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In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Trichoderma afroharzianum ( Hypocreales ) is known as an important mycoparasite and biocontrol fungus and feeds on fungal material by parasitizing other fungi. Recent studies indicate that this species is also an ear rot pathogen in Europe. Here, the complete mitochondrial genome of three T. afroharzianum strains was sequenced using next-generation sequencing and comparatively characterized by the reported Trichoderma mitogenomes. T. afroharzianum mitogenomes were varying between 29 511 bp and 29 517 bp in length, with an average A + T content of 72.32%. These mitogenomes contain 14 core protein coding genes (PCGs), 22 tRNAs, two rRNAs, one gene encoding the ribosomal protein S3, and three or four genes including conserved domains for the homing endonucleases (HEGs; GIY-YIG type and LAGLIDADG type). All PCGs are initiated by ATG codons, except for atp8, and all are terminated with TAA. A significant correlation was observed between nucleotide composition and codon preference. Four introns belonging to the group I intron class were predicted, accounting for about 14.54% of the size of the mitogenomes. Phylogenetic analyses confirmed the positions of T . afroharzianum strains within the genus of Trichoderma and supported a sister group relationship between T. afroharzianum and T. simmonsii . The recovered trees also supported the monophyly of all included families and of the genus of Acremonium . The characterization of mitochondrial genome of T. afroharzianum contributes to the understanding of phylogeny and evolution of Hypocreales., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Trichoderma spp. represent one of the most important fungal genera to mankind and in natural environments. The genus harbors prolific producers of wood-decaying enzymes, biocontrol agents against plant pathogens, plant-growth-promoting biofertilizers, as well as model organisms for studying fungal-plant-plant pathogen interactions. Pursuing highly accurate, contiguous, and chromosome-level reference genomes has become a primary goal of fungal research communities. Here, we report the chromosome-level genomic sequences and whole-genome annotation data sets of four strains used as biocontrol agents or biofertilizers ( Trichoderma virens Gv29-8, Trichoderma virens FT-333, Trichoderma asperellum FT-101, and Trichoderma atroviride P1). Our results provide comprehensive categorization, correct positioning, and evolutionary detail of both nuclear and mitochondrial genomes, including telomeres, AT-rich blocks, centromeres, transposons, mating-type loci, nuclear-encoded mitochondrial sequences, as well as many new secondary metabolic and carbohydrate-active enzyme gene clusters. We have also identified evolutionarily conserved core genes contributing to plant-fungal interactions, as well as variations potentially linked to key behavioral traits such as sex, genome defense, secondary metabolism, and mycoparasitism. The genomic resources we provide herein significantly extend our knowledge not only of this economically important fungal genus, but also fungal evolution and basic biology in general. IMPORTANCE Telomere-to-telomere and gapless reference genome assemblies are necessary to ensure that all genomic variants are studied and discovered, including centromeres, telomeres, AT-rich blocks, mating type loci, biosynthetic, and metabolic gene clusters. Here, we applied long-range sequencing technologies to determine the near-completed genome sequences of four widely used biocontrol agents or biofertilizers: Trichoderma virens Gv29-8 and FT-333, Trichoderma asperellum FT-101, and Trichoderma atroviride P1. Like those of three Trichoderma reesei wild isolates [QM6a, CBS999.97( MAT1-1 ) and CBS999.97( MAT1-2 )] we reported previously, these four biocontrol agent genomes each contain seven nuclear chromosomes and a circular mitochondrial genome. Substantial intraspecies and intragenus diversities are also discovered, including single nucleotide polymorphisms, chromosome shuffling, as well as genomic relics derived from historical transposition events and repeat-induced point (RIP) mutations.

Interactions between fungi and tardigrades have scarcely been described. The few studies that address such relationships suggest a primarily parasitic nature for various fungal taxa, including the infectious chytridiomycetes. The aim of this study was to determine the identity of a fungus growing on a tardigrade of the genus Diaforobiotus and if it could infect other tardigrade genera. Using morphological analysis and ITS barcoding, we identified a mold isolate belonging to the Trichoderma harzianum species complex and found that it infected Diaforobiotus tardigrades, as well as animals in the eutardigrade genus Milnesium, and heterotardigrade genus Viridiscus., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Laccases have high biotechnological potential in industries since they catalyze the oxidation of many chemical compounds. The production of laccases by fungi has been extensively studied due to their secretion of enzymes and rapid growth using cheap substrates. Trichoderma; the versatile fungal genus includes species of great biotechnological value and considered as a magnificent industrial cell factory of enzymes. In this study, 10 Trichoderma species were screened for laccase enzyme production by submerged cultivation. The studied species were identified by internal transcribed spacer (ITS) gene sequences. Guaiacol (0.04%) as an enzyme substrate in plate medium was used for the selection of maximum laccase-enriched Trichoderma species by formation of visual color halo intensity. This activity was evaluated by liquid submersion (flask medium) also. The absorbance of laccase contained broth was measured by a spectrophotometer (450 nm). The highest laccase production was obtained by T. atroviride (2.62 U/mL). Trichoderma cremeum and T. longipile showed medium laccase potency, while T. beinartii exhibited weak laccase secretion ability. Laccase from T. atroviride was purified by SDS-PAGE and the molecular weight was determined (57 kDa). The laccase was confirmed by their respective amino acid sequences, and the phylogenetic tree was constructed for further analysis., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In the course of investigations on peptaibol chemodiversity from marine-derived Trichoderma spp., five new 15-residue peptaibols named pentadecaibins I-V ( 1 - 5 ) were isolated from the solid culture of the strain Trichoderma sp. MMS1255 belonging to the T. harzianum species complex. Phylogenetic analyses allowed precise positioning of the strain close to T. lentiforme lineage inside the Harzianum clade. Peptaibol sequences were elucidated on the basis of their MS/MS fragmentation and extensive 2D NMR experiments. Amino acid configurations were determined by Marfey's analyses. The pentadecaibins are based on the sequences Ac-Aib 1 -Gly 2 -Ala 3 -Leu 4 -Aib/Iva 5 -Gln 6 -Aib/Iva 7 -Val/Leu 8 -Aib 9 -Ala 10 -Aib 11 -Aib 12 -Aib 13 -Gln 14 -Pheol 15 . Characteristic of the pentadecaibin sequences is the lack of the Aib-Pro motif commonly present in peptaibols produced by Trichoderma spp. Genome sequencing of Trichoderma sp. MMS1255 allowed the detection of a 15-module NRPS-encoding gene closely associated with pentadecaibin biosynthesis. Pentadecaibins were assessed for their potential antiproliferative and antimicrobial activities.

Trichoderma is a genus of soil-borne fungus with an abundance of reports of its economic importance in the agriculture industry. Thus, the correct identification of Trichoderma species is necessary for its commercial purposes. Globally, Trichoderma species are routinely identified from micro-morphological descriptions which can be tedious and prone to errors. Thus, we emphasize that the accurate identification of Trichoderma strains requires a three-pronged approach i.e. based on its morphological characteristics, multilocus gene sequences of the rDNA [internal transcribed spacer (ITS) 1 and 2 regions], translation elongation factor 1-α (TEF-1α), Calmodulin (CAL) and its lignocellulolytic activities. We used this approach to identify a total of 53 Trichoderma strains which were isolated from a wet paddy field located at Tuaran, Sabah, Malaysia. The 53 strains were positively identified as belonging to three Trichoderma species, namely T. asperellum (43 strains), T. harzianum (9 strains), and T. reesei (one strain) on the basis of its morphological characteristics and multilocus gene sequences. Phylogenetic trees constructed based on the UPGMA method of the ITS 1 and 2 regions of the rDNA, TEF-1α and CAL revealed three distinct groups with the T. asperellum, T. harzianum and T. reesei strains placed under the section of Trichoderma, Pachybasium and Longibrachiatum, respectively. In addition, the lignocellulolytic activities of the isolates were measured based on the diameters of the halo zones produced when degrading cellulose, lignin, and starch, respectively. This diagnostic assay can be used to identify Trichoderma as it produces polyphenol oxidase when Tannic Acid Media is used for the lignin test, endoglucanases when Jensen media is used for cellulose, and it hydrolyzes starch to glucose when the modified Melin-Nokrans media is used for the starch test. Accurate identification of Trichoderma species is needed as these strains can potentially be used as a biocontrol agent to prevent diseases and to increase yield in agriculture crops.

