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In this work, corn stover was studied as a lignocellulosic material with the potential of serving as a feedstock in the field of bioenergetics. To improve the enzymatic digestibility of carbohydrates in the lignocellulosic biomass, corn stover was pretreated using Myrothecium verrucaria to deplete the lignin, combined with a solid acid pretreatment method to deplete the hemicellulose. The effects of lignin degradation conditions and solid acid concentration on cellulose conversion were investigated. The two-stage pretreatment with M. verrucaria and solid acid under optimal conditions gave 81.54% cellulose conversion yield, 214.22% higher than that of untreated corn stover. Significant lignin and hemicellulose removal rates were observed with the two-stage pretreatment. Scanning electron microscope (SEM), X-ray diffractometer (XRD) and Fourier transform infrared spectroscopy (FTIR) analyses showed major structural changes in the pretreated corn stover. This study provided an efficient strategy to enhance the enzymatic saccharification of cellulose in corn stover.

The widespread use of synthetic nematicides has caused significant problems to the environment as well as human health. To address this issue, eco-friendly control measures, such as microbial nematicides, are being developed. During the screening of Myrothecium strains with nematicidal activity against the root-knot nematode (RKN) Meloidogyne incognita, we found that the acetone extract of Myrothecium sp. KACC 40321 was highly effective against hatched juveniles of M. incognita at 7 days after exposure. The fungus was identified as Meloidogyne verrucaria. Two macrocyclic trichothecenes verrucarin A and roridin A were isolated and identified as major active metabolites by bioassay-guided fractionation and instrumental analysis. When the second-stage juveniles were treated with the chemicals, no juvenile mortality was observed. However, they effectively killed juveniles from treated eggs. The hatched juvenile mortality was used to evaluate the in vitro nematicidal activity of the compounds against M. incognita. The median effective concentrations were 1.88 μg/mL for verrucarin A and 1.50 μg/mL for roridin A. Among various liquid media, commercial malt extract broth (cMEB) was found to be the best for the production of verrucarin A and roridin A, followed by potato dextrose broth. The cMEB culture filtrate effectively reduced the formation of galls and egg masses on tomato roots in a pot experiment. In addition, the culture filtrate reduced the formation of galls on the roots of melon plants and the number of RKNs in the soils under field conditions. These results suggest that M. verrucaria KACC 40321 can be used as a biocontrol agent against RKNs in various crops. To the best of our knowledge, this is the first study to report the effectiveness of verrucarin A and rorridin A against hatched juveniles of M. incognita.

A potential pulse-assisted approach was used to immobilize Myrothecium verrucaria bilirubin oxidase at planar and nanoporous gold electrodes (NPG) containing pores of ca. 20 nm and ca. 40 nm in diameter. An increase in the current due to the bioelectrocatalytic reduction of oxygen by MvBOD-modified gold electrodes obtained from a 20 μL drop by the proposed pulse-assisted approach was observed when compared to the response obtained with electrodes modified by drop-casting. This increase likely arises from a preferential orientation of MvBOD molecules at the planar gold surface obtained by fast switching of the potential pulses between opposite charges. The concomitant ion stirring effect induces the attraction of the enzymes to the charged gold surface and forces access to the internal pore volume of the NPG. Immobilization of MvBOD using the potential pulse-assisted approach significantly increases current densities by facilitating the electron transfer between the enzyme and the electrode surface.

Myco-valorization (Myco-Valtn) concept was explored using Myrothecium verrucaria ITCC-8447 via column bioreactor to remove two anthraquinone (ARq) dyes. Among the three myco-valtn approaches free cell dependent enabled more than 80% removal of Anthraquinone violet R (AVR) and Alizarin cyanine green (ACG) after 48 h at 30 ± 2 °C. Enzymatic treatment removed more than 50% AVR and ACG after 10 min. However, synthetic and natural support entrapment on Scotch-Brite® (SB) and Luffa cylindrica (LC) enabled more than 80% removal in SB-ACG and LC-ACG and more than 60% in SB-AVR and LC-AVR in 6th cycle. The regenerated column enhanced the removal of dye and allowed the column to run for multiple cycles. Further after FTIR XRD and LCMS Q-TOF, analysis it was observed that ACG and AVR were completely degraded and laccase played the key roles in the degradation of ACG and AVR and the degraded metabolites of anthraquinone dyes (ACG and AVR) exhibited much less toxicity than the parent dye using phytotoxicity assays.

