Environment  Health  Techniques Cellulases produced by Trichoderma

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Minireview The significance of cellulolytic enzymes produced by Trichoderma in opportunistic lifestyle of this fungus Judyta Strakowska, Lidia Błaszczyk and Jerzy Chełkowski Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland

The degradation of native cellulose to glucose monomers is a complex process, which requires the synergistic action of the extracellular enzymes produced by cellulolytic microorganisms. Among fungi, the enzymatic systems that can degrade native cellulose have been extensively studied for species belonging to the genera of Trichoderma. The majority of the cellulolytic enzymes described so far have been examples of Trichoderma reesei, extremely specialized in the efficient degradation of plant cell wall cellulose. Other Trichoderma species, such as T. harzianum, T. koningii, T. longibrachiatum, and T. viride, known for their capacity to produce cellulolytic enzymes, have been isolated from various ecological niches, where they have proved successful in various heterotrophic interactions. As saprotrophs, these species are considered to make a contribution to the degradation of lignocellulosic plant material. Their cellulolytic potential is also used in interactions with plants, especially in plant root colonization. However, the role of cellulolytic enzymes in species forming endophytic associations with plants or in those existing in the substratum for mushroom cultivation remains unknown. The present review discusses the current state of knowledge about cellulolytic enzymes production by Trichoderma species and the encoding genes, as well as the involvement of these proteins in the lifestyle of Trichoderma. Keywords: Cellulases / Lignocellulosis / Mycoparasitism / Plant–Trichoderma interactions / Saprotrophism Received: October 10, 2013; accepted: December 25, 2013 DOI 10.1002/jobm.201300821

Introduction Cellulose is the main component of plant biomass and thus the most abundant polysaccharide in nature. In plants, it is associated with hemicelluloses, lignin, and various extractives (protein, fats, waxes, terpenes, phenols, alcohols, and alkanes) forming the complex and rigid structure of the plant cell walls. Cellulose is a linear polymer of 7000–15,000 glucose units linked together by b-1,4-glycosidic bonds [1]. The smallest and most repetitive unit of the cellulose chain is the cellobiose, consisting of two glucose units. In native cellulose, the polymeric chains are linked together by hydrogen bonds and van der Waals forces to form highly insoluble crystalline structures [2]. In addition, it is possible to observe a small amount of less-organized Correspondence: Lidia Błaszczyk, Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland E-mail: [email protected] Phone: þ48 61 6550279 Fax: þ48 61 6550301 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

or paracrystalline regions, which form amorphous cellulose [2]. Degradation of native cellulose to glucose monomers is a complex process, which requires the synergistic action of the extracellular enzymes produced by cellulolytic microorganisms including aerobic and anaerobic bacteria besides fungi. Among bacteria, the ability to produce the cellulase complex (cellulosome) has been stated for several strains, such as Acidothermus cellulolyticus, Paenibacillus polymyxa, Cellulomonas fimi, Clostridium stercorarium, Clostridium thermocellum, Pyrococcus furiosus, Saccharophagus degradans, and Rhodospirillum rubrum [3]. The fungal cellulolytic system, typically composed of endoglucanases, exoglucanases, and b-glucosidases acting on cellulose degradation, has been described for species from the ascomycetes and basidiomycetes [3–5]. Among basidiomycetes, several white-rot fungi (Ceriporiopsis subvermispora, Dichomitus squalens, Irpex lacteus, Phanerochaete chrysosporium, Pleurotus ostreatus, Polyporus arcularius, Polyporus schweinitzii, Schizophyllum commune, Trametes versicolor), brown-rot fungi (Coniophora cerebella,

