Repeat-induced point (RIP) mutation in the industrial workhorse fungus Trichoderma reesei

Trichoderma reesei (syn. Hypocrea jecorina) is a filamentous ascomycete. Due to its capability of producing large amounts of lignocellulolytic enzymes and various heterologous proteins, this fungus has been widely used for industrial applications for over 70 years. It is also a model organism for lignocellulosic biomass degradation and metabolic engineering. Recently, we experimentally and computationally demonstrated that Trichoderma reesei exhibits high homology pairing and repeat-induced point (RIP) mutation activities at a premeiotic stage, i.e., between fertilization and karyogamy or premeiotic DNA replication. The discovery of RIP in Trichoderma reesei not only reveals significant impacts of sexual reproduction on evolution and chromosome architecture but also provides intriguing perspectives for industrial strain improvement. This review emphasizes two major points about RIP and RIP-like processes in Pezizomycotina fungi. First, the molecular mechanisms of RIP and RIP-like processes in Trichoderma reesei and other Pezizomycotina fungi are apparently distinct from those originally described in the model fungus Neurospora crassa. Second, orthologs of the rid1 (deficient in RIP-1) DNA methyltransferase gene were shown to be essential for sexual development in at least four Pezizomycotina fungi, including Trichoderma reesei. In contrast, rid1 is dispensable for Neurospora crassa sexual development. We suggest that the rid1-like gene products and/or their DNA methyltransferase activities play critical roles in promoting fungal sexual development. The Neurospora crassa rid1 gene might have lost this evolutionarily conserved function.

     

The first 100 Trichoderma species characterized by molecular data

Trichoderma species are generally abundant on decaying wood and in soil because of their success in various heterotrophic interactions, including decomposition, parasitism, and even opportunistic endophytism. Many Trichoderma species or, precisely, many individual Trichoderma strains, have various important applications in industry and human life, which led to the inclusion of Hypocrea jecorina (Trichoderma reesei), the well-known producer of industrial enzymes, in the list of organisms whose genomes have been sequenced. Trichoderma species also have been adopted as agents of biological control of plant pathogenic fungi and as antibiotic producers. Trichoderma longibrachiatum is known as an opportunistic pathogen of immunocompromised mammals, including humans, and some species are common indoor contaminants. Given these properties, correct identification at the species level is highly desirable. However, within the past decade, the number of recognized Trichoderma species has tripled, reaching 100. Therefore, Trichoderma taxonomy and species identification is a difficult issue. The abundant homoplasy in phenetic characters is likely the reason, given that the number of morphologically distinct species is significantly lower than the number of phylogenetically distinct species recognized using methods of gene sequence analysis. In this review, we introduce to the scientific community the development of modern tools for Trichoderma species identification: the oligonucleotide barcode program TrichOKEY version 1.0, and TrichoBLAST, the multilocus database of vouchered sequences powered by a similarity search tool. We also discuss the application of the Genealogic Concordance Phylogenetic Species Recognition approach. In combination, these advances make it possible to identify all known Trichoderma species based on sequence analysis.     

Use of CRISPR-Cas tools to engineer Trichoderma species

Given their lignocellulose degradability and biocontrol activities, fungi of the ubiquitously distributed genus Trichoderma have multiple industrial and agricultural applications. Genetic manipulation plays a valuable role in tailoring novel engineered strains with enhanced target traits. Nevertheless, as applied to fungi, the classic tools of genetic manipulation tend to be time-consuming and tedious. However, the recent development of the CRISPR-Cas system for gene editing has enabled researchers to achieve genome-wide gene disruptions, gene replacements, and precise editing, and this technology has emerged as a primary focus for novel developments in engineered strains of Trichoderma. Here, we provide a brief overview of the traditional approaches to genetic manipulation, the different strategies employed in establishing CRSIPR-Cas systems, the utilization of these systems to develop engineered strains of Trichoderma for desired applications, and the future trends in biotechnology.     

The Polyketide Synthase Gene pks4 of Trichoderma reesei Provides Pigmentation and Stress Resistance

Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.     

Management of palm oil mill effluent through production of cellulases by filamentous fungi

A laboratory scale study to evaluate the potentiality of filamentous fungi for the production of cellulolytic enzymes using palm oil mill effluent (POME) as a basal medium was initiated. A total of 25 filamentous fungi in which 16 filamentous fungi were isolated and purified from oil palm industrial residues and 9 strains from laboratory stock were screened using POME with 1% total suspended solids. Trichoderma reesei RUT C-30 was identified as a potential strain for cellulolytic enzyme production as compared to other genera of Aspergillus, Penicillum, Rhizopus, Phanerochaete, Trichoderma and basidiomycete groups. The results showed that T. reesei RUT C-30 gave the highest filter paper cellulase and carboxy methyl cellulase activity of 0.917 and 2.51 U/ml respectively at day 5 of fermentation. Other parameters such as growth formation, pH, filterability and total biosolids were observed to evaluate the bioconversion process.     

