SEARCH

SEARCH BY CITATION

Keywords:

  • Laccase;
  • Interspecific interactions;
  • Pleurotus ostreatus;
  • Trametes versicolor;
  • White-rot fungi

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

White-rot fungi are of interest due to their ability to degrade lignin. Lignin-degrading enzymes such as laccase can also degrade xenobiotic compounds. The effects of interspecific interactions between white-rot fungi and other microorganisms on laccase activity was studied in laboratory cultures. Laccase activity in cultures of Trametes versicolor and Pleurotus ostreatus increased significantly after the introduction of soil fungi, bacteria and yeasts or after contact with nonsterile soil. Addition of Trichoderma harzianum to cultures of T. versicolor increased laccase activity more than 40 fold, whereas addition of other soil fungi or bacteria resulted in 2–25 fold increases and the addition of soil or soil extracts led to 10–15 fold increases. No laccase induction was detected after addition of heat or filter-sterilized microbial cultures, soil or soil extract. Increased decolorization of the synthetic dye Remazol Brilliant Blue R occurred in mixed cultures. When T. versicolor was cocultured with other soil microorganisms, the number of colony forming units of the other soil microbes decreased. This effect could not be shown to be caused by laccase. In 16 of 24 species of white-rot fungi tested, laccase increased following the addition of T. harzianum. The increase was only absent in species with no or low laccase production. Co-inoculation of P. ostreatus and T. versicolor resulted in an increase of laccase in the mixed culture.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

White-rot fungi are of particular interest due to their ability to degrade lignin[1]. Lignin-degrading enzymes can also degrade various organic compounds, including xenobiotics. The potential biotechnological use of white-rot fungi has attracted considerable attention during the past few decades [2,3], but among the species tested so far, only a few are potentially useful in contaminated soil. Most in situ biodegradation experiments have used Phanerochaete chrysosporium, Trametes versicolor, or Pleurotus ostreatus[4–7]. These species are characterized by their ability to colonize soil containing an indigenous microflora [2,8].

Under natural conditions, white-rot fungi colonize lignocellulose substrates and their hyphae can penetrate the soil. Soil is also the principal environment for litter-decomposing white-rot fungi. In both cases, these fungi interact with other microorganisms, with most studies focused on interactions with other wood-rotting fungi [9,10] or biocontrol fungi, e.g. Trichoderma sp. [11,12]. Enhanced degradation of xenobiotics occurs when white-rot fungi and indigenous soil microorganisms are cocultured [13,14] and ligninolytic activity in nonsterile soil has been reported [8,15,16]. However, the influence of soil microorganisms on ligninolytic enzymes has never been directly assessed.

Although laccase is involved in the degradation of lignin[17], it seems that this enzyme has a wider range of physiological functions including the defence against stressful conditions [18–20]. Increase in laccase activity has been found during the interactions between two white-rot fungi[21] or a white-rot fungus and a biocontrol species such as Trichoderma[11,22] and it seems that laccase might be generally involved in interspecific interactions of white-rot fungi. The objectives of this study were: (i) to study if the interactions of different microorganisms and the white-rot fungi T. versicolor and P. ostreatus affect the production of laccase by white-rot fungi and their ability to degrade a model xenobiotic compound; (ii) to test the hypothesis that laccase is involved in the response of different white-rot fungi to interspecific interactions and (iii) to study whether there is a connection between laccase production and the survival of soil microorganisms.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Microbial strains and maintenance

The strains of white-rot fungi (Tables 1 and 2) were from the Culture Collection of Basidiomycetes (Institute of Microbiology ASCR, Prague, Czech Republic). The strains of filamentous fungi Acremonium sphaerospermum, Fusarium reticulatum, Penicillium rugulosum, Humicola grisea, and Trichoderma harzianum obtained from Dr. M. Gryndler (Laboratory of Experimental Mycology, Institute of Microbiology ASCR, Prague, Czech Republic) were originally isolated from the soil used in this study. All strains were maintained on GC agar plates (25 g l−1 agar)[23]. For preparation of inocula, fungi were grown on HNHC agar plates (25 g l−1 agar)[15] at 28 °C for seven days. Mycelial agar plugs 7 mm in diameter (cut from the edge of an actively growing colony) were used as inocula for liquid cultures. Escherichia coli K12 (ATCC 29427) and Bacillus subtilis (ATCC 6633) were maintained on LB agar (10 g l−1 peptone, 10 g l−1 NaCl, 5 g l−1 yeast extract, 15 g l−1 agar, pH 7.5), Endomyces magnusii (DMUP 4–1–1) was maintained on MEG agar (20 g l−1 malt extract, 20 g l−1 glucose, 1 g l−1 peptone, 15 g l−1 agar, pH 5.5). For preparation of inocula, bacteria and yeasts were grown in 250-ml Erlenmeyer flasks with 40 ml liquid LB or MEG media on a rotary shaker (28 °C, 150 rpm, dark).

Table 1.  Activity of laccase (U l−1) in mixed cultures of Trametes versicolor, Pleurotus ostreatus and other microorganisms
 Day 12Day 13Day 14Maximum activity at day(%)Induction (days)
  1. Microorganisms were added to growing cultures of T. versicolor or P. ostreatus on day 11 as described in the text. The percentages are based on laccase activity of respective mixed and individual culture at the given day when maximum activity in mixed culture was observed. Bold script indicates values significantly different from control (P < 0.02). Induction length is indicated as the time of significantly increased laccase addition (P < 0.02).