Background: Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans and is the second most common mycobacterial disease after tuberculosis in Ghana and Côte d'Ivoire. M. ulcerans produces mycolactone, an immunosuppressant macrolide toxin, responsible for the characteristic painless nature of the infection. Secondary infection of ulcers before, during and after treatment has been associated with delayed wound healing and resistance to streptomycin and rifampicin. However, not much is known of the bacteria causing these infections as well as antimicrobial drugs for treating the secondary microorganism. This study sought to identify secondary microbial infections in BU lesions and to determine their levels of antibiotic resistance due to the prolonged antibiotic therapy required for Buruli ulcer., Results: Swabs from fifty-one suspected BU cases were sampled in the Amansie Central District from St. Peters Hospital (Jacobu) and through an active case surveillance. Forty of the samples were M. ulcerans (BU) positive. Secondary bacteria were identified in all sampled lesions (N = 51). The predominant bacteria identified in both BU and Non-BU groups were Staphylococci spp and Bacilli spp. The most diverse secondary bacteria were detected among BU patients who were not yet on antibiotic treatment. Fungal species identified were Candida spp, Penicillium spp and Trichodema spp. Selected secondary bacteria isolates were all susceptible to clarithromycin and amikacin among both BU and Non-BU patients. Majority, however, had high resistance to streptomycin., Conclusions: Microorganisms other than M. ulcerans colonize and proliferate on BU lesions. Secondary microorganisms of BU wounds were mainly Staphylococcus spp, Bacillus spp and Pseudomonas spp. These secondary microorganisms were less predominant in BU patients under treatment compared to those without treatment. The delay in healing that are experienced by some BU patients could be as a result of these bacteria and fungi colonizing and proliferating in BU lesions. Clarithromycin and amikacin are likely suitable drugs for clearance of secondary infection of Buruli ulcer.

To better apply the biocontrol agent Trichoderma spp. in Northeast China, collecting and screening more suitable native Trichoderma strains is necessary. In the present study, 10 isolates were obtained from Juglans mandshurica rhizosphere soils in Heilongjiang Province, and were identified as T. asperellum (four isolates), T. harzianum (four), T. hamatum (one), T. atroviride (one). The fastest-growing isolate per species on potato dextrose agar medium were further evaluated in stress tolerance tests (salt, alkali, nutritional stress, and low temperature) and confrontation assays (eight pathogens), which showed that T. asperellum TaspHu1 possessed the best adaptation and biological control ability. Then, Solanum lycopersicum (tomato) seeds were sown and treated with a series of concentrations of TaspHu1 spore suspension, as was unsown soil. Tomato seedlings treated by TaspHu1 had a significantly greater height, stem diameter, soluble protein content and soluble sugar content. Furthermore, their nitrate reductase activity and catalase activity were significantly increased, and these promoting effects depended on the concentration of the spore suspension. Meanwhile, a decrease in chlorophyll content was observed in the tomato seedlings treated with TaspHu1. In addition, strain TaspHu1 enhanced the tomato seedlings' absorption of available nitrogen, but did not influence the soil available nitrogen content. Furthermore, the resistance of tomato seedlings against Alternaria alternata was enhanced by TaspHu1 (smaller, fewer leaf spots), the seedlings' hormone signal transduction genes JAR1, MYC2, NPR1, PR1, and GH3.2 were highly expressed. Thus, TaspHu1 is a promising biocontrol candidate for use in agriculture and forestry., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Twenty-two strains of Trichoderma spp. ( T. harzianum species complex [THSC], Trichoderma aggressivum f. europaeum, Trichoderma pleuroti, and Trichoderma pleuroticola ) causing green mold disease on edible mushrooms (button mushroom, shiitake and oyster mushroom), collected during 2004-2018 from four countries (Serbia, North Macedonia, Croatia, and Hungary) were examined. Based on their ITS (internal transcribed spacer) sequences, strains from shiitake mushroom in Serbia were identified as members of the THSC, while in samples obtained from Serbian and North-Macedonian oyster mushroom farms THSC, T. pleuroti and T. pleuroticola were detected, which represent the first findings in the region. In fungicide susceptibility tests, all examined Trichoderma strains were found to be highly sensitive to prochloraz (ED 50 <0.4 µg mL -1 ) and considerably susceptible to metrafenone (ED 50  < 4 µg mL -1 ). The most sensitive taxon to both fungicides was THSC from oyster mushroom. The toxicity of metrafenone was satisfying and strains from oyster mushroom showed the highest sensitivity (ED 50  < 1.43 µg mL -1 ), while strains originating from button mushroom and shiitake displayed similar susceptibilities (ED 50  < 3.64 µg mL -1 ). After additional in vivo trials, metrafenone might also be recommended for the control of green mold disease in mushroom farms.

Aims: Sustainable agriculture requires effective and safe biofertilizers and biofungicides with low environmental impact. Natural ecosystems that closely resemble the conditions of biosaline agriculture may present a reservoir for fungal strains that can be used as novel bioeffectors., Methods and Results: We isolated a library of fungi from the rhizosphere of three natural halotolerant plants grown in the emerging tidal salt marshes on the south-east coast of China. DNA barcoding of 116 isolates based on the rRNA ITS1 and 2 and other markers (tef1 or rpb2) revealed 38 fungal species, including plant pathogenic (41%), saprotrophic (24%) and mycoparasitic (28%) taxa. The mycoparasitic fungi were mainly species from the hypocrealean genus Trichoderma, including at least four novel phylotypes. Two of them, representing the taxa Trichoderma arenarium sp. nov. (described here) and T. asperelloides, showed antagonistic activity against five phytopathogenic fungi, and significant growth promotion on tomato seedlings under the conditions of saline agriculture., Conclusions: Trichoderma spp. of salt marshes play the role of natural biological control in young soil ecosystems with a putatively premature microbiome., Significance and Impact of the Study: The saline soil microbiome is a rich source of halotolerant bioeffectors that can be used in biosaline agriculture., (© 2020 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Antagonism of plant pathogenic fungi by endophytic fungi is a well-known phenomenon. In plate assays, the antagonism could be due to mycoparasitism, competition for space or antibiosis, involving a chemical diffusate, or a volatile organic compound (VOC). In this study, we demonstrate that besides mycoparasitism, VOCs play a major role in antagonism of pathogenic fungi by four endophytic fungi belonging to the genus Trichoderma. Using a double-plate assay, we show that all the four endophytic Trichoderma species significantly inhibited mycelial growth of three of the four pathogens, (Sclerotinia sclerotiorum-TSS, Sclerotium rolfsii-CSR and Fusarium oxysporum-CFO), while that of Macrophomina phaseolina-CMP was not affected. GC-MS analysis of the pure cultures of one of the endophytic fungi studied, namely, Trichoderma longibrachiatum strain 2 (Acc. No. MK751758) and the pathogens, F. oxysporum-CFO and M. phaseolina-CMP revealed the presence of several VOCs including hydrocarbons, alcohols, ketones, aldehydes, esters, acids, ethers and different classes of terpenes. In mixed double plates, where the endophyte was grown along with either of the two plant pathogens, F. oxysporum-CFO or M. phaseolina-CMP, there was an induction of a number of new VOCs that were not detected in the pure cultures of either the endophyte or the pathogens. Several of these new VOCs are reported to possess antifungal and cytotoxic activity. We discuss these results and highlight the importance of such interactions in endophyte-pathogen interactions., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Filamentous fungi are known as producers of a large array of diverse secondary metabolites (SMs) that aid in securing their environmental niche. Here, we demonstrated that the SMs have an additional role in fungal defence against other fungi: Trichoderma guizhouense, a mycoparasite, is able to antagonize Fusarium oxysporum f. sp. cubense race 4 (Foc4) by forming aerial hyphae that kill the host with hydrogen peroxide. At the same time, a gene cluster comprising two polyketide synthases is strongly expressed. Using functional genetics, we characterized this cluster and identified its products as azaphilones (termed as trigazaphilones). The trigazaphilones were found lacking of antifungal toxicity but exhibited high radical scavenging activities. The antioxidant property of trigazaphilones was in vivo functional under various tested conditions of oxidative stress. Thus, we conclude that the biosynthesis of trigazaphilones serves as a complementary antioxidant mechanism and defends T. guizhouense against the hydrogen peroxide that it produces to combat other fungi like Foc4., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Aims: Develop quantitative assays (qPCR) to determine the detection threshold limits, colonization and persistence of Trichoderma gamsii, Trichoderma afroharzianum and T. harzianum inoculants in cropping soils, the wheat rhizosphere and their in planta suppressive efficacy against the crown rot pathogen Fusarium pseudograminearum., Methods and Results: Trichoderma qPCR primers were designed from the internal transcribed spacer region of 5.8S rDNA and from sequences of DNA fragments diagnostic for each inoculant genotype. The minimum detection thresholds of qPCR assays varied between 1 × 10 3 (log 3) and 8 × 10 4 (log 4·9) conidia (genome) equivalents per gram of soil for multi- and single-copy target sequences, respectively and were independent of soil type. There was a strong correlation (r > 0·974) between culture-dependent and culture-independent (qPCR) quantification methods. In wheat bioassays, Trichoderma inoculants colonized rhizosphere soils and wheat roots at 56-112 days postemergence to a depth of 20 cm but were more abundant (P < 0·001) at 0-10 cm root depth, means ranging from 2 × 10 2 (log 2·3) to 4 × 10 5 (log 5·6) conidia equivalents per gram of rhizosphere soil or root tissue. Inoculants reduced (P < 0·001) F. pseudograminearum biomass in wheat crown and root tissue by up to 5754-fold and increased (P = 0·008) plant biomass, relative to the pathogen control., Conclusions: The qPCR assays provided sensitive and accurate assessment of wheat root and rhizosphere soil colonization of Trichoderma inoculants. Strains persisted through to grain maturity at levels shown to significantly suppress F. pseudograminearum in planta., Significance and Impact of the Study: The qPCR assays developed here were used to determine the wheat rhizosphere dynamics of T. harzianum, T. afroharzianum and T. gamsii inoculants and their suppressive efficacy against F. pseudograminearum in planta. These assays can be applied to monitor inoculant dynamics in suppressing crown rot and other wheat root diseases in the field., (© 2020 The Society for Applied Microbiology.)