In the present work attempts have been made to mimic the natural system of biomass delignification using laccase from Myrothecium verrucaria ITCC-8447 and synergistic effect of cellulase and xylanase from Schizophyllum commune NAIMCC-F-03379 and Aspergillus oryzae ITCC-8571 respectively for enhanced saccharification and subsequent bioethanol production. The laccase mediated pretreatment resulted in 6.7% delignification of rice straw (RS). The structural analysis of enzymatic pretreated RS using XRD and FTIR suggested high crystallinity index (CrI) and improved accessibility of cellulose and hemicelluloses as compared to untreated RS. The single step simultaneous delignification, saccharification and fermentation (SDSF) of RS using partially purified in-house enzyme cocktail with Saccharomyces cerevisiae MTCC-173 resulted in sugar yield of 26.7 ± 0.2 g/L with an experimental ethanol yield of 6.47 ± 0.16 g/L which is comparable to maximum sugar and ethanol yield of 31.2 ± 0.7 g/L and 7.34 ± 0.39 g/L respectively using commercial enzyme cocktail with S. cerevisiae MTCC-173. The SDSF was obtained as a suitable single step process for the ethanol production from RS.

Multicopper oxidases (MCOs) has a unique feature of having the presence of four Cu atoms arranged into three (Type I, II and III) spectral classification. MCOs laccase due to its broad range of substrate specificity has numerous biotechnological applications. The two types of laccases include the typical blue and the atypical white, yellow laccases which have been isolated from diverse geographical locations globally. In the present study laccases were identified using Liquid Chromatograph Mass Spectrometer Studies (LCMS) study where blue laccase exhibited homology with Trametes villosa Q99044 and Q99046 and white, yellow laccase exhibited homology with Myrothecium verrucaria OX = 1859699; Q12737 and Trametes versicolor Q12717 respectively. The spectral comparison between laccases were determined via spectroscopic analysis where UV-spectra of blue laccase from Trametes versicolor had a peak at 605 nm (Type I Cu atom) whereas in case of white and yellow laccases the peak was absent and in addition had an absorption peak at 400nm. It was followed by X-Ray Diffraction (XRD) analysis of proteins where α-helix (10°) and β-sheet (22°) structure were observed in case of all the three laccases. However, the intensity of α-helix in white and yellow laccase was stronger as compared to the blue laccase whereas the intensity of β-sheet was stronger in case of blue laccase as compared to other two laccases. Further, Fourier-transform infrared spectroscopy (FTIR) analysis was performed which enabled the analysis of proteins where α-helix (1650–1658 cm−1), β-sheets (1620–1640 cm−1), amide I (1700−1600 cm−1) amide II (bands at under 1400 cm−1) and amide A, B (bands above 3000 cm−1)., Bioinformatics; Microbiology; Structural biology; Mycology; Microorganism; Enzymology; Proteomics; Spectroscopy; Laccase; LCMS; UV-Spectra; XRD; FTIR