Trametes versicolor CCBAS6140.9 ± 0.20.9 ± 0.23.2 ± 0.3
Trichoderma harzianum16.3± 4.044.2± 6.532.1± 6.51344.2± 6.549003
Acremonium sphaerospermum2.6± 1.312.9± 7.748.0± 18.91861.8± 37.66908
Fusarium reticulatum4.8 ± 3.412.9± 7.956.9± 19.920121.3± 43.21500>10
Humicola grisea5.4 ± 5.813.9± 12.554.7± 14.11888.0± 73.1980>10
Penicillium rugulosum4.9± 1.985.4± 45.1181.4± 74.818223.2± 45.12490>10
Bacillus subtilis1.0 ± 0.21.1 ± 0.24.3 ± 0.72011.5± 3.41402
Escherichia coli2.1± 0.33.1± 0.512.1± 2.31837.2± 8.04109
Endomyces magnusii1.3± 0.21.8± 0.27.1± 1.41823.9± 6.22709
 Soil1.3 ± 0.16.8 ± 7.012.1± 4.118125.7± 104.91400>10
 Soil extract1.0 ± 0.42.2 ± 0.410.7 ± 8.723122.7± 122.21520>10
Pleurotus ostreatus CCBAS4770.8 ± 0.70.8 ± 0.71.8 ± 1.4
Trichoderma harzianum12.0± 5.66.8± 2.47.5 ± 5.91212.0± 5.614602
Acremonium sphaerospermum0.8 ± 0.57.2±3.315.4±9.71524.1 ± 6.411007
Fusarium reticulatum0.7 ± 0.19.8±4.427.0±11.51532.0 ± 13.21620>10
Humicola grisea3.6±0.613.6±5.940.6±11.71440.6 ± 11.72260>10
Penicillium rugulosum2.9±0.711.9±3.535.5±15.01435.5 ± 15.019707
Bacillus subtilis2.3 ± 1.65.9± 4.16.9± 3.81810.9± 1.51140>10
Escherichia coli1.4 ± 0.41.9 ± 1.22.6 ± 2.0203.1±0.71605
Endomyces magnusii14.1± 9.18.1± 2.515.8± 9.42319.3± 11.32320>10
 Soil2.3±0.73.5±1.77.6±2.01518.2 ± 10.48307
 Soil extract2.0±0.52.8±0.66.5±2.8146.5 ± 2.83605
Table 2.  Activity of laccase (U l−1) in mixed cultures of different white-rot fungi and Trichoderma harzianum
 Day 14Maximum activity at day(%)Induction (days)
  1. Homogenized culture of T. harzianum (3 days cultivation, 0.5 ml) was added to growing cultures of white-rot fungi on day 14. The percentages are based on laccase activity of respective individual cultures at the given day. Bold script indicates values significantly different from control (P < 0.02). Induction length is indicated as the time of significantly increased laccase addition (P < 0.02).

  2. an.d. – not detected; detection limit 0.05 U l−1.

Abortiporus biennis CCBAS5216.5 ± 1.82356.8±16.32209
Agrocybe dura CCBAS6409.8 ± 2.616359.1±138.325107
Coriolopsis occidentalis CCBAS740n.d.a2324.1±23.4>5000>10
Daedaleopsis confragosa CCBAS5304.5 ± 0.81714.8±6.55504
Dichomitus squalens CCBAS7508.7 ± 5.61517.4±13.11903
Grifola frondosa CCBAS653n.d.a
Inonotus hispidus CCBAS8100.2 ± 0.2
Irpex lacteus CCBAS6170.4 ± 0.2161.1±0.33802
Lentinus edodes CCBAS3890.6 ± 0.5173.4±1.51802
Lyophyllum ulmosum CCBAS4080.3 ± 0.3
Mycena pura CCBAS817n.d.a216.8±2.5>50006
Phellinus igniarius CCBAS5750.1 ± 0.11712.8±11.6>50004
Pholiota aurivela CCBAS8461.0 ± 0.6
Pleurotus eryngii CCBAS47120.2 ± 5.81556.7±12.42502
Pleurotus ostreatus CCBAS4771.2 ± 0.61512.0±5.614605
Polyporus squamosus CCBAS676n.d.a
Pycnoporus cinnabarinus CCBAS59511.9 ± 6.51536.1±15.93303
Pycnoporus sanguineus CCBAS59617.3 ± 5.71643.6±22.41603
Stereum hirsutum CCBAS6081.1 ± 0.7
Trametes gibbosa CCBAS806n.d.a
Trametes hirsuta CCBAS610n.d.a23357.4±24.7>5000>10
Trametes suaveolens CCBAS611119.0 ± 34.015194.8 ± 18.31301
Trametes versicolor CCBAS61425.9 ± 12.119186.4±21.01210>10
Trichaptum abietinum CCBAS53231.6 ± 4.017121.7±16.03603

2.2Soil samples

Soil samples – agricultural cambisol (pH: 6.89, carbon: 12.04 mg g−1 d.w., hot water extractable carbon: 0.26 mg g−1, nitrogen: 1.23 mg g−1, sulfur: 0.13 mg kg−1, biomass: 0.17 mg C per g d.w.)[24] were from control plots of a long-term fertilization experiments run for 45 years by the Research Institute of Crop Production (Prague, Czech Republic). The soil was stored frozen (−18 °C).

