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Axenic Cultivation of Mycelium of the Lichenized Fungus, Lobaria pulmonaria (Peltigerales, Ascomycota)
地衣型真菌肺衣(地卷目、子囊菌门)菌丝体的无菌培养   

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Abstract

Lichens are symbiotic organisms consisting of a fungal partner (the mycobiont) and one or more algal or cyanobacterial partners (the photobiont); moreover lichen thalli comprise a plethora of epi- and endobiotic bacteria and non-lichenized fungi. Genetic markers are the most promising tools for the study of fungal diversity. However, applying genetic methods to intimately admixed symbiotic organisms typically requires the development of species-specific genetic markers, since DNA extraction from environmental specimens implicates the acquirement of total DNA of all symbionts and their cohabitants. While the cultivation of the alga is straight forward, the axenic cultivation of lichen-forming fungi is more difficult due to their very slow growth, as compared with the majority of non-lichenized taxa, and the presence of saprophytic, endophytic and parasitic fungi within the lichen thallus. Moreover, lichen-forming fungi (predominantly ascomycetes, few basidiomycetes) are oligotrophic organisms and thus adapted to nutrient poor conditions; in axenic culture on nutrient-rich media, as normally used for mass production of fast-growing saprophytic fungi, they often autointoxicate. Most lichen-forming fungi are not obligately biotrophic and thus can be cultured in the non-symbiotic state.
Here, we present a protocol for the isolation of the lichen-forming ascomycete Lobaria pulmonaria into axenic culture and for mycelial mass culture as a source of pure fungal DNA. We describe the initiation of axenic cultures on agar plates from germinating ascospores and explain the optimization of the in vitro growth in liquid medium. By grinding the few dense, only centrifugally growing fungal colonies with a homogenizer we obtain lots of smaller, well growing colonies and thus higher amounts of mycelium for DNA or RNA isolation (Honegger and Bartnicki-Garcia, 1991).

Materials and Reagents

  1. Freshly collected lichen thalli of Lobaria pulmonaria bearing apothecia
  2. Alpha-cyclodextrin (Sigma-Aldrich, catalog number: C4642 )
  3. BBLTM cornmeal agar (BD, catalog number: 211132 )
  4. BactoTM casamino acids (BD, catalog number: 228830 )
  5. BactoTM malt extract (BD, catalog number: 218630 )
  6. BactoTM peptone (BD, catalog number: 211677 )
  7. BactoTM yeast extract (BD, catalog number: 212750 )
  8. CaCl2.2H2O (Merck, catalog number: 102382 )
  9. Co(NO3).6H2O (Sigma-Aldrich, catalog number: 60832 )
  10. CuSO4.5H2O (Sigma-Aldrich, catalog number: RES10395 )
  11. EDTA (Sigma-Aldrich, catalog number: ED2SS )
  12. FeSO4.7H2O (Sigma-Aldrich, catalog number: F7002 )
  13. Glucose (Fluka, catalog number: 49159 )
  14. H3BO3 (Sigma-Aldrich, catalog number: 31146 )
  15. KH2PO4 (Merck, catalog number: 1.04873 )
  16. K2HPO4 (Merck, catalog number: 1.05104 )
  17. KOH (Fluka, catalog number: 60369 )
  18. NaCl (Sigma-Aldrich, catalog number: 71376 )
  19. NaNO3 (Sigma-Aldrich, catalog number: S5506 )
  20. MgSO4.7H2O (Merck, catalog number: 5856 )
  21. MnCl2.4H2O (Merck, catalog number: 105927 )
  22. MoO3 (Sigma-Aldrich, catalog number: M0753 )
  23. ZnSO4.7H2O (Sigma-Aldrich, catalog number: Z4750 )
  24. Aluminum foil
  25. Bidistilled water
  26. Sterilized tap water
  27. Liquid nitrogen
  28. Sterilization agent for hands
  29. Germination medium (see Recipes)
  30. Lichen medium (see Recipes)
  31. Bold’s basal medium (BBM) (see Recipes)

