Selective Medium for Isolating Phanerochaete chrysosporium from Soil

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1990, p /90/ $02.00/0 Copyright 1990, American Society for Microbiology Vol. 56, No. 10 Selective Medium for Isolating Phanerochaete chrysosporium from Soil DIANE M. DIETRICH* AND RICHARD T. LAMAR Institute for Microbial and Biochemical Technology, Forest Products Laboratory, U.S. Department of Agriculture Forest Service, Madison, Wisconsin Received 11 June 1990/Accepted 6 August 1990 A selective medium was developed that is capable of isolating Phanerochaete chrysosporium from soil. This medium contains 15 ppm of benomyl (15 µg g -1 ) and 550 ppm of streptomycin sulfate in 2% malt agar and is held at 39 C after inoculation. P. chrysosporium was isolated from three nonsterile forest soils to which the fungus had been added. These soils contained large microbial populations. 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. 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). 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). 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. * Corresponding author. MATERIALS AND METHODS 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. Fungus and inoculum preparation. P. chrysosporium Burdsal1 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 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. 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 -1 ) was dissolved in sterilized, deionized, distilled water, and benomyl (1,000 mg liter -1 ) and OPP (1,000 mg liter -1 ) were dissolved in acetone. Effect of selective inhibitors on fungal growth. The maximum concentration of each compound that did not significantly affect the growth of P. chrysosporium was determined by comparing the rate of hyphal extension on 2% malt agar (20 g of malt extract, 15 g of Bacto-Agar per liter) plates that contained the compounds at various concentrations to the rate of fungal growth on plates that contained no inhibitor. The rate of hyphal extension was the average daily increase in colony diameter measured in two perpendicular directions. Inhibitor concentrations were as follows; benomyl, 1, 3088

2 VOL. 56, 1990 MEDIUM FOR ISOLATING P. CHRYSOSPORIUM FROM SOIL ,10,20,50, and 100 µg g of medium -1 (ppm); streptomycin sulfate, 40,80,200,400,440, and 550 ppm; and OPP, 5,9,18, 45, 55, and 65 ppm. There were five replicates at each inhibitor concentration. The appropriate amounts of acetone were included in the media used to prepare control plates when testing the effect of benomyl and OPP on fungal growth. Plates were inoculated by aseptically transferring asexual spores of P. chrysosporium from a slant to the center of each plate. After inoculation, the plates were incubated at 39 C, which is the optimal temperature for growth of P. chrysosporium (6). Rates of hyphal extension were determined daily for 1 week or until fungal growth reached the edge of the plate. Statistical analysis of growth rate data involved single-factor analysis of variance for each inhibitor compared. A multiple comparison of treatment means was performed by using Scheffk's test. All testing was performed at α = After the maximum concentration of each compound that did not significantly affect growth was determined, the effects of benomyl and OPP in combination with streptomycin sulfate on fungal growth were investigated. Rates of hyphal extension were determined as previously described. Soils and quantification of indigenous microbes. Three forest soils (Marshan, Batavia, and Zurich) were collected, air dried, passed through a 2-mm-pore-size sieve, and stored in plastic bags at 4 C. The Marshan and Zurich soils were sandy loams collected from the A horizon. The Batavia silty clay loam was collected from the Bt2 horizon. These soils have been previously characterized (13). Estimates of the number of bacteria, fungi, and actinomycetes in each soil were obtained by using the dilution plate method (16). Dilution plates of media selective for growth of fungi, bacteria and actinomycetes were prepared as follows. A peptoneglucose-acid-agar medium containing 1 g of KH 2PO 4, 0.5 g of MgSO 4 7H 2O, 5 g of peptone, 10 g of glucose, 20 g of agar, 1,000 ml of distilled water, and 1.0 ml of melted 0.5 N H 2SO 4 for each 100 ml of medium was used to isolate fungi. The yeast extract agar selective for bacteria contained 15 g of agar, 1.0 g of dextrose, 0.5 g of KNO 3, 1.0 g of K 2HPO 4 (after autoclaving), 0.2 g of MgSO 4 7H 2O, 0.1 g of CaCl 2, 0.1 g of NaCl, 0.01 g of FeCl 3, 1.0 g of yeast extract, and 1 liter of H 2O. The ph was adjusted to 6.8 with HCl or NaOH. Actinomycete isolation agar contained 2.0 g of sodium caseinate, 0.1 g of asparagine, 4.0 g of sodium propionate, 0.5 g of dipotassium sulfate, 0.1 g of