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.

Mycoparasitism related targets of Tmk1 indicate stimulating regulatory functions of this MAP kinase in Trichoderma atroviride.

Mycoparasitism is a key feature of Trichoderma (Hypocreales, Ascomycota) biocontrol agents. Recent studies of intracellular signal transduction pathways of the potent mycoparasite Trichoderma atroviride revealed the involvement of Tmk1, a mitogen-activated protein kinase (MAPK), in triggering the mycoparasitic response. We previously showed that mutants missing Tmk1 exhibit reduced mycoparasitic activity against several plant pathogenic fungi. In this study, we identified the most robustly regulated targets that were governed by Tmk1 during mycoparasitism using transcriptome and proteome profiling. Tmk1 mainly exerts a stimulating function for T. atroviride during its mycoparasitic interaction with the fungal plant pathogen Rhizoctonia solani, as reflected by 89% of strongly differently responding genes in the ∆tmk1 mutant compared to the wild type. Specifically, 54% of these genes showed strong downregulation in the response with a deletion of the tmk1 gene, whereas in the wild type the same genes were strongly upregulated during the interaction with the fungal host. These included the gene encoding the mycoparasitism-related proteinase Prb1; genes involved in signal transduction pathways such as a candidate coding for a conserved 14-3-3 protein, and a gene coding for Tmk2, the T. atroviride cell-wall integrity MAP kinase; genes encoding a specific siderophore synthetase, and multiple FAD-dependent oxidoreductases and aminotransferases. Due to the phosphorylating activity of Tmk1, different (phospho-)proteomics approaches were applied and identified proteins associated with cellular metabolism, energy production, protein synthesis and fate, and cell organization. Members of FAD- and NAD/NADP-binding-domain proteins, vesicular trafficking of molecules between cellular organelles, fungal translational, as well as protein folding apparatus were among others found to be phosphorylated by Tmk1 during mycoparasitism. Outstanding downregulation in the response of the ∆tmk1 mutant to the fungal host compared to the wild type at both the transcriptome and the proteome levels was observed for nitrilase, indicating that its defense and detoxification functions might be greatly dependent on Tmk1 during T. atroviride mycoparasitism. An intersection network analysis between the identified transcripts and proteins revealed a strong involvement of Tmk1 in molecular functions with GTPase and oxidoreductase activity. These data suggest that during T. atroviride mycoparasitism this MAPK mainly governs processes regulating cell responses to extracellular signals and those involved in reactive oxygen stress.

Identification and evaluation of suitable reference genes for RT-qPCR analyses in Trichoderma atroviride under varying light conditions.

BACKGROUND: Trichoderma atroviride is a competitive soil-borne mycoparasitic fungus with extensive applications as a biocontrol agent in plant protection. Despite its importance and application potential, reference genes for RT-qPCR analysis in T. atroviride have not been evaluated. Light exerts profound effects on physiology, such as growth, conidiation, secondary metabolism, and stress response in T. atroviride, as well as in other fungi. In this study, we aimed to address this gap by identifying stable reference genes for RT-qPCR experiments in T. atroviride under different light conditions, thereby enhancing accurate and reliable gene expression analysis in this model mycoparasite. We measured and compared candidate reference genes using commonly applied statistical algorithms. RESULTS: Under cyclic light-dark cultivation conditions, tbp and rho were identified as the most stably expressed genes, while act1, fis1, btl, and sar1 were found to be the least stable. Similar stability rankings were obtained for cultures grown under complete darkness, with tef1 and vma1 emerging as the most stable genes and act1, rho, fis1, and btl as the least stable genes. Combining the data from both cultivation conditions, gapdh and vma1 were identified as the most stable reference genes, while sar1 and fis1 were the least stable. The selection of different reference genes had a significant impact on the calculation of relative gene expression, as demonstrated by the expression patterns of target genes pks4 and lox1. CONCLUSION: The data emphasize the importance of validating reference genes for different cultivation conditions in fungi to ensure accurate interpretation of gene expression data.

Prokaryote communities associated with different types of tissue formed and substrates inhabited by Serpula lacrymans.