2.3Culture conditions and establishment of mixed cultures

Cultures of white-rot fungi were grown in 250-ml Erlenmeyer flasks containing 40 ml of growth medium. In the experiments with T. versicolor and P. ostreatus, CLN medium (5 g l−1 cellulose, 1 g l−1 KH2PO4, 0.5 g l−1 MgSO4.7H2O, 0.2 g l−1 ammonium tartrate, 0.2 g l−1 NaH2PO4, 50 mg l−1 CaCl2, 50 mg l−1, FeSO4.7H2O, 10 mg l−1 CuSO4.5H2O, 5 mg l−1 ZnSO4.7H2O, 5 mg l−1 MnSO4.4H2O, 25 g l−1 agar, pH 6.0) was used (the same as LNHC medium used by Katagiri et al.[25] except that glucose was replaced by microcrystalline cellulose (5 g l−1) as a carbon source). The composition of this medium reflects environmental conditions with a high C:N ratio when cellulose is the major substrate and simple carbon sources are usually not present. In experiments with different species of white-rot fungi however, HNHC medium was used since not all species could grow on CLN. After 11 d on CLN or 14 d on HNHC at 28 °C, mixed cultures were established by adding to the white-rot fungal cultures 500 μl of microbial cultures, 500 μl of soil extract, or 0.5 g nonsterile soil. These cultures were prepared as follows: (i) E. coli, B. subtilis and E. magnusii were grown in 250-ml Erlenmeyer flasks with 40 ml liquid LB media (MEG in case of E. magnusii) for 24 h on a rotary shaker (28 °C, 150 rpm). The cultures were directly used for inoculation; (ii) cultures of soil fungi were grown in 250-ml Erlenmeyer flasks with 40 ml liquid CLN or HNHC media for 5 d (28 °C, static culture). Immediately before inoculation, the cultures were blended (Ultra-Turrax, IKA, Germany) for 10 s. The homogenized culture was used directly for supplementation; (iii) agricultural soil was removed from the freezer and thawed. The water content was adjusted to 20% dry weight and soil was incubated at 28 °C for three days. Ten grams of soil were supplemented with 40 ml of 25% Ringer solution[26] and mixed. After centrifugation to remove soil particles (500 ×g, 2 min), supernatant was transferred to sterile tubes and immediately used for supplementation of cultures. Cultures supplemented with 500 μl of culture media were used as controls.

Control treatments were prepared as follows: (i) To rule out ligninolytic enzymes production by microorganisms added to white-rot fungi cultures, all microorganisms were added to flasks containing heat-sterilized cultures of the white-rot fungi. (ii) To estimate the effect of microbial biomass and products on ligninolytic enzymes production, T. versicolor and P. ostreatus cultures were supplemented with heat-sterilized (autoclaving, 121 °C, 25 min) or filter-sterilized microbial cultures. All treatments including controls were run in at least four replicates.

2.4Enzyme activity measurements and decolorization of remazol brilliant blue R

Samples (200 μl) of culture liquid were centrifuged (2 min, 12 000 ×g) and the supernatant was used for laccase measurements. Laccase activity was measured by monitoring the oxidation of ABTS[27] in citrate-phosphate (100 mM citrate, 200 mM phosphate) buffer (pH 5.0). The formation of green dye was followed spectrophotometrically (A425). Absorption was measured with a microplate reader (Sunrise, Tecan, Austria) in 1-min intervals for 6 min. The data were recorded by the Magellan program from the same manufacturer. One unit of enzyme activity (U) was defined as the amount of enzyme catalyzing the production of one micromole of colored product per min. Results were analyzed with one-way analysis of variance and t-test (StatistiXL software package, StatistiXL, USA).

In microtiter plates 100 μl of culture liquid sample were combined with 20 μl of 0.2% remazol brilliant blue R (RBBR). Purified T. versicolor laccase (100 μl, 50 U l−1) in citrate-phosphate (100 mM citrate, 200 mM phosphate) buffer (pH 5.0) was also combined with 20 μl of 0.2% RBBR. The decolorization reaction proceeded for 24 h at 28 °C in the dark and A595, the absorbance maximum of RBBR, was measured in a microplate reader (Sunrise, Tecan, Austria). Estimations proceeded in triplicates.

2.5Electrophoresis and fast protein liquid chromatography isolation of T. versicolor laccase

Samples of culture liquid for native electrophoresis were desalted and concentrated using a Microcon centrifugation filter device (Millipore, Bedford, MA). Native polyacrylamide gel chromatography (PAGE) was performed using a 10% polyacrylamide gel. Gels were activity-stained with ABTS or guaiacol[27]. For fast protein liquid chromatography (FPLC), the culture liquid from a 20-d static culture of T. versicolor grown on CLN medium was filtered and concentrated by ultrafiltration using Amicon Stirred Cell (Microcon, Bedford, MA) with 10 kDa cut-off NMWL membrane (Sigma, Deisenhofen, Germany). The concentrate was loaded onto a DEAE-Sepharose column (Pharmacia LKB, Sweden, HR 10/10) equilibrated with 20 mM phosphate buffer, pH 7.0. Proteins were eluted with a gradient from 0 to 1 M NaCl in 20 min at a flow rate of 1 ml min−1. The laccase fractions were pooled, desalted and applied to a Mono Q anion-exchange column (Pharmacia LKB, HR 5/5) and eluted with 20 mM phosphate buffer, pH 7.0 containing 1 M NaCl at a flow rate of 0.5 ml min−1. Laccase fractions were concentrated by ultrafiltration and chromatographed on Superdex 75 column (Pharmacia LKB, HR 10/30) using 20 mM phosphate buffer, pH 7.0 containing 0.15 M NaCl. In the last step, the laccase fractions were desalted and chromatographed again on a MonoQ column equilibrated with 20 mM phosphate buffer, pH 7.0 as described above. The purity of the enzyme preparation was checked using SDS–PAGE (10% polyacrylamide, Coomassie Brilliant Blue staining). The enzyme was maintained frozen at −18 °C in citrate-phosphate (100 mM citrate, 200 mM phosphate) buffer (pH 5.0).