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Coniophora puteana, Gloeophyllum sepiarium, Gloeophyllum trabeum, Fomitopsis palustris, Piptoporus betulinus, Postia placenta, Serpula incrassata), and others, such as Rhodotorula glutinis, Pisolithus tinktorius, Sclerotium rolfsii, Termitomycetes sp., and Volvariella volvacea were documented as producers of cellulolytic enzymes [6–10]. However, it should be noted that exocellulase activity has never been detected in brown-rot fungi [9, 11]. Interestingly, basidiomycetous wood-rotting fungi are also supposed to possess alternative (independent of enzymatic), radicalbased mechanisms of cellulose degradation [9]. Apart from basidiomycetes, cellulase systems have been studied extensively for ascomycetous fungi belonging to the genera of Aspergillus (A. niger, A. fumigatus), Chaetomium (Ch. thermophilum), Fusarium (F. oxysporum, F. solani), Humicola (H. insolens, H. grisea var. thermoidea), Melanocarpus (M. albomyces), Penicillium (P. echinulatum, P. purpurogenum, P. pinophilum), Rhizopus (R. oryzae), Scytalidium (S. thermophillum), Talaromyces (T. emersonii), and Thermoascus (T. aurantiacus) [3, 12]. However, the best characterized species in this respect are those of the genus Trichoderma (teleomorph Hypocrea), especially T. reesei (Hypocrea jecorina), which is known for its capacity to secrete extremely excess high amounts of extracellular cellulolytic enzymes [13]. Besides T. reesei, the other Trichoderma species, such as T. harzianum, T. koningii, T. longibrachiatum, and T. viride, have also been reported to produce cellulases and to degrade crystalline cellulosic substrates [14, 15]. In the present review, we summarize the current state of knowledge about cellulolytic enzymes produced by the Trichoderma species and their encoding genes. In addition, we attempt to trace the involvement of cellulases in the lifestyle of Trichoderma.

Cellulolytic enzymes of Trichoderma Cellulases belong to the O-glycoside hydrolases. Their Nomenclature EC-Numbers: EC 3.2.1 have been assigned by The Nomenclature Committee of the International Union of Biochemistry. They represent a widespread group of enzymes that hydrolyze the glycosidic bonds either between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Based on the amino acids sequence, cellulases have been classified into glycoside hydrolases families (GHs) [16–19]. Accordingly, they were assigned to nine families: 1, 3, 5, 6, 7, 12, 45, 61, and 74 [20]. These enzymes form a multiple enzymatic complex containing three basic types of synergically active enzymes: exo-b-1,4-glucanases, endo-b-1,4-glucanases, and b-glucosidase. All three of ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

the mentioned groups of cellulolytic enzymes catalyze the hydrolysis of b-1,4-D-glycosidic bonds of the cellulose molecule. The exoglucanases (cellobiohydrolases) preferentially hydrolyze b-1,4-glycosidic bonds from chain ends, releasing cellobiose as the main product. The endoglucanases hydrolyze internal cellulose bonds randomly, but in the more accessible amorphous regions, creating new free cellulose ends which are more accessible to cellobiohydrolases. b-Glucosidases are only active on cello-oligosaccharides and cellobiose, and release glucose monomer units, for example from the cellobiose. These enzymes have mainly been categorized as members of the T. reesei species [21, 22], termed “the crowned king of cellulolytic fungi” by Gusakov [15]. In recent years a number of studies have been performed on cellulases and the genes encoding them in many other species, such as T. harzianum, T. koningii, T. longibrachiatum, and T. viride. The cellulolytic encoding genes of Trichoderma species are summarized in Fig. 1. Exo-b-1,4-glucanases Exo-b-1,4-glucanases (EC 3.2.1.91) (Accepted Names: Exoglucanase, Cellobiohydrolases, Exo-1,4-b-D-glucan, cellobiohydrolase, CBH) hydrolyze the cellulose molecule releasing a cellobiose molecule either reducing or nonreducing end. These enzymes have been shown to create a substrate-binding tunnel with their extended loops which surround the cellulose. Cellobiohydrolases are released initially at low concentrations, but sufficient for the hydrolysis of the cellulose present in the medium. Oligosaccharides released by this way are an inducer to secrete more of the whole complex of cellulolytic enzymes [23–25]. Among cellobiohydrolases, cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II) can be distinguished. According to Shoemaker [26], these are considered the main cellulolytic enzymes secreted by fungi. The list of Trichoderma exo-b-1,4-glucanases and encoding genes is shown in Table 1. Cellobiohydrolase I (CBHI/CEL7A) is a glycoprotein with a molecular weight of 65 kDa for T. reesei [33], although some sources state that the molecular weight of CBH I for T. reesei ranges from 59 to 68 kDa [26, 31]. CBH I is built from 496 amino acids, is 10% composed of carbohydrate and its isoelectric point is 4.4 [33]. It is classified as a member of the GH7 family of GH proteins [20, 25]. Based on nucleotide sequence analysis, it has been revealed that CBH I has four sites of N-linked glycosylation, of which perhaps three are involved in the process of glycosylation. Most of the carbohydrates, however, are bonded with the O-glycosidic bonds via amino acid residues of serine and