Fungi in the genus Trichoderma are notorious producers of secondary metabolites with diverse applications, such as antibacterial, antifungal, and plant growth-promoting properties. Peptaibols are linear peptides produced by such fungi, with more than 440 compounds described to date, including tricholongins, longibrachins, trichobrachins, and trichovirins. Peptaibols are synthesized by non-ribosomal peptide synthetases and they have several biological activities. Our research group isolated four peptaibols (6DP2, 6DP3, 6DP4, and 6DP5) with antifungal activity against the plant pathogen Colletotrichum gloeosporioides and the proteasome (a cancer chemotherapy target) from Trichoderma sp. P8BDA1F1, an endophytic fungus from Begonia venosa. The ethyl acetate extract of this endophyte showed activity of 6.01% and 75% against C. gloeosporioides and the proteasome, respectively. The isolated compounds were identified by MS/MS and compared to literature data, suggesting the presence of trilongins BI, BII, BIII, and BIV, which are peptaibols containing 20 amino acid residues. The minimum inhibitory concentration against C. gloeosporioides was 40 μM for trilongin BI, 320 μM for trilongin BII, 160 μM for trilongin BIII, and 310 μM for trilongin BIV. BI-BIV trilongins inhibited proteasome ChTL activity, with IC 50 values of 6.5 ± 2.7; 4.7 ± 1.8; 6.3 ± 2.2; and 2.7 ± 0.5 μM, respectively. The compounds were tested ex vivo against the intracellular amastigotes of Leishmania (L.) infantum but showed no selectivity. It is the first report of trilongins BI-BIV with antifungal activity against C. gloeosporioides and the proteasome target.

Aspergillus is a fungal genus that strongly affects health of humans, animals, and plants worldwide. Endophytes are now widely considered as a rich source of bioproducts with potential uses in medicine, agriculture, and bioindustry. Cupressaceae plant family hosts a variety of bioactive ascomycetous endophytes. In this study, antifungal activity of a number of such endophytes were investigated against human pathogenic fungi Aspergillus fumigatus and Aspergillus niger. To this end, 16 superior bioactive endophytic fungi from Cupressaceae were used, including Alternaria alternata, Alternaria pellucida, Ascorhizoctonia sp., Aspergillus fumigatus, Aspergillus niger, Aurobasidium sp., Cladosporium porophorum, Fusarium oxysporum, Penicillium viridicatum, Phoma herbarum, Phoma sp., Pyrenochaeta sp., Trichoderma atroviride, Trichoderma atroviride and Trichoderma koningii. In vitro bioassays indicated anti-Asperilli activity of the endophytic fungi in dual cultures. Most notably, Trichoderma koningii CSE 32 and Trichoderma atroviride JCE 33 showed complete growth inhibition of both A. niger and A. fumigatus, within 3 to 7 days. Also, volatile compouds (VOCs) of T. koningii CSE 32 and T. atroviride JCE 33 exhibited 33-100% growth inhibition of A. niger, whithin 3 days. Moreover, on the day 7, growth of A. niger was less affected than that of A. fumigatus. In general, it appears that there is a direct relationship between the exposure time and the inhibitory activity of endophytes VOCs on the growth of target Aspergillus species. Furthremore, the extracellular secondary metabolites (SMs) of four selected fungal endophytes exhibited anti-Aspergillus activity at all treatment levels as shown by Agar-diffusion assay. SMs from T. koningii CSE 32 and Pyrenochaeta CSE 134 showed strongest activities against A. niger, and SMs from T. koningii CSE 32 and F. oxysporum CAE 14 showed strongest activities against A. fumigatus. In conclusion, given the globally recognized issue of antibiotic resistance and the urge to discover new antimicrobial substances, our findings provide new insights into the potential use of Cupressaceae's endophytic fungi in antifungal-based drug discovery programs., (Copyright © 2020. Published by Elsevier Masson SAS.)

Due to the rapid expansion in microbial taxonomy, precise identification of common industrially and agriculturally relevant fungi such as Trichoderma species is challenging. In this study, we introduce the online multilocus identification system (MIST) for automated detection of 349 Trichoderma species based on a set of three DNA barcodes. MIST is based on the reference databases of validated sequences of three commonly used phylogenetic markers collected from public databases. The databases consist of 414 complete sequences of the nuclear rRNA internal transcribed spacers (ITS) 1 and 2, 583 sequence fragments of the gene encoding translation elongation factor 1-alpha ( tef1 ), and 534 sequence fragments of the gene encoding RNA polymerase subunit 2 ( rpb2 ). Through MIST, information from different DNA barcodes can be combined and the identification of Trichoderma species can be achieved based on the integrated parametric sequence similarity search (blastn) performed in the manner of a decision tree classifier. In the verification process, MIST provided correct identification for 44 Trichoderma species based on DNA barcodes consisting of tef1 and rpb2 markers. Thus, MIST can be used to obtain an automated species identification as well as to retrieve sequences required for manual identification by means of phylogenetic analysis. IMPORTANCE The genus Trichoderma is important to humankind, with a wide range of applications in industry, agriculture, and bioremediation. Thus, quick and accurate identification of Trichoderma species is paramount, since it is usually the first step in Trichoderma -based research. However, it frequently becomes a limitation, especially for researchers who lack taxonomic knowledge of fungi. Moreover, as the number of Trichoderma -based studies has increased, a growing number of unidentified sequences have been stored in public databases, which has made the species identification more ambiguous. In this study, we provide an easy-to-use tool, MIST, for automated species identification, a list of Trichoderma species, and corresponding sequences of reference DNA barcodes. Therefore, this study will facilitate the research on the biodiversity and applications of the genus Trichoderma ., (Copyright © 2020 American Society for Microbiology.)