2.6Effect of laccase and laccase oxidation products on bacteria

One ml of 24-h old cultures of B. subtilis and E. coli was added to 100 ml LB agar cooled to 50 °C. After mixing, the agar was plated on Petri dishes. After solidification, a 7 mm circular well was made in the center of each plate. To each well, 50 μl of T. versicolor laccase (in water, 6000 U l−1) was added. The plates were incubated at 28 °C until bacterial growth occurred (2 d) and inspected for the formation of clear zone around the well. In control plates, 50 μl of sterile deionized water was added to each well. All treatments were performed in four replicates.

Klason lignin (0.5 g) was mixed with 100 ml of citrate-phosphate buffer for 30 min and filtered through Whatman No. 1 filter paper to obtain low molecular weight water-soluble fraction. After sterilization by autoclaving, 10 ml of the filtrate was supplemented with 500 μl of T. versicolor laccase (in water, 6000 U l−1) and incubated at 35 °C for 24 h. 250-ml Erlenmeyer flasks with 40 ml LB media were inoculated with 500 μl of 24-h culture of B. subtilis or E. coli and supplemented with (i) 1 ml of laccase-treated soluble lignin fraction, or (ii) 1 ml of nontreated soluble lignin fraction. The flasks were incubated on a rotary shaker (150 rpm, 28 °C). Samples of cultures were removed hourly for 8 h, diluted with water (1:10) and A600 measured. Specific growth rates were calculated from the absorbance data. All treatments were performed in four replicates.

2.7Counts of colony forming units

Different amounts of soil extract were added to the cultures of T. versicolor growing on CLN medium at 28 °C on day 11 of cultivation in order to follow the response of T. versicolor to different initial concentrations of the introduced soil microorganisms. The soil extract was prepared as described above. Cultures were supplemented with 100, 200, 500, and 1000 μl soil extract, respectively. T. versicolor cultures sterilized by autoclaving were used as controls and supplemented with the same amounts of soil extract. Four replicates were used for each treatment. After mixing, the static cultures were incubated at 28 °C. On days 2, 4, and 7 after addition of the soil extract, a 500 μl sample was taken from each flask. 10−1–10−7 dilutions in PG medium (1 g l−1 peptone, 0.1 g l−1 K2HPO4, 0.1 g l−1 glucose, 0.02 g l−1, FeSO4.7H2O, pH 7.2)[28] were prepared and 200 μl from each dilution was plated on a Petri dish (three plates/dilution) with PG medium solidified with 1.5% agar. The plates were incubated at 28 °C and colonies counted after two days of incubation. The rest of the culture liquid was used to measure laccase activity.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Interaction between T. versicolor and T. harzianum

Laccase production and RBBR decolorization activity was tested during cultivation of T. versicolor with T. harzianum in CLN media. During 11 days of cultivation, the mycelium mat developed in about 50% of the flask volume. Laccase activity was first detectable on day 5 and reached 0.7 U l−1 on day 11. T. harzianum was added at day 11 and developed rapidly in the mixed culture, with mycelial colonies of this fungus apparent by day 13. By day 14–16, the T. harzianum mycelium had completely overgrown the T. versicolor mycelium, to form a uniform sporulating mycelial mat on the surface of the liquid culture. The laccase activity increased immediately after the addition of T. harzianum and reached 17 U l−1 on day 12 and 45 U l−1 on day 13, compared to approximately 1 U l−1 in control flasks (Fig. 1). The elevated laccase activity persisted for 7 days, after which the activity dropped to the limit of detection. RBBR decolorization activity followed a similar course (Fig. 1) and was strongly positively correlated with the laccase activity (y= 1.33x+ 34.93, R= 0.826, P < 0.001). Also the purified laccase of T. versicolor decomposed RBBR, 71% decolorization was achieved within 24 h in the presence of 50 U l−1 laccase. The addition of a sterilized T. harzianum culture homogenate did not significantly affect the enzyme activity (Fig. 1). The same results (no laccase induction) were obtained when sterile-filtered culture liquid from T. harzianum cultures or mixed T. versicolor×T. harzianum cultures were added to the flasks with T. versicolor or when T. versicolor cultures were combined with whole T. versicolor×T. harzianum cultures sterilized by autoclaving or filtration. Laccase production was not detected in control treatments with T. harzianum and sterilized T. versicolor cultures.

image

Figure 1. Activity of laccase and decolorization activity of the culture liquid in mixed T. versicolor/T. harzianum cultures. The time course of laccase activity (full symbols) and decolorization activity of the culture liquid towards the synthetic dye RBBR (open symbols), squares: T. versicolor, circles: T. versicolor/T. harzianum, triangles: T. versicolor/sterilized mycelium of T. harzianum. Averages and standard errors of five replicates are shown.

