Specific Aspects Of Enzymology Of Basidiomycetes From Different Habitats

3.1 Decaying Wood

Most of the enzymology of saprotrophic basidiomycetes in natural resources has been studied in wood-rotting species, especially those causing white rot (Hat-akka, 2001; Martínez et al., 2005). Two main types of rot caused by basidiomycetes have been differentiated based on morphological appearance of the wood— white rot and brown rot, though they can also cause other types under certain circumstances (Table 1). Those species causing white rot and brown rot are taxonomically closely related, both often being found in the same genus. Those causing brown rot are most commonly found on conifers, and represent only 7% of wood-rotting basidiomycetes (Gilbertson, 1980). With white rot all wood components are utilized, resulting in a bleached appearance as wood decays, though different components can be removed at different rates (Table 1). With brown rot lignin is slightly modified, allowing utilization of polysaccharides, resulting in a brown material consisting of oxidized lignin, which represents a potential source of aromatic compounds for the stable humic material fraction in forest soils (Hatakka, 2001). The white-rot basidiomycetes and the brown-rotter C. puteana decreased both the relative content of lignin and the ratio of syringyl/guaiacyl units compared to undecayed wood of Eucalyptus sp. (del Rio et al., 2001).

The ability to decompose wood and the types of wood decay caused relates not only to the enzymes and other reactive species produced by the fungi, but also the location to which they are delivered. It is generally accepted that the mobility of enzymes in decaying lignocellulosic material is limited to cell lumina, the largest anatomical pores, and to pores formed during the decay process. During both white rot and brown rot of wood by P. chrysosporium and Postia placenta respectively, pores of less than 50 A were inaccessible to molecules with a molecular weight above 12 kDa, i.e. for enzymes (Flournoy et al., 1991,1993). The initiators of both cellulose and lignin breakdown are thus suggested to be small molecular mass compounds that can readily diffuse from fungal hyphae and penetrate into the wood cell walls (Hatakka, 2001; Hammel et al., 2002; Reading et al., 2003). Due to (1) limited diffusibility of enzymes, (2) the reactivity of small molecular mass compounds generated by fungi (Hammel et al., 2002), (3) the small likelihood of concerted action of peroxidases and hydrogen peroxide generating enzymes over larger distances and (4) the fact that a considerable part of the lignocellulose-degrading apparatus is cell-wall associated (Valaskova and Baldrian, 2006b) most of the wood-decay reactions apparently occur near fungal hyphae. Thus the penetration of a resource is a necessary prerequisite for the capture and utilization of the substrate (Boddy, 2000). With white rot, enzymic decomposition results in formation of erosion grooves around hyphae, which eventually coalesce causing gradual thinning of the cell wall from the lumen outwards, i.e. from the S3 layer to the S1 layer (Rayner and Boddy, 1988). With brown rot, carbohydrates are removed from the S2 layer, even though hyphae are in the lumen on the S3 layer.

Table 1 Comparison of structural and chemical features caused by saprotrophic basidiomycetes during different types of wood decay

White rot simultaneous rot

White rot selective delignification

Brown rot

Soft rot

Properties of decayed wood

Host and wood type

Cell wall constituents degraded

Bleached appearance, lighter in colour than sound wood, moist, soft, spongy, strength loss after advanced decay; brittle fracture

Hardwood, rarely softwood

Cellulose, lignin and hemicellulose

Bleached appearance, lighter in colour than sound wood, moist, soft, spongy, strength loss after advanced decay; fibrous appearance

Hardwood and softwood

Initial attack selective for hemicelluloses and lignin, later also cellulose

Anatomical features

Cell wall attacked progressively from

Lignin decomposition in

Brown, dry, crumbly, powdery, brittle consistency, breaks up into cubes, drastic loss of strength at initial stage of decay; very uniform ontogeny of wood decay Softwoods; seldom hardwoods; forest ecosystems and timber

Cellulose and hemicelluloses; lignin slightly modified; in some cases, extended decomposition of hardwood (including middle lamella) Decomposition at a great distance from

Soft consistency in wet environments; brown and crumbly in dry environments; generally uniform ontogeny of wood decay

Generally hardwoods (softwoods very slightly degraded); forest ecosystems, waterlogged woods

Cellulose and hemicelluloses, lignin slightly altered

Cell wall attack in the proximity of

Table 1 (Continued)