An endophytic member of the genus Trichoderma was isolated from the root of a healthy 3-year-old Panax notoginseng in Yunnan province, PR China. The results of phylogenetic analyses based on a combined of ITS, tef 1 and rpb 2 indicated that this isolate was distinct from other species of the genus Trichoderma and closely related to Trichoderma songyi . It can be distinguished from T. songyi by its slower growth rates on PDA and colony morphology. The novel isolate formed conidia in thick white pustules scattered mostly at the margin. Its conidiophores tended to be regularly verticillium -like, little branched, sometimes substituted by phialides singly or in whorls. Conidia are smooth, mostly broadly subglobose to ellipsoidal. In combination with the genotypic and phenotypic characteristics, all data demonstrated that the fungus studied represented a unique and distinguishable novel species of the genus Trichoderma , for which the name Trichoderma panacis sp. nov. is proposed.

Fifty four Trichoderma strains were isolated from soil samples collected from garlic and onion crops in eight different sites in Brazil and were identified using phylogenetic analysis based on combined ITS region, tef1-α, cal, act and rpb2 sequences. The genetic variability of the recovered Trichoderma species was analysed by AFLP and their phenotypic variability determined using MALDI-TOF. The strain clusters from both typing techniques coincided with the taxonomic determinations made from phylogenetic analysis. The phylogenetic analysis showed the occurrence of Trichoderma asperellum, Trichoderma asperelloides, Trichoderma afroharzianum, Trichoderma hamatum, Trichoderma lentiforme, Trichoderma koningiopsis, Trichoderma longibrachiatum and Trichoderma erinaceum, in the soil samples. We also identified and describe two new Trichoderma species, both in the harzianum clade of section Pachybasium, which we have named Trichoderma azevedoi sp. nov. and Trichoderma peberdyi sp. nov. The examined strains of both T. azevedoi (three strains) and T. peberdyi (12 strains) display significant genotypic and phenotypic variability, but form monophyletic clades with strong bootstrap and posterior probability support and are morphologically distinct from their respective most closely related species., Competing Interests: The authors have declared that no competing interests exist.

Dichlorvos (DDVP) is widely applied in the agricultural industry, and its residues are considered hazardous to the environment. Microbial bioremediation is an innovative technology with the potential to mitigate such pollution. Trichoderma atroviride strain T23, a filamentous fungus, is very efficient at degrading DDVP. Therefore, we used DDVP as a model organophosphate pesticide to study the mechanism by which Trichoderma degrades organophosphate pesticides, with the aim of attaining a global understanding of the molecular mechanism of enzymatic degradation of organophosphate pesticides by beneficial fungi. DDVP can be biodegraded via two routes, and the primary one involves hydrolysis of the P-O bond, which can result in the production of the novel degradation intermediate trichloroethanol. TaPon1-like showed continuously high expression during 120 h, and deletion of the gene decreased the efficiency of P-O bond hydrolysis. The enzyme produced by TaPon1-like had a low K m for DDVP (0.23 mM) and a high k cat (204.3 s -1 ). The enzyme was able to hydrolyze broad substrates such as organophosphate oxons and lactone and maintain stable activity in a wide range of pH and temperature values. The TaPon1-like hydrolase played an important role in the first step of DDVP degradation by strain T23 and contributed to a comprehensive understanding of the mechanism of organophosphate pesticide degradation.

Trichoderma species are abundant in different agricultural habitats, but some representatives of this genus, mainly clade Longibrachiatum members are also emerging as causative agents of various human diseases with even fatal outcome. Strains of these species frequently show resistance to commonly used azole antifungals. Based on previous data it is hypothesized that Trichoderma isolates identified in human infections derive from environmental-including agricultural-origins. We examined Trichoderma longibrachiatum Rifai and Trichoderma bissettii Sandoval-Denis & Guarro strains recovered from four novel cases of human mycoses, along with isolates from previous case reports and different agricultural habitats, using multilocus phylogenetic analysis, BIOLOG Phenotype Microarrays and Etest. Strains attributed to T. bissettii were more abundant in both clinical and agricultural specimens compared to T. longibrachiatum. The majority of the isolates of both taxa could tolerate >256, >32 and >32 μg/ml fluconazole, itraconazole and posaconazole, respectively. None of the obtained results revealed characteristic differences between strains of clinical and agricultural origin, nor between the two taxa, supporting that agricultural environments may be significant sources of infections caused by these emerging human fungal pathogens. Furthermore, based on our findings we propose the re-classification of T. bissettii as T. longibrachiatum f. sp. bissettii., (© FEMS 2019.)

Objective: Trichoderma species are found in soil and in association with plants. They can act directly or indirectly in the biological control of plant diseases and in the promotion of plant growth, being among the most used fungi in the formulation of bioproducts applied to agricultural systems. The main objective of this study was to characterize at a first-tier level a collection of 67 Trichoderma isolates from various tropical sources, based solely on sequencing of the internal transcribed spacer (ITS) region of the rRNA genes. Our goal was to provide a preliminary idea of the baseline diversity in this collection, to combine this information later with an array of other isolate-specific physiological data. This study provides a required knowledge at molecular level for assessment of this germplasm potential as a source of biotechnological products for beneficial effects in plants., Results: Sequencing of the ITS region showed that the 67 Trichoderma isolates belonged in 11 species: T. asperellum, T. atroviride, T. brevicompactum, T. harzianum, T. koningiopsis, T. longibrachiatum, T. pleuroticola, T. reesei, T. spirale, T. stromaticum and T. virens. A total of 40.3% of the isolates were very closely related to each other and similar to T. harzianum. The baseline genetic diversity found indicates that the collection has different genotypes, which can be exploited further as a source of bioproducts, aiming at providing beneficial effects to plants of interest to cope with biotic and abiotic stresses.

Trichoderma reesei is a biotechnologically important filamentous fungus with the remarkable ability to secrete large amounts of enzymes, whose production is strongly affected by both the carbon and nitrogen sources. While the carbon metabolism regulators are extensively studied, the regulation of enzyme production by the nitrogen metabolism regulators is still poorly understood. In this study, the GATA transcription factor Are1, which is an orthologue of the Aspergillus global nitrogen regulator AREA, was identified and characterized for its functions in regulation of both protease and cellulase production in T. reesei . Deletion of the are1 gene abolished the capability to secrete proteases, and complementation of the are1 gene rescued the ability to produce proteases. Quantitative RT-PCR analysis revealed that the transcripts of protease genes apw1 and apw2 were also significantly reduced in the Δ are1 strain when grown in the medium with peptone as the nitrogen source. In addition, deletion of are1 resulted in decreased cellulase production in the presence of (NH 4 ) 2 SO 4 . Consistent with the reduction of cellulase production, the transcription levels of the major cellulase genes, including cbh1 , cbh2 , egl1 , and egl2, were dramatically decreased in Δ are1 . Sequence analysis showed that all promoter regions of the tested protease and cellulase genes contain the consensus GATA elements. However, the expression levels of the major cellulase transcription activator Xyr1 and the repressor Cre1 had no significant difference between Δ are1 and the parental strain QM9414, indicating that the regulatory mechanism deserves further investigation. Taken together, these results demonstrate the important role of Are1 in the regulation of protease and cellulase production in T. reesei , although these processes depend on the kind of nitrogen sources. The findings in this study contribute to the understanding of the regulation network of carbon and nitrogen sources in filamentous fungi.

An investigation of Trichoderma biodiversity involving a large-scale environmental gradient was conducted to understand the Trichoderma distribution in China. A total of 3,999 isolates were isolated from forestry, grassland, wetland and agriculture ecosystems, and 50 species were identified based on morphological characteristics and sequence analysis of genetic markers. Trichoderma harzianum showed the largest proportion of isolates and the most extensive distribution. Hypocrea semiorbis, T. epimyces, T. konilangbra, T. piluliferum, T. pleurotum, T. pubescens, T. strictipilis, T. hunua, T. oblongisporum and an unidentified species, Trichoderma sp. MA 3642, were first reported in China. Most Trichoderma species were distributed in Jilin and Heilongjiang Provinces in northeast China and the fewest were distributed in Qinghai Province. Based on the division of ecological and geographic factors, forestry ecosystems and low-altitude regions have the greatest species biodiversity of Trichoderma.

