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.

Evolution and comparative genomics of the most common Trichoderma species.

BACKGROUND: The growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood. RESULTS: We selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function. CONCLUSIONS: Our study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist.

Volatiles from the Mandibular Gland Reservoir Content of Colobopsis explodens Laciny and Zettel, 2018, Worker Ants (Hymenoptera: Formicidae).

Forty-five volatile organic compounds (VOCs) were identified or annotated in the mandibular gland reservoir content (MGRC) of the Southeast Asian ant Colobopsis explodens Laciny and Zettel, 2018 (Hymenoptera: Formicidae), using headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography mass spectrometry (GC-MS) and liquid extraction combined with GC-MS. In extension of previous reports on VOCs of C. explodens, members of different compound classes, such as alkanes, aliphatic and aromatic carboxylic acids, and phenolics, were detected. The ketone 2-heptanone and the biochemically related phenolics benzene-1,3,5-triol (phloroglucinol, PG), 1-(2,4,6-trihydroxyphenyl)ethanone (monoacetylphloroglucinol, MAPG), 5,7-dihydroxy-2-methylchromen-4-one (noreugenin), and 1-(3-Acetyl-2,4,6-trihydroxyphenyl)ethanone (2,4-diacetylphloroglucinol, DAPG) dominated the GC-MS chromatograms. The identities of the main phenolics MAPG and noreugenin were further verified by liquid chromatography-high resolution-tandem mass spectrometry (LC-HRMS/MS). A comparative study of MGRC samples originating from three distinct field expeditions revealed differences in the VOC profiles, but the presence and relative abundances of the dominating constituents were largely consistent in all samples. Our study considerably extends the knowledge about the number and type of VOCs occurring in the MGRC of C. explodens. Based on the type of the detected compounds, we propose that the likely irritant and antibiotic phenolic constituents play a role in defense against arthropod opponents or in protection against microbial pathogens.

Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma.

We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma. To this end, we isolated 482 strains of Hypocrea/Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea/Trichoderma (TrichOKEY), sequence similarity analysis (Trichoblast) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea/Trichoderma species from sections Trichoderma and Pachybasium, comprising H. lixii/T. harzianum, T. gamsii, T. spirale, T. velutinum, T. hamatum, H. koningii/T. koningii, H. virens/T. virens, T. tomentosum, H. semiorbis, H. viridescens/T. viridescens, H. atroviridis/T. atroviride, T. asperellum, H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum-Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum. All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea/Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector.

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean 'exploding ants' belonging to the Colobopsis cylindrica (COCY) group. For this purpose, the model species C. explodens is used. Ants belonging to the minor worker caste possess distinctive hypertrophied mandibular glands (MGs). In territorial combat, they use the viscous contents of their enlarged mandibular gland reservoirs (MGRs) to kill rival arthropods in characteristic suicidal 'explosions' by voluntary rupture of the gastral integument (autothysis). We show the dissection of worker ants of this species for the isolation of the gastral portion of the wax-like MGR contents as well as listing the necessary steps required for solvent-extraction of the therein contained volatile compounds with subsequent gas chromatography-mass spectrometry (GC-MS) analysis and putative identification of metabolites contained in the extract. The dissection procedure is performed under cooled conditions and without the use of any dissection buffer solution to minimize the changes in the chemical composition of the MGR contents. After solvent-based extraction of volatile metabolites contained therein, the necessary steps for analyzing the samples via liquid-injection-GC-MS are presented. Lastly, data processing and putative metabolite identification with the use of the open-source software MetaboliteDetector is shown. With this approach, the profiling and identification of volatile metabolites in MGRs of ants belonging to the COCY group via GC-MS and the MetaboliteDetector software become possible.

A complete annotation of the chromosomes of the cellulase producer Trichoderma reesei provides insights in gene clusters, their expression and reveals genes required for fitness.

BACKGROUND: Investigations on a few eukaryotic model organisms showed that many genes are non-randomly distributed on chromosomes. In addition, chromosome ends frequently possess genes that are important for the fitness of the organisms. Trichoderma reesei is an industrial producer of enzymes for food, feed and biorefinery production. Its seven chromosomes have recently been assembled, thus making an investigation of its chromosome architecture possible. RESULTS: We manually annotated and mapped 9194 ORFs on their respective chromosomes and investigated the clustering of the major gene categories and of genes encoding carbohydrate-active enzymes (CAZymes), and the relationship between clustering and expression. Genes responsible for RNA processing and modification, amino acid metabolism, transcription, translation and ribosomal structure and biogenesis indeed showed loose clustering, but this had no impact on their expression. A third of the genes encoding CAZymes also occurred in loose clusters that also contained a high number of genes encoding small secreted cysteine-rich proteins. Five CAZyme clusters were located less than 50 kb apart from the chromosome ends. These genes exhibited the lowest basal (but not induced) expression level, which correlated with an enrichment of H3K9 methylation in the terminal 50 kb areas indicating gene silencing. No differences were found in the expression of CAZyme genes present in other parts of the chromosomes. The putative subtelomeric areas were also enriched in genes encoding secreted proteases, amino acid permeases, enzyme clusters for polyketide synthases (PKS)-non-ribosomal peptide synthase (NRPS) fusion proteins (PKS-NRPS) and proteins involved in iron scavenging. They were strongly upregulated during conidiation and interaction with other fungi. CONCLUSIONS: Our findings suggest that gene clustering on the T. reesei chromosomes occurs but generally has no impact on their expression. CAZyme genes, located in subtelomers, however, exhibited a much lower basal expression level. The gene inventory of the subtelomers suggests a major role of competition for nitrogen and iron supported by antibiosis for the fitness of T. reesei. The availability of fully annotated chromosomes will facilitate the use of genetic crossings in identifying still unknown genes responsible for specific traits of T. reesei.