2): 245-254.

简介

马铃薯胞囊线虫(PCNs; Globodera rostochiensis 和 G。pallida )是感染土豆根的毁灭性害虫。 我们描述了在组织培养的马铃薯小植株上的PCN感染的体外测定。 这种方法可用于研究马铃薯上的线虫寄生和调查马铃薯克隆/株系对PCN感染的反应。

关键字:线虫, 马铃薯金线虫, 线虫感染, 马铃薯试管苗

材料和试剂

  1. 马铃薯植物( Solanum tuberosum cv。Désirée)
  2. 马铃薯胞囊线虫( G rostochiensis )囊肿
  3. 叠氮化钠(RICCA Chemical,目录号:7144-16)
  4. 氯化汞(RICCA Chemical,目录号:4650-16)
  5. 无菌蒸馏水
  6. 庆大霉素(Fisher Scientific,目录号:61398-0010)
  7. 制霉菌素(Sigma-Aldrich,目录号:N-3503)
  8. 琼脂糖(Fisher Scientific,目录号:BP160-100)
  9. Timentin( Phyto Technology Laboratories ®,目录号:T869)
  10. Micropore胶带(Fisher Scientific,目录号:19-027-761)
  11. 灭菌溶液(0.004%氯化汞/0.004%叠氮化钠)
  12. Hoagland's溶液(Sigma-Aldrich,目录号:H2395)
  13. Amberlite XAD-4树脂(Fisher Scientific,目录号:AC20223)
  14. MS盐(Caisson Laboratories,目录号:MSP01-50LT)
  15. 肌醇(Fisher Scientific,目录号:AC122261000)
  16. 盐酸硫胺素(Sigma-Aldrich,目录号:T1270)
  17. Gelrite(Fisher Scientific,目录号:CAS 71010-52-1)
  18. 马铃薯根分泌物(PRD)(参见食谱)
  19. 传播介质(参见配方)
  20. 0.1%琼脂糖(参见配方)

设备

  1. 筛号60(250μm),200号(75μm)和500号(25μm)(Endecotts,目录号:683927,681837,691131)
  2. 无菌线虫卵孵化室(不锈钢金属锅盖和收集盘可购自Vollrath Products,尺寸为6英寸;如图3所示,在中心区域具有金属丝筛网的金属筛网盖是定制的) >
  3. 无菌6孔板(Greiner Bio-One GmbH,目录号:657185)
  4. 搅拌板
  5. 无菌镊子和手术刀
  6. 生长孵化器
  7. 30-mesh(小部件,目录号:7050-1220-000-12)
  8. 组织培养生物安全柜
  9. 离心机
  10. 孵育摇床(New Brunswick Scientific,型号:C24KC)
  11. tuberosum生长室(我们使用几种类型的生长室,如来自Percival的I-66LLVL)
  12. 橡胶塞(尺寸1号)(Fisher Scientific,目录号:14-130℃)
  13. 烧杯
  14. 铝箔
  15. 解剖显微镜

程序

  1. 植物生长
    1. 在无菌环境中,切割节段茎段(约1cm长)(图1),并将茎片牢固地推入每孔含有6至7ml的传代培养基的6孔板中,其中含有特美汀(100μg/ml) ,使茎的顶部暴露(图2)
    2. 标签板并用微孔胶带密封
    3. 在生长培养箱中,在24℃,16小时/8小时光照/黑暗循环下培养两周,直到根出现几天。

      图1.在传播介质中生长的马铃薯苗。节段如下所示。


      图2.具有嵌入传播介质中的马铃薯节段的6孔板

  2. 线虫卵孵化和灭菌(所有以下步骤在室温下进行)
    1. 在接种前一周,在室温下在用铝箔松散地覆盖的小(20-30ml)烧杯中浸泡线虫囊肿,并每天检查以确保囊肿在7天水合期间完全浸泡在水中。
    2. 使用以下方法粉碎囊肿:
      1. 将筛子放在顶部有60号筛,中间有200号筛,底部有500号筛。
      2. 将囊肿从烧杯中倒入筛子,用水冲洗烧杯以收集剩余的囊肿。
      3. 用橡胶塞,使用圆周运动通过60号筛子研磨囊肿,经常用自来水洗涤以保持囊肿离开两侧。 研磨,直到没有更多的囊肿片将通过屏幕
      4. 用水彻底冲洗60号筛。
      5. 取出顶部的筛子并丢弃内容物。 用水彻底冲洗200号筛并取出。 线虫卵收集在500号筛上
      6. 使用水瓶从500号筛子冲洗鸡蛋到一个干净的250毫升玻璃烧杯
    3. 加入叠氮化钠溶液(注意:TOXIC!戴丁腈手套)到含有线虫卵的烧杯中,并用水补足至100 ml。鸡蛋悬浮液的最终浓度应为0.02%叠氮化钠
    4. 在搅拌板上搅拌鸡蛋悬浮液20分钟。
    5. 将鸡蛋悬浮液倒在先前使用的500号筛上,用恒定流量的自来水冲洗鸡蛋约2分钟,以除去所有痕量的叠氮化钠。
    6. 设置孵化室(图3)。将大型网筛盘放在固体盘上,并在筛盘上放置30-μm网孔。使用含有庆大霉素(1.5mg/ml)和制霉菌素(0.1mg/ml)抗生素的PRD小心地将来自500号筛的线虫卵冲洗到30-μm网上。确保添加足够的PRD与抗生素进入孵化室覆盖30-μm网格。盖上一个倒置的固体盘,放在托盘中。所有的平底锅和30-μm筛网在使用前必须高压灭菌