The aim of this study was to select isolated filamentous fungi, naturally occurring in the digestive tract of Neoteredo reynei, with high potential to hydrolyze cellulolytic biomass. The selection of the fungi strains, which produce cellulases, was performed by adding carboxymethylcellulose (CMC), microcrystalline cellulose (MCC) or glucose in the media containing peptone and yeast-extract. In this case, the glucose was used as a reference standard for the growth of the mycelia. The abilities of the fungal strains, to hydrolyze cellulose, on the solid media (MCC or CMC), were evaluated. Two methods were used: the congo-red and the speed of mycelial growth. The measurements of the diameters were performed at 24 h intervals, and the speed of the mycelial growth was calculated after 72 h of cultivation. The molecular and morphological identification of the fungi were applied to the isolated strains. Statistical analysis, including ANOVA and Tukey test, and multivariate analysis were applied as tools to select the strains. Twelve strains were isolated and the results of the identification were 2 strains of Hypocrea lutea, 2 strains of Trichoderma reesei, 2 strains of Aspergillus niger; 2 genera of Aspergillus sp., 2 genera of Trichoderma sp., 1 genus of Fusarium sp. and 1 genus of Gliocadium sp. The discrimination analysis methods such as HCA (Hierarchical Cluster Analysis) and PCA (Principal Component Analysis) indicated three strains with the highest potential to hydrolyze cellulose. In this study, the selection strategy was successful, resulting in the classification of strains from the genera Trichoderma and Hypocrea. This is the first time that this kind of studying was applied to select the potential of the cellulolytic fungal strains, isolated from N. reynei, using the methods of the growth of the mycelial diameter and the statistical discrimination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Studies carried out with novel 13 strains of Trichoderma, isolated from mangrove sediments (PE, Brazil) using morphophysiological and molecular characterization, followed evaluation of biocontrol using Fusarium strains isolated from Caatinga soil (PE, Brazil). Trichoderma strains were characterized by polyphasic taxonomic approach, and the extracted DNA was amplified with primers ITS 1 and 4, and sequenced. The biocontrol evaluation was conducted at 24 and 48 h of growth intervals by Tukey test, with a significance of 5%. Antibiosis tests were assessed in vitro by dual plate and partition plate techniques against Fusarium strains., Results: Trichoderma molecular identification, sequences of 500 bp were amplified, deposited into GenBank, and used for phylogenetic analyses. The strains were identified as T. asperellum (10), as T. harzianum (2) and one as T. longibrachiatum. Growth rate presented an average of 0.1207 cm h -1 for Trichoderma and lower growth rate of 0.031 cm h -1 for Fusarium spp., respectively. Antibiosis tests presented the best antagonist level of efficiency for T. asperellum UCP 0149 against F. solani UCP 1395 (82.2%) and F. solani UCP 1075 (70.0%), followed by T. asperellum UCP 0319 against F. solani UCP1083 (73.4%) and T. asperellum UCP 0168 against F. solani UCP1098 (71.5%), respectively., Conclusions: The data obtained in this study as tool for identification of novel Trichoderma strains serve as basis for development of several sustainable use for biotechnological processes. Those Trichoderma strains found promising for the management antagonistic potential and interaction could aid the conduct of biotechnological biocontrol of contaminants, and improve environmental conditions for the health of plants.

During a biodiversity survey of Trichoderma (Ascomycota, Hypocreales, Hypocreaceae) in coastal and lake wetlands of China, a new species, Trichoderma cyanodichotomus, was isolated from Dongting Lake wetland of Hunan province. The strain TW21990-1 was characterized as having two types of conidia and producing a distinct blue-green pigment on potato dextrose agar and cornmeal dextrose agar. The taxonomic position was analyzed using three molecular markers, internal transcribed spacer rDNA, translation elongation factor 1-alpha, and RNA polymerase II subunit B, revealing less than 95.0% homology with all known Trichoderma species. The combined phylogenetic tree further identified T. cyanodichotomus as an independent subgroup belonging to Section Pachybasium, with no close relatives. In vitro antagonistic activity by dual-culture assay exhibited broad inhibition against various plant pathogens, including Botryosphaeria dothidea, Pythium aphanidermatum, Rhizoctonia solani, and Verticillium dahliae. In addition, TW21990-1 demonstrated moderate hydrolase activity of cellulase, chitinase, β-1,3-glucanase, and protease, which might be involved in mycoparasitism. Greenhouse experiments showed strong biocontrol effects against tomato damping-off incited by P. aphanidermatum, together with increased seedling height and weight gain. The identification of T. cyanodichotomus will provide useful information for sufficient utilization of fungal resources.

The study evaluates the survivability and storage stability of seven Trichoderma strains belonging to the species: T. harzianum (1), T. atroviride (4), and T. virens (2) after the lyophilization of their solid state cultures on wheat straw. Biomass of Trichoderma strains was freeze-dried with and without the addition of maltodextrin. Furthermore, in order to determine the ability of tested Trichoderma strains to preserve selected technological features, the biosynthesis of extracellular hydrolases (cellulases, xylanases, and polygalacturonases) after a 3-month storage of lyophilizates was investigated. Strains of T. atroviride (except TRS40) and T. harzianum TRS85 showed the highest viability after lyophilization process (up to 100%). After 3 months of storage, T. atroviride TRS14 exhibited the highest stability (95.23%); however, the number of active conidia remained at high level of 10 6 -10 7  cfu/g for all tested T. atroviride strains and T. harzianum TRS85. Interestingly, after a 3-month storage of lyophilized formulations, most of the tested Trichoderma strains exhibited higher cellulolytic and xylanolytic activities compared to the control, i.e., before freeze-drying process. The highest activities of these enzymes exhibited the following: T. atroviride TRS14-2.37 U/g and T. atroviride TRS25-21.47 U/g, respectively, whereas pectinolytic activity was weak for all tested strains, with the highest value of 0.64 U/g registered for T. virens TRS109.

Fourteen Trichoderma (Hypocreales) species were identified during a survey of the genus in South Africa. These include T. afroharzianum, T. asperelloides, T. asperellum, T. atrobrunneum, T. atroviride, T. camerunense, T. gamsii, T. hamatum, T. koningii, T. koningiopsis, T. saturnisporum, T. spirale, T. virens, and T. viride. Ten of these species were not known to occur in South Africa prior to this investigation. Five additional species were novel and are described here as T. beinartii, T. caeruleimontis, T. chetii, T. restrictum, and T. undulatum. These novel Trichoderma species display morphological traits that are typical of the genus. Based on molecular identification using calmodulin, endochitinase, nuc rDNA internal transcribed spacers (ITS1-5.8S-ITS2), RNA polymerase II subunit B, and translation elongation factor 1-α gene sequence data, T. beinartii, T. caeruleimontis, and T. chetii were found to belong to the Longibrachiatum clade, whereas T. restrictum is a member of the Hamatum clade. Trichoderma undulatum occupies a distinct lineage distantly related to other Trichoderma species. Strains of T. beinartii and T. chetii were isolated previously in Hawaii and Israel; however, T. caeruleimontis, T. restrictum, and T. undulatum are so far known only from South Africa.