Equipment

  1. Double edge razor blades (any brand)
  2. Platinum inoculation needles (flame-resistant)
  3. Dissection forceps, stainless steel (flame-resistant)
  4. Spatula, stainless steel (flame-resistant)
  5. Filter paper circles (ø 45-55 mm) (Schleicher & Schuell 589/2)
  6. Petri dishes (ø 100 mm, sterile) (plastic, plus 1-2 autoclavable glass dishes) (any brand)
  7. Perforated bar spoons, ca. 30 cm long, stainless steel (flame-resistant)
  8. Laboratory bottles (SCHOTT DURAN® 100 ml, 500 ml, and 1,000 ml)
  9. Erlenmeyer flasks (SCHOTT DURAN® 200 ml, wide neck)
  10. Plugs for Erlenmeyer flasks, cellulose
  11. Two glass beakers (500 ml and 200 ml)
  12. Glass pipets (20 ml, 10 ml, and 1 ml)
  13. Rubber pipet filler up to 100 ml
  14. Falcon tubes (50 ml, sterile, nonpyrogenic and DNase-/RNase-free, cryo-resistent) (liquid nitrogen)
  15. Racks for Falcon tubes (cryo-resistant) (liquid nitrogen)
  16. Dissecting microscope with an objective lens capable of resolving ascospores (10-30 µm)
  17. Precision balance with milligram resolution range
  18. Sterile Bench, ideally with UV radiation
  19. Bunsen burner
  20. Autoclave for liquid and solid material
  21. Homogenizer (either a IKA DI 18 basic or Ultraturrax T 18), mounted on a plate stand with a boss head clamp
  22. Autoclavable homogenizer rods (made from stainless steel, IKA S 18 N-19G)
  23. Cryogenic vessel big enough to contain a rack with Falcon tubes
  24. Growth chamber with (continuous) 15-16 °C±1 °C and 12 h light/12 h dark regime
    Note: The humidity must not be controlled but ensure dry conditions within the chamber.

Procedure

  1. Axenic mycelium
    1. Collect fresh lichen thalli with mature apothecia (Figure 1A), ideally in autumn to spring, but not during summer because this species does not produce sufficient viable spores during the warm and dry season. Thalli may be stored in glass Petri dishes, air-dry during several days in the refrigerator for up to one week.


      Figure 1. Thallus of Lobaria pulmonaria and arrangement of the apothecium. A. Fresh thallus with apothecia of different ages. Arrows: Mature apothecia suitable for ascospore harvesting; they are convex in the fully hydrated state and approx. 1.5-3.0 mm in diameter. B. Inverted Petri dish with apothecium on moist filter paper.

    2. Thoroughly clean the sterile bench with either 75% alcohol or 5 min UV radiation, or both.
    3. Prepare the germination medium, autoclave and pour into plastic Petri dishes. Prepare a high number of agar plates for several isolation steps (up to 50 plates). Allow the germination medium to cool down and gelatinize within the sterile bench. With a sterile inoculation needle scratch few superficial lines into the agar (this will help finding the focal plane of the agar surface), then turn the agar plates upside down and continue work.
    4. Place several filter papers into the glass Petri dish. Wrap Petri dish with aluminum foil and sterilize it in the autoclave. Place one sterile filter paper on the inner surface of the lid of one inverted Petri dish and moisten it with sterile tap water (Figure 1B).
    5. Use a pointed forceps for excising mature apothecia. Cut the lower part of the apothecia flat by means of a fresh razor blade. Work in sterile bench and place one apothecium on the moisten filter paper by using sterile forceps. The apothecium needs to be fully hydrated, but without superficial water film (this might prevent the ascospores from being ejected). Close the inverted Petri dish, including the apothecium. After having prepared 15-20 Petri dishes, each with one apothecium, incubate the inverted Petri dishes in a growth chamber at 15-16 °C in the dark. The wet paper hydrate the apothecium causing it to discharge ascospores upward onto the medium within minutes or during the first 24 h at the latest.
    6. Important! After 24 h incubation replace all lids, including filter paper and the apothecium, by new sterile lids and keep the plates at 15-16 °C in the dark.
    7. Check the Petri dishes every day under the dissecting microscope with transmitted light for bacterial (slimy) or fungal contaminants (see Note 2). Fast-growing fungal contaminants sporulate within a few days and opening the plate will disseminate the wind-dispersed spores. Thus, discard the infected plates without opening!
    8. Ascospores of L. pulmonaria will germinate within 10-15 days after having been ejected. Place a clean microscope within the sterile bench. Normally, fusiform spores are visible (approx. 18-30 µm long x 5-9 µm wide) (Figure 2A). As these spores are usually three-celled more than one cell may germinate (Figure 2A, asterisk).
    9. Ignite the Bunsen burner and adjust the blue flame. Thoroughly sterilize the inoculation needles by flaming and allow them to cool down before using. Open a plate with germinated spores under the microscope, pick out one spore using a sterile needle and transfer it onto a fresh Petri dish with germinating medium. Transfer around four spores to each plate, then close the Petri dish and incubate it at 15-16 °C in the dark. Repeat this for each germinating spore.
    10. Check the plates every second day for the growth rates of the isolates and for contaminations. If single spores are affected by contamination, either cut them out or transfer the others to new agar plates.
    11. Repeat steps A7-9 during several weeks until the plates contain clean colonies of growing mycelia of L. pulmonaria.


