Mycologia, 81(2), 1989, pp. 205-215. 1989, by The New York Botanical Garden. Bronx, NY 10458-5126

PHYSIOLOGICAL CHARACTERISTICS OF A NON-DEGRADATIVE ISOLATE OF POSTIA

(≡ PORIA) PLACENTA 1

JESSIE A. MICALES AND TERRY L. HIGHLEY

U.S. Department of Agriculture, Forest Service, Forest Products Laboratory,2

One Gifford Pinchot Drive, Madison, Wisconsin 53705-2398

ABSTRACT

Decay capacity of 14 strains of the brown-rot fungus Postia (≡ Poria) placenta was determined using soil-wood block tests. One monokaryotic isolate, ME20, was identified as being unable to degrade wood. It retained the ability to produce extracellular carbohydrate-degrading enzymes. although levels of glycosidases and carboxymethylcellulase were atypical under certain cultural conditions. The elec-trophoretic protein profile of this isolate varied from degradative strains. It produced H2O2 and oxalic acid under a variety of carbon and nitrogen regimes and did not contain double-stranded RNA. An understanding of the physiology of this isolate would further our knowledge of decay mechanisms leading to safer preservation protocols. Key Words: wood decay, brown rot, biodeterioration, Postia.

The brown-rot fungus Postia placenta (Fr.) M. J. Lars. et Lomb. (≡ Poria plancenta Fr.) is an economically important degrader of wood and wood products. This fungus and other wood-de-cay fungi are currently controlled by applying broad-spectrum biocides, many of which are re-ceiving restricted use because of their extreme toxicity. A better understanding of the physio-logical mechanisms of decay may assist in de-veloping specific metabolic controls that would target the decay fungus but would not affect other organisms.

The mechanisms of decay by brown-rot fungi are not fully understood. Brown rotters are able to metabolize the cellulose and hemicellulose of wood but are unable to substantially metabolize lignin. White-rot fungi, by contrast, metabolize all three components of wood. Brown-rot fungi create a low pH while decaying wood. primarily by producing oxalic acid (Cowling, 1961). White-rot fungi also produce oxalic acid, but they me-

1 A portion of this study was presented at the sym-posium, "Current Topics in Forest Research: Empha-sis on Contribution by Women Scientists." USDA For-est Service, S.E. Station, and Department of Forestry, University of Florida, Gainesville, Florida: November 4-6, 1986.

2 The Forest Products Laboratory, is maintained at Madison, Wisconsin, in cooperation with the Univer-sity of Wisconsin. This article was written and pre-pared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright.

tabolize it so the pH of the wood does not drop. Brown rot is characterized by a rapid depoly-merization of cellulose early in the decay process (Cowling, 1961). This is thought to be nonen-zymatic because enzymes are too large to pene-trate the wood and reach the glycosidic bonds of the cellulose (Cowling and Brown, 1969; Highley et al., 1983). One hypothesis is that depolymer-ization is caused by the generation of free radicals from the reaction of hydrogen peroxide, which is produced by the fungus, and ferrous ions, which are formed from the reduction of ferric ions in the wood by oxalic acid (Cowling and Brown, 1969; Koenigs. 1972, 1974a, b; Schmidt et al., 1981). Extracellular. carbohydrate-degrading en-zymes are also produced by these fungi and may result in the final breakdown and removal of cellulose and hemicellulose from the wood, al-though the role of these enzymes has not yet been fully explained (Kirk and Cowling, 1984).