The basidiomycete Serpula lacrymans is responsible for major timber devastation in houses. Basidiomycetes are known to harbour a diverse but poorly understood microbial community of bacteria, archaea, yeasts and filamentous fungi. In this study, we used amplicon-sequencing to analyse the abundance and composition of prokaryotic communities associated with fruiting bodies of S. lacrymans and compared them to communities of surrounding material to access the 'background' community structure. Our findings indicate that bacterial genera cluster depended on sample type and that the main driver for microbial diversity is specimen, followed by sample origin. The most abundant bacterial phylum identified in the fruiting bodies was Pseudomonadota, followed by Actinomycetota and Bacteroidota. The prokaryote community of the mycelium was dominated by Actinomycetota, Halobacterota and Pseudomonadota. Actinomycetota was the most abundant phylum in both environment samples (infested timber and underground scree), followed by Bacillota in wood and Pseudomonadota in underground samples. Nocardioides, Pseudomonas, Pseudonochardia, Streptomyces and Rubrobacter spp. were among others found to comprise the core microbiome of S. lacrymans basidiocarps. This research contributes to the understanding of the holobiont S. lacrymans and gives hints to potential bacterial phyla important for its development and lifestyle.

Co-culture Wood Block Decay Test with Bacteria and Wood Rotting Fungi to Analyse Synergism/Antagonism during Wood Degradation.

Mixed communities of fungi and bacteria have been shown to be more efficient in degrading wood than fungi alone. Some standardised protocols for quantification of the wood decay ability of fungi have been developed (e.g., DIN V ENV 12038:2002 as the legal standard to test for the resistance of wood against wood-destroying basidiomycetes in Germany). Here, we describe a step-by-step protocol developed from the official standard DIN V ENV12038 to test combinations of bacteria and fungi for their combined wood degradation ability. Equally sized wood blocks are inoculated with wood decay fungi and bacterial strains. Axenic controls allow the analysis of varying degradation rates via comparison of the wood dry weights at the end of the experiments. This protocol provides new opportunities in exploration of inter- and intra-kingdom interactions in the wood-related environment and forms the basis for microcosm experiments. Key features • Quantification of wood decay ability of mixed cultures. • Allows testing if fungi are more efficient in degrading wood when bacteria are present.

Light-Induced Changes in Secondary Metabolite Production of Trichoderma atroviride.

Many studies aim at maximizing fungal secondary metabolite production but the influence of light during cultivation has often been neglected. Here, we combined an untargeted isotope-assisted liquid chromatography-high-resolution mass spectrometry-based metabolomics approach with standardized cultivation of Trichoderma atroviride under three defined light regimes (darkness (PD), reduced light (RL) exposure, and 12/12 h light/dark cycle (LD)) to systematically determine the effect of light on secondary metabolite production. Comparative analyses revealed a similar metabolite profile upon cultivation in PD and RL, whereas LD treatment had an inhibiting effect on both the number and abundance of metabolites. Additionally, the spatial distribution of the detected metabolites for PD and RL was analyzed. From the more than 500 detected metabolites, only 25 were exclusively produced upon fungal growth in darkness and 85 were significantly more abundant in darkness. The majority were detected under both cultivation conditions and annotation revealed a cluster of substances whose production followed the pattern observed for the well-known T. atroviride metabolite 6-pentyl-alpha-pyrone. We conclude that cultivation of T. atroviride under RL can be used to maximize secondary metabolite production.

qRAT: an R-based stand-alone application for relative expression analysis of RT-qPCR data.

BACKGROUND: Reverse transcription quantitative real-time PCR (RT-qPCR) is a well-established method for analysing gene expression. Most RT-qPCR experiments in the field of microbiology aim for the detection of transcriptional changes by relative quantification, which means the comparison of the expression level of a specific gene between different samples by the application of a calibration condition and internal reference genes. Due to the numerous data processing procedures and factors that can influence the final result, relative expression analysis and interpretation of RT-qPCR data are still not trivial and often necessitate the use of multiple separate software packages capable of performing specific functions. RESULTS: Here we present qRAT, a stand-alone desktop application based on R that automatically processes raw output data from any qPCR machine using well-established and state-of-the-art statistical and graphical techniques. The ability of qRAT to analyse RT-qPCR data was evaluated using two example datasets generated in our laboratory. The tool successfully completed the procedure in both cases, returning the expected results. The current implementation includes functionalities for parsing, filtering, normalizing and visualisation of relative RT-qPCR data, like the determination of the relative quantity and the fold change of differentially expressed genes as well as the correction of inter-plate variation for multiple-plate experiments. CONCLUSION: qRAT provides a comprehensive, straightforward, and easy-to-use solution for the relative quantification of RT-qPCR data that requires no programming knowledge or additional software installation. All application features are available for free and without requiring a login or registration.

