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Trichoderma harzianum Root Colonization in Arabidopsis
哈茨木霉在拟南芥中的根部定植   

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Abstract

Trichoderma is a soil-borne fungal genus that includes species with a significant impact on agriculture and industrial processes. In this article we show a detailed protocol of Trichoderma harzianum (T. harzianum) root invasion procedure described by Alonso-Ramírez et al. (2014). Some Trichoderma strains exert beneficial effects in plants through root colonization. They promote growth and development, modify root architecture, facilitate efficient nutrient use, or stimulate defenses against pathogens, although little is known about how this interaction takes place. For this purpose, Trichoderma-Arabidopsis hydroponic cultures were grown inside Phytatray II boxes, using mycelia obtained from spores of T. harzianum and Arabidopsis thaliana (A. thaliana) seedlings. In this way changes in root architecture, such as callose deposition, promoted by the fungus can be analyzed.

Materials and Reagents

  1. Arabidopsis thaliana Col-0 ecotype seeds from Arabidopsis Information Service Collection (www.arabidopsis.info)
  2. Trichoderma harzianum CECT 2413 (Spanish Type Culture Collection, Valencia, Spain) [referred to as T34 along the paper is the strain used in this work. T. harzianum T34 is grown on Potato Dextrose Agar (PDA) and spores are maintained at -80 °C in a 30% glycerol solution]
  3. Murashige & Skoog medium (MS), including B5 vitamins (Duchefa Biochemia, catalog number: M0255.0050 )
  4. Sucrose (Applichem Panreac, catalog number: 141621.1211 )
  5. 85% potassium hydroxide pellets (KOH) (Applichem Panreac, catalog number: 141515.1210 )
  6. 0.15% agarose (Conda Pronadisa, catalog number: 8016 )
  7. 0.39% potato dextrose agar (PDA) (Sigma-Aldrich, catalog number: P2182 )
  8. 0.24% potato dextrose broth (PDB) (Sigma-Aldrich, catalog number: P6685 )
  9. Potato dextrose agar (PDA) (Conda, catalog number: 1022.00 )
  10. Potato dextrose broth (PDB) (Difco, catalog number: 254920 )
  11. Glass wool washed QP (Panreac, catalog number: 211376.1208 )
  12. Resma filter paper (420 x 500 mm) (Auxilab S.L, catalog number: 80250452 )
  13. Liquid nitrogen (Air Liquide)
  14. Ethanol (Panreac, catalog number: 161086 )
  15. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  16. Sodium hypochlorite
  17. Sterilization solution (see Recipes)
  18. MS Medium (see Recipes)
  19. Washing solution (see Recipes)

Equipment

  1. Sterile distilled water purification system (EMD Millipore, model: ELIX35 )
  2. Brand cotton roving (Sigma-Aldrich, catalog number: BR28205 )
  3. Sterile stainless steel screen (Alunet, catalog number: 174562 )
  4. Surgical Micropore tape (3M, catalog number: 1530-0 )
  5. 1.5 ml microtubes (Deltalab, catalog number: 200400P )
  6. Cold chamber
  7. Phytatray II boxes [114 mm, 86 mm, 102 mm (W x D x H)] (Sigma-Aldrich, catalog number: P5929 )
  8. Laminar flow cabinet (Telstar, model: AV-100 )
  9. Plant Growth Chamber AGP-1400-HR (Radiber SA)
  10. Shaker Certomat® R (B. Braun, model: 986302/4 )
  11. Petri dishes (90 x 14 mm) (Deltalab, catalog number: 200209 )
  12. Surgeon carbon steel (surgical blade sterile) (Jai Surgicals, catalog number: 0835147 )
  13. 1.5 ml microtubes with glass wool (homemade)
  14. 15 ml Tubes (Deltalab, catalog number: 401402 )
  15. Thoma cell counting chamber (BRAND, catalog number: 7180 05 )
  16. Coverslip EUROTUBO (22 x 22 mm) (Deltalab, catalog number: D102222 )
  17. Optical microscope (Leica Microsystems AG, model: DC300F; catalog number: 10447115 )
  18. Erlenmeyer flask (Thermo Fischer Scientific, catalog number: 11972233 )
  19. Kühner shaker (Thermo Fisher Scientific, model: ISF-1-W )
  20. Vacuum/pressure pump PALL (Life Sciences, model: DOA-P730-BN )
  21. Safety glass (vacuum flask, 10 ml pipette and gums) (homemade)
  22. X5 Graduated pipette (type1, class B, ISO 835) (Thermo Fisher Scientific, catalog number: 11912178 )
  23. Medical grade silicone tubing (1x 1.5, internal diameter x external diameter) (Deltalab, catalog number: 3500115 )
  24. Rubber plugs (VWR International, catalog number: 217-9463 )
  25. Forceps (stainless, L= 105 mm) (Thermo Fisher Scientific, catalog number: 10458242 )
  26. Vacuum flask pirex (1,000 ml) (Thermo Fisher Scientific, catalog number: 12693182 )
  27. Magnetic filter funnel (VWR International, catalog number: 516-7590 )
  28. Scissors stainless 170 mm (Thermo Fisher Scientific, catalog number: 12693182)
  29. Lyophilizer Virtis Advantage (SP Scientific)

