Plant Cell Wall Changes in Common Wheat Roots as a Result of Their Interaction with Beneficial Fungi of Trichoderma.

Plant cell walls play an important role in shaping the defense strategies of plants. This research demonstrates the influence of two differentiators: the lifestyle and properties of the Trichoderma species on cell wall changes in common wheat seedlings. The methodologies used in this investigation include microscopy observations and immunodetection. In this study was shown that the plant cell wall was altered due to its interaction with Trichoderma. The accumulation of lignins and reorganization of pectin were observed. The immunocytochemistry indicated that low methyl-esterified pectins appeared in intercellular spaces. Moreover, it was found that the arabinogalactan protein epitope JIM14 can play a role in the interaction of wheat roots with both the tested Trichoderma strains. Nevertheless, we postulate that modifications, such as the appearance of lignins, rearrangement of low methyl-esterified pectins, and arabinogalactan proteins due to the interaction with Trichoderma show that tested strains can be potentially used in wheat seedlings protection to pathogens.

Multiple heavy metal tolerance and removal by an earthworm gut fungus Trichoderma brevicompactum QYCD-6.

Fungal bioremediation is a promising approach to remove heavy-metal from contaminated water. Present study examined the ability of an earthworm gut fungus Trichoderma brevicompactum QYCD-6 to tolerate and remove both individual and multi-metals. The minimum inhibitory concentration (MIC) of heavy metals [Cu(II), Cr(VI), Cd(II) and Zn(II)] against the fungus was ranged 150-200 mg L-1 on composite medium, and MIC of Pb(II) was the highest with 1600 mg L-1 on potato dextrose (PD) medium. The Pb(II) presented the highest metal removal rate (97.5%) which mostly dependent on bioaccumulation with 80.0%, and synchronized with max biomass (6.13 g L-1) in PD medium. However, on the composite medium, the highest removal rate was observed for Cu(II) (64.5%). Cellular changes in fungus were reflected by TEM analysis. FTIR and solid-state NMR analyses indicated the involvement of different functional groups (amino, carbonyl, hydroxyl, et al.) in metallic biosorption. These results established that the earthworm-associated T. brevicompactum QYCD-6 was a promising fungus for the remediation of heavy-metal wastewater.

Molecular insights into development of Trichoderma interfusants for multistress tolerance enhancing antagonism against Sclerotium rolfsii Sacc.

The current study aimed at developing diverse Trichoderma fusants for fungicides, drought, and salt tolerance with enhanced antagonistic activity against Sclerotium rolfsii Sacc. Trichoderma virens NBAII Tvs12 (mycoparasitic) and Trichoderma koningii MTCC796 (multistress tolerant) were used as parental strains for development of interspecific protoplast fusants. A total of 36 stable fusants were used for mycoparasitism, fungicides, and abiotic stresses (drought and salt) tolerance. The results revealed 20 homozygous progenies showing characteristics of either one parental strain and 14 heterozygous mutants depicting traits of both parental strains. A novel concept of inhibition coefficient was established using growth-related key parameters that represent the pathogen biology and the biocontrol-related biophysics of Trichoderma fusants. The results indicated a differential inhibition coefficient of the test pathogen and the highest (92.88%) inhibition coefficient of S. rolfsii was observed by interstable fusant Fu21. It also grew better under fungicides and abiotic stress (drought and salt) conditions. The molecular characterization and heterozygosity analysis evidenced the highest observed heterozygosity (0.5441) and gene flow (0.3872) in stable heterozygous Fu21. Principal coordinates analysis exhibited 62.7% of total variability. The ecofriendly heterozygous Trichoderma fusant (Fu21) might be useful for biocontrol of stem rot disease under adverse conditions or as a part of integrated disease management.

Taxonomy characterization and plumbum bioremediation of novel fungi.

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 Pb2+ was 3500 mg L-1 . The fungus was identified as Trichoderma asperellum. The optimum condition for the Pb2+ 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.

Inhibition coefficient and molecular diversity of multi stress tolerant Trichoderma as potential biocontrol agent against Sclerotium rolfsii Sacc.