The objective of this study was to investigate the screening, taxonomy characterization, Pb biosorption, and application of the high Pb-resistant fungus F1 separated from the heavy metal contaminated soil. Fungus F1 was screened through metal concentration gradient ranging from 25 to 4000 mg L -1 . The internal transcribed spacers (ITS) of the strain was analyzed by molecular biotechnology. The adsorption conditions were also evaluated. The precipitation of fungus F1 was analyzed by Scanning Electron Microscopy (SEM), Fourier transformer infrared spectroscopy (FTIR) and energy dispersive X-ray (EDX) techniques. The Pb speciation was determined by BCR three-step sequential extraction. The highest concentration of fungus F1 resistance to Pb 2+ was 3500 mg L -1 . The fungus was identified as Trichoderma asperellum. The optimum condition for the Pb 2+ removal rate was discovered as follows: MTL: 3500 mg L -1 ; pH: 7; Pb2+ concentration: 800 mg L -1 ; temperature was 30 °C; initial biosorbent dosage: 6% (v/v). The surface chemical functional groups of fungus F1 were amino, hydroxyl, and carbonyl groups, which may be involved in the biosorption of Pb. Application test showed that the exchangeable, acid-and water soluble Pb were reduced, and the sulfide, organic combination state, and residual Pb were increased. With the preferable absorption capacity, fungus F1 was considered to have good prospects of bioremediation., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Trichoderma is one of the most exploited biocontrol agent for the management of plant diseases. Twenty strains of Trichoderma (six of T. harzianum, four of T. viride, three of T. virens, three of T. koningii, each one of T. hamatum, T. reesei, T. parceramosum and Trichoderma spp.) subjected to in vitro antagonism up to 12days after inoculation against Sclerotium rolfsii Sacc. causing stem rot in groundnut. A new concept was developed to determine inhibition coefficient representing pathogen biology and biocontrol related biophysical variables. Results explained differential inhibition coefficient of test pathogen by Trichoderma antagonists. The inhibition coefficient of test pathogen was examined highest (91.13%) by T. virens NBAII Tvs12 followed by T. virens MTCC 794 (89.33%) and T. koningii MTCC 796 (62.39%). Microscopic study confirmed biocontrol mechanism as mycoparasitism for Tvs12 and antibiosis for T. koningii MTCC 796. The sclerotial biogenesis of test pathogen was elevated during weak antagonism and diminished in interactions with strong antagonists. The inhibition coefficient of S. rolfsii was significantly negatively correlated with sclerotia formation and lipid peroxidation during the antagonism. Trichoderma strains were screened for fungicides (carbendazim and tebuconazole, thiram and mancozeb) and abiotic stress (drought and salt) tolerance. Results indicated that T. koningii MTCC 796 efficiently grew better than the other strains with maximum radial growth under adverse conditions. The genetic variability among the Trichoderma was determined using 34 gene specific markers which amplified 146 alleles. The SSR similarities explained substantial diversity (15 to 87%) across Trichoderma strains and pathogen S. rolfsii. Principal coordinates analysis (PCA) were comparable to the cluster analysis and first three most informative PC components explained 64.45% of the total variation. In PCA, potent antagonists appear to be distinct from other strains. Five SSR markers T1F/T1R (311) , TvCTT56f/TvCTT56r (387) , TvGAT18f/TvGAT18r (364) , TvCA39f/TvCA39r (196) and TvAG29f/TvAG29r (418) found to be unique to distinguish best antagonist strain Tvs12. However, MTCC 796 was examined most stress tolerant strain with better inhibition coefficient which might be useful to control the disease under adverse conditions or as a part of integrated pest management., (Copyright © 2017. Published by Elsevier B.V.)

Spliceosomal introns can occupy nearby rather than identical positions in orthologous genes (intron sliding or shifting). Stwintrons are complex intervening sequences, where an 'internal' intron interrupts one of the sequences essential for splicing, generating after its excision, a newly formed canonical intron defined as 'external'. In one experimentally demonstrated configuration, two alternatively excised internal introns, overlapping by one G, disrupt respectively the donor and the acceptor sequence of an external intron, leading to mRNAs encoding identical proteins. In a gene encoding a DHA1 antiporter in Pezizomycotina, we find a variety of predicted intron configurations interrupting the DNA stretch encoding a conserved peptidic sequence. Some sport a stwintron where the internal intron interrupts the donor of the external intron (experimentally confirmed for Aspergillus nidulans). In others, we found and demonstrate (for Trichoderma reesei) alternative, overlapping internal introns. Discordant canonical introns, one nt apart, are present in yet other species, exactly as predicted by the alternative loss of either of the internal introns at the DNA level from an alternatively spliced stwintron. An evolutionary pathway of 1 nt intron shift, involving an alternatively spliced stwintron intermediate is proposed on the basis of the experimental and genomic data presented., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Trichoderma is a dominant component of the soil mycoflora. During the field investigations of northern, central, and southwestern China, three new species in the Stromaticum clade were encountered from soil, and named as T. hebeiense, T. sichuanense, and T. verticillatum. Their phylogenetic positions were determined by analyses of the combined two genes: partial sequences of translation elongation factor 1-alpha and the second largest RNA polymerase subunit-encoding genes. Distinctions between the new species and their close relatives were discussed. Trichoderma hebeiense appeared as a separate terminal branch. The species is distinctive by its oblong conidia and aggregated pustules in culture. Trichoderma sichuanense features in concentric colony and produces numerous clean exudates on aerial mycelium in culture. Trichoderma verticillatum is characterized by its verticillium-like synanamorph and production of abundant chlamydospores. In vitro antagonism towards the new species was tested by dual culture technique.

The first large-scaled survey of soil-inhabiting Trichoderma is conducted in 23 provinces of China. Twenty-three new species belonging to the green-ascospored clades are discovered. Their phylogenetic positions are determined by sequence analyses of the combined partial sequences of translation elongation factor 1-alpha and the second largest RNA polymerase subunit encoding genes. Morphology and culture characteristics are observed, described and illustrated in detail. Distinctions between the new species and their close relatives are compared and discussed. They are named as: T. aggregatum, T. alpinum, T. bannaense, T. breve, T. brevicrassum, T. byssinum, T. chlamydosporicum, T. concentricum, T. ganodermatis, T. hainanense, T. hengshanicum, T. hirsutum, T. hunanense, T. ingratum, T. liberatum, T. linzhiense, T. longisporum, T. polypori, T. pseudodensum, T. simplex, T. solum, T. undatipile and T. zayuense.

Climate change is one of the biggest challenges of the twenty-first century for sustainable agricultural production. Several reports highlighted the need for better agricultural practices and use of eco-friendly methods for sustainable crop production under such situations. In this context, Trichoderma species could be a model fungus to sustain crop productivity. Currently, these are widely used as inoculants for biocontrol, biofertilization, and phytostimulation. They are reported to improve photosynthetic efficiency, enhance nutrient uptake and increase nitrogen use efficiency in crops. Moreover, they can be used to produce bio-energy, facilitate plants for adaptation and mitigate adverse effect of climate change. The technological advancement in high throughput DNA sequencing and biotechnology provided deep insight into the complex and diverse biotic interactions established in nature by Trichoderma spp. and efforts are being made to translate this knowledge to enhance crop growth, resistance to disease and tolerance to abiotic stresses under field conditions. The discovery of several traits and genes that are involved in the beneficial effects of Trichoderma spp. has resulted in better understanding of the performance of bioinoculants in the field, and will lead to more efficient use of these strains and possibly to their improvement by genetic modification. The present mini-review is an effort to elucidate the molecular basis of plant growth promotion and defence activation by Trichoderma spp. to garner broad perspectives regarding their functioning and applicability for climate resilient agriculture.

Filamentous fungi from the genus Trichoderma are commonly found in soil. They are considered facultative mycoparasites, and are antagonists of other fungi such as the cultivar of leaf-cutting ants (Leucoagaricus gongylophorus). The aim of the present study was to bioprospect Trichoderma spp. from different soils collected from Gurupi, Tocantins, Brazil, for antagonistic effects against the mutualistic fungus of leaf-cutting ants. To isolate filamentous fungi, samples were collected from six locations. Preliminarily, isolates were identified by morphological analysis as belonging to Trichoderma. Trichoderma spp. had their internal transcribed spacer region (ITS) of ribosomal RNA genes (rRNA) sequenced to confirm species-level taxonomy. L. gongylophorus was isolated from a laboratory ant colony. Antagonistic properties of seven isolates of Trichoderma against L. gongylophorus were measured using paired disks in Petri dishes with potato dextrose agar medium (PDA). All Trichoderma isolates inhibited the growth of L. gongylophorus in Petri dishes. Isolate 2 of Trichoderma spirale group exhibited slow mycelial growth in the Petri dish, and a high rate of inhibition against L. gongylophorus. This isolate is a promising fungus for field tests of biological control methods for leaf-cutting ants., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Due to its efficient broad-spectrum antimicrobial activity, Trichoderma has been established as an internationally recognized biocontrol fungus. In this study, we found and identified a novel strain of Trichoderma asperellum, named GDFS1009. The mycelium of T. asperellum GDFS1009 exhibits a high growth rate, high sporulation capacity, and strong inhibitory effects against pathogens that cause cucumber fusarium wilt and corn stalk rot. T. asperellum GDFS1009 secretes chitinase, glucanase, and protease, which can degrade the cell walls of fungi and contribute to mycoparasitism. The secreted xylanases are good candidates for inducing plant resistance and enhancing plant immunity against pathogens. RNA sequencing (RNA-seq) and gas chromatography-mass spectrometry (GC-MS) showed that T. asperellum GDFS1009 produces primary metabolites that are precursors of antimicrobial compounds; it also produces a variety of antimicrobial secondary metabolites, including polyketides and alkanes. In addition, this study speculated the presence of six antimicrobial peptides via ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS). Future studies should focus on these antimicrobial metabolites for facilitating widespread application in the field of agricultural bio-control.