One way to study the mechanisms of wood decay is to examine the physiology of fungal iso-lates that have lost their ability to degrade wood. Occasionally. stock cultures become less vigor-ous and are no longer able to decay wood. This condition may be caused by (1) mutation (Am-burgey, 1969); (2) changes in the ratio of nuclear types within an isolate (Amburgey, 1969), (3) viruses (Castanho et al., 1978; Day and Dodds, 1979; Hammar et al,. 1986; Lemke, 1977); (4) bacteria- or mycoplasma-like organisms (Wilson and Hanton, 1979); (5) specific chromosomes working with plasmids (Esser and Tudzynski,

205

206 MYCOLOGIA

TABLE I COLLECTION DATA OF POSTIA PLACENTA ISOLATES RANKED BY THEIR ABILITY TO DEGRADE SOUTHERN YELLOW

PINEa

Collec- Weight tion loss

Isolate number date Source and location (%)

TRL2556 3/50 Mineshaft support; Transvaal, Africa 68.2 RLG3760R 7/63 Betula alleghaniensis Britton; Newcomb, NY 65.9 L9138sp MD484

8/58 -

Pinus ponderosa Laws.; Coronado National Forest, AZ -

65.4 62.0

MAD575 = ATCC9891 10/20 Pseudotsuga menziesii (Mirb.) Franco; Oregon 60.8 MAD698 = ATCCl1538 12/41 Pseudotsuga menziesii; yacht "America'' 60.4 ME48 11/63 Pseudotsuga menziesii: Clark Co., WA 60.3 ME146 2/62 Telephone pole; Clark Co., WA 60.0 FP100086sp 3/49 Conifer sills and piles; Fairfield; OH 59.5 MD281 11/51 Pseudotsuga menziesii; Oregon 59.4 BTL-V-10 2/52 Philadelphia, PA 58.3 MD506 MAD4874

4/62 2/50

Pinus sp.; Madison, WI Tsuga heterophylla (Raf.) Sarg.; Madison, WI

57.2 52.5

ME20 - Floor planking; Pleasant Hill, CA 5.1 a Degradative ability determined by the ASTM soil-wood block assay.

1979; Esser et al., 1984); or (6) mitochondrial DNA (Esser et al., 1984).

In this study, a non-degradative isolate of P. placenta was identified and analyzed for its abil-ity to produce extracellular carbohydrate-de-grading enzymes, oxalic acid. and hydrogen per-oxide. Our objective was to determine whether the loss of these or other processes could be cor-related to the inability of the isolate to decay wood.

MATERIALS AND METHODS

Maintenance of fungal cultures. -All cultures were maintained on 2% malt extract agar (MEA) slants at 4 C. Pertinent data for each isolate are listed in TABLE I.

Identification of non-degradative isolates. -Iso-lates of P. placenta were evaluated for their abil-ities to degrade wood by the standard ASTM soil block method (ASTM. 1971) using southern pine blocks (25.4 x 25.4 x 3.2 mm. the long axis parallel to the grain). Soil block bottles were in-cubated at 27 C and 70% relative humidity for 12 wk. At the end of the period, the percent weight loss of each block was determined.

Determination of fungal morphology and growth rate.-Cultures of P. placenta were grown on 2% MEA. The characteristics of individual hyphae were observed by light microscopy after 3 and 7 wk. Gross colony morphology was described. Growth curves were produced by inoculating 2%

MEA plates with 5 mm diam inoculum plugs taken from the margins of 7 da old cultures that were also grown on 90 MEA. Colonies were in-cubated at 27 C for 2 wk; radial growth of each colony was measured after 2, 6, 11, and 13 da.

Growth and enzyme activity in liquid culture. -Isolates were grown in 25 ml stationary liquid cultures in 350 ml Erlenmeyer flasks with 0.5% cellobiose in a basal salts solution (Highley, 1973). Each flask was inoculated with a 5 mm diam mycelial plug taken from the margin of 7 da old colonies grown on 2% MEA.

