Suppression of Aerial Hyphae by Staling Products of Postia placenta

International Biodeterioration & Biodegradation 30 (1992) 285-302 Suppression of Aerial Hyphae by Staling Products of Postia placenta Jessie A. Micales USDA Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive. Madison, Wisconsin 53705-2398, USA (Received 1 November 1991; accepted 24 January 1992) ABSTRACT Isolate ME20 is a wild-type monokaryotic strain of the brown-rot fungus Postia placenta that does not cause significant weight losses in standard soilwood block decay tests. It also fails to form aerial hyphae in liquid and agar culture, This abnormal morphological feature may be caused by the same aberrant physiology that prevents the strain from efficiently degrading wood. Aerial hyphae formation was suppressed in MAD698, a standard floccose strain of P. placenta when spent media from 14-day-old ME20 cultures made up 40% or more of the nutrient medium. Spent media from MAD698 cultures caused a similar effect but only at 80% and 100% concentrations. This suppression does not appear to be caused by nutrient deprivation or by the activity of extracellular autolytic enzymes. The suppressive factor has a molecular weight of <10 000 Da, is resistant to boiling, and thus probably non-enzymatic. The inhibition of aerial hyphae formation is probably due to unidentified, low molecular weight staling products. INTRODUCTION Brown-rot basidiomycetes are a major cause of the decay of wood and wood products throughout the world. The rapid depolymerization of cellulose associated with brown rot results in serious strength reductions in wood early in the decay process (Cowling, 1961). The physiological and biochemical mechanisms of brown-rot decay are not well-understood (Highley, 1987, 1989). A better understanding of these 285

286 Jessie A. Micales mechanisms could lead to the development of specifically targeted wood preservatives that are disruptive to the metabolism of wood-decay fungi but are non-toxic to other life forms. Aberrant strains or mutants of wood-decay fungi that are unable to degrade wood effectively can serve as tools to identify the metabolic pathways necessary for decay development. Isolate ME20, a wild-type monokaryon of the brown-rot fungus Postia placenta, is such a strain. Many physiological characteristics of ME20 have been described (Micales & Highley, 1989, 1991; Micales et al., 1990). The strain typically displays an abnormal, appressed colony morphology in both liquid and agar culture (Micales & Highley, 1989,1991). Isolate ME20 does not grow well on wood but is able to penetrate it (Green et al., 1991). This strain can produce large amounts of mycelium in liquid culture, but its growth rate peaks and declines rapidly (Micales & Highley. 1991). Isolate ME20 generally fails to produce extracellular glucan in liquid culture (Micales & Highley, 1991; Micales et al., 1990), and scanning electron microscopy has shown that ME20 forms an abnormal, non-fibrillar hyphal sheath on both woody and inert substrates (Green et al., 1991; Micales et al., 1989).The ME20 strain often produces excessive levels of extracellular enzymes, including the autolytic enzymes laminarinase and protease (Micales& Highley, 1991). Excessive production of autolytic degradative enzymes may be responsible for the atypical. appressed colony morphology and abnormal hyphal sheath of ME20. High levels of laminarinase, which degrade the ß-1,3-glucan of the cell wall and hyphal sheath, are especially suspect. In the presence of excessive laminarinase, the fungus may digest itself before the onset of normal autolysis. If this hypothesis is true, any strain of P. placenta grown in the presence of excessive autolytic enzymes is likely to take on the appearance of ME20. The objective of this study was to grow a normal, floccose strain of P. placenta, designated MAD698, in the spent culture filtrate of ME20 to determine whether the production of aerial mycelia would be affected. Commercial enzyme preparations of laminarinase and protease were also used to assess the effect of excess levels of autolytic enzymes on the colony morphology and growth of MAD698. Fungal isolates MATERIALS AND METHODS Postia placenta (Fr.) M. Lars. et Lomb. isolates ME20 and MAD698 were maintained on 2%malt-extract agar (MEA) slants at 4 C for the duration of the study.