Options for control of northern root-knot nematode (NRKN, Meloidogyne hapla ) on vegetables are very limited currently. In this study, we characterized the parasitism of Myrothecium verrucaria strain X-16, a new nematophagous fungus, on NRKN at the stages of eggs, J2, and adult females and evaluated its biocontrol efficacy in the greenhouse. Strain X-16 produced conidia that geminated and invaded in 80 h after in contact with eggs, causing the shrinkage and depression of egg shell and blastocolysis of the embryo. The strain also attacked 2nd-stage juveniles by producing developing surface networks of hyphae on the nematode body wall. Strain X-16 attacked adult females by producing dense networks of hyphae on the nematode body wall in 120 h. Strain X-16 had lethal effects (22–71% mortality) against NRKN J2 at the concentration as low as 3.1 × 10 7 conidia/ml and with the incubation treatment time as short as 24 h. The lethal effects linearly increased with the increase of conidial concentration, with the estimated LC 50 values as low as 1.0 × 10 8 conidia/ml. Soil treatments with strain X-16 at 1%, 2% or 4% (wt/wt) induced significant reductions in J2 nematode counts in 100 g of dry soil, Pf/Pi ratios and root-know index in cucumber in the greenhouse evaluations. These studies are the first to demonstrate that M . verrucaria is able to parasitize NRKN and strain X-16 can be a potential biocontrol agent for management of NRKN.

Here we present the very first study of an enzymatic fuel cell (EFC) in a cell culture. An EFC with Corynascus thermophilus cellobiose dehydrogenase (CDH) based bioanode and Myrothecium verrucaria bilirubin oxidase (BOx) based biocathode was constructed at the bottom of a medusa cell culture plate. The constructed EFC had a power density of up to 25 μW cm(-2) at 0.5 V potential in simple buffer solution and in cell culturing medium. L929 murine fibroblast cells were seeded on top of the EFC and possible effects of the EFC on the cells and vice versa were studied. It was shown that on average the power of the EFC drops by about 70% under a nearly confluent layer of cells. The EFC appeared to have a toxic effect on the L929 cell line. It was concluded that the bioanode, consisting of CDH, produced hydrogen peroxide at toxic concentrations. However, the toxic effect was circumvented by co-immobilizing catalase on the bioanode.

6 pages; International audience; A new laccase from the filamentous fungus Podospora anserina has been isolated and identified. The 73 kDa protein containing 4 coppers, truncated from its first 31 amino acids, was successfully overexpressed in Pichia pastoris and purified in one step with a yield of 48% and a specific activity of 644 U mg-1. The kinetic parameters, kcat and KM, determined at 37 -C and optimal pH are 1372 s-1 and 307 M for ABTS and, 1.29 s-1 and 10.9 lM, for syringaldazine (SGZ). Unlike other laccases, the new protein displays a better thermostability, with a half life > 400 min at 37 °C, is less sensitive to chloride and more stable at pH 7. Even though, the new 566 amino-acid enzyme displays a large homology with Bilirubin oxidase (BOD) from Myrothecium verrucaria (58%) and exhibits the four histidine rich domains consensus sequences of BODs, the new enzyme is not able to oxidize neither conjugated nor unconjugated bilirubin.

The enzymatic and electrochemical catalytic properties of a novel white laccase from Myrothecium verrucaria NF-05 were evaluated and compared with those of commercial oxidoreductases. NF-05 laccase effectively catalyzed the oxidation of several phenols and amines, indicating its wide substrate specificity. The cyclic voltammetry curves showed direct electron transfer on carbonous electrodes due to the direct absorption of NF-05 laccase. Peroxide was bioelectrochemically reduced more efficiently on NF-05 laccase-modified electrodes than on electrodes that were modified with commercial enzymes. The results demonstrate the potential applications of this enzyme in environmental bioremediation of aromatic pollutants and preparation of electronic devices.

Myrothecium verrucaria 3.2190 is a nonligninolytic fungus that produces bilirubin oxidase. Both M. verrucaria and the extracellular bilirubin oxidase were tested for their ability to decolorize indigo carmine. The biosorption and biodegradation of the dye were detected during the process of decolorization; more than 98% decolorization efficiency was achieved after 7 days at 26°C. Additionally, the crude bilirubin oxidase can efficiently decolorize indigo carmine at 30°C~50°C, pH 5.5~9.5 with dye concentrations of 50 mg l(-1)~200 mg l(-1). Bilirubin oxidase was purified and visualized as a single band on native polyacrylamide gel electrophoresis (PAGE). Several enzymatic properties of the purified enzyme were investigated. Moreover, the identity of the purified bilirubin oxidase (BOD) was confirmed by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). These results demonstrate that the purified bilirubin oxidase in M. verrucaria strain has potential application in dye effluent decolorization.