magnesium sulfate, 1 mg of ferrous sulfate, 15 g of agar, 1,000 ml of distilled water, and 5.0 g of glycerol (after boiling). This medium was heated until boiling and then removed from heat, and glycerol was added. These plates were inoculated with water extract dilutions from the soils. A 10-1 dilution was prepared by adding 10 g of soil to 90 ml of sterile, deionized, distilled water. Decimal dilutions through 10-7 were prepared from the 10-1 dilution. Plates were streaked with 0.1 ml of the prepared dilutions. These dilutions were 10-5 to 10-8 for bacteria, 10-3, to 10-7 for fungi, and 10-4 to 10-7 for actinomycetes. The plates were held at 25 C in the dark for 4 days for bacteria and fungi and 7 days for actinomycetes. Effect of selective media on microbial growth. Several media containing combinations of the compounds that did not affect the growth of P. chrysosporium were tested for their ability to inhibit growth of microorganisms indigenous to the three soils. Three plates of each medium were inoculated with soil-water extracts from the nonsterile soils. The soil-water extracts were prepared and plates were TABLE 1. Effect of inhibitor concentration on rate of hyphal extension of P. chrysosporium Inhibitor concn Rate of hyphal (ppm) extension a (cm day -1 ) Benomyl a a a b c d d Streptomycin sulfate ef e ef e f g g OPP h h h hi ij 55 0j 65 0j a Multiple comparison of treatment means was performed by using Scheffe's test. Rates within a column followed by the same letter are not significantly different. Rates were determined from data obtained on day 2. inoculated as previously described. The plates were then held at 39 C and checked daily for microbial growth. Isolation of P. chrysosporium from soil. Media that prevented growth of the indigenous microbes were tested for their ability to isolate P. chrysosporium from soils that had been sterilized and then inoculated with the fungus or from nonsterile soils that contained the fungus. For these isolation experiments, the sterile soils were prepared by autoclaving them for 0.5 h on each of three consecutive days at 121 C and kpa. Before inoculation, the water content of both nonsterile and sterile soils was adjusted to MPa. To inoculate both sterile and nonsterile soils, petri plates were filled with soil and five inoculum chips were then aseptically placed in the soil. The plates were then incubated at 39 C for 2 weeks and checked daily for microbial growth. Soil-water extracts from both sterile and nonsterile soils were prepared to inoculate three plates per test medium. A medium was identified that selectively isolated P. chrysosporium from the three soils. To confirm that this medium was selective for P. chrysosporium, 25 replicates each of selective medium and 2% malt agar plates were inoculated with extracts from nonsterile Marshan soil that contained P. chrysosporium. All plates were then incubated at 39 C and checked daily for microbial growth. RESULTS No significant difference was observed in hyphal extension rates of P. chrysosporium at concentrations up to and including 5 ppm of benomyl (Table 1). At concentrations above 5 ppm, benomyl had an inhibitory effect on P. chrysosporium growth, which increased with increasing inhibitor concentration. At 50 ppm and above, fungal growth was negligible. The rate of hyphal extension was slightly enhanced at concentrations of 40 ppm of streptomycin

3 3090 DIETRICH AND LAMAR APPL. ENVIRON. MICROBIOL. TABLE 2. Number of bacteria, fungi, and actinomycetes in the Marshan, Zurich, and Batavia soils as determined by the dilution plate method Soil type No. of: Bacteria Fungi Actinomycetes Marshan 1.15 l Zurich Batavia 4.71 l l0 6 sulfate, in comparison to the control. Concentrations of 80 to 200 ppm of streptomycin sulfate had no significant effect on rate of hyphal extension. However, at concentrations of 440 ppm and above, streptomycin sulfate significantly decreased fungal growth (Table 1). Increasing concentrations of OPP caused a decrease in the rate of hyphal extension. However, this decrease was not significant until the concentration of OPP exceeded 18 ppm. Significant inhibition of growth was observed at 45 ppm of OPP, and at concentrations above 45 ppm, fungal growth was completely inhibited (Table 1). On the basis of these results, we chose to work with benomyl, OPP, and streptomycin sulfate at concentrations up to and including 15, 45, and 550 ppm, respectively. Because large populations of microorganisms were found in all three nonsterile soils (Table 2), we chose to work with streptomycin sulfate at the highest concentrations possible even though they significantly inhibited growth of the fungus. P. chrysosporium grew in all media containing combinations of benomyl and streptomycin sulfate (Table 3). The rate of hyphal extension