Cultural conditions Suppression of aerial hyphae by Postia placenta 287 P. placenta isolates MAD698 and ME20 were grown for 60 days at 27 C in 250-ml Erlenmeyer flasks containing 25 ml of non-aerated 1% cellobiose plus basal salts, ph 5.0 (Highley, 1973). Six to ten replicate flasks of each isolate were pooled, and the culture filtrate was collected by centrifugation (5000 g for 15 min). The pooled culture filtrate was resterilized by filtration through a 0.22 µm Millipore* filter and aliquots added aseptically to autoclaved 250-ml Erlenmeyer flasks containing fresh 1% cellobiose plus basal salts, ph 5.0. The final concentration of spent culture filtrate in each combined medium was 0%, 20%, 40%, 60%, 80%, and 100% (v/v) of the final volume of 25 ml. Control flasks contained sterile, deionized water in the place of the spent culture filtrate. At least two replicate flasks were made for each combination of isolate and concentration. The media were inoculated with 7-mm-diameter mycelial plugs of MAD698 taken from the margins of 7-day-old colonies grown on 2% (w/v) MEA. The colonies were incubated for 21 days at 27 C. The amount of aerial mycelium produced in each flask was estimated using a four-point visual-rating scale (Tables 1 and 2). Fungal mycelia were collected on tared glass-fibered filter paper by vacuum filtration. The filter papers were oven dried overnight at 55 C to determine mycelial dry weight. The culture filtrates were dialyzed overnight against deionized water, ph 5 0, to remove excess reducing sugars and then assayed for extracellular protease. laminarinase, and protein, as described later. The effect of spent culture filtrate from 6- and 10-week-old cultures of ME20 was also determined in agar culture. Increasing concentrations of spent culture filtrate were added to molten 1% cellobiose plus basal salts, ph 5.0, in 1.5% agar to yield final concentrations of 0%, 20%, 40%, 60%, 80%, and 90% (v/v). The media were poured into 55-mm-diameter plastic Petri plates and inoculated with 5-mm-diameter plugs taken from the margins of 2-week-old colonies of MAD698 on 2% MEA. Control plates contained sterile, deionized water in the place of culture filtrate. The cultures were incubated at 27 C and examined for the presence of aerial hyphae at 5, 7, and 21 days. Time-course study Isolate ME20 was grown in 10 replicate 250-ml Erlenmeyer flasks containing 25 ml of non-aerated 1% cellobiose plus basal salts, ph 5.0 (Highley, 1973) for 1,2,3,4,6, and 8 weeks at 27 C. The spent media were harvested by centrifugation and assayed for ph, protein, laminarinase, *The use of trade or firm names in this publication is for reader information and does not imply endorsement by the US Department of Agriculture of any product or service.

288 Jessie A. Micales TABLE 1 Characteristics of 60-day-old Spent Culture Filtrate from Cultures of ME20 and MAD698 and their Effect on Mycelial Growth, ph, and Autolytic Enzyme Production by MAD698 Incubated for 21 days at 27 C a

Suppression of aerial hyphae by Postia placenta 289 TABLE 2 Effect of ME20 Spent Culture Filtrate on Growth of MAD698 in Agar Culture protease, chitinase, and N-acetylglucosamine, as described later. Spent media were then incorporated into fresh media in 50-ml Erlenmeyer flasks to give final concentrations of 20%, 40%, 60%, 80%, and 100% in a final volume of 5 ml. All media were inoculated with 5-mm-diameter mycelial plugs of MAD698, and the cultures were incubated for 3 weeks at 27 C. The amount of aerial mycelium was estimated on a four-point visual-rating scale (Tables 1 and 2), and the dry weight of mycelium was determined by vacuum filtration on tared filter paper. Separation and treatment of spent culture filtrate Sixty-day-old spent culture filtrate was collected from ME20, as previously described, and separated into components of greater and less than 10 000 Da using an Amicon Diaflo ultrafiltration membrane. The spent culture filtrate was added to fresh media, ph 5 0, in three replicate