Culture filtrates were collected by vacuum fil-tration through glass-fibered filter paper, di-alyzed overnight against deionized water, and assayed for general extracellular proteins (Lowry et al., 1951) using dilutions of bovine serum al-bumin for the standard curve. The activities of α-D-galactosidase (E.C. 3.2.1.22): β-D-galacto-sidase (E.C. 3.2.1.23). and β-D-glucosidase (E.C. 3.1.1.21), measured as aryl β-D-glucosidase, were determined by the liberation of p-nitrophenol from the respective p-nitrophenol substrate (Agrawal and Bahl. 1968). One unit of enzyme activity was defined as the amount of enzyme needed to release 1 µmol of p-nitrophenol per hour at 40 C. The activities of endo-β-1.4-glu-conase (carboxymethylcellulase) (E.C. 3.2.1.4) and endo-b-1.4-xylanase (E.C. 3.2.1.37) were as-sayed by measuring the increase in reducing groups using Nelson's modification of the So-mogyi method (Nelson. 1944). One unit of en-

MICALES AND HIGHLEY:

zyme activity was defined as the amount of en-zyme needed to liberate reducing power equivalent to 1 µmol of glucose per hour at 40 C.

Cellulolytic enzyme activity was also mea-sured by the degradation of cellulose azure as described by Highley (1983). Degradative and non-degradative isolates were grown in vertical tubes of a basal salts medium (Highley, 1973) in which sterilized cellulose azure had been asep-tically placed onto the agar surface. Enzyme ac-tivity was estimated by the amount of dyed cel-lulose degradation products released into the agar medium. Color intensity was estimated on a 0 to 4 scale, with 0 representing no coloration (i.e., no cellulase activity) and 4 representing an in-tense blue (i.e., high levels of cellulase activity).

Production of H2O2.-The production of H2O2

was detected using a modification of Mueller’s (1984) procedure. A filter-sterilized stock solu-tion of 2,2'-azino-di(ethylbenzthiazoline-6-sul-phonic acid) (ABTS) and horseradish peroxidase was added to 50 ml Erlenmeyer flasks containing 5 ml of a sterilized nutrient broth, pH 5.8, for a final concentration of 0.024 mg/ml peroxidase and 0.64 mM ABTS. The nutrient broth con-sisted of 55.5 or 5.5 mM glucose or mannose, or 66.6 or 6.1 mM xylose or arabinose in a basal salts solution, with or without nitrogen (Highley, 1973). Each flask was inoculated with 0.2 ml of a hyphal suspension, which was prepared by blending 10 da old mycelium grown in 1% cel-lobiose + basal salts in 100 ml sterile, deionized water.

Production ofoxalic acid.-Selected isolates were grown in 25 mi stationary cultures in 250 ml Erlenmeyer flasks with 1.0% carboxymethylcel-lulose in basal salts. After 3 wk, culture filtrates were collected by vacuum filtration. Oxalic acid content of the filtrates was detected at 230 nm by HPLC chromatography using an HPX-87 (Bio-Rad) column at 75 C. Samples containing oxalic acid were used as standards.

Isolation of dsRNA. - Degradative isolates (MAD698, RLG3760R, TRL2556, L9 138sp) and a non-degradative isolate (ME20) of P. placenta were tested for the presence of double-stranded RNA (dsRNA). Each degradative isolate was grown on 1% cellobiose + basal salts agar over-laid with cellophane in 135 mm diam Petri plates. The non-degradative isolate (ME20) was grown in liquid culture (1% cellobiose + basal salts in

NON-DEGRADATIVE POSTIA 207

2 L Erlenmeyer flasks) because it did not produce aerial hyphae. The cultures were incubated for 20 da at 27 C.

Double-stranded RNA was extracted using Morris and Dodds’ (1979) procedure. Gels (5% acrylamide/0.4% bis-methylene-acrylamide, 0.75 mm thick) were run on a Protean II vertical slab gel apparatus (Bio-Rad). Nucleic acids were stained with ethidium bromide (1 µg/ml) and viewed on a UVP Transilluminator (254 nm). The extraction procedure was repeated two ad-ditional times for each isolate. Positive dsRNX controls were barley infected with barley mosaic virus and Phytophthora infestans (Mont.) deBary isolates 519 and 547 (USDA-ARS Foreign Dis-ease and Weed Science Research Unit, Freder-ick, Maryland).