Download figure to PowerPoint

3.2Interactions between T. versicolor and other soil fungi

Strains of A. sphaerospermum, F. reticulatum, H. grisea and P. rugulosum also were tested for their ability to increase laccase activity in mixed cultures with T. versicolor. Growth of these fungi in the mixed cultures was apparent by day 16, 5 days after addition to T. versicolor cultures, for all species except A. sphaerospermum that grew slower. Mycelia of H. grisea and P. rugulosum completely covered the liquid media in mixed cultures within 6 days and the latter fungus began sporulating. Laccase activities were significantly increased in all mixed cultures (Table 1) within a few days of introduction and reached maxima of 690–2500% of the control value. Laccase induction lasted longer than in the T. harzianum cultures since the growth of T. versicolor probably was not completely inhibited. Again, addition of heat-sterilized mycelia did not induce significant changes in the T. versicolor cultures (results not shown).

Individual fungal cultures, nonsterile soil and soil extracts were screened for laccase production after addition to sterilized cultures of T. versicolor. In all cases except P. rugulosum, no detectable laccase activity was recorded; P. rugulosum produced 1–3 U l−1 laccase on days 18–24. The culture liquids from all mixed cultures were concentrated and analyzed by native PAGE. The intensity of individual laccase isoenzymes relative to the T. versicolor control varied, but all of them ran at positions characteristic of laccases produced by T. versicolor in pure culture. No new bands that might indicate production of laccases by the challenging fungi or production of other laccase isoenzymes by T. versicolor were ever observed.

3.3Interactions between T. versicolor and other microorganisms

A series of experiments was conducted to demonstrate if bacteria, yeast and soil microorganisms are also able to increase laccase activity in T. versicolor cultures and if – on the other hand –T. versicolor and its laccase might affect soil microorganisms. Laccase activity was increased when T. versicolor cultures were supplemented with E. magnusii, B. subtilis, and E. coli. The increase in laccase activity was significant in all cases although less than with the filamentous fungi (Table 1). In mixed cultures of T. versicolor and nonsterile soil, development of soil microbial populations were apparent by days 13–14, 2–3 days after inoculation. When autoclaved soil was used, no changes occurred except that the white-rot fungus grew on the surface of soil particles. Laccase activity began to increase in mixed cultures on days 13–14 (Fig. 2) peaking around 120 U l−1 on day 18 in the soil treatment and on day 21 in the nonsterile soil extract treatment. Later on, laccase activity remained relatively stable at least until day 28. The activity in control flasks and flasks with sterile soil/soil extract also increased but reached only 6–12 U l−1 in the same time period (Table 1).

image

Figure 2. Activity of laccase in the culture of T. versicolor with sterile and nonsterile soil and soil extract. Averages of five replicates are shown.

Download figure to PowerPoint

To study the effect of T. versicolor on the soil microflora, 11-day cultures of T. versicolor were supplemented with 0, 100, 200, 500 and 1000 μl of soil extract [1.4 × 103−1.4 × 104 ml−1 colony forming units (CFU)]. The CFU count in the control flasks increased during incubation and was independent of the initial inoculum size (Table 3). Laccase activity in mixed cultures differed in the cultures with different initial dose of soil extract but was substantially higher than in the control treatment without soil extract. The highest laccase activity occurred in flasks supplemented with 200 μl of soil extract.

Table 3.  Number of colony forming units (107 ml−1) and laccase activity (U l−1) in T. versicolor cultures (living or heat-sterilized) supplemented with soil extract (0–1.35 × 104 ml−1 CFU)
Initial dose (CFU)Day 13Day 15Day 18
CFULaccaseCFULaccaseCFULaccase 
  1. Soil extract was added to T. versicolor cultures on day 11.

  2. an.d. – not detected.

Trametes versicolor
0n.d.a16 ± 2n.d. a28 ± 4n.d.a21 ± 11
1.35 × 103 ml−110 ± 0134 ± 318 ± 1171 ± 1914 ± 2178 ± 33
2.70 × 103 ml−111 ± 1169 ± 618 ± 4259 ± 2810 ± 3383 ± 42
6.75 × 103 ml−17 ± 0138 ± 717 ± 1191 ± 2513 ± 1139 ± 39
1.35 × 104 ml−16 ± 068 ± 416 ± 484 ± 1310 ± 1048 ± 23
Trametes versicolor (dead)
1.35 × 103 ml−19 ± 1n.d.a26 ± 1n.d.a26 ± 2n.d.a
2.70 × 103 ml−113 ± 2n.d.a31 ± 3n.d.a28 ± 1n.d.a
6.75 × 103 ml−115 ± 3n.d.a28 ± 0n.d.a36 ± 0n.d.a
1.35 × 104 ml−113 ± 1n.d.a26 ± 1n.d.a39 ± 8n.d.a

To confirm the possible inhibitory effect of laccase or the products of laccase oxidation, the purified enzyme was added to a well in a Petri dish containing agar with B. subtilis or E. coli. No clearing zone was observed around the well indicating that direct contact on the enzyme did not inhibit the tested bacteria. In another experiment, samples of oxidation products of soluble lignin fractions obtained during the oxidation with purified T. versicolor laccase were added to liquid cultures of B. subtilis or E. coli. No inhibitory effect on bacterial growth was observed in treatments with laccase-treated samples. In contrast, growth of bacteria was slightly (but not significantly) slower in the control treatments supplemented by nontreated soluble lignin fraction.