      Figure 2. Ascospores and mycelium of L. pulmonaria on agar. A. Germinating ascospores 10 days after spore discharge. Asterisk: Spore with several germ tubes. B. The mycelium of L. pulmonaria grows very slowly. Axenic mycelium 85 days after spore discharge.

  2. Propagation in liquid Lichen Medium
    1. Prepare 1 L of Lichen Medium and autoclave it with the program for liquids. Wrap cellulose plugs and dissection needles in aluminum foil. Close Erlenmeyer flasks and glass beakers openings with aluminum foil. Autoclave all instruments with the program for solid material.
    2. Store the Lichen Medium and material in the cleaned sterile bench and wait until they have attained room temperature! Place a microscope into the sterile bench and clean all of its surfaces thoroughly with 75% EtOH.
    3. Fill 50-100 ml of Lichen Medium into each Erlenmeyer flask. Under the microscope dissect one or several mycelial colonies from the agar surface with a sterile inoculation needle and transfer them to the liquid medium in the Erlenmeyer flask.
    4. Gently flame the opening of the Erlenmeyer flask, close the flask and keep the aluminum foil on the outer part of the plug intact. Incubate at 15-16 °C with a 12 h/12 h light/dark cycle during a few months, thus allowing the mycelium to grow into a colony visible to the naked eye.
    5. Wrap each stainless steel rod of the homogenizer with aluminum foil prior to autoclaving; mark the opening of the rod, which will fit to the homogenizer, and keep the blade side covered until it is used. Mount the sterile rod on the homogenizer in a 45° angle and remove the aluminum foil immediately prior to use (Figure 3A). Adjust the homogenizer to level 2 (bigger colonies) or higher (smaller colonies).
    6. Insert the homogenizer rod into the Erlenmeyer flask with liquid medium and fungal colony. Switch the homogenizer on. Turn the flask slowly during the grinding process, thus making sure that the fungal colonies are getting finely disrupted. This process will kill many cells by disruption, but the majority stays intact and will continue growth. After approx. 10-20 sec homogenization, switch the homogenizer off (Figure 3C). Flame the opening of the Erlenmeyer flask and the cotton plug and seal the flask. Replace the used homogenizer rod by a sterile one for the next flask.
    7. Incubate at 15-16 °C with 12 h light and 12 h dark during several months, allowing the mycelium pieces to grow visible to the naked eye.


      Figure 3. Accelerating fungal growth by homogenization of the mycelium of L. pulmonaria. A. Homogenizer with mounted rod in a 45° angle. B. Erlenmeyer flask containing whitish and brownish mycelium of L. pulmonaria. The dark brown mycelium was overfed on agar plates. It has begun to produce light colored mycelium on the surface of each hyphae clump after transfer into liquid medium (see Note 1). C. Whitish mycelium of L. pulmonaria after homogenization.

  3. Harvesting mycelium for DNA or RNA isolation
    1. Introduce a rack containing a Falcon tube into a cryogenic vessel and fill it with liquid nitrogen. The cryogenic vessel must not be sterile.
    2. Mount a sterile rod on the homogenizer. Ignite the Bunsen burner. Remove the plug from the Erlenmeyer flask. Harvest some mycelium by using a sterile bar spoon with perforations. Immediately, introduce the mycelium into liquid nitrogen in the Falcon tube by pushing it down from the spoon with a sterile spatula.
    3. After harvesting, decant the old medium slowly into the 500 ml glass beaker without losing the remaining mycelium and without touching the glass beaker. Refill 50-100 ml fresh medium into the Erlenmeyer flask. Remove the aluminum foil from the homogenizer and grind the mycelium as mentioned above. Flame the Erlenmeyer opening and the cotton plug prior to closing. Repeat steps C1-3 as many times as needed.
    4. Keep Falcon tubes always filled with liquid nitrogen. Store Falcon tubes at -80 °C. Important: do not completely close the tube; instead, open one twist of the screw thread to let evaporating nitrogen escape.