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Cellulases produced by Trichoderma

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Figure 1. The inventory of cellulolytic encoding genes in Trichoderma species. Numbers on the plots indicate number of nucleotide sequences deposited in NCBI GenBank database (Table 1).

threonine [34]. The 3D-structures of the catalytic cores of CBH I and of the cellulose binding domains have been solved [35, 36]. The CBH I molecule, with a length of 18 nm, is composed of three domains: domain A (CBD – cellulose binding domain) with a weight of 11 kDa that

allows adsorption of an enzyme on cellulose, strongly glycosylated domain B (hinge domain) connecting the adsorption domain to the catalytic domain C (catalytic core domain) of an ellipsoidal structure and a molecular weight of 45 kDa [37]. As a result of the crystallization of

Table 1. Exo-b-1,4-glucanases (cellobiohydrolases, EC 3.2.1.91) and encoding genes in Trichoderma species. Enzyme

Gene

Species

GenBank accession no.

Reference

Cellobiohydrolase I

cbh1

T. viride

AB021656 AY368686 AF223252 FR714421 JF343545 JF343546 JF969305 HM053612 HQ896446 JF969295 JQ309039 JQ309040 EU872026 GL985084 P62694 AY368688 DQ864992 AF302657 GU724763 M16190 M55080 AF315681 DQ504304 DQ660372 GU144297

http://www.ncbi.nlm.nih.gov/genbank [27] [28] http://www.ncbi.nlm.nih.gov/genbank [29] [29] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [29] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [30] [26] [27] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [31] [32] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank

T. harzianum

T. pleuroticola T. virens T. reesei Cellobiohydrolase II

cbh2

T. viride T. reesei

T. koningii T. longibrachiatum

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catalytic domain CBH I, it has been discovered that it formed two antiparallel b-structures containing a tunnel with a length of 4 nm at the active center [36]. The enzyme is coded by the cbh1/cel7a gene and it has been cloned for the following fungi from the Trichoderma species: T. harzianum, T. pleuroticola, T. reesei, T. virens, and T. viride [26]. Cellobiohydrolase II (CBH2/CEL6A) is classified as a member of the GH6 protein family. For T. reesei, this cellobiohydrolase has a weight of 50–58 kDa and an isoelectric point of 5.0 [38]. This enzyme accounts for up to 20% of the proteins secreted into the culture medium by T. reesei. It contains 8% carbohydrate of its molecule. The structure of CBH II is very similar to the structure of CBH I, but it reveals the presence of the double linking region B, resulting in the elongation of the molecule in comparison with CBH I. The 3D-structures of the catalytic core of these enzymes have been solved [39]. The CBH II catalytic domain adopts a b-barrel structure, formed of seven parallel turns, six of which are connected to the a-helices, while the connection between twists 6 and 7 creates an irregular structure [39]. The gene encoding cellobiohydrolases II is cbh2/cel6a and it has been discovered for T. viride, T. parceramosum, T. koningii, and T. longibrachiatum. Endo-b-1,4-glucanases The second important group of enzymes which are part of a cellulolytic complex are Endo-b-1,4-glucanases (EC 3.2.1.4) (Alternative Names: Cellulase, Avicelase, b-1,4endoglucanhydrolase, b-1,4-glucanase, Carboxymethylcellulase, Celludextrinase, Endo-1,4-b-D-glucanase, Endo1,4-b-D-glucanohydrolase, Endo-1,4-b-glucanase, Endoglucanase, EG). These enzymes, during the enzymatic hydrolysis of cellulose, randomly cleave the b-1,4glycosidic bonds in the amorphous regions of cellulose, resulting in the formation of cellulodextrines with different chain lengths. There are two main endoglucanases – EG I and EG III. Additionally, the following are included in this group of cellulases: EGII, EGIV, EG45, CEL74A, CEL61B, and CEL5B. The list of Trichoderma endob-1,4-glucanases and encoding genes is shown in Table 2. Endoglucanase I (CEL7B) represents 6–10% of the total secreted protein (value determined for T. reesei). This endoglucanase has a molecular weight of 54 kDa and an isoelectric point of 4.7 for T. reesei. Usually, the weight of this protein ranges between 50 and 55 kDa. This enzyme belongs to the GH7 protein family. It contains about 4% carbohydrates, of which 70% are linked to a protein by O-glycosidic bonds. The 3D-structures of the catalytic cores of EG I and of the cellulose binding domains have been solved [49, 50]. The gene encoding EG I is eglI/cel7b. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