was only slightly lower on the medium containing 550 ppm of streptomycin sulfate than on the medium containing 80 ppm of the antibiotic. Combinations of OPP and streptomycin sulfate proved to be more inhibitory to the fungus (Table 3). The fungus grew well in the medium that contained 30 ppm of OPP except when OPP was combined with 550 ppm of streptomycin sulfate. At the 45-ppm level, growth occurred slowly, if at all. Therefore, combinations of 30 ppm of OPP with 80, 440, or 550 ppm of streptomycin sulfate were chosen for further study. A medium containing 15 ppm of benomyl, 550 ppm of streptomycin sulfate, and 45 ppm of OPP was also prepared. Fungal growth was severely inhibited in this medium. Malt agar plates containing 15 ppm of benomyl and 550 ppm of streptomycin sulfate or containing 30 ppm of OPP with 80, 440, or 550 ppm of streptomycin sulfate were prepared. These plates were inoculated with soil-water extracts prepared from sterile soil that had been inoculated with P. chrysosporium after sterilization or with extracts TABLE 3. Effect of combinations of benomyl, streptomycin sulfate, and OPP on rate of hyphal extension of P. chrysosporium Benomyl Inhibitor (ppm) Streptomycin sulfate OPP Fungal growth a (cm day -1 ) a Fungal growth rates were determined from data obtained on day 1. prepared from nonsterile soils. Growth of the fungus was observed on all plates inoculated with sterile soil-water extracts, indicating that the fungus could be isolated from soil via soil extracts. In plates inoculated with extracts from the nonsterile soils, growth of many native organisms was observed on the plates containing the OPP and streptomycin sulfate combinations, indicating that this combination could not be used as a selective medium. In the plates containing the benomyl and streptomycin sulfate medium, no growth was observed on any of the plates inoculated with nonsterile soil-water extracts, which indicated that this medium completely inhibited the growth of microbes indigenous to the nonsterile soils. Soil-water extracts from nonsterile soils containing the fungus were then used to inoculate plates containing benomyl and streptomycin sulfate. This combination successfully isolated P. chrysosporium from nonsterile soils. In the 25 repeated inoculations of this medium with soil-water extracts from the nonsterile Marshan soil that contained the fungus, pure cultures of P. chrysosporium were obtained, confirming that this medium was selective for the fungus. In all 25 malt agar plates inoculated with this same soil extract, bacteria and several fungi grew over the entire plate (Fig. 1). DISCUSSION A selective medium was developed that is capable of isolating P. chrysosporium from three nonsterile forest soils that contained abundant indigenous microbial populations. This medium contained 15 ppm of benomyl and 550 ppm of streptomycin sulfate in 2% malt agar; the medium was held at 39 C after inoculation. Although P. chrysosporium is a mesophile, its optimum temperature for growth is 39 C (6). This temperature is probably close to the maximum growth temperature for many microbes found in the soils used in our study. Therefore, incubation at 39 C may also confer some selective advantage to P. chrysosporium. Our results are similar to those reported for other media selective for wood-rotting basidiomycetes. Benomyl at 15 ppm in 2% malt agar was useful in the isolation of wood decay fungi (17). Benomyl at 1 ppm completely inhibited three non-wood decay fungi, but it did not inhibit growth of wood decay fungi (14). Malt agar plates inoculated with soil-water extracts from each of the three nonsterile soils indicated the presence of large bacterial populations. Thus, the addition of streptomycin sulfate to the medium was necessary to control bacterial growth. The concentration of streptomycin used in selective media has varied greatly depending on the source of inocula. Uscuplic and Pawsey (20) reported using 2,666 ppm streptomycin. Their source of inoculum was soil suspensions. Wood chips used as a source of inoculum required smaller concentrations of streptomycin. Streptomycin at 50 to 100 ppm has been reported in such cases. Taylor (19) reported the use of 0.05 ppm of streptomycin and diseased roots for inoculum. The selective medium reported in this paper contains an intermediate level of streptomycin sulfate. Previous reports have indicated that the usefulness of OPP in a selective medium depends greatly upon the basidiomycete being selected. Uscuplic and Pawsey (20) developed a medium containing OPP that could isolate two kinds of basidiomycetes but could not isolate ten others. Cary and Hull (7) developed a selective medium for white rot basidiomycetes that contained OPP and benomyl. They found that all of their unidentified white rot fungi were tolerant to OPP up to 30 ppm in a medium that also contained 10 ppm of