White rot simultaneous White rot selective Brown rot Soft rot rot delignification

Table 1 (Continued)

White rot simultaneous White rot selective Brown rot Soft rot rot delignification

lumen; erosion

middle lamella and

hyphae (diffusion

hyphae starts from

furrows associated

secondary wall;

mechanism); entire

cell lumen;

with hyphae

middle lamella

cell wall attacked

longitudinal

dissolved by

rapidly with cracks

cylindrical cavities

diffusible agents

and clefts

in secondary wall

(not in contact with

or secondary wall

hyphae), radial

erosions from cell

cavities in cell wall

lumen

Examples of causal

Basidiomycetes (e.g.

Basidiomycetes (e.g.

Basidiomycetes

Mainly ascomycetes;

agents

Trametes versicolor,

Ganoderma australe,

exclusively (e.g.

some white-rot

Irpex lacteus,

Phlebia tremellosa,

Gloeophyllum spp.,

(Inonotus hispidus)

Phanerochaete

Ceriporiopsis

Laetiporus

and brown-rot

chrysosporium,

subvermispora,

sulphureus,

(Rigidoporus

Heterobasidion

Pleurotus spp.,

Piptoporus betulinus,

crocatus)

annosum) and some

Phellinus pini)

Postia placenta,

basidiomycetes

xylariaceous

Serpula lacrymans,

cause facultative

ascomycetes

Coniophora puteana)

soft-rot decay

Source: Based on Eriksson et al. (1992), Zabel and Morell (1992) and Schwarze et al. (2000).

Source: Based on Eriksson et al. (1992), Zabel and Morell (1992) and Schwarze et al. (2000).

All models suggested that brown rot decay involves generation of hydroxyl radicals, which are small enough to enter the wood cell wall (Hammel et al., 2002; Goodell, 2003). However, none of the proposed models has been fully verified experimentally. Many of the proposed low molecular mass compounds have been isolated from cultures of both brown-rot and white-rot fungi which makes it difficult to understand their specific role in brown rot decay. These compounds can be phenolates or other types of iron-chelating compounds (siderophores), oxalate and simple aromatic compounds (Hatakka, 2001; Goodell, 2003).

3.2 Litter and Soil

Compared to wood, far less is known about the occurrence, properties and roles of enzymes in forest litter and soil. Soil and litter is a heterogeneous environment which may hamper detection and estimation of enzyme activities. Another problem is the difficulty of linking enzyme activities in soil/litter to specific species producing them. There have been a few studies of ligninolytic activities and enzymes of litter-decomposing basidiomycetes. Basidiomycetes in the genera Stropharia and Agrocybe mineralized 14C-(ring)-labelled synthetic lignin at about half the rate of wood-inhabiting white-rot fungi (Steffen et al., 2000). The main ligninolytic enzymes in litter-decomposing fungi such as Agaricus bisporus (Bonnen et al., 1994), Collybia dryophila (Steffen et al., 2002a) and Stropharia rugosoannulata (Steffen et al., 2002b) are laccase and MnP. Many other litter-decomposing species produce laccase but the production of ligninolytic per-oxidases has not been reported till date (Baldrian, 2006).

Unlike the activity of other extracellular enzymes, ligninolytic enzymes can be linked to the presence of fungi, although not only to basidiomycetes. Relatively high activities of laccase—the dominant ligninolytic enzyme—were detected in angiosperm and coniferous forest litter and soils, compared to agricultural or meadow soils (Rosenbrock et al., 1995; Criquet et al., 2000; Ghosh et al., 2003). Several laccase enzymes (most of them from basidiomycetes) were usually responsible for the measured activity in temperate forest soil (Luis et al., 2004). Laccase activity reflects the course of decomposition of organic substances and thus varies in time. It increased during leaf litter decomposition in Mediterranean broadleaved litter (Fioretto et al., 2000) and the pattern of detected isoenzymes varied during the succession (Di Nardo et al., 2004). Laccase actually also reflects the presence of mycelia. In evergreen oak litter, laccase activity reflected the annual dynamics of fungal biomass that is probably driven by microclimate, while Mn-peroxidase occurred only in autumn (Criquet et al., 2000). Significantly increased activity of oxidases and peroxidases occurs in the soil under fairy rings of saprobic basidiomycetes, e.g. Marasmius oreades or Agaricus arvensis, compared to soil devoid of visible mycelia (Gramss, 1997; Gramss et al., 2005). Laccase activity and the diversity of laccase gene sequences decreases with depth in soil profiles, associated with decrease in fungal biomass (Luis et al., 2004). Laccase activity has a high small-scale variability in soil, and the distribution of mRNA transcripts does not always reflect the laccase gene pool (Luis et al., 2005a, 2005b).