Herbicides and spray adjuvants were evaluated for compatibility with the bioherbicidal fungus, Myrothecium verrucaria. Several commercial formulations of glyphosate were found to be compatible for tank mixing with M. verrucaria, including Touchdown and RoundUp HiTech. Others, such as Accord XRT II and RoundUp WeatherMAX killed all the spores of M. verrucaria immediately after mixing at only 10% the maximum labeled application rate. Many herbicides, which were not suitable for co-application with M. verrucaria, did not inhibit the growth of the fungus when added directly to media at up to 1% concentration, indicating that these products could be compatible with M. verrucaria as sequential applications in an integrated weed management system. Several commercially available spray adjuvants and polyoxyethylene tridecyl ether (TDA) formulations were tested in vitro for their efficiency in dispersing spores and in a plant bioassay for bioherbicidal activity. All of the products improved the activity of M. verrucaria over the water-only treatments and TDA formulations with a hydrophilic–lipophilic balance (HLB) number of 8 or 10 had the highest activity. The mechanism for improved bioherbicidal activity with these adjuvants was investigated in vitro, and TDA HLB 8 and 10 did not significantly improve conidia dispersal or accelerate spore germination relative to other surfactants. It is possible that the role of the surfactant is in the alteration of the plant cuticle or otherwise preparing the infection court. Better adjuvant selection and integration with affordable synthetic herbicides should aid in the development of more cost-effective biological control of weeds.

Met467, the axial ligand to type I Cu in a multicopper oxidase, Myrothecium verrucaria bilirubin oxidase was substituted with a non-coordinating Phe and Leu to transform the spectral and magnetic properties and oxidase activities of the enzyme into those of fungal laccases, but the mutated type I Cu center showed properties characteristic of phytocyanins, blue copper proteins with an axial coordination of Gln, due to compensatory binding of the distal Asn459 as evidenced by a double mutation.

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was >650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.

Most studies of cellulose hydrolysis have been carried out on three components of the cellulolytic systems, viz, endoglucanases, exoglucanases, and cellobiases. Little attention has been paid to the fragmentation activity of certain cellulolytic systems. We have noticed that despite being a more powerful degrader of modified cellulose (CMC), the 7-day grown culture filtrate of Myrothecium verrucaria was less effective than that of Trichoderma reesei at degrading pure unmodified cellulose. Scanning electron microscopy imaging showed that one distinguishing feature of the latter is its ability to fragment (macerate) the cellulose. Cellulose particle size decreased with time as it was incubated in the culture filtrate of T. reesei at 37 °C. This was used as a pre-treatment. Pre-treated cellulose was then washed and incubated with fresh T. reesei or M. verrucaria culture filtrates. Pre-treatment increased liberation of reducing sugars during subsequent incubation of cellulose in T. reesei culture filtrate but not in subsequent incubation in M. verrucaria culture filtrate. It was hypothesized that fragmentation activity of the pre-treatment opened up attack sites for further hydrolysis, but these were not available for attack by other enzyme systems.

Galactomyces geotrichum and Myrothecium verrucaria were grown on rye straw and phenol oxidase activity (peroxidase, laccase and tyrosinase) and after 20, 30 and 60 days lignin degradation was investigated. The amount of lignin in rye straw was reduced more by G. geotrichum than M. verrucaria (48% vs. 42% relative to controls). For the former, the greatest peroxidase activity was recorded after 60 days and tyrosinase after 30 days.