It has been identified for T. viride, T. longibrachiatum, T. pseudokoningii, and T. reesei. Endoglucanase II (CEL5A) is an endonuclease that belongs to the HG5 protein family. Its molecular mass for T. reesei is 48 kDa and composed of 48 amino acids [43]. The protein is coded by the egl2/cel5a gene, which has been identified for such species as: T. orientalis, T. reesei, and T. viride. Endoglucanase III (EGIII/CEL12A) is the second best studied (after EGI) and the most important endoglucanase from fungi of the genus Trichoderma. It has been classified as a member of the GH protein family number 12 [20, 25]. For T. reesei, the molecular mass of this enzyme is 25 kDa and composed of 234 amino acids [51, 52], although most recent studies for the T. viride strain AS 3.3711 have shown that this enzyme has a molecular mass close to 44.1 kDa. Additionally, it has been shown that it is composed of 418 amino acids, of which 21 serve as a signal peptide in the N-terminal. The structure of catalytic cores of endoglucanase III has been solved [53]. Studies have claimed that the protein is consisted of 17 strongly basic, 25 strongly acidic, 179 polar, and 129 hydrophobic amino acids. It has an isoelectric point of 4.8 [54]. The gene coding this endoglucanase is egl3/cel12a. It has been identified so far for the following species: T. citrinoviride, T. harzianum, T. koningii, T. reesei, and T. viride. For T. viride strain AS 3.3711, this gene has a length of 1257 bp [54]. Endoglucanase IV (EGIV/CEL61A) is an endoglucanase that belongs to the glycosyl hydrolase GH61 family [20, 25]. For the T. viride AS 3.3711 strain, the molecular mass is to be 35.5 kDa, with 344 amino acids and an isoelectric point of 5.29 [45]. The gene encoding endoglucanase IV is eg4 and it has been cloned for T. reesei, T. saturnisporum, and T. viride. For the T. viride AS 3.3711 strain, eg4 has 1297 bp and contains 1035 bp open reading frame. Expression of this gene can be induced by different sources of sucrose, e.g., microcrystalline cellulose and corn straw, but significant expression is induced by carboxymethylcellulose (CMC). The transcription of eg4 can be inhibited by glucose and fructose [45]. Endoglucanase V (EGV/EG45/CEL45A) has been classified in the GH45 protein family [20, 25] and is the smallest of all the cellulases with a mass of 23 kDa. It is composed of 242 amino acids [46]. This enzyme is coded by egl5/ cel45a which has been isolated from T. reesei. CEL74A from the HG 7 (glycosyl hydrolase) and CEL61B from the GH 61 protein families are two enzymes that also belong to endoglucanase group [20, 25]. CEL74A is an endoglucanase with a molecular mass of 87 kDa and composed of 838 amino acids [47]. The gene encoding the CEL74A protein is cel74a and it has been cloned for

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Table 2. Endo-b-1,4-glucanases (endoglucanases, EC 3.2.1.4) and encoding genes in Trichoderma species. Enzyme

Gene

Species

GenBank accession no.