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

Stress-Activated Protein Kinase Signalling Regulates Mycoparasitic Hyphal-Hyphal Interactions in Trichoderma atroviride.

Trichoderma atroviride is a mycoparasitic fungus used as biological control agent against fungal plant pathogens. The recognition and appropriate morphogenetic responses to prey-derived signals are essential for successful mycoparasitism. We established microcolony confrontation assays using T. atroviride strains expressing cell division cycle 42 (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1) interactive binding (CRIB) reporters to analyse morphogenetic changes and the dynamic displacement of localized GTPase activity during polarized tip growth. Microscopic analyses showed that Trichoderma experiences significant polarity stress when approaching its fungal preys. The perception of prey-derived signals is integrated via the guanosine triphosphatase (GTPase) and mitogen-activated protein kinase (MAPK) signalling network, and deletion of the MAP kinases Trichoderma MAPK 1 (Tmk1) and Tmk3 affected T. atroviride tip polarization, chemotropic growth, and contact-induced morphogenesis so severely that the establishment of mycoparasitism was highly inefficient to impossible. The responses varied depending on the prey species and the interaction stage, reflecting the high selectivity of the signalling process. Our data suggest that Tmk3 affects the polarity-stress adaptation process especially during the pre-contact phase, whereas Tmk1 regulates contact-induced morphogenesis at the early-contact phase. Neither Tmk1 nor Tmk3 loss-of-function could be fully compensated within the GTPase/MAPK signalling network underscoring the crucial importance of a sensitive polarized tip growth apparatus for successful mycoparasitism.

Microbiota Associated with Different Developmental Stages of the Dry Rot Fungus Serpula lacrymans.

The dry rot fungus Serpula lacrymans causes significant structural damage by decaying construction timber, resulting in costly restoration procedures. Dry rot fungi decompose cellulose and hemicellulose and are often accompanied by a succession of bacteria and other fungi. Bacterial-fungal interactions (BFI) have a considerable impact on all the partners, ranging from antagonistic to beneficial relationships. Using a cultivation-based approach, we show that S. lacrymans has many co-existing, mainly Gram-positive, bacteria and demonstrate differences in the communities associated with distinct fungal parts. Bacteria isolated from the fruiting bodies and mycelia were dominated by Firmicutes, while bacteria isolated from rhizomorphs were dominated by Proteobacteria. Actinobacteria and Bacteroidetes were less abundant. Fluorescence in situ hybridization (FISH) analysis revealed that bacteria were not present biofilm-like, but occurred as independent cells scattered across and within tissues, sometimes also attached to fungal spores. In co-culture, some bacterial isolates caused growth inhibition of S. lacrymans, and vice versa, and some induced fungal pigment production. It was found that 25% of the isolates could degrade pectin, 43% xylan, 17% carboxymethylcellulose, and 66% were able to depolymerize starch. Our results provide first insights for a better understanding of the holobiont S. lacrymans and give hints that bacteria influence the behavior of S. lacrymans in culture.

Monitoring the volatile language of fungi using gas chromatography-ion mobility spectrometry.

Fusarium oxysporum is a plant pathogenic fungus leading to severe crop losses in agriculture every year. A sustainable way of combating this pathogen is the application of mycoparasites-fungi parasitizing other fungi. The filamentous fungus Trichoderma atroviride is such a mycoparasite that is able to antagonize phytopathogenic fungi. It is therefore frequently applied as a biological pest control agent in agriculture. Given that volatile metabolites play a crucial role in organismic interactions, the major aim of this study was to establish a method for on-line analysis of headspace microbial volatile organic compounds (MVOCs) during cultivation of different fungi. An ion mobility spectrometer with gas chromatographic pre-separation (GC-IMS) enables almost real-time information of volatile emissions with good selectivity. Here we illustrate the successful use of GC-IMS for monitoring the time- and light-dependent release of MVOCs by F. oxysporum and T. atroviride during axenic and co-cultivation. More than 50 spectral peaks were detected, which could be assigned to 14 volatile compounds with the help of parallel gas chromatography-mass spectrometric (GC-MS) measurements. The majority of identified compounds are alcohols, such as ethanol, 1-propanol, 2-methyl propanol, 2-methyl butanol, 3-methyl-1-butanol and 1-octen-3-ol. In addition to four ketones, namely acetone, 2-pentanone, 2-heptanone, 3-octanone, and 2-octanone; two esters, ethyl acetate and 1-butanol-3-methylacetate; and one aldehyde, 3-methyl butanal, showed characteristic profiles during cultivation depending on axenic or co-cultivation, exposure to light, and fungal species. Interestingly, 2-octanone was produced only in co-cultures of F. oxysporum and T. atroviride, but it was not detected in the headspace of their axenic cultures. The concentrations of the measured volatiles were predominantly in the low ppbv range; however, values above 100 ppbv were detected for several alcohols, including ethanol, 2-methylpropanol, 2-methyl butanol, 1- and 3-methyl butanol, and for the ketone 2-heptanone, depending on the cultivation conditions. Our results highlight that GC-IMS analysis can be used as a valuable analytical tool for identifying specific metabolite patterns for chemotaxonomic and metabolomic applications in near-to-real time and hence easily monitor temporal changes in volatile concentrations that take place in minutes.