Control of the bean rust fungus <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> by means of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates

Several species of the fungal genus Trichoderma act as antagonists of other fungi. A number of strains from the Trichoderma species T. harzianum Rifai are used as biological control agents for the control of soilborne as well as foliar plant pathogens. Six T. harzianum strains, five of them isolated from commercial preparations, were evaluated for their capability to control the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger. Different kinds of leaf disc assays were performed with conidial spore suspensions and sterile culture filtrates of the T. harzianum strains. Great differences were observed concerning the efficacy of the Trichoderma strains to reduce the number of the uredial pustules developing after rust inoculation which followed the application of the particular Trichoderma strains. Efficacy values ranged from 1 to over 50%. Increasing spore or culture filtrate concentrations of the two most effective isolates T12 and T_U led to decreases in the number of developing uredial pustules. Culture filtrate applications had a protective but no curative effect. T12 spore suspensions maintained their disease reducing activity even when autoclaved. This and some other evidence for an antibiotic interaction between T. harzianum and U. appendiculatus are discussed. Handling Editor: Reijo Karjalainen.     

Peculiarities of <Emphasis Type="Italic">Pycnoporus</Emphasis> species for applications in biotechnology

The genus Pycnoporus forms a cosmopolitan group of four species belonging to the polyporoid white-rot fungi, the most representative group of homobasidiomycetes causing wood decay. Pycnoporus fungi are listed as food- and cosmetic-grade microorganisms and emerged in the early 1990s as a genus whose biochemistry, biodegradation and biotechnological properties have since been progressively detailed. First highlighted for their original metabolic pathways involved in the functionalization of plant cell wall aromatic compounds to yield high-value molecules, e.g. aromas and antioxidants, the Pycnoporus species were later explored for their potential to produce various enzymes of industrial interest, such as hydrolases and oxidases. However, the most noteworthy feature of the genus Pycnoporus is its ability to overproduce high redox potential laccase—a multi-copper extracellular phenoloxidase—as the predominant ligninolytic enzyme. A major potential use of the Pycnoporus fungi is thus to harness their laccases for various applications such as the bioconversion of agricultural by-products and raw plant materials into valuable products, the biopulping and biobleaching of paper pulp and the biodegradation of organopollutants, xenobiotics and industrial contaminants. All the studies performed in the last decade show the genus Pycnoporus to be a strong contender for white biotechnology. In this review, we describe the properties of Pycnoporus fungi in relation to their biotechnological applications and potential.     

Effect of Trichoderma Fungi on Soil Micromycetes That Cause Infectious Conifer Seedling Lodging in Siberian Tree Nurseries

Soils in the tree nurseries studied were characterized by a lower species diversity of fungi than adjacent virgin soils. In particular, the relative abundances of representatives of the genera Mucor, Chaetomium, and Trichoderma in the nursery soil were two times lower than in adjacent virgin soils. On the other hand, the nursery soil exhibited greater abundances of fungi of the genus Fusarium, which are causative agents of many diseases of conifer seedlings. To appreciate the efficiency of biocontrol of the infectious diseases of conifer seedlings, we introduced several indigenous Trichoderma strains into the nursery soil and found that this affected the species composition of soil microflora considerably. Changes in the species composition of mycobiota beneficially influenced the phytosanitary state of soils and reduced the infectious lodging of conifer seedlings.     

The toolbox of Trichoderma spp. in the biocontrol of Botrytis cinerea disease

Botrytis cinerea is a necrotrophic fungal pathogen causing disease in many plant species, leading to economically important crop losses. So far, fungicides have been widely used to control this pathogen. However, in addition to their detrimental effects on the environment and potential risks for human health, increasing fungicide resistance has been observed in the B. cinerea population. Biological control, that is the application of microbial organisms to reduce disease, has gained importance as an alternative or complementary approach to fungicides. In this respect, the genus Trichoderma constitutes a promising pool of organisms with potential for B. cinerea control. In the first part of this article, we review the specific mechanisms involved in the direct interaction between the two fungi, including mycoparasitism, the production of antimicrobial compounds and enzymes (collectively called antagonism), and competition for nutrients and space. In addition, biocontrol has also been observed when Trichoderma is physically separated from the pathogen, thus implying an indirect systemic plant defence response. Therefore, in the second part, we describe the consecutive steps leading to induced systemic resistance (ISR), starting with the initial Trichoderma–plant interaction and followed by the activation of downstream signal transduction pathways and, ultimately, the defence response resulting in ISR (ISR‐prime phase). Finally, we discuss the ISR‐boost phase, representing the effect of ISR priming by Trichoderma spp. on plant responses after additional challenge with B. cinerea.     

