Mycoparasitic Fungus Trichoderma Research Articles

Demonstrating the Applicability of Proton Transfer Reaction Mass Spectrometry to Quantify Volatiles Emitted by the Mycoparasitic Fungus Trichoderma atroviride in Real Time: Monitoring of Trichoderma-Based Biopesticides.

The present study aims to explore the potential application of proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS) for real-time monitoring of microbial volatile organic compounds (MVOCs). This investigation can be broadly divided into two parts. First, a selection of 14 MVOCs was made based on previous research that characterized the MVOC emissions of Trichoderma atroviride, which is a filamentous fungus widely used as a biocontrol agent. The analysis of gas-phase standards using PTR-ToF-MS allowed for the categorization of these 14 MVOCs into two groups: the first group primarily undergoes nondissociative proton transfer, resulting in the formation of protonated parent ions, while the second group mainly undergoes dissociative proton transfer, leading to the formation of fragment ions. In the second part of this investigation, the emission of MVOCs from samples of T. atroviride was continuously monitored over a period of five days using PTR-ToF-MS. This also included the first quantitative online analysis of 6-amyl-α-pyrone (6-PP), a key MVOC emitted by T. atroviride. The 6-PP emissions of T. atroviride cultures were characterized by a gradual increase over the first two days of cultivation, reaching a plateau-like maximum with volume mixing ratios exceeding 600 ppbv on days three and four. This was followed by a marked decrease, where the 6-PP volume mixing ratios plummeted to below 50 ppbv on day five. This observed sudden decrease in 6-PP emissions coincided with the start of sporulation of the T. atroviride cultures as well as increasing intensities of product ions associated with 1-octen-3-ol and 3-octanone, whereas both these MVOCs were previously associated with sporulation in T. atroviride. The study also presents the observations and discussion of further MVOC emissions from the T. atroviride samples and concludes with a critical assessment of the possible applications and limitations of PTR-ToF-MS for the online monitoring of MVOCs from biological samples in real time.

The histone deacetylase Hda1 affects oxidative and osmotic stress response as well as mycoparasitic activity and secondary metabolite biosynthesis in Trichoderma atroviride.

The mycoparasitic fungus Trichoderma atroviride is applied in agriculture as a biostimulant and biologic control agent against fungal pathogens that infest crop plants. Secondary metabolites are among the main agents determining the strength and progress of the mycoparasitic attack. However, expression of most secondary metabolism-associated genes requires specific cues, as they are silent under routine laboratory conditions due to their maintenance in an inactive heterochromatin state. Therefore, histone modifications are crucial for the regulation of secondary metabolism. Here, we functionally investigated the role of the class II histone deacetylase encoding gene hda1 of T. atroviride by targeted gene deletion, phenotypic characterization, and multi-omics approaches. Deletion of hda1 did not result in obvious phenotypic alterations but led to an enhanced inhibitory activity of secreted metabolites and reduced mycoparasitic abilities of T. atroviride against the plant-pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The ∆hda1 mutants emitted altered amounts of four volatile organic compounds along their development, produced different metabolite profiles upon growth in liquid culture, and showed a higher susceptibility to oxidative and osmotic stress. Moreover, hda1 deletion affected the expression of several notable gene categories such as polyketide synthases, transcription factors, and genes involved in the HOG MAPK pathway.IMPORTANCEHistone deacetylases play crucial roles in regulating chromatin structure and gene transcription. To date, classical-Zn2+ dependent-fungal histone deacetylases are divided into two classes, of which each comprises orthologues of the two sub-groups Rpd3 and Hos2 and Hda1 and Hos3 of yeast, respectively. However, the role of these chromatin remodelers in mycoparasitic fungi is poorly understood. In this study, we provide evidence that Hda1, the class II histone deacetylases of the mycoparasitic fungus Trichoderma atroviride, regulates its mycoparasitic activity, secondary metabolite biosynthesis, and osmotic and oxidative stress tolerance. The function of Hda1 in regulating bioactive metabolite production and mycoparasitism reveals the importance of chromatin-dependent regulation in the ability of T. atroviride to successfully control fungal plant pathogens.