Electrophoresis of fungal proteins.-The electro-phoretic banding patterns of intracellular pro-teins were compared between degradative (MAD698, TRL2556, RLG3760R) and non-degradative (ME20) isolates of P. placenta. The fungi were grown with aeration in 1% cellobiose + basal salts for 7 da. Mycelia were harvested by centrifugation and frozen in liquid N2. Cells were broken by grinding the frozen mycelium with a cold mortar and pestle. Enzymes were extracted from the hyphae with 0.1 ml 0.5 M Tris-C1 buffer (pH 6.8); the crushed hyphae pro-vided additional liquid for a final concentration of approximately 0.05 M Tris-C1. The suspen-sions were centrifuged (2000 × g) for 20 min. Glycerol, β-mercaptoethanol and SDS were added to the supernatant to make final concentrations of 10%, 0.1% and 1.0%, respectively. Samples were boiled for 2 min and 5 µl of 1% bromo-phenol blue were added to each sample. Vertical slab gel electrophoresis was conducted using the Laemmli (1970) buffer system; 200 µl of each sample was loaded onto the gel. General proteins were detected by staining the gels with 0.1% Coomassie blue as described by Hames (1981). Polysaccharides were visualized with the period-ic acid-Schiffstain (Johnston and Thorpe, 1982). Electrophoretic patterns were traced with a Shi-madzu Dual-wavelength Thin-layer Chromato Scanner (Model CS-930).

RESULTS

Identification of non-degradative isolates. -The source of P. placenta isolates and their ability to

208 MYCOLOGIA

FIG. 1. Culture morphology of MAD698 (above) and ME20 (below) on 2% malt extract agar after 6 wk at 27 C.

decay southern yellow pine are presented in TA-BLE I. One isolate. ME20, was unable to degrade wood. We termed this isolate non-degradative and used it in subsequent tests with selected deg-radative isolates.

Colony morphology and growth rate.-The gross colony morphology of ME20 was very different from that of the other cultures, as shown in FIG. 1. Degradative isolates were white, robust, and floccose; they produced so many aerial hyphae that a thick mat was formed. Isolate ME20 was thin. appressed, and did not produce white cot-ton!. mats. Aerial growth was so sparse that the colony took on the color of the medium.

Studies with light microscopy showed that ME20 was monokaryotic; it lacked the clamp connections found in the dikaryotic, degradative

ML88 5406

FIG. 2. Radial growth (mm/da) of degradative and non-degradative isolates of P. placenta on 2% malt extract agar. Solid line represents the degradative iso-lates; broken line represents the non-degradative iso-late.

strains. All cultures produced chlamydospores by 7 wk. Isolate ME20 lacked many of the thin, generative hyphae that made up the majority of the mycelium of the other strains. Its hyphae were generally bigger and involved in chlamydo-spore production.

We paired isolate ME20 with the monokar-yotic standards of P. placenta, L-8035 A and B mating types, to confirm its identification. A di-karyote was formed with the A mating type. Iso-late ME20 also formed a dikaryotic-monokar-yotic cross with isolate MAD698.

We found that the growth rate of P. placenta isolates in culture was not related to their ability to degrade wood. All isolates of P. placenta ex-hibited similar growth rates on 2% MEA (FIG. 2).