The GPI-Anchored GH76 Protein Dfg5 Affects Hyphal Morphology and Osmoregulation in the Mycoparasite Trichoderma atroviride and Is Interconnected With MAPK Signaling.

The fungal cell wall is composed of a cross-linked matrix of chitin, glucans, mannans, galactomannans, and cell wall proteins with mannan chains. Cell wall mannans are directly attached to the cell wall core, while the majority of mannoproteins is produced with a glycosylphosphatidylinositol (GPI) anchor and then transferred to ß-1,6-glucan in the cell wall. In this study, we functionally characterized the transmembrane protein Dfg5 of the glycoside hydrolase family 76 (GH76) in the fungal mycoparasite Trichoderma atroviride, whose ortholog has recently been proposed to cross-link glycoproteins into the cell wall of yeast and fungi. We show that the T. atroviride Dfg5 candidate is a GPI-anchored, transmembrane, 6-hairpin member of the GH76 Dfg5 subfamily that plays an important role in hyphal morphology in this mycoparasite. Alterations in the release of proteins associated with cell wall remodeling as well as a higher amount of non-covalently bonded cell surface proteins were detected in the mutants compared to the wild-type. Gene expression analysis suggests that transcript levels of genes involved in glucan synthesis, of proteases involved in mycoparasitism, and of the Tmk1 mitogen-activated protein kinase (MAPK)-encoding gene are influenced by Dfg5, whereas Tmk3 governs Dfg5 transcription. We show that Dfg5 controls important physiological properties of T. atroviride, such as osmotic stress resistance, hyphal morphology, and cell wall stability.

Resistance Marker- and Gene Gun-Mediated Transformation of Trichoderma reesei.

Transformation enables the transfer of DNA into fungal cells for subsequent integration into the genome. Due to its versatility in industrial application, transformation is of utmost importance in Trichoderma reesei and hence continuously optimized. As one of the most crucial obstacles in fungal transformation efforts, removal of the cell wall is required to efficiently target genome modification cassettes to the genome. Here we describe resistance marker-mediated gene gun (biolistic) transformation of fungal spores of T. reesei as an alternative to protoplast transformation.

Chemotropism Assays for Plant Symbiosis and Mycoparasitism Related Compound Screening in Trichoderma atroviride.

Trichoderma atroviride is a mycoparasitic fungus used as biological control agent to protect plants against fungal pathogens. Successful biocontrol is based on the perception of signals derived from both the plant symbiont and the fungal prey. Here, we applied three different chemotropic assays to study the chemosensing capacity of T. atroviride toward compounds known or suspected to play a role in the mycoparasite/plant or host/prey fungal interactions and to cover the complete spectrum of T. atroviride developmental stages. Purified compounds, including nutrients, the fungal secondary metabolite 6-amyl-α-pyrone (6-pentyl-α-pyrone, 6-PP) and the plant oxylipin 13-(s)-HODE, as well as culture supernatants derived from fungal preys, including Rhizoctonia solani, Botrytis cinerea and Fusarium oxysporum, were used to evaluate chemotropic responses of conidial germlings, microcolonies and fully differentiated mycelia. Our results show that germlings respond preferentially to compounds secreted by plant roots and T. atroviride itself than to compounds secreted by prey fungi. With the progression of colony development, host plant cues and self-generated signaling compounds remained the strongest chemoattractants. Nevertheless, mature hyphae responded differentially to certain prey-derived signals. Depending on the fungal prey species, chemotropic responses resulted in either increased or decreased directional colony extension and hyphal density at the colony periphery closest to the test compound source. Together these findings suggest that chemotropic sensing during germling development is focused on plant association and colony network formation, while fungal prey recognition develops later in mature hyphae of fully differentiated mycelium. Furthermore, the morphological alterations of T. atroviride in response to plant host and fungal prey compounds suggest the presence of both positive and negative chemotropism. The presented assays will be useful for screening of candidate compounds, and for evaluating their impact on the developmental spectrum of T. atroviride and other related species alike. Conidial germlings proved particularly useful for simple and rapid compound screening, whereas more elaborate microscopic analysis of microcolonies and fully differentiated mycelia was essential to understand process-specific responses, such as plant symbiosis and biocontrol.