Procedure

  1. Arabidopsis thaliana seeds have to be washed and sterilized superficially before sowing. For this purpose, approximately 200 seeds are placed in a 1.5 ml microtube, and 1 ml of washing solution is added. Shake seeds for 30 min at room temperature in a shaker (Kühner shaker). Remove washing solution. Add 1 ml of sterilization solution. Shake again for 10 min at room temperature. Remove sterilization solution and wash seeds by tube inverting four times with sterile distilled water. Finally, seeds are placed in 1.5 ml microtubes and kept in stratification at 4 °C in the cold chamber for 3 days in order to break seed dormancy and synchronize the germination.
  2. Place Arabidopsis seeds inside Phytatray II boxes with 50 ml of liquid MS media on a sterile gauze sheet over a sterile stainless steel screen (Figures 1 and 2) in a laminar flow cabinet, using an aqueous agarose solution (0.15%) to sow seeds individually.


    Figure 1. Set up of a sterile gauze sheet over a sterile stainless steel screen in Phytatray II boxes


    Figure 2. Pictures showing the hydroponic cultures in the Phytatray II boxes

  3. For the propagation of the T. harzianum strain, incubate a fungal plug of 5 mm of diameter, grown and sporulated on a petri dish with PDA, at least 7 days at room temperature, to achieve full coverage of the plate surface with spores.
  4. To harvest the spores, add 5 ml of sterile distilled water to the plate. Filter this suspension in 1.5 ml microtubes with glass wool, placed on 15 ml tubes, in order to remove traces of mycelium (Figure 3 and 4). Spores are maintained at 4 °C no more than 1 week. Finally, determine spore concentration in a Thoma cell counting chamber by pipetting 100 μl of a 1:100 dilution, using the following formula:

    : It is the mean of the spores counted in four different quadrants.
    Finally, keep spores at 4 °C until use.


    Figure 3. Set up for the glass wool in a microtube placed on a 15 ml tube


    Figure 4. Harvest of T. harzianum spores

  5. Use spores of T. harzianum (107 spores) to inoculate 250 ml flasks containing 100 ml of PDB. Maintain cultures at 25 °C and 200 rpm for 48 h. Harvest mycelia (approximately 250 mg) by filtration (filtration system: vacuum/pressure pump PALL, safety glass, rubber plugs, vacuum flask pirex and magnetic filter funnel). Wash the mycelia through the magnetic funnel with 100 ml of sterile water (Figure 5). Use the washed mycelia to inoculate the Phytatray boxes containing Arabidopsis plants grown for 21 days. Inoculate mycelia by lifting the stainless steel screen where the Arabidopsis seedlings are, with the help of sterile forceps (Figure 6). Place mycelia on MS media and shake (shaker Certomat® R) in order to promote dispersion.


    Figure 5. Set up of the filtration system to harvest the spores


    Figure 6. Procedure to inoculate the mycelia into the Phytatray boxes

  6. Finally, keep hydroponic cultures for 20 days at 80 rpm and 22 °C in a plant growth chamber with 40% humidity under long daylight conditions (16 h light/8 h dark) (light intensity of 80 to 100 µE/µm2/s). Afterwards, roots are recovered using sterile scissors on a laminar flow cabinet. Wash the roots with sterile water to remove mycelia traces. Dry the roots on filter paper and finally freeze them in liquid nitrogen. Lyophilize using a Lyophilizer Virtis Advantage until the water from the roots is entirely removed. Collected samples are ready for nucleic acid extraction or qPCR procedures. Success of root invasion is analyzed by qPCR. The corresponding protocols are described in Alonso-Ramírez et al. (2014).

Recipes

  1. Sterilization solution
    2.5% sodium hypochlorite
    0.005% Triton X-100
    dH2O
  2. MS medium
    9 g of Murashige & Skoog medium, including B5 vitamins, for 1 L
    1% sucrose
    Adjust pH to 5.7 with KOH
    Qs. dH2O 1 L
    Sterilized for 20 min at 120 °C/1 atm using an autoclave
  3. Washing solution
    70% ethanol
    1% Triton X-100
    dH2O

Acknowledgments

Research project funding was from Junta de Castilla y León (SA260A11-2) and Spanish national projects MICINN (AGL2009-13431-C02) and MINECO (AGL2012-40041-C02-01).