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.

A novel study based on adaptive metal tolerance behavior in fungi and SEM-EDX analysis.

Four fungal isolates: Simplicillium chinense (iso 9, accession no. KX425621), Penicillium simplicissimum (iso 10, KP713758), Trichoderma asperellum (iso 11, KP792512), and Coriolopsis sp. (1c3, KM403574) were subjected to a series of induced-tolerance training under high metal concentrations to determine if greater tolerance could be achieved from constant exposure to such conditions. Adaptive tolerance assay (Tolerance Index, TI) and Field-Emission Scanning Electron Microscopy with Energy Dispersive X-ray (SEM-EDX) characterized their metal tolerance. "Untrained" S. chinense, P. simplicissimum and T. asperellum showed tolerance towards 4000-4500ppm Al(III) (TI: 0.64-0.71), 1000ppm Cr(III) (0.52-0.83) and Pb(II) (0.32-0.88). With tolerance training, tolerance towards 2000-6000ppm Al(III), 500-3000ppm Pb(II) and 2000-3000ppm Cr(III) were achieved (TI: 0.01-0.82) compared to untrained cultures (0.00-0.59). In contrast, tolerance training for Coriolopsis sp. and P. simplicissimum was less successful, with TI values similar or lower than untrained cultures. SEM-EDX analysis proposed biosorption and bioaccumulation as mechanisms for metal removal. The latter was demonstrated with the removal of Cr(III) and Pb(II) by S. chinense (12.37 and 11.52mgg-1, respectively) and T. asperellum (10.44 and 7.50mgg-1). Induced-tolerance training may render benefit in the long run, but this delicate approach is suggestively species and metal dependent.

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

Ultrastructural features of the early secretory pathway in Trichoderma reesei.

We have systematically analysed the ultrastructure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase-overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24 h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24 h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls; also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFP fusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.

An ethanolamine kinase Eki1 affects radial growth and cell wall integrity in Trichoderma reesei.

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1.82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The functions of eki genes that encode ethanolamine kinase have been intensively studied in mammalian cells, fruit flies and yeast. However, the role of the eki gene has not yet been characterized in filamentous fungi. In this study, Treki1, an ortholog of Saccharomyces cerevisiae EKI1, was identified and functionally characterized using a target gene deletion strategy in Trichoderma reesei. A Treki deletion mutant was less sensitive to cell wall stressors calcofluor white and Congo red and released fewer protoplasts during cell wall digestion than the parent strain QM9414. Further transcription analysis showed that the expression levels of five genes that encode chitin synthases were drastically increased in the ΔTreki1 mutant. The chitin content was also increased in the null mutant of Treki1 comparing to the parent strain. In addition, the ΔTreki1 mutant exhibited defects in radial growth, conidiation and the accumulation of ethanolamine. The results indicate that Treki1 plays a key role in growth and development and in the maintenance of cell wall integrity in T. reesei.

Novel Strategies for Genomic Manipulation of Trichoderma reesei with the Purpose of Strain Engineering.

The state-of-the-art procedure for gene insertions into Trichoderma reesei is a cotransformation of two plasmids, one bearing the gene of interest and the other a marker gene. This procedure yields up to 80% transformation efficiency, but both the number of integrated copies and the loci of insertion are unpredictable. This can lead to tremendous pleiotropic effects. This study describes the development of a novel transformation system for site-directed gene insertion based on auxotrophic markers. For this purpose, we tested the applicability of the genes asl1 (encoding an enzyme of the l-arginine biosynthesis pathway), the hah1 (encoding an enzyme of the l-lysine biosynthesis pathway), and the pyr4 (encoding an enzyme of the uridine biosynthesis pathway). The developed transformation system yields strains with an additional gene at a defined locus that are prototrophic and ostensibly isogenic compared to their parental strain. A positive transformation rate of 100% was achieved due to the developed split-marker system. Additionally, a double-auxotrophic strain that allows multiple genomic manipulations was constructed, which facilitates metabolic engineering purposes in T. reesei. By employing goxA of Aspergillus niger as a reporter system, the influence on the expression of an inserted gene caused by the orientation of the insertion and the transformation strategy used could be demonstrated. Both are important aspects to be considered during strain engineering.