Comparison of different cellulolytic fungi for bioconversion of apple distillery waste

Summary The suitability of three ascomycetous fungi, Aspergillus niger, A. awamori and Trichoderma reesei, as well as two basidiomycetes, Pleurotus ostreatus and Phanerochaete chrysosporium, for bioconversion of apple distillery slop was compared. Trichoderma and Phanerochaete degraded raw fiberes by 20%, producing filter cakes with 17% to 22% raw protein contents. Aspergillus spp. were superior in filtration time and COD reduction and were of the same efficiency in protein synthesis as Trichoderma and Phanerochaete, but did not degrade fibres. Pleurotus ostreatus did not degrade lignin under fermentation conditions used and could not compete with other fungi due to its slower growth.     

Trichoderma Xylanases,Their Properties and Application

Abstract

Trichoderma spp. are known to produce enzymes with high xylanolytic activity. Different xylanases and various components of their xylanolytic system have been identified and purified. Some of the xylanases have been characterized extensively with respect to their physicochemical, hydrolytic, and molecular properties. Cellulase-free xylanase preparations have been tested successfully in industrial applications such as the prebleaching of kraft pulps in the pulp and paper industry. Future work on understanding the functional significance of xylanase multiplicity, the mechanisms of xylanase prebleaching, and the structural conformation of xylanases could lead to improved or alternative applications of Trichoderma xylanases.     

PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resilied in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.     

Biotransformation of Trichoderma spp. and Their Tolerance to Aromatic Amines,a Major Class of Pollutants

Trichoderma spp. are cosmopolitan soil fungi that are highly resistant to many toxic compounds. Here, we show that Trichoderma virens and T. reesei are tolerant to aromatic amines (AA), a major class of pollutants including the highly toxic pesticide residue 3,4-dichloroaniline (3,4-DCA). In a previous study, we provided proof-of-concept remediation experiments in which another soil fungus, Podospora anserina, detoxifies 3,4-DCA through its arylamine N-acetyltransferase (NAT), a xenobiotic-metabolizing enzyme that enables acetyl coenzyme A-dependent detoxification of AA. To assess whether the N-acetylation pathway enables AA tolerance in Trichoderma spp., we cloned and characterized NATs from T. virens and T. reesei. We characterized recombinant enzymes by determining their catalytic efficiencies toward several toxic AA. Through a complementary approach, we also demonstrate that both Trichoderma species efficiently metabolize 3,4-DCA. Finally, we provide evidence that NAT-independent transformation is solely (in T. virens) or mainly (in T. reesei) responsible for the observed removal of 3,4-DCA. We conclude that T. virens and, to a lesser extent, T. reesei likely utilize another, unidentified, metabolic pathway for the detoxification of AA aside from acetylation. This is the first molecular and functional characterization of AA biotransformation in Trichoderma spp. Given the potential of Trichoderma for cleanup of contaminated soils, these results reveal new possibilities in the fungal remediation of AA-contaminated soil.     

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and -octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.     

EVOLUTION OF HABITAT PREFERENCE AND NUTRITION MODE IN A COSMOPOLITAN FUNGAL GENUS WITH EVIDENCE OF INTERKINGDOM HOST JUMPS AND MAJOR SHIFTS IN ECOLOGY

Host jumps by microbial symbionts are often associated with bursts of species diversification driven by the exploitation of new adaptive zones. The objective of this study was to infer the evolution of habitat preference (decaying plants, soil, living fungi, and living plants), and nutrition mode (saprotrophy and mycoparasitism) in the fungal genus Trichoderma to elucidate possible interkingdom host jumps and shifts in ecology. Host and ecological role shifts were inferred by phylogenetic analyses and ancestral character reconstructions. The results support several interkingdom host jumps and also show that the preference for a particular habitat was gained or lost multiple times. Diversification analysis revealed that mycoparasitism is associated with accelerated speciation rates, which then suggests that this trait may be linked to the high number of species in Trichoderma. In this study it was also possible to infer the cryptic roles that endophytes or soil inhabitants play in their hosts by evaluating their closest relatives and determining their most recent ancestors. Findings from this study may have implications for understanding certain evolutionary processes such as species radiations in some hyperdiverse groups of fungi, and for more applied fields such as the discovery and development of novel biological control strategies.     