Trichoderma koningiopsis (Hypocreaceae) has the smallest mitogenome of the genus Trichoderma.

Fungal mitogenomes exhibit remarkable variation in conformation, size, gene content, arrangement and expression, including their intergenic spacers and introns. The complete mitochondrial genome sequence of the mycoparasitic fungus Trichoderma koningiopsis was determined using the Illumina next-generation sequencing technology. We used data from our recent Illumina NGS-based project of T. koningiopsis genome sequencing to study its mitochondrial genome. The mitogenome was assembled, annotated, and compared with other fungal mitogenomes. T. koningiopsis strain POS7 mitogenome is a circular molecule of 27,560 bp long with a GC content of 27.80%. It harbors the whole complement of the 14 conserved mitochondrial protein-coding genes (PCG) such as atp6, atp8, atp9, cox1, cox2, cox3, cob, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6, also found in the same gene order to other Hypocreales. The mitogenome also contains 26 transfer RNA genes (tRNAs), 5 of them with more than one copy. Other genes also present in the assembled mitochondrial genome are a small rRNA subunit and a large rRNA subunit containing ribosomal protein S3 gene. Despite the small genome size, two introns were detected in the T. koningiopsis POS7 mitogenome, one of them in cox3 gene and the other in rnl gene, accounting 7.34% of this mitogenome with a total size of 2,024 bp. A phylogenetic analysis was done using the 14 PCGs genes of T. koningiopsis strain POS7 mitogenome to compare them with those from other fungi of the Subphyla Pezizomycotina and Saccharomycotina. T. koningiopsis strain POS7 was clustered together with other representatives of Trichoderma lineage, within the Hypocreales group, which is also supported by previous phylogenetic studies based on nuclear markers. The mitochondrial genome of T. koningiopsis POS7 will allow further investigations into the taxonomy, phylogenetics, conservation genetics, and evolutionary biology of this important genus as well as other closely related species.

Potential use of entomopathogenic and mycoparasitic fungi against powdery mildew in aquaponics

Aquaponics has the potential to produce sustainable and accessible quality food through the integration of hydroponics and aquaculture. Plants take up dissolved nutrients in fish wastewater, allowing water reuse for fish. However, the simultaneous presence of fish and plants in the same water loop has made phytosanitary treatments of diseases such as powdery mildew problematic due to risks of toxicity for fish and beneficial bacteria, limiting its commercialization. Entomopathogenic and mycoparasitic fungi have been identified as safe biological control agents for a broad range of pests. This study aimed to investigate the efficacy of entomopathogenic fungi, Lecanicillium attenuatum (LLA), Isaria fumosorosea (IFR), and mycoparasitic fungus Trichoderma virens (TVI) against Podosphaera xanthii. Also, we investigated the possible harmful effects of the three fungal biocontrol agents in aquaponics by inoculating them in aquaponics water and monitoring their survival and growth. The findings showed that the three biocontrol agents significantly suppressed the powdery mildew at 107 CFU/ml concentration. Under greenhouse conditions (65-73% relative humidity (RH)), a significant disease reduction percentage of 85% was recorded in L. attenuatum-pretreated leaves. IFR-treated leaves had the least AUDPC (area under disease progress curve) of ~434.2 and disease severity of 32% under 65-73% RH. In addition, L. attenuatum spores were the most persistent on the leaves, the spores population increased to 9.54 × 103 CFUmm-2 from the initial 7.3 CFUmm-2 under 65-73%. In contrast, in hydroponics water, the LLA, IFR, and TVI spores significantly reduced by more than 99% after 96 hrs. Initial spore concentrations of LLA of 107 CFU/ml spores were reduced to 4 x 103 CFU after 96 hrs. Though the results from this study were intended for aquaponics systems, relevance of the results to other cultivation systems are discussed.

Sequencing and Analysis of the Entire Genome of the Mycoparasitic Bioeffector Fungus Trichoderma asperelloides Strain T 203 (Hypocreales).

ABSTRACTThe filamentous mycoparasitic fungus Trichoderma asperelloides (Hypocreales, Ascomycota, Dikarya) strain T 203 was isolated from soil in Israel by the Ilan Chet group in the 1980s. As it has been the subject of laboratory, greenhouse, and field experiments and has been incorporated into commercial agricultural preparations, its genome has been sequenced and analyzed.