In addition to ligninolysis, ligninolytic enzymes in soils are also involved in the transformation of soil humic compounds: humic acids (HA), fulvic acids and humin (Kastner and Hofrichter, 2001). Extracellular peroxidase activities correlate with HA decomposition (Kastner and Hofrichter, 2001). MnP may have a more important role than LiP in the decomposition process (Dehorter and Blondeau, 1992), being able to depolymerize and mineralize HA (Steffen et al., 2000, 2002a; Hofrichter, 2002). The interaction of laccases with humic substances probably leads both to depolymerization of humic substances and their synthesis from monomeric precursors, and the balance of these two processes can be influenced by the nature of the humic compounds (Zavarzina et al., 2004). Fakoussa and Frost (1999) observed the decolourization and decrease of molecular weight of HA, accompanied by the formation of fulvic acids during the growth of T. versicolor cultures producing mainly laccase, while HA synthesis occurred in vitro with laccase (Katase and Bollag, 1991). Adsorption of laccases to soil humic substances or inorganic soil constituents changes their temperature activity profiles (Criquet et al., 2000) and inhibits their activity (Claus and Filip, 1990; Zavarzina et al., 2004; Baldrian, 2006).

There are unfortunately only a few studies of enzyme production by litter-decomposing basidiomycetes in leaf litter, though production of, e.g. cellulases, is often attributed to them (Criquet et al., 2002). Laccase, Mn-peroxidase, mannanase and xylanase are produced by Mycena galopus in Picea sitchensis litter (Ghosh et al., 2003). This species is particularly vigorous in degrading lignin and cellulose, leading to the formation of a "white-rot" litter (Frankland, 1998). The litter-decomposer Lepista nuda produced laccase, endoglucanase, b-glucosidase and b-xylosidase in Fagus sylvantica buried leaves in soil (Colpaert and van Laere, 1996).

3.3 Biopolymer-Degrading Enzymes in Ectomycorrhizal Fungi

ECM fungi have some saprotrophic abilities. Several attempts have been made to detect ligninolytic enzymes including laccases in ECM fungi (Cairney and Burke, 1998; Burke and Cairney, 2002). Gene fragments with high similarity to laccase from wood-rotting fungi have been found in species in several genera including Amanita, Cortinarius, Hebeloma, Lactarius, Paxillus, Piloderma, Russula, Tylospora and Xerocomus (Chen et al., 2003; Luis et al., 2004). Laccases have been purified from Cantharellus cibarius, Lactarius piperatus, Russula delica and Thelephora terre-stris (Baldrian, 2006). However, syringaldazine oxidation was rare (Burke and Cairney, 2002) and the laccase activities are much less than for white-rotters and litter decomposers (Baldrian, 2006). Activity of another ligninolytic enzyme, Mn-peroxidase, has to date been confirmed only in Tylospora fibrillosa, a species also containing a putative sequence of laccase and possibly also lignin peroxidase (Chambers et al., 1999; Chen et al., 2001, 2003).

T. terrestris and Suillus bovinus colonizing beech litter produced significant activities of b-glucosidase and b-xylosidase but not endoglucanase; the sap-rotroph L. nuda, however, always produced more (Colpaert and van Laere, 1996). Cellulolytic and xylanolytic activity also occurs, e.g. with Pisolithus tinctorius (Cao and Crawford, 1993; Colpaert and van Laere, 1996), cellobiohydrolase and glucuronidase has been detected in mycorrhizal root tips (Courty et al., 2005). Chitinases are also produced by some mycorrhizal basidiomycetes but less than by saprotophic or parasitic root-colonizers (Hodge et al., 1995). Extracellular enzyme systems provide an additional means of nutrient acquisition by my-corrhizal species but probably are of far less importance to decomposition processes than are those of saprotrophs.

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