Growth and extracellular enzyme activity in liq­uid culture -Mycelial weight, pH, protein and enzyme activities of isolates grown in 0.5% cel-lobiose + basal salts are presented in TABLE II. All cultures dramatically lowered the pH of the culture filtrates from an initial reading of 4.5. The extent of this decrease was usually related

MICALES AND HIGHLEY: NON-DEGRADATIVE POSTIA 209

TABLE II

ENZYME PRODUCTION OF POSTIA PLACENTA ISOLATES IN 0.5% CELLOBIOSE + BASAL SALTS SOLLITIONa

Dryweight Enzyme production (units/mg protein)c

Isolate (mg) pH Proteinb β-GLU α-GAL β-GAL XYL CMC

TRL2556 22cd 3.37a 1.56bc 2.230b 4.626a 0.341cd 293a 94a RLG3760R 27b 3.02c 1.26c 0.538c 1.141d 0.000d 301a 70b L9138sp MAD698

48a 26b

2.98c 3.27b

0.90d 1.78b

0.312c 0.570c

2.473c 0.963d

0.698bc 0.957b

197b 226b

54b 53b

ME20 30b 2.55d 3.46a 5.328a 3.700b 5.962a 144c 27c a Cultures incubated at 27 C for 4 wk. All readings represent an average of five replications. Isolates arranged

in descending order of decay capacity. b Protein production expressed as µg/mg dry weight.

Abbreviations represent activity of the following enzymes: β-GLU = β-D-glucosidase: α-GAL = α-D-galac-tosidase; β-GAL = β-D-galactosidase: XYL = β-1,4-D-xylanase; CMC = β-1,3-D-glucanase (carboxymethyl-cellulase).

d Readings with the same letter within a column are by the Student-Newman-Keuls test (SNK) at p = 0.05

to mycelial growth; those isolates with the great-est mycelial weight generally produced the lowest pH. The non-degradative isolate was one excep-tion: it was the most acidic, although it did not produce the greatest amount of mycelium. Iso-late ME20 yielded much higher quantities of gen-eral extracellular proteins per unit dry weight of mycelium than the degradative isolates. This was observed in the elevated levels of the glycosi-

not significantly different from each other when analyzed

dases. β-D-glucosidase and α- and β-D-galacto-sidase. In some cases, extracellular glycosidase production of ME20 was 10 to 20 times that of the degradative isolates. This was not true for the glycanases, xylanase and carboxymethylcel-lulase, in which extracellular levels were similar to the degradative isolates.

Degradative and non-degradative isolates of P. placenta also grew in a basal salt medium

TABLE III ENZYME PRODUCTION BY ISOLATES OF POSTIA PLACENTA ON SWEETGUM SAWDUST (1%) ± 0.5% CELLOBIOSEa

Pro- Enzyme production (units/ml)c Treat- tein mentb Isolate PH (µg/ml) β-GLU α-GAL β-GAL XYL CMC

A TRL2556 4.30abd 20a 0.005a 0.032a 0.005a 0.7a 0.4a RLG3760R 4.44a 20a 0.005a 0.060a 0.005a 1.0a 0.3a MAD698 4.45a 20a 0.005a 0.018a 0.005a 0.7a 0.0a ME10 4.83a 20a 0.005a 0.005a 0.005a 0.2a 0.0a

B TRL2556 4.41a 20a 0.026a 0.437abc 0.111ab 15.5de 1.5ab RLG3760R 427ab 21a 0.023a 0.627bc 0.114ab 12.7cd 3.0bc MAD698 4.83a 20a 0.050ab 0.245ab 0.307c 11.9c 0.3a ME20 1.94a 20a 0.053ab 0.195ab 0.133abc 8.5b 0.6a

C TRL2556 2.86c 80b 0.105b 0.815cd 0.247bc 18.8ef 5.1d RLG3760R 2.93c 79b 0.005a 0.190ab 0.011a 14.2cd 4.4cd MAD698 3.30c 80b 0.197c 1.940e 1.580d 19.2f 3.2bcd ME20 3.54bc 100c 0.870d 1.260d 1.935e 18.1ef 1.0a

a Cultures incubated at 27 C for 4 wk. Each reading represents the average of three replications. Isolates arranged in descending order of decay ability.

b Treatments: A = basal salts solution: B = basal salts solution + 1% sweetgum sawdust: C = basal salts solution + 1% sweetgum sawdust + 0.5% cellobiose.