Low Virulence of the Fungi Escovopsis and Escovopsioides to a Leaf-Cutting Ant-Fungus Symbiosis.

Eusocial insects interact with a diversity of parasites that can threaten their survival and reproduction. The amount of harm these parasites cause to their hosts (i.e., their virulence) can be influenced by numerous factors, such as the ecological context in which the parasite and its host are inserted. Leaf-cutting ants (genera Atta, Acromyrmex and Amoimyrmex, Attini: Formicidae) are an example of a eusocial insect whose colonies are constantly threatened by parasites. The fungi Escovopsis and Escovopsioides (Ascomycota: Hypocreales) are considered a highly virulent parasite and an antagonist, respectively, to the leaf-cutting ants’ fungal cultivar, Leucoagaricus gongylophorus (Basidiomycota: Agaricales). Since Escovopsis and Escovopsioides are common inhabitants of healthy colonies that can live for years, we expect them to have low levels of virulence. However, this virulence could vary depending on ecological context. We therefore tested two hypotheses: (i) Escovopsis and Escovopsioides are of low virulence to colonies; (ii) virulence increases as colony complexity decreases. For this, we used three levels of complexity: queenright colonies (fungus garden with queen and workers), queenless colonies (fungus garden and workers, without queen) and fungus gardens (without any ants). Each was inoculated with extremely high concentrations of conidia of Escovopsis moelleri, Escovopsioides nivea, the mycoparasitic fungus Trichoderma longibrachiatum or a blank control. We found that these fungi were of low virulence to queenright colonies. The survival of queenless colonies was decreased by E. moelleri and fungus gardens were suppressed by all treatments. Moreover, E. nivea and T. longibrachiatum seemed to be less aggressive than E. moelleri, observed both in vivo and in vitro. The results highlight the importance of each element (queen, workers and fungus garden) in the leaf-cutting ant-fungus symbiosis. Most importantly, we showed that Escovopsis may not be virulent to healthy colonies, despite commonly being described as such, with the reported virulence of Escovopsis being due to poor colony conditions in the field or in laboratory experiments.

Sequencing and Analysis of the Entire Genome of the Mycoparasitic Fungus Trichoderma afroharzianum.

Trichoderma sp. is a globally occurring fungal ascomycete. The genus Trichoderma belongs to the order of Hypocreales in the class of Sordariomycetes. Due to its importance as a mycoparasite and biocontrol fungus that antagonizes phytopathogenic and mycotoxin-producing fungi, the genome of the Trichoderma afroharzianum strain BFE349 from the fungal strain collection of the Max Rubner-Institut was sequenced and analyzed.

Functional characterization of the AGL1 aegerolysin in the mycoparasitic fungus Trichoderma atroviride reveals a role in conidiation and antagonism

Aegerolysins are small secreted pore-forming proteins that are found in both prokaryotes and eukaryotes. The role of aegerolysins in sporulation, fruit body formation, and in lysis of cellular membrane is suggested in fungi. The aim of the present study was to characterize the biological function of the aegerolysin gene agl1 in the mycoparasitic fungus Trichoderma atroviride, used for biological control of plant diseases. Gene expression analysis showed higher expression of agl1 during conidiation and during growth in medium supplemented with cell wall material from the plant pathogenic fungus Rhizoctonia solani as the sole carbon source. Expression of agl1 was supressed under iron-limiting condition, while agl1 transcript was not detected during T. atroviride interactions with the prey fungi Botrytis cinerea or R. solani. Phenotypic analysis of agl1 deletion strains (Δagl1) showed reduced conidiation compared to T. atroviride wild type, thus suggesting the involvement of AGL1 in conidiation. Furthermore, the Δagl1 strains display reduced antagonism towards B. cinerea and R. solani based on a secretion assay, although no difference was detected during direct interactions. These data demonstrate the role of AGL1 in conidiation and antagonism in the mycoparasitic fungus T. atroviride.