Biodegradation of sulfosulphuron in agricultural soil by Trichoderma sp.

UNLABELLED: Sulfosulphuron-degrading fungus was isolated by enrichment technique from the sulfosulphuron-contaminated soil of wheat rhizosphere. To assess the biodegradation potential of isolated Trichoderma sp., minimal potato dextrose agar broth with different levels of sulfosulphuron (up to 2 g l(-1) ) was evaluated in the growth and biotransformation experiments. ESI LC-MS/MS analysis revealed the presence of degradation products 2-amino-4,6-dimethoxypyrimidine (I) and 2-ethylsulfonyl imidazo{1,2-a} pyridine-3-sulfonamide-2-ethylsulfonyl imidazo{1,2-a} pyridine-3-sulfonamide (II) indicating the cleavage of the urea bridge and the presence of the by-product N-(4,6-dimethoxypyrimidin-2-yl)urea (III) indicating the degradation of sulfonylamide linkage. Two other metabolites, N-(4,6-dimethoxypyrimidin-2-yl)-N'-hydroxyurea (IV) and N, N'-bis(4,6-dimethoxypyrimidin-2-yl)urea (V), were also identified. From the previous reports, it was found that the degradation of sulfonyl urea herbicides took place through the chemical degradation of the sulfonylurea bridge followed by microbial degradation. During this investigation, Trichoderma sp. grew well with and degraded sulfosulphuron via both the decarboxylation on the sulphonyl urea bridge and the hydrolytic cleavage of the sulfonylamide linkage as demonstrated by the formation of metabolites. Trichoderma is nonphytopathogenic in nature, and some species of it restrict the growth of soil-dwelling phytopathogens. Therefore, it is a promising candidate for the decontamination of soil from sulfosulphuron residues. SIGNIFICANCE AND IMPACT OF THE STUDY: The degradation of sulfosulphuron by any individual fungus is being reported for the first time. Trichoderma sp. isolated from wheat-rhizospheric soil could survive in minimal broth rich in sulfosulphuron. Previous reports have described the complete degradation of any sulfonyl urea herbicides by micro-organisms only after the pH-dependent chemical hydrolysis of the sulfonyl urea bridge of the herbicide. This study demonstrates the novel result that the Trichoderma sp. utilized the sulfosulphuron as a sole carbon source and degraded it by cleaving sulfonyl urea bridge and sulfonylamide linkage. Thus, the application of Trichoderma sp., which is nonphytopathogenic, has the potential to decontaminate agricultural soil from sulfosulphuron load.

Bioremediation from wastewater and extracellular synthesis of copper nanoparticles by the fungus Trichoderma koningiopsis.

This is the first study describing the rapid extracellular production of copper nanoparticles by dead biomass of Trichoderma koningiopsis. The production and uptake of copper nanoparticles by dead biomass of Trichoderma koningiopsis were characterized by investigating physicochemical factors, equilibrium concentrations and biosorption kinetics, combined with scanning electron microscopy (SEM), energy dispersive X-ray (EDS), transmission electron microscopy (TEM), and X-ray photoelectron spectroscopy (XPS). A successful route for the metallic copper nanoparticles synthesis was achieved, and followed a Langmuir isotherm where a high biosorption capacity was observed, 21.1 mg g(-1). The kinetic analysis showed that copper biosorption followed a pseudo-second-order model. The nanoparticles mainly exhibited a spherical shape, with an average size of 87.5 nm, and were synthesized extracellularly. The presence of proteins as stabilizing agents of the nanoparticles was demonstrated. The extracellular biosynthesis and uptake of copper nanoparticles using dead fungal biomass is a low-cost green processes, and bioremediation of impacted local.

The effect of gas double-dynamic on mass distribution in solid-state fermentation.