3.4Laccase activity during interactions with other white-rot species

Twenty four species of white-rot fungi (Table 2) were tested for laccase induction in mixed cultures with T. harzianum. P. squamosus and G. frondosa did not produce laccase. Laccase activity on day 14 ranged from 0.05 to 120 U l−1 in individual species; six species had not produced laccase at that time (Table 2). Significant increase of laccase activity after the introduction of T. harzianum occurred in 16 species including C. occidentalis, M. pura and T. hirsuta where laccase was not detectable in control cultures. No increase in activity was only observed in species with little or no laccase activity (<1.5 U l−1). Peak laccase activity in mixed cultures was usually detected within the first three days of coculture. Laccase activity in mixed cultures was 2–25 times higher than in individual cultures depending on the species. Higher laccase activities in mixed cultures were detected for 2–6 days in cultures if T. harzianum rapidly overgrew the host. In C. occidentalis, T. hirsuta and T. versicolor, laccase increased laccase activity lasted for more than 10 days suggesting that the white-rot fungus was competing with T. harzianum.

P. ostreatus was also tested for induction of laccase activity mediated by soil fungi, yeast and bacteria, or by soil and soil extract. As with T. versicolor, laccase activity increased significantly in all mixed cultures tested (Table 1).

Cocultures of T. versicolor and P. ostreatus had significantly higher laccase activities than did individual cultures of either fungus. The interpretation of this result is difficult since both fungi produce laccase. It seems, that on the first day of coculture only T. versicolor increased laccase production, but in the later stages, both T. versicolor and P. ostreatus laccase was present (data not shown).

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

In the natural environment, white-rot fungi interact with different microorganisms and compete with them for resources and space. The interactions among wood-rotting fungi have attracted considerable attention of fungal ecologists[9] and some studies have focused on the effect of interactions on the production of extracellular enzymes by fungi. It was noticed, that laccase is sometimes produced during interspecific interactions[29] and that it might be preferentially located in the interaction zones[21]. Attention has also been paid to the interactions of white-rot fungi with potential mycoparasites, especially from the genus Trichoderma. Trichoderma species have been shown to inhibit several species of white-rot fungi including T. versicolor[11,30]. Compared to individual cultures, activity of extracellular enzymes are changed during interspecific interactions between Trichoderma and white-rot fungi[11]. Laccase was induced during the interaction of mycoparasitic Trichoderma strains isolated from mushroom compost and L. edodes[31,32]. The same was found in this study for the interaction between a soil isolate of T. harzianum and T. versicolor. However, unlike in L. edodes, cell-free extract from T. harzianum was not able to increase laccase activity in T. versicolor.

Competitive abilities of white-rot fungi are necessary for their survival in soil as well as their applicability to in situ biodegradation. Soil is the living environment for litter-decomposing and saprophytic white-rotters, e.g., Agrocybe, Stropharia or Agaricus[33]. However, typical wood-colonizing species differ in their ability to colonize nonsterile soil [34–36]. The reason is that the interactions with soil microbes are mostly combative – the growth inhibition of white-rot fungi by soil fungi and bacteria is a usual phenomenon [29,37–39]. On the other hand some white-rot fungi, e.g., P. ostreatus are able to prevent soil microbes from utilizing lignocellulose substrate added into soil [14,40] or decrease the number of soil bacteria [30,41]. In this study reduced CFU counts were observed in T. versicolor cultures with soil microorganisms compared to controls with the killed fungus. Despite different size of soil microbial inocula, the CFU counts in mixed cultures reached the same value in all treatments (Table 3). The mycelia of all soil fungi tested overgrew the well-established culture of their white-rot rival and MnP activity in mixed cultures decreased or even disappeared (data not shown).

Ligninolytic activity of white-rot fungi in nonsterile soil is primarily dependent on the establishment of the fungus in the soil environment [15,42]. Although a distinct peak of laccase activity during the initial colonization of nonsterile soil by P. ostreatus was reported[8], the direct effect of nonsterile soil or soil microorganisms on ligninolytic enzymes has never been tested. This study showed, that the addition of nonsterile soil or soil extract as well as individual strains of isolated soil fungi and soil-associated bacterial and yeast species increased laccase formation by P. ostreatus and T. versicolor. The inability of sterilized soil and soil extract to affect ligninolytic activities showed, that living microorganisms are required for the increase of activity. Laccase activity increased also after a short homogenization of T. versicolor mycelia grown on CLN (data not shown). However, the temporal profile of the activity (2–2.5 fold increase on days 19–25) seems to be different from the induction by T. harzianum or other treatments used.

In the case of many contaminants like PAH or polychlorinated biphenyls, the degradation is a two-step process where both white-rot fungus and indigenous soil microbes perform sequential reactions resulting in the transformation of xenobiotics [8,14]. As shown here for RBBR, xenobiotics transformation can also be enhanced due to the increase of laccase activity during interspecific interactions.

This study showed that the increase of laccase activity is a general response to interactions. It was even found during interaction with Escherichia coli, a bacterium, which is completely absent in the environment of white-rot fungi. Performed screening demonstrated that laccase induction is a typical phenomenon in white-rot fungi including both litter decomposers (A. dura, M. pura) and typical wood-associated species interesting from the biotechnological viewpoint (D. squalens, I. lacteus, P. eryngii, P. ostreatus, T. versicolor).