The Lipoxygenase Lox1 Is Involved in Light- and Injury-Response, Conidiation, and Volatile Organic Compound Biosynthesis in the Mycoparasitic Fungus Trichoderma atroviride.

The necrotrophic mycoparasite Trichoderma atroviride is a biological pest control agent frequently applied in agriculture for the protection of plants against fungal phytopathogens. One of the main secondary metabolites produced by this fungus is 6-pentyl-α-pyrone (6-PP). 6-PP is an organic compound with antifungal and plant growth-promoting activities, whose biosynthesis was previously proposed to involve a lipoxygenase (Lox). In this study, we investigated the role of the single lipoxygenase-encoding gene lox1 encoded in the T. atroviride genome by targeted gene deletion. We found that light inhibits 6-PP biosynthesis but lox1 is dispensable for 6-PP production as well as for the ability of T. atroviride to parasitize and antagonize host fungi. However, we found Lox1 to be involved in T. atroviride conidiation in darkness, in injury-response, in the production of several metabolites, including oxylipins and volatile organic compounds, as well as in the induction of systemic resistance against the plant-pathogenic fungus Botrytis cinerea in Arabidopsis thaliana plants. Our findings give novel insights into the roles of a fungal Ile-group lipoxygenase and expand the understanding of a light-dependent role of these enzymes.

The Evolutionary and Functional Paradox of Cerato-platanins in Fungi.

Cerato-platanins (CPs) form a family of fungal small secreted cysteine-rich proteins (SSCPs) and are of particular interest not only because of their surface activity but also their abundant secretion by fungi. We performed an evolutionary analysis of 283 CPs from 157 fungal genomes with the focus on the environmental opportunistic plant-beneficial and mycoparasitic fungus Trichoderma Our results revealed a long evolutionary history of CPs in Dikarya fungi that have undergone several events of lateral gene transfer and gene duplication. Three genes were maintained in the core genome of Trichoderma, while some species have up to four CP-encoding genes. All Trichoderma CPs evolve under stabilizing natural selection pressure. The functional genomic analysis of CPs in Trichoderma guizhouense and Trichoderma harzianum revealed that only epl1 is active at all stages of development but that it plays a minor role in interactions with other fungi and bacteria. The deletion of this gene results in increased colonization of tomato roots by Trichoderma spp. Similarly, biochemical tests of EPL1 heterologously produced by Pichia pastoris support the claims described above. Based on the results obtained, we conclude that the function of CPs is probably linked to their surfactant properties and the ability to modify the hyphosphere of submerged mycelia and, thus, facilitate the nutritional versatility of fungi. The effector-like functions do not sufficiently describe the diversity and evolution of these proteins in fungi, as they are also maintained, duplicated, or laterally transferred in the genomes of nonherbivore fungi.IMPORTANCE Cerato-platanins (CPs) are surface-active small proteins abundantly secreted by filamentous fungi. Consequently, immune systems of plants and other organisms recognize CPs and activate defense mechanisms. Some CPs are toxic to plants and act as virulence factors in plant-pathogenic fungi. Our analysis, however, demonstrates that the interactions with plants do not explain the origin and evolution of CPs in the fungal kingdom. We revealed a long evolutionary history of CPs with multiple cases of gene duplication and events of interfungal lateral gene transfers. In the mycoparasitic Trichoderma spp., CPs evolve under stabilizing natural selection and hamper the colonization of roots. We propose that the ability to modify the hydrophobicity of the fungal hyphosphere is a key to unlock the evolutionary and functional paradox of these proteins.

Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion

In this study, a chitinase gene, Chit46 from a mycoparasitic fungus Trichoderma harzianum was successfully expressed in Pichia pastoris with a high heterologous chitinase production of 31.4 U/mL, much higher than the previous reports. The active center and substrate binding pocket of the recombinant Chit46 (rChit46) were analyzed and the effects of pH, temperature, metal ions and glycosylation on its activity were tested. rChit46 effectively hydrolyzed colloidal chitin with a high conversion rate of 80.5% in 3 h and the chitin hydrolysates were mainly composed of (GlcNAc)2 (94.8%), which make it a good candidate for the green recycling of chitinous waste. rChit46 could also significantly inhibit growth of the phytopathogenic fungus Botrytis cinerea, which endowed it with the potential as a biocontrol agent.