Reference

Endoglucanase I

eg1

T. viride

AY343986 AY526093 EU587012 JN247430 JF309108 JF682772 JQ796067 GU144298 X60652 EF185865 JN208864 HM641862 M15665 E00390 EU149644 JQ238607 DQ178347 AB021657 AF435068 JQ923478 AF435069 M19373 AF435070 AY343987 Y11113 GU290062 HM222525 EU518929 Z33381 AY343989 DD182177 DD182176 AY281371 AY281372 EU518927 DD181298 DD181297 AY281373 DD181300 DD181299 EU518928

http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [40] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [41] [42] [41] [43] [41] [44] [21] http://www.ncbi.nlm.nih.gov/genbank [44] [45] [46] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [47] [47] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [47] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [48]

T. longibrachiatum

T. pseudokoningii T. reesei

Endoglucanase II

eg2

T. T. T. T. T. T. T. T.

orientalis reesei viride citrinoviride harzianum koningii reesei viride

Endoglucanase III

eg3

Endoglucanase IV

eg4

T. reesei T. saturnisporum T. viride

Endoglucanase V

eg5

Endoglucanase VI

eg6

T. reesei T. viride T. reesei

CEL74A CEL61B EGVII

cel74a cel61b eg7

T. T. T. T.

CEL5B (EGVIII)

cel5b

T. reesei

reesei reesei viride reesei

T. viride

T. reesei. CEL61B, in turn, has a molecular mass of 27 kDa and comprise 249 amino acids [47]. CEL61B is encoded by the cel61b gene which was identified, with other mentioned cellulase genes, for T. reesei. Endoglucanase VIII is a protein composed of 438 amino acids with a molecular mass of 46.86 kDa and isoelectric point of 4.32 (data for T. viride AS 3.3711). This enzyme has been recognized as a member of the glycosyl hydrolase family number 5 (GH5). At the N-terminal, CEl5B has a signal peptide sequence composed of 19 amino acids, which suggests that this a protein secreted extracellularly. The gene encoding this cellulase is cel5b and its size for the T. viride AS 3.3711 strain is 1317 bp [48]. In addition to T. viride AS 3.3711, this gene ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

has been cloned for T. reesei (Table 2). The transcription of such gene for T. viride AS 3.3711 can be induced by carboxymethyl cellulose sodium (CMC-Na), sucrose, microcrystalline cellulose, and corn stalk, and inhibited by glucose and fructose [48]. b-Glucosidases b-Glucosidase (EC 3.2.1.21) (Other Names: Cellobiase, b-Dglucoside glucohydrolase) catalyzes the hydrolysis of short chain oligosaccharides and cellobiose (resulting from the synergistic action of endoglucanases and cellobiohydrolases) into glucose. b-Glucosidase makes up only 0.5–1% of the extracellular protein secreted by T. reesei. This is an enzyme with a molecular weight of

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73–150 kDa [33]. The weight of the native protein isolated from T. citrinoviride is 94 kDa [55]. It is associated with the fungal cell wall, and only less than half of the b-glucosidase is released to the medium. It is suggested that b-glucosidase plays an important role in the induction of other components of the cellulolytic complex. More specifically, b-glucosidase is not a cellulase, because it does not act on the cellulose chain. It plays a very important role in the regulation of the activity of other cellulase components because it prevents the accumulation of cellobiose. Since cellobiose inhibits other cellulolytic enzymes such as cellobiohydrolases and endoglucanases, its presence inhibits the complete hydrolysis of cellulose to glucose molecules [56–60]. It has been shown that construction of mutants with high b-glucosidase activity can be very beneficial for bioethanol production [61]. The primary structure of this enzyme consists of four potential glycosylation sites and the purified protein, there are no carbohydrate chains [33]. Seven proteins belonging to b-glucosidases have been identified. Kubicek et al. [25] mentioned the following enzymes for T. reesei: BGLI/ CEL3A, BGLII/CEL1A, CEL3B, CEL3C, CEL1B, CEL3D, and CEL3E. The list of Trichoderma b-glucosidases and encoding genes is given in Table 3. b-Glucosidase I (BGLI/CEL3A) is a protein that belongs to the glycosyl hydrolase GH3 family [20, 25]. It has a molecular mass of 75 kDa for T. reesei and is composed of 744 amino acids [62, 64]. The gene encoding the BGLI is bgl1/cel3a and it has been cloned for T. viride and T. reesei. Another b-glucosidase, that is classified to the GH 1 family, is b-glucosidase II (BGLII/CEL1A) [20, 25]. This enzyme, in turn, is composed of 466 amino acids giving it a molecular mass of 52 kDa for T. reesei [63, 65]. The gene encoding CEL1A is bgl2/cel1a, which has been identified for T. viride, T. harzianum, T. reesei, and T. longibrachiatum. Another member of the GH 1 protein family is CEL1B