      Figure 4. Falcon tube (a) with harvested mycelium (1) in liquid nitrogen (2)

Notes

  1. After establishing an axenic mycelium on agar, switch to culture in liquid medium three months after germination at the latest. Do not wait longer! L. pulmonaria is adapted to extremely oligotrophic conditions; thus the mycelium will be overfed on the agar medium and eventually die! Overfed mycelium becomes brownish to dark brown (Figure 3B).
  2. The decisive criterion to discriminate a fungal contamination from germinating ascospores of L. pulmonaria is the rapidity of growth (Figure 2B; mycelium growth after 85 days). Fungal contaminations produce a dense network of hyphae within a few days and soon start asexual sporulation. Once asexual spores are formed by the contaminant it is almost impossible to rescue the culture, contaminant spores being wind-dispersed already while the lid of the Petri dish is opened.
  3. To verify the taxonomical belonging of the cultured sample, perform a polymerase chain reaction and sequence the nuclear intragenic transcribed spacer (ITS) rDNA region as described in Cornejo and Scheidegger (2010). The sequence can be checked with BLASTn since the ITS marker of L. pulmonaria is well documented in GenBank.
  4. The cultures obtained with this method are multispore cultures; in L. pulmonaria, a heterothallic (cross-fertilized) species, each spore might have had different genetic background. If single-spore isolates are required, e.g. for the analysis of the progeny of meiosis, individual spores, as ejected by single asci, can be manually separated and cultured separately (Honegger et al., 2004).

Recipes

  1. Germination medium (Denison, 2003)
    1.0% alpha-cyclodextrin
    1.5% cornmeal agar
    Preparation of 1 L Germination medium:
    Fill in a bottle 15 g cornmeal agar, 10 g cyclodextrin and 1 L bidistilled H2O and autoclave the medium afterwards. The slightly cooled agar can be poured into Petri dishes.
  2. Lichen medium (Honegger, 1985)
    BBM 940 ml
    Malt extract 0.875 g
    Proteose pepton 0.125 g
    Yeast extract 0.0625 g
    Casamino acids 0.0625 g
    Glucose 0.9375 g
  3. Bold’s basal medium (BBM) (Deason and Bold, 1960)
    1. Stock solutions
      Flask label
      Compound
      Dilution of stock solution
      Dilution of BBM
      A
      NaNO3
      20 g/800 ml
      20 ml
      B
      CaCl2
      20 g/800 ml
      10 ml
      C
      MgSO4.7H2O
      20 g/800 ml
      10 ml
      D
      K2HPO4
      20 g/800 ml
      10 ml
      E
      KH2PO4
      7 g/400 ml
      10 ml
      F
      NaCl
      1 g/400 ml
      10 ml
    2. Trace elements
      0.157 g/100 ml
      Flask label
      Compound
      Dilution of trace elements
      Dilution of BBM
      g
      H3BO3
      1.142 g/100 ml
      1 ml
      h*
      FeSO4.7H2O
      0.498 g/100 ml
      1 ml

      ZnSO4.7H2O
      0.882 g/100 ml


      MnCl2.4H2O
      0.144 g/100 ml

      i*
      MoO3
      0.071 g/100ml
      1 ml

      CuSO4.5H2O


      CoNO3.6H2O
      0.049 g/100 ml

      j*
      EDTA
      5.000 g/100 ml
      1 ml

      KOH
      3.100 g/100 ml

      Note: *Several elements are mixed in one bottle.
    3. How to prepare BBM
      Mix stock solutions and trace elements (second and third columns). Autoclave stock solutions and trace elements and store them in the refrigerator for future use.
      To prepare BBM, mix the amounts of the fourth column and adjust the volume with bidistilled H2O to 1 L. Adjust the pH to 6.0.

Acknowledgments

The Swiss National Science Foundation kindly supported this study: projects 31003A-105830 and 31003A-127346 to CS, and projects 3100-064079.00 and 3100A0-103860/1 to RH.

References

  1. Cornejo, C. and Scheidegger, C. (2010). Lobaria macaronesica sp. nov., and the phylogeny of Lobaria sect. Lobaria (Lobariaceae) in Macaronesia. Bryologist 113: 590-604.
  2. Deason, T. R. and Bold, H. C. (1960). Phycological studies I. Exploratory studies of Texas soil algae. University of Texas Publications, Nr. 6022, 70.
  3. Denison, W. C. (2003). Apothecia and ascospores of Lobaria oregana and Lobaria pulmonaria investigated. Mycologia 95(3): 513-518.
  4. Honegger, R. (1985). The hyphomycetous anamorph of Coniocybe furfuracea. The Lichenologist 17(03): 273-279.
  5. Honegger, R. and Bartnicki-Garcia, S. (1991). Cell wall structure and composition of cultured mycobionts from the lichens Cladonia macrophylla, Cladonia caespiticia, and Physcia stellaris (Lecanorales, Ascomycetes). Mycological Res 95(8): 905-914.
  6. Honegger, R., Zippler, U., Gansner, H. and Scherrer, S. (2004). Mating systems in the genus Xanthoria (lichen-forming ascomycetes). Mycol Res 108(Pt 5): 480-488.