Massive lateral transfer of genes encoding plant cell wall-degrading enzymes to the mycoparasitic fungus Trichoderma from its plant-associated hosts.

Unlike most other fungi, molds of the genus Trichoderma (Hypocreales, Ascomycota) are aggressive parasites of other fungi and efficient decomposers of plant biomass. Although nutritional shifts are common among hypocrealean fungi, there are no examples of such broad substrate versatility as that observed in Trichoderma. A phylogenomic analysis of 23 hypocrealean fungi (including nine Trichoderma spp. and the related Escovopsis weberi) revealed that the genus Trichoderma has evolved from an ancestor with limited cellulolytic capability that fed on either fungi or arthropods. The evolutionary analysis of Trichoderma genes encoding plant cell wall-degrading carbohydrate-active enzymes and auxiliary proteins (pcwdCAZome, 122 gene families) based on a gene tree / species tree reconciliation demonstrated that the formation of the genus was accompanied by an unprecedented extent of lateral gene transfer (LGT). Nearly one-half of the genes in Trichoderma pcwdCAZome (41%) were obtained via LGT from plant-associated filamentous fungi belonging to different classes of Ascomycota, while no LGT was observed from other potential donors. In addition to the ability to feed on unrelated fungi (such as Basidiomycota), we also showed that Trichoderma is capable of endoparasitism on a broad range of Ascomycota, including extant LGT donors. This phenomenon was not observed in E. weberi and rarely in other mycoparasitic hypocrealean fungi. Thus, our study suggests that LGT is linked to the ability of Trichoderma to parasitize taxonomically related fungi (up to adelphoparasitism in strict sense). This may have allowed primarily mycotrophic Trichoderma fungi to evolve into decomposers of plant biomass.

The Putative Protein Methyltransferase LAE1 of Trichoderma atroviride Is a Key Regulator of Asexual Development and Mycoparasitism

In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of lae1 gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a Δ lae1 and 30–50% increased in lae1-overexpressing (OElae1) strains. In darkness, Δ lae1 strains did not sporulate, and the OElae1 strains produced as much spores as the parent strain. Loss-of-function of lae1 also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of lae1 also increased the sensitivity of T. atroviride to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the OElae1 strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the Δ lae1 strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows T. atroviride LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.

Evaluation of the effect of chitin-rich residues on the chitinolytic activity of Trichoderma harzianum: In vitro and greenhouse nursery experiments

The chitin-rich residues obtained from several industries have numerous applications in diverse fields such as biotechnology, pharmacology, medicine and agriculture. In this study, three different chitin-rich residues were evaluated with regard to enhancement of the chitinolytic activity of the mycoparasitic fungus Trichoderma harzianum, used for biological control of Fusarium oxysporum in greenhouse nurseries. Chitosan (CHIT), shrimp shell powder (SSP) and mushroom wastes (MW) activated the expression of the T. harzianum genes encoding the NAGase and chitinase activities (exc1, exc2, chit42, chit33 and chit37), the highest in vitro activities of T. harzianum being seen with MW. Under greenhouse nursery conditions, the treatments involving amendment with these residues and with T. harzianum increased the NAGase activity (between 5- and 20-times higher than peat) and the chitinase activity (between 3- and 6-times higher than peat) of the growing media and maintained the shoot dry weight of plants infected with F. oxysporum. Moreover, the incorporation of these residues into the growing media enhanced the growth (first leaf length, stem length and shoot dry weight) of muskmelon seedlings. The use of these residues with T. harzianum as amendments of growing media enhanced the growth of muskmelon seedlings and decreased the weight loss due to the pathogen, giving an added-value to these residues and enhancing their management.