290 Jessie A. Micales 50-ml Erlenmeyer flasks, each containing 5 ml of media. for final concentrations of 20%, 40%, 60%, 80%, and 100% (v/v). Control flasks contained spent culture filtrate that had not been separated. All media were inoculated with 5-mm-diameter mycelial plugs of MAD698, and the cultures were incubated for 3 weeks at 27 C. The amount of aerial mycelium was estimated on the four-point scale (previously described). Sixty-day-old filter-sterilized spent culture filtrate from ME20 was boiled for 10 min before it was added to fresh media, ph 5.0, in 50-ml Erlenmeyer flasks, as described above. Control flasks contained spent media that had not been boiled. All media were inoculated with 5-mmdiameter mycelial plugs of MAD698, and the cultures were incubated for 3 weeks at 27 C. The amount of aerial mycelium was again estimated on the four-point scale. Dosage-response curves of commercial laminarinase and protease The effect of commercial preparations of laminarinase and protease on the aerial growth of MAD698 was examined by the formation of dosageresponse curves. Increasing concentrations of laminarinase or protease were added to 5 ml 1% cellobiose plus basal salts, ph 54, in 50-ml Erlenmeyer flasks to yield concentrations of 0-50 000 units/ml or 0-100 units/ml, respectively. Three replicate flasks were prepared for each treatment. The media were inoculated with 5-mm-diameter mycelial plugs of MAD698 and incubated for 3 weeks at 27 C. The amount of aerial mycelium was estimated on the four-point scale. and the dry weight of mycelium was determined by vacuum filtration on tared filter paper. Enzyme assays General extracellular proteinase activity was determined by the hydrolysis of azocasein using the procedure of Prestidge et al. (1971) as described by Venables and Watkinson (1989). Culture filtrates were separated by centrifugation at 5000 g for 15 min. All assays were run in 0 1M citrate-phosphate buffer, ph 4.5, for 3 h. Enzyme activity was expressed as that equivalent to the amount of azocasein hydrolyzed per hour by a known quantity of a commercial protease (Type XIX from Aspergillus sojae, Sigma Chemical Company). Chitinase (E.C. 3.2.1.14) activity was determined using the procedure of Reissig et al. (1955) to detect N-acetylglucosamine. Purified chitin from crab shells (Sigma Chemical Company) was suspended in 0 1M McIlvaine buffer, ph 5 1, at the rate of 0 8 mg/ml. Enzyme activity was

Suppression of aerial hyphae by Postia placenta 291 determined by incubating 1 ml culture filtrate with 2 ml of the chitin suspension for 2 h at 37 C. The reaction was stopped by boiling, and the reaction mixture colorimetrically assayed for the presence of N acetylglucosamine (Reissig et al., 1955). Culture filtrate was boiled before the addition of substrate as a control for background coloration. One unit of enzyme activity was defined as the amount of enzyme needed to release 1 µg N-acetylglucosamine per hour at 37 C and ph 5 1. The concentration of N-acetylglucosamine in spent culture filtrate was calculated using the same procedure but without incubating the sample in the presence of chitin. Dilutions of N-acetylglucosamine (l-200µg/ml, Sigma Chemical Company) were used to form the standard curve for both tests. The activity of endo-ß-1,3-glucanase (laminarinase) (E.C. 3.2.1.6) was assayed by measuring the increase in reducing groups using Nelson's modification of the Somogyi method (Nelson, 1944).One unit of enzyme activity was defined as the amount of enzyme needed to liberate reducing power equivalent to 1 µg of glucose per hour at 40 C. The Lowry technique (Lowry et al., 1951) was used to determine the concentration of extracellular proteins. Dilutions of bovine serum albumin (20-350µg/ml) were used to form the standard curve. RESULTS The formation of aerial mycelium by MAD698 was inhibited when spent culture filtrate from ME20 was incorporated into a basal medium at concentrations of >20% (Table 1, Fig. 1). Inhibition was severe at 40%, and aerial hyphae formation was totally prevented at 60%,80%and 100%. The inhibitory properties of spent culture filtrate from ME20 varied somewhat from preparation to preparation, but severe inhibition of aerial hyphae formation always occurred at concentrations of >60%. The production of aerial hyphae by MAD698 was also greatly inhibited in the presence of its own spent media. Aerial hyphae formation was suppressed when spent culture filtrate from 60-day-old cultures of MAD698 was incorporated into the medium at 80% and 100% concentrations (Fig. 2). Dilution of the growth medium with water did not inhibit aerial hyphae development; a few aerial hyphae even formed in pure water (Fig. 3). Fungal dry weight and extracellular protein levels decreased when water made up 80 and 100%of the medium. Protease and laminarinase concentrations did not vary significantly when the medium was diluted with water. Spent media from 60-day-old cultures of ME20 were not as acidic as