References

  1. Alonso-Ramirez, A., Poveda, J., Martin, I., Hermosa, R., Monte, E. and Nicolas, C. (2014). Salicylic acid prevents Trichoderma harzianum from entering the vascular system of roots. Mol Plant Pathol 15(8): 823-831.

简介

<木霉属是土壤传播的真菌属,包括对农业和工业过程具有显着影响的物种。 在本文中,我们展示了由Alonso-Ramírez等人(2014年)所描述的哈茨木霉的(哈茨木霉)根侵入程序的详细方案 )。 一些木霉菌株通过根定植在植物中发挥有益效果。 它们促进生长和发育,改变根结构,促进有效的营养物利用或刺激对病原体的防御,尽管关于如何发生这种相互作用了解甚少。 为此目的,使用从T孢子获得的菌丝体,在Phytatray II盒内培养木霉属拟南芥水培培养物。 harzianum 和拟南芥(拟南芥)幼苗。 以这种方式,可以分析由真菌促进的根构造的变化,例如胼cal质沉积。

材料和试剂

  1. 拟南芥信息服务集合中的拟南芥 Col-0生态型种子( www .arabidopsis.info
  2. 哈茨木霉CECT 2413(西班牙型培养物保藏中心,西班牙瓦伦西亚)[本文中称为T34是本工作中使用的菌株。 T。 harzianum T34在马铃薯葡萄糖琼脂(PDA)上生长,孢子在-80℃下在30%甘油溶液中维持]/
  3. Murashige& Skoog培养基(MS),包括B5维生素(Duchefa Biochemia,目录号:M0255.0050)
  4. 蔗糖(Applichem Panreac,目录号:141621.1211)
  5. 85%氢氧化钾粒料(KOH)(Applichem Panreac,目录号:141515.1210)
  6. 0.15%琼脂糖(Conda Pronadisa,目录号:8016)
  7. 0.39%马铃薯葡萄糖琼脂(PDA)(Sigma-Aldrich,目录号:P2182)
  8. 0.24%马铃薯葡萄糖肉汤(PDB)(Sigma-Aldrich,目录号:P6685)
  9. 马铃薯葡萄糖琼脂(PDA)(Conda,目录号:1022.00)
  10. 马铃薯葡萄糖肉汤(PDB)(Difco,目录号:254920)
  11. 玻璃棉洗涤QP(Panreac,目录号:211376.1208)
  12. Resma滤纸(420×500mm)(Auxilab S.L,目录号:80250452)
  13. 液氮(液空)
  14. 乙醇(Panreac,目录号:161086)
  15. Triton X-100(Sigma-Aldrich,目录号:T8787)
  16. 次氯酸钠
  17. 灭菌溶液(参见配方)
  18. MS Medium(请参阅配方)
  19. 洗涤液(见配方)

设备

  1. 无菌蒸馏水纯化系统(EMD Millipore,型号:ELIX35)
  2. 品牌棉粗纱(Sigma-Aldrich,目录号:BR28205)
  3. 无菌不锈钢筛(Alunet,目录号:174562)
  4. 外科微孔胶带(3M,目录号:1530-0)
  5. 1.5ml微管(Deltalab,目录号:200400P)
  6. 冷室
  7. Phytatray II盒[114mm,86mm,102mm(W×D×H)](Sigma-Aldrich,目录号:P5929)
  8. 层流柜(Telstar,型号:AV-100)
  9. 植物生长室AGP-1400-HR(Radiber SA)
  10. Shaker Certomat R(B.Braun,型号:986302/4)
  11. 培养皿(90×14mm)(Deltalab,目录号:200209)
  12. 外科医生碳钢(手术刀无菌)(Jai Surgicals,目录号:0835147)
  13. 1.5毫升玻璃棉(自制)微管
  14. 15ml管(Deltalab,目录号:401402)
  15. Thoma细胞计数室(BRAND,目录号:7180 05)
  16. 盖玻璃EUROTUBO(22×22mm)(Deltalab,目录号:D102222)
  17. 光学显微镜(Leica Microsystems AG,型号:DC300F;目录号:10447115)
  18. 锥形瓶(Thermo Fischer Scientific,目录号:11972233)
  19. Kühner摇床(Thermo Fisher Scientific,型号:ISF-1-W)
  20. 真空/压力泵PALL(生命科学,型号:DOA-P730-BN)
  21. 安全玻璃(真空瓶,10ml移液管和树胶)(自制)
  22. X5刻度移液管(类型1,B类,ISO 835)(Thermo Fisher Scientific,目录号:11912178)
  23. 医用级硅胶管(1x 1.5,内径x外径)(Deltalab,目录号:3500115)
  24. 橡胶塞(VWR International,目录号:217-9463)
  25. 钳(不锈钢,L = 105mm)(Thermo Fisher Scientific,目录号:10458242)
  26. 真空瓶pirex(1,000ml)(Thermo Fisher Scientific,目录号:12693182)
  27. 磁性过滤漏斗(VWR International,目录号:516-7590)
  28. 不锈钢剪刀170mm(Thermo Fisher Scientific,目录号:12693182)
  29. 冻干器Virtis Advantage(SP Scientific)