Hydrophobins are small secreted cysteine-rich proteins exclusively found in fungi. They are able to self-assemble in single molecular layers at hydrophobic-hydrophilic interfaces and can therefore be directly involved in establishment of fungi in their habitat. The genomes of filamentous mycotrophic fungi Trichoderma encode a rich diversity of hydrophobins, which are divided in several groups based on their structure and evolution. Here we describe a new member of class II hydrophobins, HFB7, that has a taxonomically restricted occurrence in Harzianum and Virens clades of Trichoderma. Evolutionary analysis reveals that HFB7 proteins form a separate clade distinct from other Trichoderma class II hydrophobins and that genes encoding them evolve under positive selection pressure. Homology modelling of HFB7 structure in comparison to T. reesei HFB2 reveals that the two large hydrophobic patches on the surface of the protein are remarkably conserved between the two hydrophobins despite significant difference in their primary structures. Expression of hfb7 gene in T. virens increases at interactions with other fungi and a plant and in response to a diversity of abiotic stress conditions, and is also upregulated during formation of aerial mycelium in a standing liquid culture. This upregulation significantly exceeds that of expression of hfb7 under a strong constitutive promoter, and T. virens strains overexpressing hfb7 thus display only changes in traits characterized by low hfb7 expression, i.e. faster growth in submerged liquid culture. The hfb7 gene is not expressed in conidia. Our data allow to conclude that this protein is involved in defence of Trichoderma against a diversity of stress factors related to the oxidative stress. Moreover, HFB7 likely helps in the establishment of the fungus in wetlands or other conditions related to high humidity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max  = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes.

Development of reliable and low-cost requirement for large-scale eco-friendly biogenic synthesis of metallic nanoparticles is an important step for industrial applications of bionanotechnology. In the present study, the mycosynthesis of spherical nano-Ag (12.7 ± 0.8 nm) from extracellular filtrate of local endophytic T. harzianum SYA.F4 strain which have interested mixed bioactive metabolites (alkaloids, flavonoids, tannins, phenols, nitrate reductase (320 nmol/hr/ml), carbohydrate (25 μg/μl) and total protein concentration (2.5 g/l) was reported. Industrial mycosynthesis of nano-Ag can be induced with different characters depending on the fungal cultivation and physical conditions. Taguchi design was applied to improve the physicochemical conditions for nano-Ag production, and the optimum conditions which increased its mass weight 3 times larger than a basal condition were as follows: AgNO 3 (0.01 M), diluted reductant (10 v/v, pH 5) and incubated at 30 °C, 200 rpm for 24 hr. Kinetic conversion rates in submerged batch cultivation in 7 L stirred tank bioreactor on using semi-defined cultivation medium was as follows: the maximum biomass production (X max ) and maximum nano-Ag mass weight (P max ) calculated (60.5 g/l and 78.4 g/l respectively). The best nano-Ag concentration that formed large inhibition zones was 100 μg/ml which showed against A.alternate (43 mm) followed by Helminthosporium sp. (35 mm), Botrytis sp. (32 mm) and P. arenaria (28 mm).

The ITS (nuclear ribosomal internal transcribed spacer) RefSeq database at the National Center for Biotechnology Information (NCBI) is dedicated to the clear association between name, specimen and sequence data. This database is focused on sequences obtained from type material stored in public collections. While the initial ITS sequence curation effort together with numerous fungal taxonomy experts attempted to cover as many orders as possible, we extended our latest focus to the family and genus ranks. We focused on Trichoderma for several reasons, mainly because the asexual and sexual synonyms were well documented, and a list of proposed names and type material were recently proposed and published. In this case study the recent taxonomic information was applied to do a complete taxonomic audit for the genus Trichoderma in the NCBI Taxonomy database. A name status report is available here: https://www.ncbi.nlm.nih.gov/Taxonomy/TaxIdentifier/tax&#95;identifier.cgi. As a result, the ITS RefSeq Targeted Loci database at NCBI has been augmented with more sequences from type and verified material from Trichoderma species. Additionally, to aid in the cross referencing of data from single loci and genomes we have collected a list of quality records of the RPB2 gene obtained from type material in GenBank that could help validate future submissions. During the process of curation misidentified genomes were discovered, and sequence records from type material were found hidden under previous classifications. Source metadata curation, although more cumbersome, proved to be useful as confirmation of the type material designation. Database URL:http://www.ncbi.nlm.nih.gov/bioproject/PRJNA177353, (© Crown copyright 2017.)

To investigate the biodiversity of Trichoderma (Hypocreaceae) and their relation to sediment physical and chemical properties, we collected a total of 491 sediment samples from coastal wetlands (tidal flat and wetland) in Southeast China. Further, we applied two types of molecular approaches such as culture dependent and independent methods for identification of Trichoderma spp. A total of 254 isolates were obtained and identified to 13 species such as T. aureoviride, T. asperellum, T. harzianum, T. atroviride, T. koningiopsis, T. longibrachiatum, T. koningii. T. tawa, T. viridescens, T. virens, T. hamatum, T. viride, and T. velutinum by the culture-dependent (CD) method of these, T. tawa was newly described in China. Subsequently, the culture indepented method of 454 pyrosequencing analysis revealed a total of six species such as T. citrinoviride, T. virens, T. polysporum, T. harzianum/Hypocrea lixii and two unknown species. Notably, T. citrinoviride and T. polysporum were not found by the CD method. Therefore, this work revealed that the combination of these two methods could show the higher biodiversity of Trichoderma spp., than either of this method alone. Among the sampling sites, Hangzhou, Zhejiang Province, exhibited rich biodiversity and low in Fengxian. Correlation and Redundancy discriminant analysis (RDA) revealed that sediment properties of temperature, redox potential (Eh) and pH significantly influenced the biodiversity of Trichoderma spp., Competing Interests: The authors have declared that no competing interests exist.

Some species of Trichoderma are fungicolous on fungi and have been extensively studied and commercialized as biocontrol agents. Multigene analyses coupled with morphology, resulted in the discovery of T. hypoxylon sp. nov., which was isolated from surface of the stroma of Hypoxylon anthochroum. The new taxon produces Trichoderma- to Verticillium-like conidiophores and hyaline conidia. Phylogenetic analyses based on combined ITS, TEF1-α and RPB2 sequence data indicated that T. hypoxylon is a well-distinguished species with strong bootstrap support in the polysporum group. Chemical assessment of this species reveals a richness of secondary metabolites with trichothecenes and epipolythiodiketopiperazines as the major compounds. The fungicolous life style of T. hypoxylon and the production of abundant metabolites are indicative of the important ecological roles of this species in nature.