292 Jessie A. Micales Fig. 1. Growth of MAD698 in liquid medium supplemented with spent culture filtrate from 60-day-old cultures of ME20. Concentration of spent culture filtrate (left to right): 100%, 80%, 60%, 40%, 20%, and 0% (v/v). Fig. 2. Growth of MAD698 in liquid medium supplemented with spent culture filtrate from 60-day-old cultures of MAD698. Concentration of spent culture filtrate (left to right): 100%, 80%, 60%, 40%, 20%, and 0% (v/v). were those from MAD698 (Table 1). The ME20 culture filtrate contained larger quantities of protease than did spent media from MAD698; laminarinase activity and extracellular protein concentration were equivalent in both preparations. The growth of MAD698 in the spent culture filtrates of both isolates generally increased the ph of the media. Cultures of MAD698 growing in its own spent media produced more extracellular protein and lower ph values than did those growing in

Suppression of aerial hyphae by Postia placenta 293 Fig. 3. Growth of MAD698 in liquid medium diluted with sterile. deionized water. Concentration of water (left to right): 100%, 80%, 60%, 40%, 20%, and 0% (v/v). spent culture filtrate from ME20. The addition of culture filtrate from ME20 did not significantly change the amount of extracellular proteins produced by MAD698, but the addition of culture filtrate from MAD698 was stimulatory. Laminarinase and protease levels were fairly uniform after incubation. Protease levels in cultures amended with spent media from ME20 decreased from those quantities initially present in the undiluted spent culture filtrate. The amount of mycelial dry weight obtained from MAD698 grown in increasing concentrations of spent media from either isolate did not necessarily correlate with the amount of observed aerial mycelia (Table 1). For example, when spent culture filtrate from MAD698 made up 40-60% of the growth medium, aerial hyphae formation only slightly decreased but dry weight decreased 50-75%. In contrast, colonies of MAD698 growing in 60%,80%,and 100%of spent media from ME20 produced no aerial mycelia but still formed measurable amounts of total mycelial weight. This mycelial mass is from submerged, rather than aerial, hypha e. The effect of spent culture filtrate from ME20 and MAD698 on aerial growth in agar culture is shown in Table 2 and Figure 4. The addition of the spent medium from either culture did not inhibit the growth rate (measured as colony diameter) of MAD698; in fact, spent medium appeared to stimulate the growth in 5-day-old cultures. At 7 days, all plates were overgrown. The addition of ME20 spent media inhibited the development of aerial hyphae when the spent culture filtrate made up >60% of the medium. The colony morphology of these cultures resembled the appressed appearance of ME20. Inhibition of aerial hyphae occurred with spent media from both 6- or 10-week-old cultures. A slight inhibition of aerial growth was also observed in the plates diluted with water, but the effect was not as severe.

294 Jessie A. Micales Fig. 4. Growth of MAD698 on agar medium supplemented with spent culture filtrate from 10-week-old cultures of ME20. Top row (left to right): 0%, 60%, 80%, and 90% (v/v) ME20 spent culture filtrate. Bottom row (left to right). ME20 on non-amended growth medium: MAD698 on 60%, 80%, and 90% sterile, deionized water. The length of time that ME20 grew in the medium affected its inhibitory properties (Table 3). Little inhibition of aerial mycelia formation occurred when MAD698 was grown in undiluted (100%)spent media collected from 7-day-old cultures of ME20. Inhibitory effects were observed with spent culture filtrate derived from 14-day-old cultures of ME20. This inhibition was not as pronounced when spent media were obtained from 21- or 28-day-old cultures except at concentrations of 80% or 100%.Spent culture filtrates from 6- and 8-week-old cultures were more inhibitory than were those from younger cultures and repeated the patterns of inhibition displayed in Table 1. The ph, extracellular protein concentration. and autolytic enzyme activity of spent culture filtrate from ME20 varied with the length of time that ME20 grew in the medium (Table 4). The ph declined rapidly, dropping from an initial reading of 5 00 before inoculation to 2 55 on day 14. After this time. the ph gradually returned to 3 0 at 4 weeks and remained at this level for the remainder of the experiment. The concentration of extracellular proteins and proteases detected in the ME20 spent culture filtrate also gradually increased until week 4 and then levelled off. Laminarinase activity was highest at weeks 2, 6, and 8, which corresponds to the times in which the spent media was the most inhibitory. Extracellular chitinase activity was not detected in any culture filtrates. Quantities of N-acetylglucosamine, indicative of autolytic decomposition of fungal cell walls, gradually increased in the culture filtrate throughout the experiment (Table 4). Boiling the spent culture filtrate from ME20 did not alter its ability to inhibit the formation of aerial hyphae in MAD698 (Table 5). The inhibitory properties of the spent medium were partitioned when it was put through a 10 000 molecular weight cut-off filter; the inhibition of