c Abbreviations represent activities of the following enzymes: β-GLU = β-D-glucosidase; α-GAL = α-D-galactosidase; β-GAL = β-D-galactosidase; XYL = β-1,4-D-xylanase; CMC = β-1,3-D-glucanase (carboxy-methylcellulase).

d Readings with the same letter within a column are not significantly different from each other when analyzed by the General Linear Models procedure at p = 0.05.

c

210 MYCOLOGIA

TABLE IV BREAKDOWN OF CELLULOSE AZURE BY ISOLATES OF POSTIA PLACENTA

Cellulase activity at various times after inoculation

7 da 14 da 21 da 28 da 42 da Isolatesb -C +Cc -C +C -C +C -C +C -C +C

TRL2556 1.0 1.0 1.5 1.5 2.0 1.5 2.0 1.5 2.0 2.0 RLG3760R 2.0 2.5 2.5 2.5 3.0 3.0 3.0 3.0 4.0 4.0 19138sp 1.0 1.0 1.5 2.0 2.0 2.5 2.5 3.0 2.5 4.0 MD484 2.0 1.0 2.0 1.0 3.0 1.0 3.0 1.5 4.0 2.5 MAD575 1.0 1.0 1.5 1.5 2.0 1.5 2.0 2.0 2.5 3.0 MAD698 1.0 1.0 1.5 1.5 2.0 2.0 3.0 3.0 3.0 3.5 ME48 2.0 2.0 3.0 2.5 3.5 2.5 3.5 3.5 4.0 3.5 ME146 2.0 2.0 3.0 3.0 3.5 3.5 4.0 4.0 4.0 4.0 FP100086sp 1.0 1.0 1.5 1.5 2.0 2.0 3.0 3.0 3.5 3.5 MD281 2.0 2.0 3.0 3.0 3.5 3.0 4.0 4.0 4.0 4.0 BTL-V-10 2.0 2.0 3.0 2.5 3.0 2.5 3.0 3.0 3.0 3.5 MD506 1.0 1.0 1.5 1.0 2.0 1.0 2.5 1.5 2.5 2.5 MAD4874 1.0 1.0 1.0 1.0 1.5 1.0 1.5 1.5 1.5 1.5 ME20 0.0 1.0 0.0 0.0 0.0 1.0 0.0 2.0 0.0 1.5

a Activity estimated as following: 0 = no dye release; 1 = very slight color visible; 2 = slight color visible: 3 = moderate color visible; 4 = deep color visible.

b Isolates arranged in descending order of decay ability. c -C= no cellobiose in medium; +C = 0.1% cellobiose in medium.

supplemented by 1% sweetgum sawdust (TABLE 111). Hyphae were observed growing on and around the sawdust particles and were able to gather the sawdust into a loosely connected mat. The presence of sawdust in the medium signifi-cantly increased the amount of xylanase pro-duced by all four isolates. Concentrations of re-maining enzymes, as well as pH and general protein production. were not largely affected.

Addition of 0.5% cellobiose to the sawdust medium increased enzyme production for all cul-tures. It is not known whether this increase was caused by additional mycelial growth or whether greater quantities of enzymes were induced; saw-dust in the medium prevented the measurement of mycelial dry weight. In the presence of cel-lobiose. isolate ME20 formed significantly higher quantities of extracellular proteins than did the other isolates. It also produced elevated quan-tities of glycosidases, as did isolate MAD698. No significant amount ofcarboxymethylcellulase was produced in the presence of cellobiose; the re-maining isolates formed much larger quantities under these conditions. Xylanase levels pro-duced by ME20 were similar to those of the other isolates.

The non-degradative isolate produced a very weak reaction with cellulose azure, but so did certain isolates with good decay ability (MAD4874, TRL3556) (TABLE IV). Unlike the

degradative isolates, ME20 could not degrade cellulose without the addition of 0.1% cellobiose to the medium.