Trichoderma strains exhibit enormous potential for applications in biotechnology, in particular as biocontrol agents against pathogens. However, little is known about the diversity of plant-associated Trichoderma communities at a global scale and their antagonistic spectrum. In order to gather information about structure and function, we compared Trichoderma biomes of endemic (Aeonium, Diospyros, Hebe, Rhododendron) and cosmopolitan plants (Zea mays) in a global study encompassing the area Northwest Africa to New Zealand via the European Alps and Madagascar. At the quantitative level we found no differences between cosmopolitan and endemic plants. Statistically significant differences were detected at the qualitative level: Trichoderma populations of endemic plants were highly specific and diverse with hot spots appearing in Madagascar and New Zealand. By contrast, maize plants from all sites shared the majority of Trichoderma species (65.5%). Interestingly, the high above ground biodiversity in ecosystems containing endemic plants was confirmed by a high below ground Trichoderma diversity. Despite the differences, we found a global Trichoderma core community shared by all analysed plants, which was dominated by T. koningii and T. koningiopsis. Amplicon-based network analyses revealed a high similarity between maize Trichoderma grown world-wide and distinct populations of endemic plants. Furthermore, Trichoderma strains from endemic plants showed a higher antagonistic activity against fungal pathogens compared to maize-associated strains. Our results showed that endemic plants are associated with a specific Trichoderma microbiome which possesses a high antagonistic activity indicating that it has potential to be used for biocontrol purposes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We surveyed the Trichoderma (Hypocreales, Ascomycota) biodiversity in agricultural fields in four major agricultural provinces of East China. Trichoderma strains were identified based on molecular approaches and morphological characteristics. In three sampled seasons (spring, summer and autumn), 2078 strains were isolated and identified to 17 known species: T. harzianum (429 isolates), T. asperellum (425), T. hamatum (397), T. virens (340), T. koningiopsis (248), T. brevicompactum (73), T. atroviride (73), T. fertile (26), T. longibrachiatum (22), T. pleuroticola (16), T. erinaceum (16), T. oblongisporum (2), T. polysporum (2), T. spirale (2), T. capillare (2), T. velutinum (2), and T. saturnisporum (1). T. harzianum, T. asperellum, T. hamatum, and T. virens were identified as the dominant species with dominance (Y) values of 0.057, 0.052, 0.048, and 0.039, respectively. The species amount, isolate numbers and the dominant species of Trichoderma varied between provinces. Zhejiang Province has shown the highest diversity, which was reflected in the highest species amount (14) and the highest Shannon-Wiener diversity index of Trichoderma haplotypes (1.46). We observed that relative frequencies of T. hamatum and T. koningiopsis under rice soil were higher than those under wheat and maize soil, indicating the preference of Trichoderma to different crops. Remarkable seasonal variation was shown, with summer exhibiting the highest biodiversity of the studied seasons. These results show that Trichoderma biodiversity in agricultural fields varies by region, crop, and season. Zhejiang Province (the southernmost province in the investigated area) had more T. hamatum than Shandong Province (the northernmost province), not only in isolate amounts but also in haplotype amounts. Furthermore, at haplotype level, only T. hamatum showed a gradient distribution from south to north in correspondence analysis among the four dominant species. The above results would contribute to the application of Trichoderma biocontrol strains.

The aim of this study was to explore the species diversity of Trichoderma obtained from samples of wood collected in the forests of the Gorce Mountains (location A), Karkonosze Mountains (location B) and Tatra Mountains (location C) in Central Europe and to examine the cellulolytic and xylanolytic activity of these species as an expression of their probable role in wood decay processes. The present study has led to the identification of the following species and species complex: Trichoderma atroviride P. Karst., Trichoderma citrinoviride Bissett, Trichoderma cremeum P. Chaverri & Samuels, Trichoderma gamsii Samuels & Druzhin., Trichoderma harzianum complex, Trichoderma koningii Oudem., Trichoderma koningiopsis Samuels, C. Suárez & H.C. Evans, Trichoderma longibrachiatum Rifai, Trichoderma longipile Bissett, Trichoderma sp. (Hypocrea parapilulifera B.S. Lu, Druzhin. & Samuels), Trichoderma viride Schumach. and Trichoderma viridescens complex. Among them, T. viride was observed as the most abundant species (53 % of all isolates) in all the investigated locations. The Shannon's biodiversity index (H), evenness (E), and the Simpson's biodiversity index (D) calculations for each location showed that the highest species diversity and evenness were recorded for location A-Gorce Mountains (H' = 1.71, E = 0.82, D = 0.79). The preliminary screening of 119 Trichoderma strains for cellulolytic and xylanolytic activity showed the real potential of all Trichoderma species originating from wood with decay symptoms to produce cellulases and xylanases-the key enzymes in plant cell wall degradation.

The study was performed to examine 11 isolates of Trichoderma for their bio-control potentials against Sclerotium rolfsii Sacc. causing stem rot in groundnut. The antagonists Trichoderma were subjected to sequence related amplified polymorphism (SRAP) based molecular diversity analysis and compared with their hardness to S. rolfsii with respect to secretary antifungal and antioxidant profile. T. virens NBAII Tvs 12 evident highest (87.91 %) growth inhibition of test pathogen followed by T. koningii MTCC 796 (67.03 %) at 7 days after inoculation (DAI). Microscopic study confirmed biocontrol mechanism as mycoparasitism for Tvs 12 and antibiosis for MTCC 796. The growth inhibition of test pathogen was significantly negatively correlated with sclerotia formation and lipid peroxidation during antagonism due to release of secretary bioactive antioxidants by antagonists to terminate oxidative burst generated by S. rolfsii and causing inhibition of sclerotium formation. The GC-MS profile identified antifungal and antioxidant constituents hexadecane, 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester, 1-hexadecanesulfonyl chloride, and octadecane in potent antagonists Tvs 12; and nonacosane and octadecane in MTCC 796 along with two novel compounds 1-pentadecene and 1-heneicosyl formate for biocontrol activity. Molecular diversity of Trichoderma isolates associated with antagonistic activity was assessed by SRAP markers. The 115 primer combinations generate total 1328 amplified products of which, 1095 are shared polymorphic and 199 are unique polymorphic. The 15 SRAP combinations produced 18 bands to diagnose best antagonist Tvs 12 and 13 SRAP combinations generated 19 unique bands for identification of MTCC 796. The mycoparasitic antagonist Tvs 12 would be the best antagonist and released unique antifungal and antioxidant constituents to combat pathogen infection. The SRAP based genetic diversity indicates Tvs12 strain clustered with T. viride NBAII Tv23 and shared only 52 % similarity with other isolates of Trichoderma. The SRAP similarities explained substantial diversity (19-68 %) across Trichoderma isolates.

Light-oxygen-voltage (LOV) domain-containing proteins function as small light-activated modules capable of imparting blue light control of biological processes. Their small modular nature has made them model proteins for allosteric signal transduction and optogenetic devices. Despite intense research, key aspects of their signal transduction mechanisms and photochemistry remain poorly understood. In particular, ordered water has been identified as a possible key mediator of photocycle kinetics, despite the lack of ordered water in the LOV active site. Herein, we use recent crystal structures of a fungal LOV protein ENVOY to interrogate the role of Thr(101) in recruiting water to the flavin active site where it can function as an intrinsic base to accelerate photocycle kinetics. Kinetic and molecular dynamic simulations confirm a role in solvent recruitment to the active site and identify structural changes that correlate with solvent recruitment. In vivo analysis of T101I indicates a direct role of the Thr(101) position in mediating adaptation to osmotic stress, thereby verifying biological relevance of ordered water in LOV signaling. The combined studies identify position 101 as a mediator of both allostery and photocycle catalysis that can impact organism physiology., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Defining syntenic relationships among orthologous gene clusters is a frequent undertaking of biologists studying organismal evolution through comparative genomic approaches. With the increasing availability of genome data made possible through next-generation sequencing technology, there is a growing need for user-friendly tools capable of assessing synteny. Here we present SimpleSynteny, a new web-based platform capable of directly interrogating collinearity of local genomic neighbors across multiple species in a targeted manner. SimpleSynteny provides a pipeline for evaluating the synteny of a preselected set of gene targets across multiple organismal genomes. An emphasis has been placed on ease-of-use, and users are only required to submit FASTA files for their genomes and genes of interest. SimpleSynteny then guides the user through an iterative process of exploring and customizing genomes individually before combining them into a final high-resolution figure. Because the process is iterative, it allows the user to customize the organization of multiple contigs and incorporate knowledge from additional sources, rather than forcing complete dependence on the computational predictions. Additional tools are provided to help the user identify which contigs in a genome assembly contain gene targets and to optimize analyses of circular genomes. SimpleSynteny is freely available at: http://www.SimpleSynteny.com., (Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

More than 200 recent collections of Trichoderma from China were examined and 16 species belonging to the Viride clade were identified based on integrated studies of phenotypic and molecular data. Among them, seven wood-inhabiting new species, T. albofulvopsis, T. densum, T. laevisporum, T. sinokoningii, T. sparsum, T. sphaerosporum and T. subviride, are found. They form trichoderma- to verticillium-like conidiophores, lageniform to subulate phialides and globose to ellipsoidal conidia, but vary greatly in colony features, growth rates, and sizes of phialides and conidia. To explore their taxonomic positions, the phylogenetic tree including all the known species of the Viride clade is constructed based on sequence analyses of the combined RNA polymerase II subunit b and translation elongation factor 1 alpha exon genes. Our results indicated that the seven new species were well-located in the Koningii, Rogersonii and Neorufum subclades as well as a few independent terminal branches. They are clearly distinguishable from any existing species. Morphological distinctions and sequence divergences between the new species and their close relatives were discussed.