Influence of Different Light Regimes on the Mycoparasitic Activity and 6-Pentyl-α-pyrone Biosynthesis in Two Strains of Trichoderma atroviride.

The ascomycete Trichoderma atroviride is well known for its mycoparasitic lifestyle. Similar to other organisms, light is an important cue for T. atroviride. However, besides triggering of conidiation, little is known on the physiological responses of T. atroviride to light. In this study, we analyzed how cultivation under different light wavelengths and regimes impacted the behavior of two T. atroviride wild-type strains: IMI206040 and P1. While colony extension of both strains was slightly affected by light, massive differences in their photoconidation responses became evident. T. atroviride P1 colonies conidiated under all conditions tested including growth in complete darkness, while IMI206040 required white, blue or green light to trigger asexual reproduction. Interestingly, deletion of the stress-activated MAP kinase-encoding gene tmk3 abolished the ability of strain P1 to conidiate in red and yellow light as well as in darkness. Furthermore, light-dependent differences in the mycoparasitic activity and in the biosynthesis of the secondary metabolite 6-pentyl-α-pyrone (6-PP) became evident. 6-PP production was highest upon dark incubation, while light, especially exposure to white light as light/dark cycles, had an inhibitory effect on its biosynthesis. We conclude that the response of T. atroviride to light is strain-dependent and impacts differentiation, mycoparasitism, and 6-PP production; hence, this should be considered in experiments testing the mycoparasitic activity of these fungi.

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.

Single-Molecule Localization Microscopy to Study Protein Organization in the Filamentous Fungus Trichoderma atroviride.

Single-molecule localization microscopy has boosted our understanding of biological samples by offering access to subdiffraction resolution using fluorescence microscopy methods. While in standard mammalian cells this approach has found wide-spread use, its application to filamentous fungi has been scarce. This is mainly due to experimental challenges that lead to high amounts of background signal because of ample autofluorescence. Here, we report the optimization of labeling, imaging and data analysis protocols to yield the first single-molecule localization microscopy images of the filamentous fungus Trichoderma atroviride. As an example, we show the spatial distribution of the Sur7 tetraspanin-family protein Sfp2 required for hyphal growth and cell wall stability in this mycoparasitic fungus.

The Trichoderma atroviride Strains P1 and IMI 206040 Differ in Their Light-Response and VOC Production.

Trichoderma atroviride is a strong necrotrophic mycoparasite antagonizing and feeding on a broad range of fungal phytopathogens. It further beneficially acts on plants by enhancing growth in root and shoot and inducing systemic resistance. Volatile organic compounds (VOCs) are playing a major role in all those processes. Light is an important modulator of secondary metabolite biosynthesis, but its influence has often been neglected in research on fungal volatiles. To date, T. atroviride IMI 206040 and T. atroviride P1 are among the most frequently studied T. atroviride strains and hence are used as model organisms to study mycoparasitism and photoconidiation. However, there are no studies available, which systematically and comparatively analyzed putative differences between these strains regarding their light-dependent behavior and VOC biosynthesis. We therefore explored the influence of light on conidiation and the mycoparasitic interaction as well as the light-dependent production of VOCs in both strains. Our data show that in contrast to T. atroviride IMI 206040 conidiation in strain P1 is independent of light. Furthermore, significant strain- and light-dependent differences in the production of several VOCs between the two strains became evident, indicating that T. atroviride P1 could be a better candidate for plant protection than IMI 206040.

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.