TABLE 3 Effect of ME20 Spent Culture Filtrate Age on Aerial Hyphae Formation and Mycelial Dry Weight of MAD698 a

296 Jessie A. Micales TABLE 4 Characteristics of ME20 Spent Culture Filtrate with Time a TABLE 5 Formation of Aerial Mycelium by MAD698 in Liquid Culture Grown in Increasing Concentrations of Treated and Untreated 60-day-old Spent Culture Filtrate of ME20 and MAD698 a aerial mycelium was strongest in the fraction that had a molecular weight of less than 10 000 Da (Table 5). The pattern of inhibition obtained from this fraction matched that of the unpartitioned control. The fraction that had a molecular weight greater than 10 000 Da displayed slight inhibitory activity at 60% and 80%. This pattern matches

Suppression of aerial hyphae by Postia placenta 297 the inhibitory properties of spent culture filtrate from MAD698 and control media diluted with water. Partitioning the spent media from cultures of MAD698 did not change its inhibitory properties (Table 5). Dosage-response curves were produced to determine the effect of commercial preparations oflaminarinase (Table 6) and protease (Table 7) on the growth and aerial mycelia production of MAD698. The presence of laminarinase in the medium had little or no effect on mycelial growth even at excessively high concentrations. Aerial hyphae formation was slightly decreased at 30 000 units/ml; however, this was far in excess of laminarinase concentrations in the spent culture filtrate from ME20. Mycelial dry weight production was slightly stimulated in the presence of excessive laminarinase (5000-30 000 units/ml). The commercial laminarinase may have broken down rapidly during incubation; only minor amounts of enzyme activity were detected in the culture filtrate at the end of the experiment. Protease from Aspergillus sojae dramatically inhibited the formation of aerial mycelium in MAD698 at concentrations of >10 units/ml. Although the formation of aerial mycelium was inhibited in the presence of protease, the production of submerged TABLE 6 Dosage-Response Curve of MAD698 to Commercial Laminarinase a

298 Jessie A. Micales TABLE 7 Dosage-Response Curve of MAD698 to Commercial Protease hyphae was greatly stimulated (a 550% increase in dry weight at a protease concentration of 100 units/ml). DISCUSSION The spent culture filtrate from ME20 inhibited or prevented the development of aerial mycelium in isolate MAD698, a normally floccose strain ofp. placenta. This phenomenon also occurred with spent culture filtrate from MAD698, but only at high concentrations. The suppressive agent has a molecular weight less than 10 000 Da and is resistant to boiling. The appressed morphology of ME20 does not seem to be caused by the excessive production of the autolytic enzymes laminarinase and protease. Commercial preparations of laminarinase in excessivequantities (<20000 units/ml) did not affect the formation of aerial hyphae. In addition, ME20 and MAD698 produced comparable quantities of this enzyme at 60 days, thus eliminating laminarinose as a possible cause of the inhibitory properties of ME20. The spent culture filtrate from ME20

