Thursday, 29 November 2012

Selective Media for Isolation of Entomogenous Fungi

(from: Bioassays of Entomopathogenic Microbes and Nematodes CABI)

Veen’s agar medium (1 l) (Veen and Ferron, 1966)

35 g Mycological agar (Difco) or 10 g Oxoid neutralized soya peptone, 10 g
dextrose, 15 g No. 1. agar (or Bacto-agar), 1 g chloramphenicol (store 4°C),
and 0.5 g cycloheximide (= Actidione; store 4°C). Add 1 l distilled water, stir,
and cover. Autoclave for 10–15 min at 18–20 psi. Cool to c. 52°C and pour
plates in laminar flow cabinet.

Oatmeal dodine agar (Beilharz et al., 1982)

1. Antibiotic stock solution: add 4 g penicillin G (Sigma) and 10g streptomycin
sulphate (Sigma) to 40 ml sterile distilled water under sterile conditions.
Store at 4°C.
2. Crystal violet stock solution: add 0.1 g crystal violet (Sigma) to 200 ml distilled
water. Store in the dark.
3. Add 17.5 g oatmeal agar (Difco) and 2.5 g agar (Fisons) slowly to 0.5 l
distilled water while stirring vigorously and heat to boil.
4. Add 0.5 ml of the fungicide dodine (N-dodecylguanidine monoacetate;
Cyprex 65WP, American Cyanamid Co.) and 5 ml crystal violet stock solution
to the medium.
5. Autoclave for 20 min at 15 psi.
6. Allow medium to cool to 50–55°C and add 2 ml of antibiotic stock solution
under sterile conditions.
7. Swirl flask well to ensure thorough mixing of compounds and pour while
warm. There should be enough media for twenty 9-cm diameter Petri

Selective agar medium (1 l) (Kerry et al., 1993)

37.5 mg carbendazim, 37.5 mg thiabendazole, 75 mg rose bengal, 17.5 g
NaCl, 50 mg each of streptomycin sulphate, aureomycin and chloramphenicol,
3 ml Triton X-100, and 17 g corn meal agar (Oxoid) in 1 l distilled water.
This medium is appropriate for selecting some Paecilomyces spp. and
Verticillium spp. from soil.

Paecilomyces lilicanus medium (Mitchell et al., 1987)

To prepare 1 l of medium, mix the following: 39 g PDA, 10–30 g NaCl, 1 g
Tergitol, 500 mg pentachloronitrobenzene, 500 mg benomyl, 100 mg streptomycin
sulphate, and 50 mg chlorotetracycline hydrochloride.

Wheat germ based selective agar medium (1 l) (Sneh, 1991)

1. Prepare an aqueous extract of wheat germ – mix 30 g wheat germ in 1 l
water, autoclave for 10 min and filter through four layers of cheesecloth.
2. Mix wheat germ extract (1 l) with 0.25 g chloramphenicol (heat stable) +
0.8 mg benlate (50% benomyl), 0.3 g dodine (65% n-dodecyl-guanidine
acetate), 10 mg crystal violet and 15 g agar.
3. Autoclave and pour into plates.

Copper-based selective agar medium (1 l) (Baath, 1991)

2% malt extract (Oxoid), 1.5% Agar (Difco) amended with 2–4 mg
CuSO4·5H2O per litre. Cordyceps militaris and Paecilomyces farinosus are
tolerant of high Cu levels (400 mg/L, followed by Metarhizium anisopliae
and Beauveria bassiana. Most other soil-borne fungi including
nematophagous species of Verticillium were less tolerant.

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