程序

  1. 拟南芥种子必须在播种前进行表面洗涤和灭菌。为此,将约200粒种子置于1.5ml微量管中,并加入1ml洗涤溶液。在室温下在振荡器(Kühner摇床)中摇动种子30分钟。取出洗涤液。加入1ml灭菌溶液。在室温下再摇动10分钟。取出灭菌溶液,用无菌蒸馏水倒置四次,洗涤种子。最后,将种子置于1.5ml微管中,并在4℃在冷室中保持分层3天,以便打破种子休眠并同步发芽。
  2. 在层流箱中,在无菌不锈钢筛网(图1和2)上的无菌纱布片上用50ml液体MS培养基将Phytatray II箱内的拟南芥种子放置在琼脂糖水溶液中0.15%)以单独播种种子。


    图1.在Phytatray II盒中的无菌不锈钢筛网上设置无菌纱布片


    图2.显示Phytatray II盒中水培培养物的照片

  3. 用于传播 T。在具有PDA的培养皿上生长并形成孢子,在室温下培养至少7天,以实现用孢子完全覆盖板表面。
  4. 为了收获孢子,向平板中加入5ml无菌蒸馏水。过滤此悬浮液在1.5毫升微玻璃棉管,置于15毫升管,以去除痕量的菌丝体微管(图3和4)。孢子保持在4℃不超过1周。最后,通过使用下式通过用100μl的1:100稀释液移液来确定Thoma细胞计数室中的孢子浓度:

    :这是在四个不同象限中计数的孢子的平均值。
    最后,将孢子保持在4℃直至使用

    图3.在放置在15 ml管上的微量管中设置玻璃棉


    图4. T的收获。 harzianum 孢子

  5. 使用 T的孢子。 harzianum(10×10 6孢子)接种到含有100ml PDB的250ml烧瓶中。保持培养在25℃和200 rpm下48小时。通过过滤收集菌丝体(约250mg)(过滤系统:真空/压力泵PALL,安全玻璃,橡胶塞,真空烧瓶小瓶和磁性过滤漏斗)。通过磁性漏斗用100ml无菌水洗涤菌丝体(图5)。使用洗过的菌丝体接种含有生长21天的拟南芥植物的Phytatray盒。通过在无菌镊子的帮助下提起拟南芥幼苗所在的不锈钢筛网接种菌丝体(图6)。将菌丝体放在MS培养基上并摇动(振荡器Certomat R)以促进分散。


    图5.设置收获孢子的过滤系统


    图6.将菌丝体接种到Phytatray盒中的程序

  6. 最后,在长日照条件下(16小时光照/8小时黑暗),在具有40%湿度的植物生长室中在80rpm和22℃下保持水培培养20天(光强度为80至100μE/2 /s)。然后,使用无菌剪刀在层流柜上回收根。用无菌水清洗根以除去菌丝痕迹。在滤纸上干燥根,最后在液氮中冷冻。冻干使用a 冻干机Virtis优点,直到根部的水被完全清除。 收集的样品准备用于核酸提取或qPCR程序。 根入侵的成功通过qPCR分析。 相应的方案在Alonso-Ramírez等人(2014)中描述。

食谱

  1. 灭菌溶液
    2.5%次氯酸钠
    0.005%Triton X-100 dH 2 2 O
  2. MS介质
    9g Murashige& Skoog培养基,包括B5维生素,1 L
    1%蔗糖 用KOH调节pH至5.7 Qs。 dH <2> O 1 L
    使用高压灭菌器在120℃/1atm下灭菌20分钟
  3. 洗涤溶液
    70%乙醇
    1%Triton X-100 dH 2 2 O

致谢

研究项目资金来自Junta de Castilla yLeón(SA260A11-2)和西班牙国家项目MICINN(AGL2009-13431-C02)和MINECO(AGL2012-40041-C02-01)。

参考文献

  1. Alonso-Ramirez,A.,Poveda,J.,Martin,I.,Hermosa,R.,Monte,E。和Nicolas,C。 水杨酸可防止木霉的木霉进入根系的血管系统。/a> Mol Plant Pathol 15(8):823-831。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Alonso-Ramírez, A., Poveda, J., Martín, I., Hermosa, R., Monte, E. and Nicolás, C. (2015). Trichoderma harzianum Root Colonization in Arabidopsis. Bio-protocol 5(13): e1512. DOI: 10.21769/BioProtoc.1512.
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