4 VOL. 56, 1990 MEDIUM FOR ISOLATING P. CHRYSOSPORIUM FROM SOIL 3091 FIG. 1. Microbial growth on selective medium and malt agar plates after 3 days at 39 C. Plates were inoculated with a water suspension of nonsterile Marshan soil containing chips inoculated with P. chrysosporium. Plate a was essentially a pure culture of coalesced colonies of P. chrysosporium. Plate b contained a variety of bacterial and fungal colonies. benomyl. Above 30 ppm of OPP, growth of all fungi was inhibited, except for one fungus that grew in the medium containing 120 ppm of OPP. We determined that P. chrysosporium was able to grow in the presence of 45 ppm of OPP, but upon adding 80 to 550 ppm of streptomycin sulfate, the fungal tolerance to OPP dropped to 30 ppm. The combinations of OPP and streptomycin sulfate allowed the growth of soil microorganisms indigenous to the three soils, thus making this combination inappropriate for our selective medium. The fungus could not be isolated from the malt agar plates inoculated with soil-water extract from nonsterile soil containing P. chrysosporium because of large populations of native soil microorganisms. Selective medium inoculated with identical extracts contained pure cultures of P. chrysosporium. The absence of other microorganisms on the selective medium confirmed that this medium was selective for P. chrysosporium. ACKNOWLEDGMENTS We thank T. Kent Kirk and Frances F. Lombard for critical review of the manuscript. This research was supported by grant DW , U.S. Environmental Protection Agency. LITERATURE CITED 1. Arjmand, M., and H. Sandermann, Jr Mineralization of chloroaniline/lignin conjugates and of free chloroanilines by the white-rot fungus Phanerochaete chrysosporium. J. Agric. Food Chem. 33: Arjmand, M., and H. Sandermann, Jr Plant biochemistry of xenobiotics. Mineralization of chloroaniline/lignin metabolites from wheat by the white-rot fungus, Phanerochaete chrysosporium. Z. Naturforsch. Sect. C 41: Bumpus, J. A Biodegradation of polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 55: Bumpus, J. A., and S. A. Aust Biodegradation of DDT [1,1,l-trichloro-2,2-bis(4-chlorophenyl)ethane] by the white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 53: Bumpus, J. A., M. Tien, D. Wright, and S. D. Aust Oxidation of persistent environmental pollutants by a white rot fungus. Science 228: Burdsall, H. H., and W. E. Eslyn A new Phanerochaete with a Chrysosporium imperfect state. Mycotaxon 1: Cary, J. K., and A. V. Hull A selective medium for the isolation of wood-rotting basidiomycetes. Int. Biodeterior. 25: Chang, H.-M., T. W. Joyce, A. G. Campbell, E. D. Gerrard, V. B. Huynh, and T. K. Kirk Fungal decolorization of bleach plant effluents, p In T. Higuchi, H.-M. Chang, and T. K. Kirk (ed.), Recent advances in lignin biodegradation. Uni Publishers Co., Ltd., Tokyo. 9. Coggins, C. R., and D. H. Jennings Selective medium for the isolation of Serpula lacrymans. Trans. Br. Mycol. Soc. 65: Eaton, D. C Mineralization of polychlorinated biphenyls by Phanerochaete chrysosporium: a ligninolytic fungus. Enzyme Microb. Technol. 7: Hale, M. D. C., and J. G. Savory Selective agar media for the isolation of basidiomycetes from wood-areview. Int. Biodeterior. Bull. 12: Kuhlman, E. G., and F. F. Hendrix, Jr A selective medium for the isolation of Fomes annosus. Phytopathology 52: Lamar, R. T., J. A. Glaser, and T. K. Kirk Fate of pentachlorophenol (PCP) in sterile soils inoculated with the white-rot basidiomycete Phanerochaete chrysosporium: mineralization, volatilization, and depletion of PCP. Soil Biol. Biochem. 22: Maloy, O. C Benomyl-malt agar for the purification of cultures of wood decay fungi. Plant Dis. Rep. 58: Mileski, G. J., J. A. Bumpus, M. A. Jurek, and S. D. Aust Biodegradation of pentachlorophenol by white rot fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 54: Parkinson, D. T., R. G. Gray, and S. T. Williams Methods for studying the ecology of soil microorganisms. IBP handbook no. 19. Blackwell Scientific Publications Ltd., Oxford. 17. Raabe, R. D., and J. H. Hurlimann A selective medium for isolation of Armillaria mellea. California Plant Pathology,

5 3092 DIETRICH AND LAMAR APPL. ENVIRON. MICROBIOL. No. 3. University of California Agricultural Extension Service, Br. Mycol. Soc. 56: Sacramento. 20. Uscuplic, M., and R. G. Pawsey A selective medium for 18. Russell, P A selective medium for the isolation of basid- the isolation of Polyporus schweinitzii. Trans. Br. Mycol. Soc. iomycetes. Nature (London) 177: : Taylor, J. B A selective medium for the isolation of 21. Vaartaja, O Selectivity of fungicidal materials in agar basidiomycetes for diseased roots, mycorrhizas and soil. Trans. cultures. Phytopathology 50: