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Trichoderma virens colonizes roots and develops a symbiotic relationship with plants where the fungal partner derives nutrients from plants and offers defence, in return. Tsp1, a small secreted cysteine-rich protein, was earlier found to be upregulated in co-cultivation of T. virens with maize roots. Tsp1 is well conserved in Ascomycota division of fungi, but none of its homologs have been studied yet. We have expressed and purified recombinant Tsp1, and resolved its structure to 1.25 A resolutions, from two crystal forms, using Se-SAD methods. The Tsp1 adopts a β barrel fold and forms dimer in structure as well as in solution form. DALI based structure analysis revealed the structure similarity with two known fungal effector proteins: Alt a1 and PevD1. Structure and evolutionary analysis suggested that Tsp1 belongs to a novel effector protein family. Tsp1 acted as an inducer of salicylic acid mediated susceptibility in plants, rendering maize plants more susceptible to a necrotrophic pathogen Cochliobolus heterostrophus, as observed using plant defence assay and RT-qPCR analysis.

In the present study

Beneficial interaction of members of the fungal genus

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.

Trichoderma speies are known for their diverse applications as potent bio-control organisms. 34 isolates of Trichoderma were obtained from various geographical locations in India and were subjected to phenotypic evaluation. Species identification if based on only morphology, may lead to sometimes erroneous, uncertain or remains unclear due to overlapping characters. Therefore, molecular characterization was also performed based on ITS region sequence analysis for reliable identification using both morphological and molecular characters. The isolates were identified into 20 different species viz., T. aggressivum, T. asperellum, T. atroviride, T. brevicompactum, T. citrinoviride, T. crassum, T. erinaceum, T. ghanense, T. hamatum, T. harzianum, T. koningiopsis, T. longibrachiatum, T. longipile (Syn. Hypocrea longipilosa), T. minutisporum, T. pubscenes, T.reesei, T. saturnisporum, T. spirale, T. tomentosum and T. virens based on the integrated approach of both morphological and molecular characterisation. ß-tub gene sequence analysis was also performed to find out the phylogenetic relationship of these species. The ß-tub gene sequences differentiated all the 20 different species of Trichoderma better than the ITS region sequences. The morphological characters viz., colony, conidiophores, phialides, conidia and chlamydospores of all the twenty species of Trichoderma were described along with photomicrographs. The ITS and β-tub gene sequences of all the 20 species studied have been deposited in the Genbank.

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is widely used as a cellulase producer in the industry. Herein, we describe the rational engineering of the publicly available T. reesei QM9414 strain to achieve a remarkable high-level production of cellulase on glucose. Overexpression of the key cellulase regulator XYR1 by the copper-repressible promoter Ptcu1 was first implemented to achieve a full cellulase production in the context of catabolite repression (CCR) while eliminating the requirement of inducing sugars for enzyme production. The T. reesei bgl1 gene was further overexpressed to compensate for its low β-glucosidase activity on glucose. This overexpression resulted in a 102% increase in FPase activity compared with the CCR-released RUT-C30 strain cultured on Avicel. Moreover, the saccharification efficiency toward pretreated corncob residues by crude enzymes from the engineered strain on glucose increased by 85% compared with that treated by enzymes from RUT-C30 cultivated on Avicel. The engineered T. reesei strain thus shows great potential as a viable alternative to deliver commercial cellulases after further optimization for efficient saccharification of agricultural waste.

This review summarizes the findings on the use of the Penicillium verruculosum В1-537 (ΔniaD) recipient strain for the development of producers of enzymes—β-glucosidase, polysaccharide monooxygenase, and xylanases—that complement the basic cellulase complex and enhance its effectiveness. A special feature of the P. verruculosum strain is the secretion of a basic cellulase complex consisting of cellobiohydrolases and endoglucanases (EGs) that, in terms of their hydrolytic ability, surpass Hypocrea (Trichoderma) strains that are widely used in the laboratory and in industry. The capabilities of the P. verruculosum В1-537 (ΔniaD) expression system can be used to complement the basic cellulase complex with new enzymes, which are required to enhance its hydrolytic activity, and to develop new enzyme preparations tailored to the conversion of different types of renewable plant biomass (RPB). The use of an expression system based on P. verruculosum fungus makes it possible to enhance the ability of basic cellulase complex to convert different types of RPB.

The current research aims to find out different effective and suitable fungicides, botanical extracts and bio-control agents against Cladosporium cladosporioides, the causal agent of Cladosporium rot in grapes. For this purpose, different fungicides including, Antracol, Alliette, Melody duo, Cabriotop and Topsin-M at the rates of 100, 200 and 300 ppm, as well as botanical extracts including, Garlic, Ginger, Onion, Eucalyptus and Neem at the rates of 5, 10 and 15% were tested using food poisoning method. Bio-control agents such as Pestalotiopsis sp., Neurospora sp., Fusarium sp., Arthrinium sp. and Hypocrea lixii were also evaluated against the pathogen. Pathogenicity test was also performed to see the disease severity in grapes. Minimum linear colony growth of C. cladosporioides was observed as 39, 30.5 and 21 mm for Melody duo, respectively followed by Antracol (38.5, 30.5 and 23), Alliette (39, 31.5 and 23.5), Topsin-m (42, 33 and 27.5) and cabriotop (43.5, 36.5 and 26), as compared with the control, which was 90 mm. Whereas Minimum linear colony growth of C. cladosporioides was observed as 50, 32.5 and 19 for Neem, respectively followed by Ginger (56.5, 36.5 and 22.5), Garlic (61, 39 and 24), euclayptus (62, 41.5 and 25) and Onion (64, 43 and 25), maximum linear colony growth (90) was observed under control. Whereas, the minimum linear colony growth of C. cladosporioides was observed for Neurospora sp. (3.7), followed by Arthrinium sp. (7.5), Pestalotiopsis sp. (9), Hypocrea Lixii (9) and Fusarium sp. (9.5), maximum linear colony growth (90) was recorded in control. This study could be helpful for researchers and farming community in future for better management of this disease.

Benzoic acid (BA) is potentially toxic to humans and recalcitrant in the environment. Although it may be degraded by fungi, potential degradation pathways has received limited study. In the present...

Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi–host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a β-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Å resolution and belonged to the orthorhombic space group P212121 (R meas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (V M) of the crystal is 2.32 Å3 Da−1, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.

The pharmaceutical industry has a high demand for glycoprotein production. The glycoform of glycoproteins is crucial for pharmacological activity. However, in general, cells produce glycoproteins with a heterologous glycoform, which is unfavorable for making uniform, efficacious therapeutic proteins. Here, to produce more glycoproteins with N-glycan uniformity, we applied the GlycoDelete strategy, in which endo-β-N-acetylglucosaminidase (ENGase) from the fungus Hypocrea jecorina (EndoT) is expressed at the Golgi membrane to cleave N-glycan from secretory glycoproteins, to Aspergillus oryzae cells. First, we selected candidate transmembrane domains to target EndoT to the Golgi membrane in A. oryzae cells, generated constructs for expressing the transmembrane-fused EndoT proteins and produced four potential AoGlycoDelete strains. We then confirmed that these strains produced α-amylase with a molecular weight lower than that of native α-amylase without an effect on growth. To test whether the A. oryzae α-amylase proteins had been cleaved by EndoT, we expressed and purified HA-tagged α-amylase AmyB and glucoamylase GlaA proteins from the AoGlycoDelete strain. MS and N-glycan analyses of the intact proteins confirmed neither AmyB-HA nor GlaA-HA produced from the AoGlycoDelete strain contained N-glycan. Lastly, we determined the enzymatic activities of the amylases produced by the AoGlycoDelete strain, which showed that the lack of N-glycan did not affect their activity under the conditions tested. Collectively, our findings demonstrate successful generation of an AoGlycoDelete strain that might be a good candidate for producing pharmaceutical glycoproteins with a uniform N-glycan structure.

Trichoderma reeseiβ-glucosidase (Bgl1) is one of four enzymes demonstrated to act synergistically to degrade cellulose both in vitro and in vivo. Our work attempted to better understand the substrate specificity and potential biotechnological applications of Bgl1. T. reesei Bgl1H cleaves over 80% of the β-(1-4) and β-(1-3) linkages in β-glucan and 14% of the β-(1-4) linkages in amorphous cellulose, significantly more than any tested bacterial β-glucosidase. Bgl1H cleaves 50% of the β-(1-4) linkages in xyloglucan when supplemented with cellulase and α-xyloside. Approximately 20% conversion to glucose was obtained from insoluble β-(1,3)-linked curdlan using only Bgl1H; addition of a curdlanase resulted in conversion of approximately 70% of the curdlan to glucose. Bgl1H also produces xylose from xylooligosaccharides and debranched xylans. For both glucans and xylans, the relative rates of hydrolysis increase with increasing polysaccharide chain lengths. Bgl1H is able to partially degrade β-glucan in a variety of grain components; addition of endo-acting enzymes improved the enzyme’s performance on these grain components. The ability of this enzyme to produce monosaccharides from undigestible polysaccharides suggest it may have potential in improving utilization of carbohydrates in animal feed, fermentations, and other biotechnological applications.

The aim of present study was to decipher the effect of salinity stress on growth and antagonistic potential of Trichoderma and Hypocrea isolates against tomato root rot pathogen (Rhizoctonia solani AG-4) under saline soil conditions. In vitro salinity assays, dual plate confrontation, and volatile metabolite assays were employed to establish the antagonistic potential of Trichoderma and Hypocrea isolates against R. solani AG-4 under varied salt gradients. Potential Trichoderma and Hypocrea isolates were evaluated against tomato root rot disease in greenhouse conditions under salt stress condition. Polymerase chain reaction (PCR) assay was performed to confirm the presence of endochitinase gene in salt-tolerant antagonists. Enzyme and biochemical assays were conducted to define the role of compatible solutes and defense-related enzymes in controlling tomato root rot under saline soil conditions. Trichoderma and Hypocrea isolates were capable to grow and sporulate up to 250 mM NaCl and also showed strong antagonism against R. solani. Enzymatic estimation of hydrolytic enzymes and amplification of endochitinase gene suggested that the test isolates are potent antagonistic agents. Under greenhouse evaluation, Trichoderma and Hypocrea fortified tomato plants showed significant reduction in tomato root rot disease in saline soils over untreated control. Significant increase in total phenol, polyphenol oxidase, peroxidase, β-1,3-glucanase, phenylalanine lyase, chitinase, proline, reducing sugar, and total soluble sugar displayed direct association with salt stress tolerance. Application of salt-tolerant Trichoderma and Hypocrea isolates emerged as a simple, safe, and cheap method for the biological management of tomato root rot under saline condition.

Endophytic fungi have received much attention as plant growth promoters as well as biological control agents against many plant pathogens. In this study, 30 endophytic fungal species, isolated from various plants in China, were evaluated using in vitro dual culture assay against Fusarium oxysporum f. sp. cucumerinum, causing wilt in cucumber. The results of the present study clearly showed that all the 30 endophytic fungal isolates were highly capable of inhibiting the mycelial colony growth of Fusarium oxysporum f. sp. cucumerinum with inhibition % over 66% as compared to control treatments. Among all of them, 5 isolates were highly effective such as, Penicillium sp., Guignardia mangiferae, Hypocrea sp., Neurospora sp., Eupenicillium javanicum, and Lasiodiplodia theobromae, respectively. The Penicillium sp. and Hypocrea sp. were highly effective as compared to other isolates. From in vitro results 10 best isolates were selected for greenhouse studies. The results of the greenhouse studies showed that among all of them 3 endophytic fungal isolates successfully suppressed wilt severity when co-inoculation with pathogen Fusarium. oxysporum f. sp. cucumerinum. The endophytic fungi also enhanced plant growth parameters of the host plants, the antagonistic fungal isolates increased over all plant height, aerial fresh, and dry weight as compared to control.

A new strain of Trichoderma reesei (teleomorph Hypocrea jecorina) with high cellulase production was obtained by exposing the spores from T. reesei QM9414 to an ultraviolet light followed by selecting fast-growing colonies on plates containing CMC (1% w/v) as the carbon source. The mutant T. reesei RP698 reduced cultivation period to 5 days and increased tolerance to the end-products of enzymatic cellulose digestion. Under submerged fermentation conditions, FPase, CMCase, and Avicelase production increased up to 2-fold as compared to the original QM9414 strain. The highest levels of cellulase activity were obtained at 27 °C after 72 h with Avicel®, cellobiose, and sugarcane bagasse as carbon sources. The temperature and pH activity optima of the FPase, CMCase, and Avicelase were approximately 60 °C and 5.0, respectively. The cellulase activity was unaffected by the addition of 140 mM glucose in the enzyme assay. When T. reesei RP698 crude extract was supplemented by the addition of β-glucosidase from Scytalidium thermophilum, a 2.3-fold increase in glucose release was observed, confirming the low inhibition by the end-product of cellulose hydrolysis. These features indicate the utility of this mutant strain in the production of enzymatic cocktails for biomass degradation.

In the scope of mitigating the negative impacts of pesticide use and managing greenhouse whiteflies, Trialeurodes vaporariorum sustainably, 16 endophytic fungal isolates from five different genera (Beauveria, Trichoderma, Hypocrea, Bionectria, and Fusarium) were screened for their ability to colonise two preferred host plant species, namely, tomato (Solanum lycopersicum L.) and French bean (Phaseolus vulgaris L.), through seed inoculation. Seven and nine isolates were endophytic to P. vulgaris and S. lycopersicum, respectively, where significant differences in the endophytic colonisation rates were observed among the fungal isolates in P. vulgaris and its plant parts, with a significant interaction between the isolates and plant parts in S. lycopersicum. Hypocrea lixii F3ST1, Trichoderma asperellum M2RT4, Trichoderma atroviride F5S21, and T. harzianum KF2R41 successfully colonised all the plant parts of both hosts and therefore were selected and further evaluated for their endophytic persistence, effect on plant growth, and pathogenicity to T. vaporariorum adults and F1 progeny. The four endophytes remained in both host plants for the 5-week assessment with varied colonisation rates related to the strong interaction with the time, isolates, and plant parts in both hosts. The effect of the same endophytes on the different host growth parameters varied in P. vulgaris and S. lycopersicum, with T. asperellum M2RT4 not boosting the growth in both host plants while T. atroviride F5S21 resulted in enhanced shoot biomass in S. lycopersicum. T. atroviride F5S21 and T. harzianum KF2R41 inoculated S. lycopersicum plants and H. lixii F3ST1, T. asperellum M2RT4, and T. harzianum KF2R41 inoculated P. vulgaris plants had significantly lower oviposition, while nymph development in both hosts was significantly prolonged in all the endophytically–colonised plants. The endophytes H. lixii F3ST1 and T. asperellum M2RT4 significantly reduced the longevity/survival of the exposed T. vaporariorum adults and the progeny in both S. lycopersicum and P. vulgaris. The findings demonstrate the attributes of the various endophytes in host plant growth promotion as well as their effects on the life-history parameters of T. vaporariorum and could consequently be developed as potential endophytic fungal-based biopesticides for the sustainable management of the pest in S. lycopersicum and P. vulgaris cropping systems.

Endophytic fungal isolates Hypocrea lixii F3ST1 and Beauveria bassiana G1LU3 were evaluated for their potential to endophytically colonize and induce active compounds in Phaseolus vulgaris, as a defense mechanism against pea leafminer (Liriomyza huidobrensis) and fall armyworm (Spodoptera frugiperda). Endophytic colonization was achieved through seed inoculation with the volatile emissions from P. vulgaris plants being analyzed using GC-MS. The crude extracts of P. vulgaris obtained using methanol and dichloromethane were assayed against leafminer and fall armyworm larvae using leaf dipping and topical application, respectively. The two isolates successfully colonized the entire host plant (roots, stems, and leaves) with significant variation (p &lt, 0.001) between fungal isolates and the controls. The results showed qualitative differences in the volatile profiles between the control plants, endophytically colonized and insect-damaged plants attributed to fungal inoculation and leafminer damage. The crude methanol extracts significantly reduced the percentage pupation of 2nd instar leafminer larvae (p &lt, 0.001) and adult-flies emergence (p &lt, 0.05). The survival of the 1st instar fall armyworm larvae was also significantly reduced (p &lt, 0.001) compared to the controls. This study demonstrated the high potential of endophytic fungi H. lixii and B. bassiana in inducing mainly specific defense compounds in the common bean P. vulgaris that can be used against pea leafminer and fall armyworm.

Bahry MS, Radjasa OK, Trianto A. 2021. Potential of marine sponge-derived fungi in the aquaculture system. Biodiversitas 22: 2883-2892. Organic waste from aquaculture is one of the triggers of disease outbreaks and a decrease in water quality that urgently needs to be resolved. Indonesia has a high diversity of sponges including their associated microorganisms that potential in the field of biotechnology. This study aimed to determine the enzymatic and anti-vibrio activity of fungi associated with marine sponges and identify potential fungi. The specimen of sponges was collected from Samalona Island, South Sulawesi, Indonesia. The enzymatic and anti-vibrio assay was conducted by using the plug method and the activity was determined by a clear zone around the fungal isolates. Fungal identification was carried out molecularly using universal primers ITS1 and ITS4 and phylogenetic tree analysis. The fungal isolates were screened for the extracellular enzyme activity (amylase, cellulase, protease) and anti-vibrio activity against Vibrio parahaemolyticus, V. harveyi, and V. vulnificus). A total of three fungal isolates have been isolated from the sponge Monanchora sp. Isolate SL 3 SP 3.3 had potential enzymatic activities with Enzymatic Indeks (EI) 3.95±0.17 on amylase, 3.75±0.36 on cellulase, 5.38±0.30 on protease. The highest anti-vibrio activity was obtained against V. harveyi with an inhibition zone diameter of 4.82 ±0.37 mm. The results of fungal identification showed that isolate SL3SP3.3 had a sequence length of 638 bp and was closely related to Trichoderma reesei a.k.a Hypocrea jecorina with a similarity value of 99.69%.

Biotransformation of viridin, an antifungal produced by biocontrol agent, with non-viridin producing microorganisms is studied. The results show that some environmental non-targeted microorganisms are able to reduce it in the known phytotoxin viridiol, and its 3-epimer. Consequently, this reduction, which happens in some cases by detoxification mechanism, could be disastrous for the plant in a biocontrol of plant disease. However, a process fermentation/biotransformation could be an efficient approach for the preparation of this phytotoxin.

Production of amylases by fungi under solid-state fermentation is considered the best methodology for commercial scaling that addresses the ever-escalating needs of the worldwide enzyme market. Here response surface methodology (RSM) was used for the optimization of process variables for α-amylase enzyme production from Trichoderma virens using watermelon rinds (WMR) under solid-state fermentation (SSF). The statistical model included four variables, each detected at two levels, followed by model development with partial purification and characterization of α-amylase. The partially purified α-amylase was characterized with regard to optimum pH, temperature, kinetic constant, and substrate specificity. The results indicated that both pH and moisture content had a significant effect (P 0.05) on α-amylase production (880 U/g) under optimized process conditions at a 3-day incubation time, moisture content of 50%, 30 °C, and pH 6.98. Statistical optimization using RSM showed R

Abstract Fungi cause diverse, serious socio-economic problems, including biodeterioration of valuable products and materials that spawns a biocides industry worth ~$11 billion globally. To help combat environmental fungi that commonly colonise material products, this study tested the hypothesis that combination of an approved fungicide with diverse agents approved by the FDA (Food and Drug Administration) could reveal potent combinatorial activities with promise for fungicidal applications. The strategy to use approved compounds lowers potential development risks for any effective combinations. A high-throughput assay of 1280 FDA-approved compounds was conducted to find those that potentiate the effect of iodopropynyl-butyl-carbamate (IPBC) on the growth of Trichoderma virens; IPBC is one of the two most widely used Biocidal Products Regulations–approved fungicides. From this library, 34 compounds in combination with IPBC strongly inhibited fungal growth. Low-cost compounds that gave the most effective growth inhibition were tested against other environmental fungi that are standard biomarkers for resistance of synthetic materials to fungal colonisation. Trifluoperazine (TFZ) in combination with IPBC enhanced growth inhibition of three of the five test fungi. The antifungal hexetidine (HEX) potentiated IPBC action against two of the test organisms. Testable hypotheses on the mechanisms of these combinatorial actions are discussed. Neither IPBC + TFZ nor IPBC + HEX exhibited a combinatorial effect against mammalian cells. These combinations retained strong fungal growth inhibition properties after incorporation to a polymer matrix (alginate) with potential for fungicide delivery. The study reveals the potential of such approved compounds for novel combinatorial applications in the control of fungal environmental opportunists. Key points • Search with an approved fungicide to find new fungicidal synergies in drug libraries. • New combinations inhibit growth of key environmental fungi on different matrices. • The approach enables a more rapid response to demand for new biocides.

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.

Background Exploration of microbes isolated from north western Himalayas for bioactive natural products. Results A strain of Trichoderma lixii (IIIM-B4) was isolated from Bacopa monnieri L. The ITS based rDNA gene sequence of strain IIIM-B4 displayed 99% sequence similarity with different Trichoderma harzianum species complex. The highest score was displayed for Hypocrea lixii strain FJ462763 followed by H. nigricans strain NBRC31285, Trichoderma lixii strain CBS 110080, T. afroharzianum strain CBS124620 and Trichoderma guizhouense BPI:GJS 08135 respectively. Position of T. lixii (IIIM-B4) in phylogenetic tree suggested separate identity of the strain. Microbial dynamics of T. lixii (IIIM-B4) was investigated for small peptides. Medium to long chain length peptaibols of 11 residue (Group A), 14 residue (Group B) and 17 residue (Group C) were identified using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometer. Optimization is undeniably a desideratum for maximized production of desirable metabolites from microbial strain. Here optimization studies were carried out on T. lixii (IIIM-B4) using different growth media through Intact Cell Mass Spectrometry (ICMS). A multifold increase was obtained in production of 11 residue peptaibols using rose bengal medium. Out of these, one of them named as Tribacopin AV was isolated and sequenced through mass studied. It was found novel as having unique sequence Ac-Gly-Leu-Leu-Leu-Ala-Leu-Pro-Leu-Aib-Val-Gln-OH. It was found to have antifungal activity against Candida albicans (25 μg/mL MIC). Conclusion In this study, we isolated a strain of T. lixii (IIIM-B4) producing medium and long chain peptaibols. One of them named as Tribacopin AV was found novel as having unique sequence Ac-Gly-Leu-Leu-Leu-Ala-Leu-Pro-Leu-Aib-Val-Gln-OH, which had antifungal properties. Electronic supplementary material The online version of this article (10.1186/s12866-019-1477-8) contains supplementary material, which is available to authorized users.

The glycoside hydrolase family 3 β-glucosidase Cel3A from Neurospora crassa (NcCel3A) is highly specific for cellobiose. The crystal structure of NcCel3A has been solved to 2.25 Å resolution. The structure is a dimer and exhibits a high degree of N-glycosylation., The glycoside hydrolase family 3 (GH3) β-glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β-Glucosidases hydrolyse the β-1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an R cryst and R free of 0.18 and 0.22, respectively. NcCel3A is an extensively N-glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β-glucosidase from Rasamsonia emersonii. NcCel3A is a three-domain protein with a number of extended loops that deepen the active-site cleft of the enzyme. These structures characterize this subfamily of GH3 β-glucosidases and account for the high cellobiose specificity of this subfamily.