[20, 25]. This protein has a mass of 55 kDa and is built by 484 amino acids. These data were obtained for T. reesei, for which gene cel1b has also been cloned [47]. The next enzyme that has been classified as a member of the cellobiase group is CEL3B, which belongs to the GH3 glycosyl hydrolase family [20, 25]. For T. reesei, it has a molecular weight of 94 kDa and is composed of 874 amino acids [47]. This enzyme is encoded by the cel3b gene. b-Glucosidase CEL3C has also been classified as a member of the GH3 (glycosyl hydrolase) family [20, 25]. An enzyme with a molecular mass of 91 kDa is composed of 833 amino acids [47]. The gene encoding this protein is cel3c and cloned for T. reesei. The last two b-glucosidases, i.e., CEL3D and CEL3E, have been considered as members of the GH 3 protein family [20, 25]. CEL3D for T. reesei has a molecular weight of 77 kDa and is 700 amino acid; while CEL3E, with a molecular weight of 83 kDa, is composed of 765 amino acids [47]. Genes encoding these two cellobioses are: cel3d and cel3e, respectively.

The role of Trichoderma cellulolytic enzymes in the colonization of different ecological niches The determined ability of the Trichoderma species to produce cellulolytic enzymes and the genetic predisposition of this species to utilize cellulose as a carbon source are reflected in the capacity of Trichoderma to colonize those ecological niches where this polysaccharide is available. Benefits of soil Soil has been considered to be both the most common and the natural habitat of these fungi and the occurrence of Trichoderma spp. in various soils, such as agricultural, forest, prairie, salt marsh, and desert of all climatic zones, has been the subject of several investigations [66].

Table 3. b-Glucosidases (cellobiases, EC 3.2.1.21) and encoding genes in Trichoderma species. Enzyme

Gene

Species

GenBank accession no.

Reference

b-Glucosidase I

bgl1

T. viride

b-Glucosidase II

bgl2

T. reesei T. viride T. harzianum

FJ882071 AY368687 U09580 AY343988 EF426299 EF426298 AB003110 GU144296 AY281374 AY281375 AY281377 AY281378 AY281379

http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [62] http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank http://www.ncbi.nlm.nih.gov/genbank [63] http://www.ncbi.nlm.nih.gov/genbank [47] [47] [47] [47] [47]

CEL3B CEL3C CEL1B CEL3D CEL3E

cel3b cel3c cel1b cel3d cel3e

T. T. T. T. T. T. T.

reesei longibrachiatum reesei reesei reesei reesei reesei

ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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However, recent studies based on high-throughput sequencing [67, 68] and metagenomic studies [69–71] have revealed that soil is inhabited by only a relatively small portion of Hypocrea/Trichoderma species, notably T. asperellum, T. harzianum, T. hamatum, T. atroviride, T. virens, T. longibrachiatum, T. gamsii, T. citrinoviride, T. koningiopsis, T. spirale, and T. koningii, which are known to generally have outstandingly high opportunistic potential. These species success in various heterotrophic interactions, exhibiting saprotrophic, mycoparasitic (necrotrophic hyperparasitism, mycotrophy), and biotrophic lifestyle. As saprotrophs Trichoderma spp. have been considered to make a contribution to the degradation of lignocellulosic plant material – plant debris and wood [72]. It has been reported that Trichoderma species are not able to decompose non-decayed wood lignocelluloses [73, 74]. This means that Trichoderma, as soft rot fungi, can utilize cellulose and hemicelluloses but not lignified cell wall components as their primary carbon source. For those fungi with weak or without any ligninolytic activity, preliminary delignification (removing the lignin-rich middle lamell of plant cells as well as the primary cell wall) by white rot (Basidiomycota) fungi and the exposure of the holocellulose-rich secondary cell wall is required [75]. Interestingly, besides saprotrophic nutrition on rotting wood, Trichoderma species are found to interfere antagonistically with primary wood decomposers (basidio- and ascomycetes) either via competition for nutrients and space substrate colonization or by using mechanisms of antibiosis and mycoparasitism (interfungal necrotrophic hyperparasitism) [76, 77]. Recent data from genome, secretome, and transcriptome, comparative analyses of the three species: T. atroviride, T. virens, and T. reesei, indicate mycoparasitism as the ancestral lifestyle of Trichoderma [78, 80]. Therefore, these studies support an earlier hypothesis that mycoparasites on wood-decaying basidiomycetes were primary trophic strategies which resulted in the future specialization of Trichoderma towards saprophytism on pre-degraded wood. This speculation has also been adapted to T. reesei, the best known saprobic fungus from the Trichoderma genus [81–83]. T. reesei – the cellulase producer of questionable origin T. reesei (teleomorph Hypocrea jecorina) was originally isolated during World War II at Guadalcanal on the Solomon Islands and identified as the cause of a massive infection in canvas and other cellulose-containing materials at a US army camp [84]. This wild-type isolate, labeled QM 6a, has been used for creating numerous biotechnologically important mutants, particularly those ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

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involved in the production of enzymes for lignocellulose hydrolysis, also on an industrial scale, and for genome sequencing projects [25, 83, 85]. It is interesting that T. reesei has subsequently been very rarely detected in the natural environment. Only its teleomorphic stages (H. jecorina) and associated T. parareesei nom. prov. have been found worldwide in pantropical climatic zones [82, 86]. The ecophysiological characterization of H. jecorina and T. parareesei nom. prov. indicate the adaptation of these two species to different ecological niches in the vertical profile of tropical forests [82], but do not explain the extreme specialization of T. reseei to the efficient degradation of plant cell wall cellulose and hemicellulose. Trichoderma in mushroom farms Another example of the nutritional specialization and specific relationship between Trichoderma and basidiomycetes is the existence of Trichoderma spp. as mushroom pathogens. Trichoderma aggressivum, as well as T. pleurotum and T. pleuroticola, are respectively the causal agents of the Agaricus and Pleurotus green mould disease in mushroom farms [87–91]. Trichoderma aggressivum has so far never been isolated from the natural environment. As observed in previous studies, the major source of T. aggressivum infection was compost and the origin of its constituents [88, 92]. Similarly, T. pleurotum has been found only on cultivated P. ostreatus and the substratum (e.g., cereal straw) for mushroom cultivation [89, 91–93]. In contrast, T. pleuroticola has been found both on wild and cultivated P. ostreatus, as well as on the natural and productive substratum of the oyster mushroom [88, 89, 92, 93]. Additionally, T. pleuroticola has been isolated from soil and wood in Canada, the United States, Europe, Iran, and New Zealand [93]. However, the natural habitat for all these species and their primary trophic strategies have not been revealed. It is noteworthy that, besides T. aggressivum or T. pleurotum and T. pleuroticola, other Trichoderma species, such as T. asperellum, T. atroviride, T. ghanense, T. harzianum, and T. longibrachiatum, have been isolated from substratum for Agaricus and Pleurotus cultivation [88, 89, 92]. Moreover, several of these species, namely T. pleuroticola, T. harzianum, T. atroviride, T. longibrachiatum, and T. asperellum, have been isolated from the basidiomes of wild-grown P. ostreatus in Hungary [88]. In connection with the recognition of mycoparasitism as the ancestral lifestyle of Trichoderma, it can therefore be suggested that the Trichoderma species, with its antagonistic capacity towards wild-grown Pleurotus (or Agaricus) species, specialized as their pathogen in mushroom farms and that the substratum for mushroom cultivation (plant biomass) could be the only infection source.