简介

地衣是由真菌伴侣(分枝杆菌)和一个或多个藻类或蓝藻合作伙伴(photobiont)组成的共生生物;此外,地衣thalli包括过多的表皮和内生菌和非地衣真菌。遗传标记是真菌多样性研究最有希望的工具。然而,将遗传方法应用于紧密混合的共生生物通常需要开发物种特异性遗传标记,因为从环境样品中提取DNA涉及获得所有共生体及其同居者的总e DNA。虽然藻类的培养是直接的,但是与大多数非地衣类群相比,由于其生长非常缓慢,形成地衣的真菌的无性繁殖更加困难,并且在内部存在腐生,内生和寄生真菌地衣thallus。此外,形成地衣的真菌(主要是子囊菌,少数担子菌)是营养不良的生物,因此适应营养不良的条件;在营养丰富的培养基中,如通常用于大规模生产快速生长的腐生真菌的无菌培养中,它们经常自动毒害。大多数形成地衣的真菌不是专性生物营养的,因此可以在非共生状态下培养。
在这里,我们提出了一种用于分离形成地衣的子囊菌无菌培养和作为纯真菌DNA来源的菌丝体培养。我们描述从发芽子囊孢子在琼脂平板上的无菌培养的开始,并解释在液体培养基中的体外生长的优化。通过研磨几个密集的,只有离心生长的真菌菌落,用匀浆器,我们获得许多较小的,生长良好的菌落,因此更高量的菌丝体用于DNA或RNA分离(Honegger和Bartnicki-Garcia,1991)。

材料和试剂

  1. 新鲜收集的具有apothecia的 Lobaria pulmonaria的地衣thalli
  2. α-环糊精(Sigma-Aldrich,目录号:C4642)
  3. BBL TM 玉米粉琼脂(BD,目录号:211132)
  4. Bacto TM supamin酪蛋白氨基酸(BD,目录号:228830)
  5. Bacto TM supaltium提取物(BD,目录号:218630)
  6. Bacto TM胨蛋白胨(BD,目录号:211677)
  7. Bacto TM酵母提取物(BD,目录号:212750)
  8. (Merck,目录号:102382)
  9. Co(NO 3)6+ 6H 2 O(Sigma-Aldrich,目录号:60832)
  10. CuSO 4·6H 2 O(Sigma-Aldrich,目录号:RES10395)
  11. EDTA(Sigma-Aldrich,目录号:ED2SS)
  12. FeSO 4 Aldrich,目录号:F7002)
  13. 葡萄糖(Fluka,目录号:49159)
  14. H sub 3 BO 3(Sigma-Aldrich,目录号:31146)

  15. (Merck,目录号:1.04873)

  16. (Merck,目录号:1.05104)
  17. KOH(Fluka,目录号:60369)
  18. NaCl(Sigma-Aldrich,目录号:71376)
  19. NaNO 3(Sigma-Aldrich,目录号:S5506)
  20. MgSO 4 .7H 2 O(Merck,目录号:5856)
  21. (Merck,目录号:105927)
  22. MoO 3(Sigma-Aldrich,目录号:M0753)
  23. ZnSO 47H 2 O(Sigma-Aldrich,目录号:Z4750)
  24. 铝箔
  25. 蒸馏水
  26. 灭菌自来水
  27. 液氮
  28. 手用灭菌剂
  29. 萌发培养基(参见配方)
  30. 地衣媒介(见配方)
  31. Bold的基础培养基(BBM)(参见食谱)

设备

  1. 双刃剃刀刀片(任何品牌)
  2. 铂接种针(阻燃)
  3. 解剖钳,不锈钢(阻燃)
  4. 铲刀,不锈钢(阻燃)
  5. 滤纸圆(φ45-55mm)(Schleicher& Schuell 589/2)
  6. 培养皿(ø100 mm,无菌)(塑料,加1-2个可高压灭菌的玻璃皿)(任何品牌)
  7. 穿孔酒吧匙,约。 30厘米长,不锈钢(阻燃)
  8. 实验室瓶(SCHOTT DURAN 100ml,500ml和1,000ml)
  9. 锥形瓶(SCHOTT DURAN 200ml,宽颈)
  10. 锥形烧瓶,纤维素插头
  11. 两个玻璃烧杯(500ml和200ml)
  12. 玻璃吸管(20ml,10ml和1ml)
  13. 橡胶吸管填充剂高达100 ml
  14. Falcon管(50ml,无菌,无致热和无DNase/RNase,低温抗性)(液氮)
  15. Falcon管(耐低温)(液氮)机架
  16. 具有能够分辨子囊孢子(10-30μm)的物镜的解剖显微镜
  17. 具有毫克分辨率范围的精密天平
  18. 无菌台,理想的紫外线辐射
  19. 本生灶
  20. 液体和固体物质的高压灭菌
  21. 均质器(IKA DI 18 basic或Ultraturrax T 18),安装在带凸台头夹具的板台上
  22. 可高压灭菌的均化杆(由不锈钢制成,IKA S 18 N-19G)
  23. 低温容器大到足以容纳一个带有Falcon管的支架
  24. 生长室具有(连续)15-16℃±1℃和12小时光/12小时黑暗状态 注意:湿度不能控制,但确保室内的干燥条件。

程序

  1. 轴菌丝体
    1. 理想地,用成熟apothecia(图1A)收集新鲜地衣thalli 在秋天到春天,但不是在夏天,因为这个种类没有   在温暖和干燥的季节产生足够的活的孢子。 Thalli   可以储存在玻璃培养皿中,风干几天   冰箱长达一周。


      图1. Lobaria的Thallus 肺炎和的安排。 A.新鲜的thallus与 不同年龄的apothecia。箭头:成熟apothecia适合 子囊收获;它们在完全水合状态下凸起 约。直径1.5-3.0mm。 B.倒置培养皿与apothecium 在湿滤纸上
    2. 用75%酒精或5分钟紫外线辐射或两者彻底清洁无菌工作台。
    3. 准备发芽培养基,高压灭菌器并倒入塑料 培养皿。准备大量的琼脂板几个隔离  步骤(最多50个板)。让发芽培养基冷却下来 在无菌台中凝胶化。用无菌接种针 划伤几条浅表线进入琼脂(这将有助于发现 琼脂表面的焦平面),然后将琼脂平板倒置 并继续工作
    4. 将几张滤纸放入玻璃杯中 培养皿。 包裹与铝箔的培养皿和消毒它 高压釜。 在内表面放置一张无菌滤纸 盖一个倒置培养皿,并用无菌自来水润湿 (图1B)。
    5. 使用尖锐的钳子切除成熟apothecia。   用新鲜的剃刀切下药店的下半部分 刀。 在无菌台架上工作,并将一个药剂置于湿润处 滤纸使用无菌镊子。 药剂需要完全 水合,但没有表面水膜(这可能防止 子囊孢子被排出)。 关闭倒置的培养皿,包括   药剂。 准备15-20个培养皿后,每个有一个   在生长室中孵育倒置的培养皿 15-16°C在黑暗中。 湿纸水化物使药物 并继续工作
    6. 将几张滤纸放入玻璃杯中 培养皿。 包裹与铝箔的培养皿和消毒它 高压釜。 在内表面放置一张无菌滤纸 盖一个倒置培养皿,并用无菌自来水润湿 (图1B)。
    7. 使用尖锐的钳子切除成熟apothecia。   用新鲜的剃刀切下药店的下半部分 刀。 在无菌台架上工作,并将一个药剂置于湿润处 滤纸使用无菌镊子。 药剂需要完全 水合,但没有表面水膜(这可能防止 子囊孢子被排出)。 关闭倒置的培养皿,包括   药剂。 准备15-20个培养皿后,每个有一个   在生长室中孵育倒置的培养皿 15-16°C在黑暗中。 湿纸水化物使药物... Place a clean microscope within the sterile bench. Normally, fusiform spores are visible (approx. 18-30 µm long x 5-9 µm wide) (Figure 2A). As these spores are usually three-celled more than one cell may germinate (Figure 2A, asterisk).
    8. Ignite the Bunsen burner and adjust the blue flame. Thoroughly sterilize the inoculation needles by flaming and allow them to cool down before using. Open a plate with germinated spores under the microscope, pick out one spore using a sterile needle and transfer it onto a fresh Petri dish with germinating medium. Transfer around four spores to each plate, then close the Petri dish and incubate it at 15-16 °C in the dark. Repeat this for each germinating spore.
    9. Check the plates every second day for the growth rates of the isolates and for contaminations. If single spores are affected by contamination, either cut them out or transfer the others to new agar plates.
    10. Repeat steps A7-9 during several weeks until the plates contain clean colonies of growing mycelia of L. pulmonaria.


      Figure 2. Ascospores and mycelium of L. pulmonaria on agar. A. Germinating ascospores 10 days after spore discharge. Asterisk: Spore with several germ tubes. B. The mycelium of L. pulmonaria grows very slowly. Axenic mycelium 85 days after spore discharge.

  2. Propagation in liquid Lichen Medium
    1. Prepare 1 L of Lichen Medium and autoclave it with the program for 液体。 包裹纤维素插头和解剖针在铝箔。 关闭锥形瓶和玻璃烧杯开口与铝箔。 用固体材料程序高压灭菌所有仪器。
    2. 将地衣培养基和材料存放在清洁的无菌台架上 等到他们达到室温! 放置显微镜 无菌台架,并用75%的水清洗所有表面, EtOH
    3. 在每个锥形瓶中加入50-100ml的地衣培养基。   在显微镜下从一个或几个菌丝体解剖 琼脂表面用无菌接种针转移到 液体介质在锥形瓶中
    4. 轻轻打开开口 的锥形瓶,关闭烧瓶并保持铝箔 插头的外部完好无损。 在15-16℃下孵育12小时/12小时   光/暗周期在几个月,从而允许菌丝体 成长为肉眼可见的殖民地
    5. 包裹每个不锈钢 在高压灭菌之前用铝箔的均化器的钢棒; 标记杆的开口,这将适合均化器,并保持   刀片侧覆盖直到它被使用。 安装无菌棒 以45°角均化,并立即除去铝箔 (图3A)。 将匀浆器调整到2级(更大 殖民地)或更高(较小的殖民地)。
    6. 插入均质器 杆放入带有液体培养基和真菌菌落的锥形瓶中。 打开匀浆器。 在研磨过程中缓慢转动烧瓶 过程,从而确保真菌菌落变得精细 中断。 这个过程将通过破坏杀死许多细胞,但是 大多数保持完好并将继续增长。约后。 10-20秒 均质化,关闭均化器(图3C)。火焰 打开锥形瓶和棉塞并密封烧瓶。 将用过的均化杆更换为无菌瓶,用于下一个烧瓶
    7. 在15-16℃下孵育12小时光照和12小时黑暗几个 几个月,使菌丝体变得肉眼可见

      图3.通过菌丝体的均质化加速真菌生长 的。肺动脉瓣。 A.均质器与安装杆成45°角。乙。 锥形瓶含有白色和棕色的菌丝体。 肺肺炎。将暗褐色菌丝体在琼脂平板上过量喂养。它有 开始在每个菌丝的表面上产生浅色菌丝体 转移到液体介质中后(见注1)。 C.白化 菌丝体。肺匀浆。

  3. 收获菌丝体用于DNA或RNA分离
    1. 将包含Falcon管的架子引入低温容器中 用液氮填充。 低温容器不能是无菌的
    2. 在匀浆器上安装无菌棒。 点燃本生灯。 从锥形瓶中取出塞子。 收获一些菌丝体 使用带有穿孔的无菌酒勺。 立即,介绍 菌丝体通过推动它在Falcon管中转化为液氮   用无菌铲子的匙子。
    3. 收获后,倾析 旧培养基慢慢倒入500ml玻璃烧杯中不失去 剩余的菌丝体并且不接触玻璃烧杯。 加注50-100 ml新鲜培养基倒入锥形瓶中。 从中取出铝箔   均化器并如上所述研磨菌丝体。 火焰 锥形开口和棉塞在关闭前。 重复步骤 C1-3所需的次数。
    4. 保持猎鹰管总是充满 与液氮。 将Falcon管存放在-80°C。 重要:不要 完全关闭管; 而是打开螺纹的一个扭曲 让蒸发氮气逃逸

      图4. Falcon管(a)与在液氮中收获的菌丝体(1)(2)

笔记

  1. 在琼脂上建立无菌菌丝体后,在最迟于萌发后三个月转换到液体培养基中培养。不要等待更长时间! L。肺部适应极度微营养条件;因此菌丝体将在琼脂培养基上过度喂养并最终死亡!过量的菌丝体变成棕色至深棕色(图3B)
  2. 区分真菌污染与发芽的子囊孢子的决定性标准。肺部生长是生长的快速(图2B; 85天后的菌丝体生长)。真菌污染在几天内产生密集的菌丝网,并且很快开始无性孢子形成。一旦无性孢子由污染物形成,几乎不可能拯救培养物,污染物孢子在已经打开陪替氏培养皿的盖的情况下已经风分散。
  3. 为了验证培养样品的分类学属性,进行聚合酶链反应,并按照Cornejo和Scheidegger(2010)所述对核内基因转录间隔区(ITS)rDNA区域进行测序。该序列可以用BLASTn检查,因为ITS标记为 L。肺炎病毒在GenBank中有详细记录
  4. 用该方法获得的培养物是多孢子培养物;在异种金属(交叉受精)物种中,每个孢子可能具有不同的遗传背景。如果需要单孢子分离物,例如用于分析减数分裂后代的个体孢子,则可以手动分离并单独培养单个孢子(Honegger等人/em>,2004)。

食谱

  1. 萌发培养基(Denison,2003)
    1.0%α-环糊精 1.5%玉米粉琼脂 制备1L发芽培养基:
    在瓶中装入15g玉米粉琼脂,10g环糊精和1L双蒸水H 2 O,然后将培养基高压灭菌。 稍微冷却的琼脂可以倒入培养皿中
  2. 地衣培养基(Honegger,1985)
    BBM 940 ml
    麦芽提取物0.875g
    蛋白酶0.125克
    酵母提取物0.0625克
    酪蛋白氨基酸0.0625g
    葡萄糖0.9375克
  3. Bold的基础培养基(BBM)(Deason和Bold,1960)
    1. 库存解决方案
      瓶标签
      化合物
      稀释储备液
      稀释BBM
      A
      NaNO 3
      20克/800毫升
      20ml
      B
      CaCl <2>
      20克/800毫升
      10 ml
      C
      MgSO 4。 。 O
      20克/800毫升
      10 ml
      D
      K 2 HPO 4
      20克/800毫升
      10 ml
      E
      KH 2 PO 4
      7 g/400 ml
      10 ml
      F
      NaCl
      1 g/400 ml
      10 ml
    2. 微量元素
      0.157g/100ml
      瓶标签
      化合物
      稀释痕量元素
      稀释BBM
      g
      H 3 BO 3
      1.142g/100ml
      1 ml
      h *
      FeSO 4 7H <2> O
      0.498g/100ml
      1 ml

      ZnSO 4 7H <2> 0.882g/100ml


      MnCl 2 4H O
      0.144克/100毫升
      i *
      MoO 3
      0.071g/100ml
      1 ml

      CuSO 4 5H sub 2 O


      CoNO 3 6H 2 O
      0.049g/100ml

      j *
      EDTA
      5.000g/100ml 1 ml

      KOH
      3.100g/100ml

      注意:*一瓶中混合了几种元素。
    3. 如何准备BBM
      混合储备溶液和微量元素(第二和第三列)。 高压灭菌储备溶液和微量元素,并将它们存储在 冰箱供将来使用。
      为了制备BBM,混合第四柱的量并用双蒸水H 2 O调节体积至1L。将pH调节至6.0。
  4. 3.100g/100ml

    注意:*一瓶中混合了几种元素。
  5. 如何准备BBM
    混合储备溶液和微量元素(第二和第三列)。 高压灭菌储备溶液和微量元素,并将它们存储在 冰箱供将来使用。
    为了制备BBM,混合第四柱的量并用双蒸水H 2 O调节体积至1L。将pH调节至6.0。
...
  • Denison, W. C. (2003). Apothecia and ascospores of Lobaria oregana and Lobaria pulmonaria investigated. Mycologia 95(3): 513-518.
  • Honegger, R. (1985). The hyphomycetous anamorph of Coniocybe furfuracea. The Lichenologist 17(03): 273-279.
  • Honegger, R. and Bartnicki-Garcia, S. (1991). Cell wall structure and composition of cultured mycobionts from the lichens Cladonia macrophylla, Cladonia caespiticia, and Physcia stellaris (Lecanorales, Ascomycetes). Mycological Res 95(8): 905-914.
  • Honegger, R., Zippler, U., Gansner, H. and Scherrer, S. (2004). Mating systems in the genus Xanthoria (lichen-forming ascomycetes). Mycol Res 108(Pt 5): 480-488.
    • English
    • 中文翻译
    免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
    Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
    引用:Cornejo, C., Scheidegger, C. and Honegger, R. (2015). Axenic Cultivation of Mycelium of the Lichenized Fungus, Lobaria pulmonaria (Peltigerales, Ascomycota). Bio-protocol 5(13): e1513. DOI: 10.21769/BioProtoc.1513.
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