地衣型真菌肺衣（地卷目、子囊菌门）菌丝体的无菌培养

Carolina Cornejo; Christoph Scheidegger; Rosmarie Honegger

doi:10.21769/BioProtoc.1513

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Peer-reviewed

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

Carolina Cornejo email

CS Christoph Scheidegger

Rosmarie Honegger

发布: 2015年07月05日第5卷第13期 DOI: 10.21769/BioProtoc.1513 浏览次数: 10207

评审: Fanglian HeChong HeAnonymous reviewer(s)

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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

Freshly collected lichen thalli of Lobaria pulmonaria bearing apothecia
Alpha-cyclodextrin (Sigma-Aldrich, catalog number: C4642 )
BBL^TM cornmeal agar (BD, catalog number: 211132 )
Bacto^TM casamino acids (BD, catalog number: 228830 )
Bacto^TM malt extract (BD, catalog number: 218630 )
Bacto^TM peptone (BD, catalog number: 211677 )
Bacto^TM yeast extract (BD, catalog number: 212750 )
CaCl₂^.2H₂O (Merck, catalog number: 102382 )
Co(NO₃)^.6H₂O (Sigma-Aldrich, catalog number: 60832 )
CuSO₄^.5H₂O (Sigma-Aldrich, catalog number: RES10395 )
EDTA (Sigma-Aldrich, catalog number: ED2SS )
FeSO₄^.7H₂O (Sigma-Aldrich, catalog number: F7002 )
Glucose (Fluka, catalog number: 49159 )
H₃BO₃ (Sigma-Aldrich, catalog number: 31146 )
KH₂PO₄ (Merck, catalog number: 1.04873 )
K₂HPO₄ (Merck, catalog number: 1.05104 )
KOH (Fluka, catalog number: 60369 )
NaCl (Sigma-Aldrich, catalog number: 71376 )
NaNO₃ (Sigma-Aldrich, catalog number: S5506 )
MgSO₄^.7H₂O (Merck, catalog number: 5856 )
MnCl₂^.4H₂O (Merck, catalog number: 105927 )
MoO₃ (Sigma-Aldrich, catalog number: M0753 )
ZnSO₄^.7H₂O (Sigma-Aldrich, catalog number: Z4750 )
Aluminum foil
Bidistilled water
Sterilized tap water
Liquid nitrogen
Sterilization agent for hands
Germination medium (see Recipes)
Lichen medium (see Recipes)
Bold’s basal medium (BBM) (see Recipes)

Equipment

Double edge razor blades (any brand)
Platinum inoculation needles (flame-resistant)
Dissection forceps, stainless steel (flame-resistant)
Spatula, stainless steel (flame-resistant)
Filter paper circles (ø 45-55 mm) (Schleicher & Schuell 589/2)
Petri dishes (ø 100 mm, sterile) (plastic, plus 1-2 autoclavable glass dishes) (any brand)
Perforated bar spoons, ca. 30 cm long, stainless steel (flame-resistant)
Laboratory bottles (SCHOTT DURAN^® 100 ml, 500 ml, and 1,000 ml)
Erlenmeyer flasks (SCHOTT DURAN^® 200 ml, wide neck)
Plugs for Erlenmeyer flasks, cellulose
Two glass beakers (500 ml and 200 ml)
Glass pipets (20 ml, 10 ml, and 1 ml)
Rubber pipet filler up to 100 ml
Falcon tubes (50 ml, sterile, nonpyrogenic and DNase-/RNase-free, cryo-resistent) (liquid nitrogen)
Racks for Falcon tubes (cryo-resistant) (liquid nitrogen)
Dissecting microscope with an objective lens capable of resolving ascospores (10-30 µm)
Precision balance with milligram resolution range
Sterile Bench, ideally with UV radiation
Bunsen burner
Autoclave for liquid and solid material
Homogenizer (either a IKA DI 18 basic or Ultraturrax T 18), mounted on a plate stand with a boss head clamp
Autoclavable homogenizer rods (made from stainless steel, IKA S 18 N-19G)
Cryogenic vessel big enough to contain a rack with Falcon tubes
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

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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.