Suppression of aerial hyphae by Postia placenta 299 contained higher levels of protease than did that of MAD698. Protease activity may be partially responsible for the suppression of aerial hyphae by the ME20 culture filtrate. Commercial preparations of protease greater than 10 units/ml inhibited or prevented aerial hyphae formation in MAD698; the spent culture filtrate of ME20 contained 17 units/ml of protease. However, some factor other than protease production must be involved because the suppressive agent was unaffected by boiling. Fungal proteases cannot survive 10 min of boiling (Micales, unpublished data) and have a molecular weight greater than 10 000 Da (North, 1982). The suppressive agent (or agents) is apparently non-enzymatic. The inhibition of aerial hyphae formation by the spent culture filtrates in this series of experiments is probably caused by several different factors. The prevention of growth at high concentrations of spent media from MAD698 is probably due to diluting the growth medium with material low in nutrients, as observed in control flasks diluted with deionized water. This would also explain why MAD698 failed to form aerial hyphae in the fraction of ME20 spent media greater than 10 000 Da. The prevention of aerial hyphae formation by ME20 is likely due to the accumulation of a low molecular weight, suppressive staling product (or products) in the culture filtrate. Production of the suppressive agent by ME20 cultures is apparently somewhat variable, as demonstrated by the slightly different inhibitory levels among experiments. Significant amounts of the suppressive agent are present in 14-day-old cultures. This corresponds to the beginning of autolysis in the ME20 growth curve (Micales & Highley, 1989). The inhibitory agent appears to be fairly stable because it is evident in spent media from 10-week-old cultures. In recent years. little research has been conducted on the topic of fungal staling. Reviewers agree that it is a complex process that may involve different, interacting factors (Griffin, 1981; Hawker, 1968; Prosser, 1983). The phenomenon was first described in the 1920s, when Brown (1923) differentiated between staling fungi that showed a decline in radial growth with age and non-staling fungi that were not so affected. The staling process has been ascribed to bicarbonates (Pratt, 1924a,b) and various acids (Park, 1961), including amino acids (Plunkett. 1966). succinic acid, and fatty acids (Pratt, 1924a; Robinson, 1972). The ph of the spent culture filtrate from ME20 (ph 2 8) was consistently greater than that of the spent culture filtrate of MAD698 (ph 2.4) (Table 1). This disparity of 0 4 ph units represents a 2 5-fold difference in the concentration of hydrogen ions. The ability to form an extremely acidic ph may be critical for the development of brown rot. It has long been known that brown-rot fungi acidify wood. primarily by the

300 Jessie A. Micales production of oxalic acid (Cowling, 1961). A ph of 3.4 was reported (Cowling, 1961) for hot water extracts of wood decayed by P. placenta. The ph of liquid fungal cultures can be much more extreme, perhaps because of the absence of the buffering capacity present in wood. Shimada et al. (1991) reported a ph of 1.9 in 3-week-old culture filtrates of Fomitopsis palustris (Berk. & Curt.) Gilbn & Ryv. Isolate ME20 may be unable to produce the high acidity necessary for the development of brown rot. Micales and Highley (1989) demonstrated that ME20 produced large amounts of oxalic acid in 1%) carboxymethylcellulose plus basal salts. Preliminary experiments (Green, unpublished data) suggest that ME20 may not be able to form oxalic acid in the presence of other carbon sources. Pratt (1924a) reported that oxalic acid in staled cultures of Fusarium sp. was not as effective in inhibiting the germination of Botrytis spores as were other organic acids, including acetic, propionic. and butyric acids. Experiments are in progress to determine the concentration and composition of organic acids produced by ME20 in the presence of a variety of nutrients. The appressed morphology of ME20 may be related to aberrant chemical and physical features of its hyphal sheath. Preliminary studies with scanning electron microscopy (Green et al.. 1991)have revealed that ME20 forms a hyphal sheath that does not contain abundant, organized mycofibrils, as observed in MAD698, but that does contain atypical sheet-like structures of unknown composition. An aberrant hyphal sheath may be responsible for the appressed colony morphology of ME20. A disfunctional sheath may also prevent the strain from effectively decaying wood, either by failing to protect the organism from desiccation or by preventing the orderly delivery of enzymes and other degradative agents to the substrate. More research is needed to characterize the staling products that suppress aerial hyphae formation by ME20 in liquid and agar culture and to determine whether they are involved with the compromised decay ability of ME20. These agents may act as relatively non-toxic inhibitors of wood-decay fungi. REFERENCES

Suppression of aerial hyphae by Postia plancenta 301

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