Production of H2O2. -Hydrogen peroxide was produced by degradative and non-degradative isolates of P. placenta under a variety of carbon and nitrogen conditions. We found no correla-tion between H2O2 production and the ability of the isolates to degrade wood. All isolates retained the capacity to produce H2O2, although certain cultures would do so only under limited carbon and nitrogen regimes; MAD698 did not form H2O2 in the presence of 1% mannose or xylose, and TRL2556 produced it only under conditions of low nitrogen and carbohydrate.

Production of oxalic acid. -Oxalicacid produc-tion was not related to the ability of the isolates to degrade wood (TABLE V).

Isolation of dsRNA. -Degradative (MAD698, RLG3760R, TRL2556) and non-degradative (ME20) isolates of P. placenta were assayed for dsRNA. No dsRNA was detected in these cul-tures. although it was detected in barley infected with brome mosaic virus and Phytophthora in­festans isolates 519 and 547.

Electrophoresis of funpal proreins. - Densito-metric scans of the electrophoretic profiles of in-tracellular proteins and carbohydrates for P. pla-

MICALES AND HIGHLEY: NON-DEGRADATIVE POSTIA 211

centa isolates TRL3556, RLG3460R, MAD698 TABLE V and ME90 are presented in FIGS. 3 and 4, re-spectively Although there was some intraspecific variation among the degradative isolates of P. placenta, isolate ME70 showed a strikingly dif-ferent protein profile and was missing or showed reduced quantities of the dominant bands in the center of the gel. This was also true for the car-bohydrate profile. Many proteins of all four iso-lates were glycosylated because similar banding patterns were detected by both stains.

DISCUSSION

The ability of isolates of wood-decay Basid-iomycetes to actually degrade wood can be quite variable, especially among derived monokar-yotic strains. This has been shown for Gloeo­phyllium trabeum (Pers. : Fr.) Murr. (Amburgey, 1967); Flammulina velutipes (Fr.) Karst. (Aschan and Norkrans, 1953); Piptoporus betulinus (Bull.: Fr.) Karst. (Bell and Burnett, 1966); and Heter­ohasidion annosum (Fr.) Bref. (Cowling and Kel-man, 1964). Variation has been shown to in-crease with the number of years of the parent dikaryon in culture (Amburgey, 1969). One iso-late of P. placenta (ME20) in our culture collec-tion exhibited a lack of decay capacity. This iso-late was monokaryotic and produced thin, appressed hyphae. Sparse, appressed hyphae have also been described for non-degradative forms of G. trabeum (Amburgey, 1967). The mono-karyotic state of ME20 should not necessarily affect its ability to decay wood. Monokaryons usually retain their degradative ability and may cause greater weight losses than dikaryotes (Am-burgey, 1967, 1969; Aoshima, 1954). Monokar-yons of Bjerkandera adusta (Willd.: Fr.) Karst. and Coriolus versicolor (L. : Fr.) Quél. were iso-lated from Fagus sylvatica L. logs 2 yr after cut-ting, thus demonstrating that monokaryons can extensively colonize wood (Coates and Rayner, 1985; Williams et al., 1981).

Isolate ME20 retained the ability to produce the extracellular enzymes most frequently asso-ciated with wood decay. Similar results were ob-tained by Amburgey (1969) who was unable to correlate levels of β-D-glucosidase and cellulo-lytic activity with decay capacity of G. trabeum isolates. The regulation of extracellular enzyme production may be altered in ME20. The isolate produced elevated levels of glycosidases and de-creased amounts of carboxymethylcellulase in the presence of cellobiose and was unable to degrade cellulose azure in the absence of cellobiose. More

OXALIC ACID PRODUCTION BY ISOLATES OF POSTIA

PLACENTAa

Mycelial weight Oxalic acid

Isolate (mg) (mg/ml)

TRL2556 10 0.250ab

RLG3760R 8 0.056c LY138sp 7 0.009e MAD698 9 0.033d MAD4874 11 0.061c ME20 12 0.110b

a Cultures grown for 3 wk at 27 C in 1.0% carboxy-methylcellulose + basal salts. Each reading represents the average of three replications. Isolates listed in order of their abilities to cause decay.

b Readings with the same letter are not significantly different from each other when analyzed by the Stu-dent-Newman-Keuls test (SNK) at p = 0.05.

studies are needed to understand the effect of simple sugars on the induction of extracellular enzymes by this isolate.