Structural and Phylogenetic Analysis of Laccases from Trichoderma: A Bioinformatic Approach

The genus Trichoderma includes species of great biotechnological value, both for their mycoparasitic activities and for their ability to produce extracellular hydrolytic enzymes. Although activity of extracellular laccase has previously been reported in Trichoderma spp., the possible number of isoenzymes is still unknown, as are the structural and functional characteristics of both the genes and the putative proteins. In this study, the system of laccases sensu stricto in the Trichoderma species, the genomes of which are publicly available, were analyzed using bioinformatic tools. The intron/exon structure of the genes and the identification of specific motifs in the sequence of amino acids of the proteins generated in silico allow for clear differentiation between extracellular and intracellular enzymes. Phylogenetic analysis suggests that the common ancestor of the genus possessed a functional gene for each one of these enzymes, which is a characteristic preserved in T. atroviride and T. virens. This analysis also reveals that T. harzianum and T. reesei only retained the intracellular activity, whereas T. asperellum added an extracellular isoenzyme acquired through horizontal gene transfer during the mycoparasitic process. The evolutionary analysis shows that in general, extracellular laccases are subjected to purifying selection, and intracellular laccases show neutral evolution. The data provided by the present study will enable the generation of experimental approximations to better understand the physiological role of laccases in the genus Trichoderma and to increase their biotechnological potential.     

Changes in hyphal morphology and activity of phenoloxidases during interactions between selected ectomycorrhizal fungi and two species of <Emphasis Type="Italic">Trichoderma</Emphasis>

Patterns of phenoloxidase activity can be used to characterize fungi of different life styles, and changes in phenoloxidase synthesis were suspected to play a role in the interaction between ectomycorrhizal and two species of Trichoderma. Confrontation between the ectomycorrhizal fungi Amanita muscaria and Laccaria laccata with species of Trichoderma resulted in induction of laccase synthesis, and the laccase enzyme was bound to mycelia of ectomycorrhizal fungi. Tyrosinase release was noted only during interaction of L. laccata strains with Trichoderma harzianum and T. virens. Ectomycorrhizal fungi, especially strains of Suillus bovinus and S. luteus, inhibited growth of Trichoderma species and caused morphological changes in its colonies in the zone of interaction. In contrast, hyphal changes occurred less often in the ectomycorrhizal fungi tested. Species of Suillus are suggested to present a different mechanism in their interaction with other fungi than A. muscaria and L. laccata.     

Foaming and enzyme activity in fungal cultures grown on sugar beet cosette

Summary Enzyme synthesis, foaming behaviour and its effects were studied using two common cellulolytic fungi;Trichoderma reesei andSporotrichum pulverulentum in a medium containing sugar beet cosette as a cellulosic substrate. Cellulase enzyme activities in the culture broth were found to be higher than the enzyme activities in natural and experimentally forced foam layers.     

The Production of Multiple Small Peptaibol Families by Single 14‐Module Peptide Synthetases in Trichoderma/Hypocrea

The most common sequences of peptaibiotics are 11‐residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14‐residue peptaibols sharing partial sequence identity. Genome sequencing projects of three Trichoderma strains of the major clades reveal the presence of up to three types of nonribosomal peptide synthetases with 7, 14, or 18–20 amino acid‐adding modules. Here, we provide evidence that the 14‐module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina), and T. atroviride produces both 11‐ and 14‐residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid‐activating domains and modules. The sequences of these peptides may be predicted from the gene sequences and have been confirmed by analysis of families of 11‐ and 14‐residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 sequences determined, 2 new), and the recently established trichovirins A from T. virens. The distribution of 11‐ and 14‐residue products is strain‐specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.     

Endophytes and mycoparasites associated with an indigenous forest tree, <Emphasis Type="Italic">Theobroma gileri</Emphasis>, in Ecuador and a preliminary assessment of their potential as biocontrol agents of cocoa diseases

The main constraint and continuing threat to cocoa production in Latin America is disease caused by two basidiomycete fungi belonging to the genus Crinipellis (Agaricales), both of which are currently on an invasive front. Classical biological control, in which coevolved natural enemies are considered to offer the most potential for sustainable control of invasive alien organisms, is investigated as a management strategy. Our initial approach has been to search for the suspected coevolved host (Theobroma gileri) of one of these pathogens, Crinipellis roreri, the causal agent of frosty pod rot, in the mesic forests of north-west Ecuador; to isolate the endophytes and mycoparasites associated with both host and pathogen; to screen selected fungi in the greenhouse and laboratory, in order to determine their biocontrol potential. Endophytes were isolated directly in the field from healthy tree boles and pods, and over 40 genera were recorded, mainly representing anamorphs of Hypocreales in the genera Acremonium, Clonostachys, Trichoderma and Verticillium, as well as basidiomycetes belonging to the Agaricales and Polyporales. Of the endophytic fungi tested, most can be inoculated into and recovered from asymptomatic cocoa seedlings. Twenty-eight mycoparasitic species were isolated from diseased pods and selected mycoparasites as well as endophytes were screened against C. roreri using the pre-colonised plate method. Consistently high mycoparasitism of the C. roreri pseudostroma was demonstrated, particularly by a complex of Clonostachys and Trichoderma species.