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Discrete plant – Trichoderma interactions To understand the importance and role of cellulolytic enzymes in the life of Trichoderma, it is also necessary to look at the interaction of these fungi with plants. Some groups of Trichoderma species are associated with plant roots, where they can form a symbiotic relationship or occur as plant endophytes [66, 94]. Penetration of the root tissue by hyphae of Trichoderma is usually limited to the intercellular spaces of roots alone and is restricted to epidermis [95]. It has been shown that plant root colonization efficiency in Trichoderma is increased by the production of swollenin [65, 96]. This protein, identified in T. reesei and T. asperellum, has a modular structure typical of fungal cellulases and some hemicellulase. The characteristic region of swollenin is an N-terminal cellulose binding domain (CBD), whose role is to mediate the binding of the enzyme to the insoluble substrate and modify the plant cell wall structure. It has also been observed that the swollenin can act as MAMP (microbe-associated molecular patterns) and induce a local defence response in plants [96]. Furthermore, the cellulases produced by T. longibrachiatum have been found as elicitors of melon defence, activating salicylic acid (SA) and ethylene (ET) signaling pathways [97]. Besides the intercellular root colonization, several Trichoderma species, classified as T. amazonicum, T. caribbaeum var. aequatoriale, T. evansii, T. hamatum, T. koningiopsis, T. martiale, T. ovalisporum, T. paucisporum, T. scalesiae, T. stilbohypoxyli, T. taxi, T. theobromicola, are capable of more intimate endophytic associations with plants [66, 98–100]. However, there is very little information on how Trichodema species penetrate plant tissue when establishing the endophytic relationship. As these studies are very recent, nothing can be inferred about the role of cellulases in these interactions. The place of Trichoderma cellulolytic enzymes in agriculture and industry The natural potential of Trichoderma species to attack other fungi via mycoparasitism and antibiosis as well as the exictence of this fungus as an efficient plant growth promoter, inductor of plant defence, or endophytic plant symbiont contributed to the recognition of this fungus as a significant biopesticide and an enhancer of crop productivity [101–105]. Furthermore, the documented role of Trichoderma in the bioremediation of environmental (soil) pollutants (heavy metals, hydrocarbons, pesticides) through biosorption, bioaccumulations, biovolatization, and phytobial remediation as well as in improving soil quality has expanded the possibilities of Trichoderma application in agriculture [106–110]. However, the capacity of Trichoderma species, especially ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim

T. reseei, to produce cellulolytic enzymes has mainly resulted in the application of this fungus and its enzymes in biotechnology and consequently in various industrial fields [10, 25]. The wild-type T. reesei QM6a, recognized as a good producer of cellulases, has been employed for over 50 years to generate the high cellulase-producing mutants via extensive mutagenesis and screening programs [25, 111, 112]. The most successful mutants (for example, RUT-C30 and CL487) have been used for the production of cellulolytic (especially CBHI) and hemicellulolytic enzymes as well as recombinant proteins, such as entopeptidases, glucoamylases, endochitinases, laccases, and xylanases [25, 79, 83, 112, 113]. Finally, the cellulolytic enzymes secreted by mutated Trichoderma strains find wide applications in the pulp, paper, textile, food, feed, and detergent industries [25, 114–116]. At present, these enzymes are being investigated for potential application in the processes of the conversion of lignocellulolsic material into industrially useful products, such as biofuel [3].

Conclusion Among Trichoderma species, T. reesei is thought to be a saprotrophic fungus, extremely specialized in efficient degradation of plant cell wall cellulose and hemicellulose. However, the mechanism of this specialization is still unknown. It is noteworthy that an opposing nutritional strategy was chosen by T. aggressivum as well as T. pleurotum and T. pleuroticola, which specialized in aggressive mycoparasitism. The other Trichoderma species with known ability to produce cellulolytic enzymes have been found in various ecological niches (soil, plant debris, dead wood, rhizosphere, or substratum for mushroom cultivation). These species are characterized by a high opportunistic potential enabling them to adapt to local changes in the environment and competition with other organisms as well as to access new ecological niches. Assuming, that mycoparsitism is one of the ancestral trophic strategies of Trichoderma, the ability of these fungi to adopt a saprotrophic lifestyle and thereby degrade plant cell wall cellulose is probably an additional (complementary) trait, activated under specific environmental conditions. With this in mind, there is the need to identify the key elements determining switching between these different trophic strategies.

Conflict of interest statement The authors declare no conflict of interest.

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