The electrophoretic protein profile of ME20 differed from those of degradative strains of P. placenta. This may reflect differences in the in-duction and repression of certain enzymes when the organism is growing in 1% cellobiose + basal salts. Other explanations are also possible, par-ticularly since ME20 is monokaryotic. Fewer proteins should be produced by ME20 because it contains only half the genome of the other isolates, which were all dikaryotic. Only one pa-rental band would be observed for proteins cod-ed by heterozygous loci; a dikaryotic strain should contain two bands. Heteromeric bands, formed for dimeric and tetrameric enzymes by the com-bination of proteins coded by different alleles, would also be missing. Isozyme analysis of ME20 and degradative strains of P. placenta is needed to determine the banding patterns of individual enzymes. especially those involved with wood decay.

The non-degradative isolate produced large quantities of H2O2. Although this test with sim-ple sugars may not reflect H2O2 production in vivo, it shows that ME20 can form H2O2 from a variety of carbon and nitrogen reames. We plan additional testing to determine whether ME20 can depolymerize cellulose.

Oxalic acid has been hypothesized to play a role in cellulose degradation by brown-rot fungi (Schmidt et al.. 1981). In this study, the non-degradative isolate, ME70, produced as much or more oxalic acid in liquid culture than all deg-

212 MYCOLOGIA

ML88 5407

FIG 3. Densitometric tracing of electrophoretic protein pattern of degradative and non-degradative isolates of P. placenta a) ME20L; b) MAD698; c) RLG3760R; d) TRL2556.

radative isolates tested with only one exception Neither ME20 nor the degradative isolates of In fact, some of the most potent degradative so- P. placenta contained dsRNA, the nuclear ma-lates produced ven low amounts of oxalic acid terial most frequently associated with fungal vi-on the CMC medium ruses. The presence of dsRNA has not been re-

MICALES AND HIGHLEY: NON-DEGRADATIVE POSTIA 213

ML88 5408

FIG. 4. Densitometric tracing of electrophoretic carbohydrate pattern of degradative and non-degradative isolates of P. placenta. a) ME20; b) MAD698; c) RLG3760R; d) TRL2556.

ported in wood-decay fungi although it has been detected in other Basidiomycetes, and causes a debilitating disease of the commercial mush-room, Agaricus brunnescens Peck (Manno et al., 1976). Amburgey (1969) used crossing expen-ments to demonstrate that cytoplasmic agents, such as dsRNA, were not responsible for the low degradative abilities of certain isolates of G. tra­beum.

Further experiments are planned with the non-degradative isolate. We are particularly interest-ed in whether it produces an extracellular matrix which has been described for many decay fungi and which may be necessary for decay to occur (Palmer et al., 1983a, b). Culture filtrates of ME20

are not as viscous as those of the degradative strains; this may reflect aberrations in the extra-cellular carbohydrate metabolism of the fungus. Discovery of this non-degradative isolate pro-vides an exciting opportunity to unravel the complex processes of decay.

ACKNOWLEDGMENTS

The authors wish to thank Drs. A. D Hewings and P. W. Tooley for their assistance in isolating dsRNA. Mating studies were conducted by Dr. M. J. Larsen and Ms. L. A. Poulle. The manuscript was reviewed by Drs. M. J. Larsen and L. E. Leightley. The authors would also like to thank Mr. A. L. Richter for his excellent technical assistance.

214 MYCOLOGIA

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