The role, which laccase can play during interactions with soil microorganisms, is not clear. Although direct inhibition was not confirmed in this study, this possibility cannot be completely ruled out under natural conditions. It has to be noted, that laccase is an enzyme with a wide scale of physiological functions ranging from the degradation or polymerization of phenols to differentiation and fruiting body development [18,19].

It can be concluded that among lignin-degrading enzymes of P. ostreatus and T. versicolor, only laccase activity and not MnP (data not shown) is changed after the contact with other microorganisms including soil fungi, bacteria or yeasts. Since laccase participates in the degradation of some organic pollutants, the interaction of white-rot fungi and other microorganisms also affects the biodegradation. The induction of laccase activity is a typical response of a wide range of white-rot fungi to interspecific interactions. Although T. versicolor was able to influence the counts of soil bacteria in mixed cultures, an involvement of laccase in this phenomenon was not observed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This study was supported by the Grant Agency of the Czech Academy of Sciences (B5020202).

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
  • [1]
    Rayner, A.D.M., Boddy, L. (1988) Decomposition of Wood: Its Biology and Ecology. John Wiley, Chichester, UK.
  • [2]
    Paszczynski, A., Crawford, R.L. (2000) Recent advances in the use of fungi in environmental remediation and biotechnology. Soil Biochem. 10, 379422.
  • [3]
    Pointing, S.B. (2001) Feasibility of bioremediation by white-rot fungi. Appl. Microbiol. Biotechnol. 57, 2033.
  • [4]
    Bogan, B.W., Lamar, R.T., Burgos, W.D., Tien, M. (1999) Extent of humification of anthracene, fluoranthene, and benzo[α]pyrene by Pleurotus ostreatus during growth in PAH-contaminated soils. Lett. Appl. Microbiol. 28, 250254.
  • [5]
    Canet, R., Birnstingl, J.G., Malcolm, D.G., Lopez-Real, J.M., Beck, A.J. (2001) Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by native microflora and combinations of white-rot fungi in a coal-tar contaminated soil. Biores. Technol. 76, 113117.
  • [6]
    Eggen, T., Majcherczyk, A. (1998) Removal of polycyclic aromatic hydrocarbons (PAH) in contaminated soil by white rot fungus Pleurotus ostreatus. Int. Biodeter. Biodegrad. 41, 111117.
  • [7]
    Tuomela, M., Lyytikåinen, M., Oivanen, P., Hatakka, A. (1999) Mineralization and conversion of pentachlorophenol (PCP) in soil inoculated with the white-rot fungus Trametes versicolor. Soil Biol. Biochem. 31, 6574.
  • [8]
    Baldrian, P., in der Wiesche, C., Gabriel, J., Nerud, F., Zadrazil, F. (2000) in der Influence of cadmium and mercury on activities of ligninolytic enzymes and degradation of polycyclic aromatic hydrocarbons by Pleurotus ostreatus in soil. Appl. Environ. Microbiol. 66, 24712478.
  • [9]
    Boddy, L. (2000) Interspecific combative interactions between wood-decaying basidiomycetes. FEMS Microbiol. Ecol. 31, 185194.
  • [10]
    Holmer, L., Stenlid, J. (1993) The importance of inoculum size for the competitive ability of wood decomposing fungi. FEMS Microbiol. Ecol. 12, 169176.
  • [11]
    Freitag, C.M., Morrell, J.J. (1992) Changes in selected enzyme activities during growth of pure and mixed cultures of the white-rot fungus Trametes versicolor and the potential biocontrol fungus Trichoderma harzianum. Can. J. Microbiol. 38, 317323.
  • [12]
    Highley, T.L. (1997) Control of wood decay by Trichoderma (Gliocladium) virens I. Antagonistic properties. Mater. Org. 31, 7989.
  • [13]
    Fernandez-Sanchez, J.M., Rodriguez-Vazquez, R., Ruiz-Aguilar, G., Alvarez, P.J.J. (2001) PCB biodegradation in aged contaminated soil: Interactions between exogenous Phanerochaete chrysosporium and indigenous microorganisms. J. Environ. Sci. Health A 36, 11451162.
  • [14]
    in der Wiesche, C., Martens, R., Zadrazil, F. (1996) Two-step degradation of pyrene by white-rot fungi and soil microorganisms. Appl. Microbiol. Biotechnol. 46, 653659.
  • [15]
    Lang, E., Eller, G., Zadrazil, F. (1997) Lignocellulose decomposition and production of ligninolytic enzymes during interaction of white rot fungi with soil microorganisms. Microb. Ecol. 34, 110.
  • [16]
    Novotny, C., Erbanova, P., Sasek, V., Kubatova, A., Cajthaml, T., Lang, E., Krahl, J., Zadrazil, F. (1999) Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi. Biodegradation 10, 159168.
  • [17]
    Leonowicz, A., Cho, N.S, Luterek, J., Wilkolazka, A., Wojtas-Wasilewska, M., Matuszewska, A., Hofrichter, M., Wesenberg, D., Rogalski, J. (2001) Fungal laccase: properties and activity on lignin. J. Basic Microbiol. 41, 185227.
  • [18]
    Thurston, C.F. (1994) The structure and function of fungal laccases. Microbiology 140, 1926.