Purification and characterization of an extracellular 24 kDa chitobiosidase from the mycoparasitic fungus Trichoderma saturnisporum

A Trichoderma saturnisporum Hamill isolate GITX-Panog (C) exhibiting strong chitinolytic and antifungal activity against Fusarium oxysporum f.sp. dianthi, the causal agent of vascular wilt in carnation was used to purify extracellular chitobiosidase using Czapek-Dox broth amended with the fungal mycelium as the carbon source. The protein was purified by precipitation with ammonium sulphate, followed by DEAE-Cellulose anion-exchange and Sephacryl S-200 high resolution gel filtration chromatography. The purity of the enzyme was determined by SDS-PAGE, with an estimated molecular mass of 24 kDa. In native gel assay with 4-methylumbelliferyl -N,N ' diacetyl-β-D-chitobioside (4-Mu-(GluNAc)(2) , the purified chitobiosidase was visualized as single fluorescent band. Enzyme activity towards short oligomeric natural substrates indicated that the enzyme has properties that are characteristic to exochitinases. The enzyme was active up to 60 °C and at pH 4.0, and displayed maximum stability at 50 °C. Mn(2+) and Zn(2+) stimulated the enzyme activity by 63% and 41%, respectively. The K(m) and V(max) values of the purified enzyme for 4-Mu-(GluNAc)(2) were 338.9 μM ml(-1) and 0.119 μM ml(-1) min(-1) , respectively. This appears to be the first report of characterization of a chitobiosidase from antagonistic Trichoderma saturnisporum.

Use of a non-homologous end-joining-deficient strain (delta-ku70) of the biocontrol fungus Trichoderma virens to investigate the function of the laccase gene lcc1 in sclerotia degradation

The aim of this study was to apply a generated Δtku70 strain with increased homologous recombination efficiency from the mycoparasitic fungus Trichoderma virens for studying the involvement of laccases in the degradation of sclerotia of plant pathogenic fungi. Inactivation of the non-homologous end-joining pathway has become a successful tool in filamentous fungi to overcome poor targeting efficiencies for genetic engineering. Here, we applied this principle to the biocontrol fungus T. virens, strain I10, by deleting its tku70 gene. This strain was subsequently used to delete the laccase gene lcc1, which we found to be expressed after interaction of T. virens with sclerotia of the plant pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Lcc1 was strongly upregulated at early colonization of B. cinerea sclerotia and steadily induced during colonization of S. sclerotiorum sclerotia. The Δtku70Δlcc1 mutant was altered in its ability to degrade the sclerotia of B. cinerea and S. sclerotiorum. Interestingly, while the decaying ability for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum sclerotia was even enhanced, suggesting the operation of different mechanisms in the mycoparasitism of these two types of sclerotia by the laccase LCC1.

Chitosan increases conidiation in fungal pathogens of invertebrates

Antifungal activity of chitosan on plant pathogenic fungi has been widely studied, but little is known about the effect of chitosan on fungal biocontrol agents. In this work, we characterize the increase of conidiation induced by chitosan in fungal pathogens of invertebrates (FPI). Chitosan increased conidiation of FPI, including Beauveria bassiana, widely used as mycoinsecticide, and did not affect conidia viability or pathogenicity. Increased conidiation induced by chitosan is shown to be concentration dependent and is not associated to growth inhibition as observed for the mycoparasitic fungus Trichoderma harzianum. Real-time reverse transcription polymerase chain reaction was used to study transcript levels of two genes involved in conidiation in B. bassiana, the regulatory G protein signaling gene Bbrgs1 and the hydrophobin gene hyd1, at different chitosan concentrations. Higher levels of Bbrgs1 and hyd1 transcripts were detected on chitosan-amended media. No correlation with chitosan concentration was observed for expression of Bbrgs1 unlike hyd1. Bbrgs1 deletion mutant Bbrgs1 showed that chitosan-induced conidiation is independent of Bbrgs1, suggesting an alternative mechanism controlling conidiation in B. bassiana. Our data supports that sporulation increases by chitosan, with spores retaining their viability and pathogenicity, which makes chitosan a suitable compound to increase conidia production in fungi with applications in fungal biotechnology.

Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey

BackgroundCombating the action of plant pathogenic microorganisms by mycoparasitic fungi has been announced as an attractive biological alternative to the use of chemical fungicides since two decades. The fungal genus Trichoderma includes a high number of taxa which are able to recognize, combat and finally besiege and kill their prey. Only fragments of the biochemical processes related to this ability have been uncovered so far, however.ResultsWe analyzed genome-wide gene expression changes during the begin of physical contact between Trichoderma atroviride and two plant pathogens Botrytis cinerea and Rhizoctonia solani, and compared with gene expression patterns of mycelial and conidiating cultures, respectively. About 3000 ESTs, representing about 900 genes, were obtained from each of these three growth conditions. 66 genes, represented by 442 ESTs, were specifically and significantly overexpressed during onset of mycoparasitism, and the expression of a subset thereof was verified by expression analysis. The upregulated genes comprised 18 KOG groups, but were most abundant from the groups representing posttranslational processing, and amino acid metabolism, and included components of the stress response, reaction to nitrogen shortage, signal transduction and lipid catabolism. Metabolic network analysis confirmed the upregulation of the genes for amino acid biosynthesis and of those involved in the catabolism of lipids and aminosugars.ConclusionThe analysis of the genes overexpressed during the onset of mycoparasitism in T. atroviride has revealed that the fungus reacts to this condition with several previously undetected physiological reactions. These data enable a new and more comprehensive interpretation of the physiology of mycoparasitism, and will aid in the selection of traits for improvement of biocontrol strains by recombinant techniques.

The β-N-acetylglucosaminidases NAG1 and NAG2 are essential for growth of Trichoderma atroviride on chitin

The chitinolytic enzyme machinery of fungi consists of chitinases and beta-N-acetylglucosaminidases. These enzymes are important during the fungal life cycle for degradation of exogenous chitin, which is the second most abundant biopolymer, as well as fungal cell-wall remodelling. In addition, involvement of chitinolytic enzymes in the lysis of the host cell wall in mycoparasitic Trichoderma spp. has been reported. In view of the fact that fungi have on average 15-20 chitinases, but only two beta-N-acetylglucosaminidases, the question arises how important the latter enzymes actually are for various aspects of chitin degradation. In this study, the role of two beta-N-acetylglucosaminidases, NAG1 and NAG2, was analysed in the mycoparasitic fungus Trichoderma atroviride. No beta-N-acetylglucosaminidase activity was detected in T. atrovirideDeltanag1Deltanag2 strains, suggesting that NAG1 and NAG2 are the only enzymes in T. atroviride that possess this activity. Deltanag1Deltanag2 strains were not able to grow on chitin and chitobiose, but the presence of either NAG1 or NAG2 was sufficient to restore growth on chitinous carbon sources in solid media. Our results demonstrated that T. atroviride cannot metabolize chitobiose but only the monomer N-acetylglucosamine, and that N-acetylglucosaminidases are therefore essential for the use of chitin as a nutrient source. NAG1 is predominantly secreted into the medium, whereas NAG2 mainly remains attached to the cell wall. No physiological changes or reduction of the mycoparasitic potential of T. atroviride was detected in the double knockout strains, suggesting that the use of chitin as carbon source is only of minor importance for these processes.

Chemical properties of compost extracts inhibitory to germination of Sclerotium rolfsii

Suppression of Sclerotium rolfsii by compost has been previously related to colonization of sclerotia by mycoparasitic fungi. Compost which has undergone prolonged curing was shown to lose its suppressive traits. In this work we sought to decouple the effects of the chemical conditions in the compost from the biological effect of antagonists inhabiting it. This was done by examining the direct effect of sterile compost extracts on the germination of sclerotia, and on the colonization of sclerotia by antagonistic fungal isolates. Sterile extracts of non-cured compost were found to completely inhibit sclerotia germination whereas extracts of cured compost allowed germination. Using buffered solutions which mimic the pH, NH 4 + concentration and buffering capacity of the extracts, it was shown that germination inhibition was related to effective NH 3 concentration in the extracts. A concentration of 0.5 mM NH 3 in compost extracts was identified as a threshold level above which sclerotia germination was completely inhibited. Sclerotia placed on compost extracts inhibitory to their germination were more vulnerable to attack by mycoparasites. The opportunistic antagonists Penicillium and Petriella exhibited enhanced colonization of sclerotia on these extracts, while the mycoparasitic fungus Trichoderma was found to behave as a primary antagonist, colonizing sclerotia regardless of the environmental conditions.