The mass distribution regularity in substrate of solid-state fermentation (SSF) has rarely been reported due to the heterogeneity of solid medium and the lack of suitable instrument and method, which limited the comprehensive analysis and enhancement of the SSF performance. In this work, the distributions of water, biomass, and fermentation product in different medium depths of SSF were determined using near-infrared spectroscopy (NIRS) and the developed models. Based on the mass distribution regularity, the effects of gas double-dynamic on heat transfer, microbial growth and metabolism, and product distribution gradient were systematically investigated. Results indicated that the maximum temperature of substrate and the maximum carbon dioxide evolution rate (CER) were 39.5°C and 2.48mg/(hg) under static aeration solid-state fermentation (SASSF) and 33.9°C and 5.38mg/(hg) under gas double-dynamic solid-state fermentation (GDSSF), respectively, with the environmental temperature for fermentation of 30±1°C. The fermentation production (cellulase activity) ratios of the upper, middle, and lower levels were 1:0.90:0.78 at seventh day under SASSF and 1:0.95:0.89 at fifth day under GDSSF. Therefore, combined with NIRS analysis, gas double-dynamic could effectively strengthen the solid-state fermentation performance due to the enhancement of heat transfer, the stimulation of microbial metabolism and the increase of the homogeneity of fermentation products.

Two new antibiotic pyridones produced by a marine fungus, Trichoderma sp. strain MF106.

Two unusual pyridones, trichodin A (1) and trichodin B (2), together with the known compound, pyridoxatin (3), were extracted from mycelia and culture broth of the marine fungus, Trichoderma sp. strain MF106 isolated from the Greenland Seas. The structures of the new compounds were characterized as an intramolecular cyclization of a pyridine basic backbone with a phenyl group. The structure and relative configuration of the new compounds were established by spectroscopic means. The new compound 1 and the known compound 3 showed antibiotic activities against the clinically relevant microorganism, Staphylococcus epidermidis, with IC50 values of 24 µM and 4 µM, respectively.

Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation.

Effect of fungi and light on seed germination of three Opuntia species from semiarid lands of central Mexico.

Fungal attack under light reduces mechanical resistance of the testa of Opuntia seeds, making it easier for the embryo to emerge. However, the effect of fungi on Opuntia seed germination in darkness is unknown. We evaluated the combined effects of light and inoculation with Phoma medicaginis, Trichoderma harzianum, Trichoderma koningii, and Penicillium chrysogenum on germination of O. streptacantha, O. leucotricha, and O. robusta seeds, from central Mexico. We also evaluated the combined effects of seed age (2-, 3-, and 12-year-old seeds) and presence of fungi on the testa on O. streptacantha germination. All fungal species eroded the funicular envelope and promoted seed germination for O. leucotricha and O. streptacantha, but did more so in light than in darkness. For the latter species, younger seeds inoculated with fungi had lower germination than older ones. For O. robusta, we found that seeds inoculated with P. medicaginis and T. harzianum had similar germination in light and in darkness. Our results strongly indicate that deterioration of the testa by fungi is higher in light than in darkness.

Dissecting and reconstructing synergism: in situ visualization of cooperativity among cellulases.

Cellulose is the most abundant biopolymer and a major reservoir of fixed carbon on earth. Comprehension of the elusive mechanism of its enzymatic degradation represents a fundamental problem at the interface of biology, biotechnology, and materials science. The interdependence of cellulose disintegration and hydrolysis and the synergistic interplay among cellulases is yet poorly understood. Here we report evidence from in situ atomic force microscopy (AFM) that delineates degradation of a polymorphic cellulose substrate as a dynamic cycle of alternating exposure and removal of crystalline fibers. Direct observation shows that chain-end-cleaving cellobiohydrolases (CBH I, CBH II) and an internally chain-cleaving endoglucanase (EG), the major components of cellulase systems, take on distinct roles: EG and CBH II make the cellulose surface accessible for CBH I by removing amorphous-unordered substrate areas, thus exposing otherwise embedded crystalline-ordered nanofibrils of the cellulose. Subsequently, these fibrils are degraded efficiently by CBH I, thereby uncovering new amorphous areas. Without prior action of EG and CBH II, CBH I was poorly active on the cellulosic substrate. This leads to the conclusion that synergism among cellulases is morphology-dependent and governed by the cooperativity between enzymes degrading amorphous regions and those targeting primarily crystalline regions. The surface-disrupting activity of cellulases therefore strongly depends on mesoscopic structural features of the substrate: size and packing of crystalline fibers are key determinants of the overall efficiency of cellulose degradation.

Hypocrea britdaniae and H. foliicola: two remarkable new European species.

Two new species of Hypocrea are added here to the European funga. Hypocrea britdaniae, a fungus with unknown anamorph and large, conspicuous stromata resembling basidiomata of a corticiaceous fungus, is a sister species to the Longibrachiatum clade, while H. foliicola, a leaf-dwelling species that forms pulvinate stromata, is recognized as an additional member of the pachybasium core group. Hypocrea foliicola sporulates in culture in a reduced verticillium-like manner, while it produces a white, typical pachybasium-like anamorph in nature. Ecologically H. foliicola is remarkable in inhabiting leaves, a substrate rarely recorded for Hypocrea. All relevant morphological teleomorphic and anamorphic traits are given. The phylogenetic placement of the new species within Hypocrea/Trichoderma was determined with combined analyses of rpb2 and tef1 exon sequences.

Trichoderma matsushimae and T. aeroaquaticum: two aero-aquatic species with Pseudaegerita-like propagules.

Four isolates tentatively identified as Pseudaegerita matsushimae on the basis of the morphology of bulbil-like propagules were collected from substrates submerged in water in Thailand and Japan. In culture studies the two Thai isolates were found to produce phialoconidia on conidiogenous cells and phialoconidiophores whose morphology was similar to that of Trichoderma. Phylogenetic analysis based on D1/D2 regions of LSU rDNA sequences showed that the four isolates were nested in Hypocrea/Trichoderma (Hypocreales) while P. corticalis, the type species of Pseudaegerita, belongs to Hyaloscypha (Helotiales). Preliminary analysis by ISTH Web tools based on 5.8S-ITS rDNA and phylogenetic analysis based on rpb2 and tef1-int4 genes showed that the isolates have specific sequences of Trichoderma (anchors 1-5) and belong to the Hamatum clade but they grouped apart from any known species of Trichoderma. The sequences of the tef1-int4 gene, which were amplified from the authentic specimen of P. matsushimae (IMI 266915), also showed that it belongs to the Hamatum clade closely clustering with T. yunnanense but separate from our four isolates. The morphology of P. matsushimae (IMI 266915), especially the sizes of phialides and phialoconidia, were different from T. yunnanense. Thus, we conclude that IMI 266915 and our isolates are to be assigned to two different species in the Hamatum clade of Trichoderma, although both species have similar morphology of bulbils and phialoconidia. Morphology and molecular data revealed that P. matsushimae should be assigned to the genus Trichoderma as T. matsushimae and the Thai and Japanese isolates are placed in T. aeroaquaticum sp. nov. This finding supports the interpretation that aero-aquatic fungi have evolved from terrestrial fungi. We assume that these fungi probably were derived from typically soil-inhabiting species of Trichoderma; an adaptation to aquatic environments is shown by formation of bulbil-like propagules floating on water.

Hyphal surface architecture and cell morphology of Trichoderma reesei.

The filamentous fungus Trichoderma reesei which is known to secrete high amounts of cellulolytic enzymes was found to produce a massive amount of fibrous material at the outer surface of the cell wall as observed by ultrahigh-resolution low-voltage scanning electron microscopy. Using transmission electron microscopy, the cell wall ornamentation of the hyper-cellulosic mutant PC-3-7 was found to be less massive and much thinner than for QM9414. A significant amount of fibrous material was produced in Avicel-grown cultures that were less abundant in glucose-grown cultures and Avicel was occasionally found entangled within the cell wall-associated fibrous layer.