  • [19]
    Mayer, A.M., Staples, R.C. (2002) Laccase: new functions for an old enzyme. Phytochemistry 60, 551565.
  • [20]
    Baldrian, P. (2003) Interactions of heavy metals with white-rot fungi. Enzyme Microb. Technol. 32, 7891.
  • [21]
    Iakovlev, A., Stenlid, J. (2000) Spatiotemporal patterns of laccase activity in interacting mycelia of wood-decaying basidiomycete fungi. Microb. Ecol. 39, 236245.
  • [22]
    Savoie, J.-M., Mata, G. (1999) The antagonistic action of Trichoderma sp. hyphae to Lentinula edodes hyphae changes lignocellulotytic activities during cultivation in wheat straw. World J. Microbiol. Biotechnol. 15, 369373.
  • [23]
    Baldrian, P., Gabriel, J. (2003) Lignocellulose degradation by Pleurotus ostreatus in the presence of cadmium. FEMS Microbiol. Lett. 220, 235240.
  • [24]
    Gryndler, M., Hrselova, H., Klir, J., Kubat, J., Votruba, J. (2003) Long-term fertilization affects the abundance of saprotrophic microfungi degrading resistant forms of soil organic matter. Folia Microbiol. 48, 7682.
  • [25]
    Katagiri, N., Tsutsumi, Y., Nishida, T. (1995) Correlation of brightening with cumulative enzyme activity related to lignin biodegradation during biobleaching of kraft pulp by white rot fungi in the solid-state fermentation system. Appl. Environ. Microbiol. 61, 617622.
  • [26]
    Lorch, H.J., Benckieser, G., Ottow, J.C.G. (1995) Basic methods for counting microorganisms in soil and water. In: Methods in Applied Soil Microbiology and Biotechnology (Alef, K., Nannipieri, P., Eds.), pp.146–161 Academic Press, London.
  • [27]
    Niku-Paavola, M.L., Raaska, R., Itåvaara, M. (1990) Detection of white-rot fungi by a non-toxic stain. Mycol. Res. 94, 2731.
  • [28]
    Kölbel-Boelke, J., Tienken, B., Nehrkorn, A. (1988) Microbial communities in the saturated groundwater environment. 1. Methods of isolation and characterization of heterotrophic bacteria. Microb. Ecol. 16, 1729.
  • [29]
    White, N.A., Boddy, L. (1992) Extracellular enzyme localization during interspecific fungal interactions. FEMS Microbiol. Lett. 98, 7579.
  • [30]
    Otieno, W., Jeger, M., Termorshuizen, A. (2003) Effect of infesting soil with Trichoderma harzianum and amendment with coffee pulp on survival of Armillaria. Biol. Control 26, 293301.
  • [31]
    Savoie, J.-M., Mata, G., Billette, C. (1998) Extracellular laccase production during hyphal interactions between Trichoderma sp. and Shiitake, Lentinula edodes. Appl. Microbiol. Biotechnol. 49, 589593.
  • [32]
    Savoie, J.-M., Mata, G., Mamoun, M. (2001) Variability in brown line formation and extracellular laccase production during interaction between white-rot basidiomycetes and Trichoderma harzianum biotype Th2. Mycologia 93, 243248.
  • [33]
    Steffen, K.T., Hofrichter, M., Hatakka, A. (2000) Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi. Appl. Microbiol. Biotechnol. 54, 819825.
  • [34]
    Martens, R., Zadrazil, F. (1998) Screening of white-rot fungi for their ability to mineralize polycyclic aromatic hydrocarbons in soil. Folia Microbiol. 43, 97103.
  • [35]
    Andersson, B.E., Welinder, L., Olsson, P.A., Olsson, S., Henrysson, T. (2000) Growth of inoculated white-rot fungi and their interactions with the bacterial community in soil contaminated with polycyclic aromatic hydrocarbons, as measured by phospholipid fatty acids. Biores. Technol. 73, 2936.
  • [36]
    Tornberg, K., Bååth, E., Olsson, S. (2003) Fungal growth and effects of different wood decomposing fungi on the indigenous bacterial community of polluted and unpolluted soils. Biol. Fert. Soils 37, 190197.
  • [37]
    Nicolotti, G., Varese, G.C. (1996) Screening of antagonistic fungi against air-borne infection by Heterobasidion annosum on Norway spruce. Forest Ecol. Manag. 88, 249257.
  • [38]
    Radtke, C., Cook, W.S., Anderson, A. (1994) Factors affecting antagonism of the growth of Phanerochaete chrysosporium by bacteria isolated from soils. Appl. Microbiol. Biotechnol. 41, 274280.
  • [39]
    Stahl, P.D., Christensen, M. (1992) In vitro mycelial interactions among members of a soil microfungal community. Soil Biol. Biochem. 24, 309316.
  • [40]
    Lang, E., Kleeberg, I., Zadrazil, F. (2000) Extractable organic carbon and counts of bacteria near the lignocellulose-soil interface during the interaction of soil microbiota and white rot fungi. Biores. Technol. 75, 5765.
  • [41]
    Beltran-Garcia, M.J., Estarron-Espinosa, M., Ogura, T. (1997) Volatile compounds secreted by the oyster mushroom (Pleurotus ostreatus) and their antibacterial activities. J. Agric. Food Chem. 45, 40494052.
  • [42]
    Lang, E., Kleeberg, I., Zadrazil, F. (1997) Competition of Pleurotus sp. and Dichomitus squalens with soil microorganisms during lignocellulose decomposition. Biores. Technol. 60, 9599.