Phanerochaete chrysosporium Burdsall in Burds. and Eslyn is a white rot basidiomycete that has the ability to mineralize a group of structurally heterogeneous xenobiotics. A partial list of this group includes low-molecular-weight chlorinated phenols from kraft pulp mill bleach effluents (8); Aroclor 1254 (10); DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane], 3,4,3',4'- tetrachlorobiphenyl, 2,4,5,2',4', 5'-hexachlorobiphenyl, 2, 3, 7, 8 - tetrachloro - dibenzo [p] dioxin, and lindane (4, 5); chlorinated anilines (1, 2); pentachlorophenol (15); and phenanthrene (3). The ability to oxidize these normally recalcitrant compounds makes this fungus an attractive candidate as an agent for the bioremediation of soils that are contaminated with hazardous organic materials. Our work focuses on using P. chrysosporium and related fungi to remediate soils at hazardous waste sites. A selective medium for the isolation of this fungus from soil would be useful to determine the growth and survival of P. chrysosporium in soil growth and xenobiotic degradation studies

1. Selective Medium for Isolating
Phanerochaete chrysosporium
from Soil
By sreeremya.s

2. • Phanerochaete chrysosporium Burdsall in Burds. and
• Eslyn is a white rot basidiomycete that has the ability to
• mineralize a group of structurally heterogeneous xenobiotics.
• A partial list of this group includes low-molecular-weight
• chlorinated phenols from kraft pulp mill bleach effluents
• (8); Aroclor 1254 (10); DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)
• ethane], 3,4,3',4'- tetrachlorobiphenyl, 2,4,5,2',4',
• 5'-hexachlorobiphenyl, 2, 3, 7, 8 - tetrachloro - dibenzo [p] dioxin,
• and lindane (4, 5); chlorinated anilines (1, 2); pentachlorophenol
• (15); and phenanthrene (3). The ability to
• oxidize these normally recalcitrant compounds makes this
• fungus an attractive candidate as an agent for the bioremediation
• of soils that are contaminated with hazardous organic
• materials. Our work focuses on using P. chrysosporium and
• related fungi to remediate soils at hazardous waste sites. A
• selective medium for the isolation of this fungus from soil
• would be useful to determine the growth and survival of P.
• chrysosporium in soil growth and xenobiotic degradation
• studies

3. • The first report of a selective medium for isolation of white
• rot basidiomycetes was on the isolation of Heterobasidion
• annosum (Fr.) Brev. and four other basidiomycetes from
• wood pulp (18). The fungicide orthophenylphenol (OPP)
was
• the selective inhibitor. OPP has also been used to isolate
• Phaeolus schweinitzii (Fr.) Pat. and many other
basidiomycetes
• from wood chips and soil extracts (20, 21). Recently,
• OPP was used in a selective medium to isolate
• several white rot basidiomycetes from wood (7).

4. • Other
• selective inhibitors that have been found to be useful
for the
• isolation of wood rot basidiomycetes from a variety of
• substrates are the antibiotic streptomycin and the
fungicide
• benomyl. Streptomycin was used in selective media in
• combination with other inhibitors to isolate
Heterobasidion
• annosum from wood chips (12) and to isolate Phaeolus
• schweinitzii from a soil extract (20).

5. • Streptomycin was used
• in combination with benomyl to isolate wood-rotting
basidiomycetes
• from diseased roots (19), plaster (9), and wood
• chips and cultures (11). Benomyl was used alone in
selective
• media to isolate Armillaria mellea from wood chips (17)
and
• to isolate various basidiomycetes from cultures (14). In this
• paper, we report on the development of a selective
medium
• for isolation of P. chrysosporium from soil.

6. • Approach. The protocol for development of a selective
• medium for isolating P. chrysosporium from soil was as
• follows. (i) Streptomycin sulfate, OPP, and benomyl were
• selected to be tested as possible ingredients in a selective
• medium. The maximum concentration of these compounds
• that did not decrease growth of P. chrysosporium on 2% malt
• agar was determined (i.e., the concentration at which growth
• was not significantly different from that on media that did not
• include the inhibitor). (ii) Combinations of the three compounds
• that did not significantly affect growth of P. chrysosporium
• were then identified. (iii) Finally, these combinations
• were evaluated for their ability to isolate P.
• chrysosporium from soils.

7. • Fungus and inoculum preparation. P. chrysosporium Burdsall
• in Burds. and Eslyn (BKM F-1767; ATCC 24725) was
• grown on 2% malt agar slants at 39°C for 1 week and then
• stored at 4°C. Soil inoculum consisted of aspen (Populus
• tremuloides Michx.) pulpwood chips (ca. 1.5 by 0.5 by 0.25
• cm) grown through with P. chrysosporium and prepared as
• follows. Chips were sterilized by autoclaving in aluminum
• foil-covered 500-ml Erlenmeyer flasks at 121°C and 103.5
• kPa for 1 h. The moisture content of the chips was then
• adjusted to 60% by the addition of sterile, deionized, distilled
• water. Sterile chips were inoculated by incubating them with
• pieces of malt agar from P. chrysosporium slants. Inoculated
• chips were held at 39°C for 1 week.

8. • Inhibitors. Benomyl (50% wettable powder) was obtained
• from Du Pont Chemical Co. (Wilmington, Del.), and
streptomycin
• sulfate and OPP were obtained from Sigma Chemical
• Co. (St. Louis, Mo.). Stock solutions were made as
• follows: streptomycin sulfate (4,000 mg liter-') was
dissolved
• in sterilized, deionized, distilled water, and benomyl
• (1,000 mg liter-') and OPP (1,000 mg liter-') were dissolved
• in acetone.