To expand the range of soluble carbon sources for our enzyme production system, we investigated the properties of sucrose utilization and its effect on cellulase production by Trichoderma reesei M2-1. We performed batch cultivation of T. reesei M2-1 on sucrose and related sugars along with cellobiose, which was used as a cellulase inducer. The results clearly revealed that the hydrolysis products of sucrose, i.e. glucose and fructose, but not sucrose, can be used as a carbon source for enzyme production. In a 10-day continuous feeding experiment using invertase-treated sucrose/cellobiose, the fungal strain produced cellulases with a filter paper-degrading activity of 20.3 U/mL and production efficiency of 254 U/g-carbon sources. These values were comparable with those of glucose/cellobiose feeding (21.2 U/mL and 265 U/g-carbon sources, respectively). Furthermore, the comparison of the specific activities clearly indicated that the compositions of both produced enzymes were similar. Therefore, enzymatically hydrolyzed sucrose can be utilized as an alternative carbon source to glucose in our enzyme production system with T. reesei M2-1.

Trichoderma virens produces viridin/viridiol, heptelidic (koningic) acid, several volatile sesquiterpenes and gliotoxin (Q strains) or gliovirin (P strains). We earlier reported that deletion of the terpene cyclase vir4 and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH, designated as vGPD) associated with the "vir" cluster abrogated the biosynthesis of several volatile sesquiterpene metabolites. Here we show that, the deletion of this GAPDH also impairs the biosynthesis of heptelidic acid (a non-volatile sesquiterpene), viridin (steroid) and gliovirin (non-ribosomal peptide), indicating regulation of non-volatile metabolite biosynthesis by this GAPDH that is associated with a secondary metabolism gene cluster. To gain further insights into the details of this novel form of regulation, we identified the terpene cyclase gene responsible for heptelidic acid biosynthesis (hereafter designated as has1) and prove that the expression of this gene is regulated by vGPD. Interestingly, deletion of has1 impaired biosynthesis of heptelidic acid (HA), viridin and gliovirin, but not of volatile sesquiterpenes. Deletion of the vir cluster associated terpene cyclase gene (vir4), located next to the vGPD gene, did not impair biosynthesis of HA, viridin or gliovirin. We thus unveil a novel circuitry of regulation of secondary metabolism where an HA-tolerant GAPDH isoform (vGPD) regulates HA biosynthesis through the transcriptional regulation of the HA-synthase gene (which is not part of the "vir" cluster). Interestingly, impairment of HA biosynthesis leads to the down-regulation of biosynthesis of other non-volatile secondary metabolites, but not of volatile secondary metabolites. We thus provide evidence that the "vir" cluster associated, HA-tolerant GAPDH in T. virens participates in the biosynthesis of volatile sesquiterpenes as a biosynthetic enzyme, and regulates the production of non-volatile metabolites via regulation of HA biosynthesis. The orthologue of the "vir" cluster in Aspergillus oryzae was earlier reported to synthesize HA by another group. Our study thus proves that the same gene cluster can code for unrelated metabolites in different species.

Malaria remains a worldwide threat, afflicting over 200 million people each year. The emergence of drug resistance against existing therapeutics threatens to destabilize global efforts aimed at controlling Plasmodium spp. parasites, which is expected to leave vast portions of humanity unprotected against the disease. To address this need, systematic testing of a fungal natural product extract library assembled through the University of Oklahoma Citizen Science Soil Collection Program has generated an initial set of bioactive extracts that exhibit potent antiplasmodial activity (EC(50) < 0.30 μg/mL) and low levels of toxicity against human cells (less than 50% reduction in HepG2 growth at 25 μg/mL). Analysis of the two top-performing extracts from Trichoderma sp. and Hypocrea sp. isolates revealed both contained chemically diverse assemblages of putative peptaibol-like compounds that were responsible for their antiplasmodial actions. Purification and structure determination efforts yielded 30 new peptaibols and lipopeptaibols (1–14 and 28–43), along with 22 known metabolites (15–27 and 44–52). While several compounds displayed promising activity profiles, one of the new metabolites, harzianin NPDG I (14), stood out from the others due to its noteworthy potency (EC(50) = 0.10 μM against multi-drug-resistant P. falciparum line Dd2) and absence of gross toxicity toward HepG2 at the highest concentrations tested (HepG2 EC(50) > 25 μM, selectivity index > 250). The unique chemodiversity afforded by these fungal isolates serves to unlock new opportunities for translating peptaibols into a bioactive scaffold worthy of further development.