      图3.线虫卵孵化室组装

    7. 让鸡蛋在室温下孵化3-4天,确保有足够的PRD(使用适当的抗生素)
    8. 将孵出的幼鱼(J2)(图4)倒入小玻璃烧杯中数小时,然后使用,让蠕虫在烧杯底部沉降。


      图4.阴影线虫幼稚(J2)

    9. 检查解剖范围内的J2悬浮液是否有严重的细菌或真菌污染的迹象。 如果严重污染,不要用于接种工作
    10. 使用玻璃吸管尽可能多地取出PRD(不干扰底部沉降的J2),并在1.5ml低保留离心管中在13,400×g下旋转J2s 2分钟。 将J2s重悬于1ml无菌水中
    11. 通过进行1:10稀释和计数J2s在5个10-μl滴中计数J2s。根据10μl液滴中J2的平均数计算1ml中J2的总数,并在13,400×g 下旋转2分钟所需的J2s量(旋转更多J2s比感染所需的,因为一些线虫在灭菌后会死亡)。继续灭菌。
    12. 灭菌
      1. 向含有J2s的每个管中加入1ml稀释的灭菌溶液(0.004%氯化汞/0.004%叠氮化钠;注意:TOXIC!戴丁腈手套)。关闭管并在摇动摇床上孵育10分钟
      2. 旋转J2s在13,400 x g 2分钟。移除灭菌溶液,并丢弃在特殊的废物容器中(含汞溶液不得在水槽中丢弃)。
      3. 用1ml无菌蒸馏水洗涤J2,在摇动摇床上孵育5分钟,然后离心除去水。重复洗涤步骤三次以上。
      4. 重悬在J2毫升无菌蒸馏水中,取出10微升的悬浮液计数。计算获得所需的感染J2数所需的卷。
    13. 离心机旋转下来J2s,尽可能多地除去水。加入计算体积的0.1%琼脂糖以获得所需的J2s感染密度(建议使用每μl1 - 3 J2)。

  3. 感染。 tuberosum with potato cyst nematode G。 rostochiensis
    1. 使用无菌技术切割6孔板中的马铃薯苗的顶部
    2. 取决于实验,用100-200个线虫J2接种每个马铃薯苗。吸取所需体积的J2琼脂糖悬浮液,并将吸头尖插入培养基,并释放线虫悬液周围的根尖(执行接种后半小时后J2s悬浮在0.1%琼脂糖,因为长悬浮在这种琼脂糖溶液中降低线虫感染性)
    3. 用微孔带和返回板在生长室中密封(24℃,黑暗条件)
    4. 监控 G的开发。 rostochiensis 在马铃薯小根根每周在接下来的四到五个星期。两周的感染后,J3期的线虫应该是可见的
    5. 计数线虫女性感染后四至五周
      1. 取出每个苗(包括培养基)并放在培养皿(直径100mm)的盖子上。切除苗的顶部,只是保持根,在板上加几滴水,盖上另一半的培养皿在上面。轻轻按下顶部培养皿,直到培养基和根部在底部培养皿上完全扩展(图5)。
      2. 在解剖显微镜下计数此板上的女性

        图5.在马铃薯根上形成的线虫雌性(一些由于处理而从根分离)

食谱

  1. 马铃薯根分泌物(PRD)
    将土豆(具有芽苗的块茎)种植在填充有珍珠岩并在底部装配管的4L倒置的琥珀色玻璃瓶中,在温室内生长。植物用1/2强度的Hoagland溶液受精,并在整个生长季节保持湿润。以每周的间隔,从种植后三至十周开始,向盆中装入蒸馏水,静置1小时,然后排出并收集含有根扩散物的水。将该物质通过Amberlite XAD-4树脂的柱床,用500ml水冲洗,然后用500ml甲醇洗脱。将甲醇洗脱液转子蒸发至最小体积(通常为2ml),转移至小瓶中,用液氮冷冻,并置于-80℃冰箱中。用无菌蒸馏水稀释1:800,用于线虫卵孵化
  2. 繁殖培养基(1 L)
    4.3克MS盐
    0.17g NaH 2 PO 4 subO 2。 H O 0.10克肌醇
    0.4mg硫胺素HCl 30克蔗糖 2.5克gelrite
    用KOH和高压釜将pH调节至6.0
  3. 0.1%琼脂糖
    0.1g琼脂糖在100ml蒸馏水中的溶液 高压灭菌并在室温下贮存

致谢

这项工作得到了美国农业部的资助。

参考文献

  1. Baum,T.J.,Wubben,M.J.,Hardyy,K.A.,Su,H。和Rodermel,S.R。(2000)。 拟南芥突变体的筛选,其对 Heterodera的敏感性改变schachtii。 J Nematol 32(2):166-173。
  2. Brodie,B.B。(1996)。 初始线虫密度对抗性栽培品种和非寄主的管理 Globodera rostochiensis 的影响。 J Nematol 28:510-519。
  3. Brodie,B.B.,Plaisted,R.L.and de Scurrah,M.M。(1991)。 将 Globodera pallida 的抗性整合到适应北美的马铃薯种质中。 J 68:1-11
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引用:Chronis, D., Chen, S., Lang, P., Tran, T., Thurston, D. and Wang, X. (2014). In vitro Nematode Infection on Potato Plant. Bio-protocol 4(1): e1016. DOI: 10.21769/BioProtoc.1016.
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