A series of fungal laccases (Polyporus pinsitus, Rhizoctonia solani, Myceliophthora thermophila, Scytalidium thermophilum) and one bilirubin oxidase (Myrothecium verrucaria) have been studied to determine their redox potential, specificity, and stability. Polyporus and Rhizoctonia laccases possess potentials near 0.7-0.8 V (vs. NHE), while other oxidases have potentials near 0.5 V. It is observed that higher redox potential correlates with higher activity. By EPR, no significant change in the geometry of type 1 copper (II) site is observed over this series. At the optimal pH, the two substrates studied, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine, show Km values ranging form 10 to 120 and from 1 to 45 microM; and kcat values ranging from 50 to 16 000 and 200 to 3000 per min, respectively. The enzymes are more stable in the neutral-alkaline pH range. The thermal stability is in the order of bilirubin oxidase equivalent to Myceliophthora laccase equivalent to Scytalidium laccase > Polyporus laccase > Rhizoctonia laccase. Based on these results and the sequence alignments made against Zucchini ascorbate oxidase it is speculated that structural differences in the substrate-activation site (a 'blue', type 1 copper center) control the redox potential range as well as substrate specificity, and the cystine content contributes to stability.

Myrothecium verrucaria bilirubin oxidase (EC 1.3.3.5) is an enzyme catalyzing the oxidation of bilirubin to biliverdin and other substrates. We have purified bilirubin oxidase from the medium of M. verrucaria and determined its partial amino acid sequence and isolated cDNA fragment amplified by polymerase chain reaction using oligonucleotide primers designed on the basis of the partial amino acid sequence. The gene for bilirubin oxidase has been cloned from a genomic library using the cDNA fragment as a probe. The gene encodes a precursor of bilirubin oxidase consisting of 572 amino acid residues, which comprises the prepro-region of 38 amino acid residues and the mature enzyme of 534 amino acid residues containing one cysteine. Five introns were found within the coding region. Sequence comparison of bilirubin oxidase with other blue copper proteins (laccase, ascorbate oxidase, human ceruloplasmin, plastocyanin, and azurin) revealed the presence of four domains corresponding to potential copper ligands. We have expressed this bilirubin oxidase gene in Saccharomyces cerevisiae under the repressible acid phosphatase promotor and found an active recombinant bilirubin oxidase, establishing the functional identity of the gene.

A low-molecular-weight endo-β-1,4- d -xylanase (endo-β-1,4- d -xylan xylanohydrolase; E.C. 3.2.1.8) isolated from solid-state cultures of Myrothecium verrucaria has M r and pI values of 15,900 and 4.35, respectively, and a carbohydrate content of 11% (w/w). The enzyme is most active at pH 5.5 and 45°C, and has a half-life of 16 min at pH 5, 50°C. It catalyzes the hydrolysis of various β-1,4-linked and mixed (1,3; 1,4)-linked β-glucans, but kinetic parameters showed it to be primarily a xylanase. Inhibition studies suggested the possible involvement of arginine, cysteine, histidine, tryptophan, and a carboxylate(s) in binding or catalysis. The pattern of products of hydrolysis of various xylans and of xylopentaose clearly demonstrated the purified enzyme to be an endo-β-1,4-xylan xylanohydrolase (E.C. 3.2.1.8).

Soil in two Persea americana orchards infested with Phytophthora cinnamomi was fumigated with methyl bromide (mb) and subsequently infested with Myrothecium verrucaria or Streptomyces sp. to attempt to prevent the reinvasion of the treated soil by the pathogen. Chemical measurements of the soil atmosphere during fumigation indicated that ED90 doses for P. cinnamomi were attained in most areas. Natural reinfestation by P. cinnamomi was facilitated by planting highly susceptible Persea indica along the outside upper border of the plots. After fumigation, and up to 3 months later, P. cinnamomi was not recovered from the treated areas. Streptomyces spp. and M. verrucaria were successfully introduced into the soils and populations remained high for approximately 6 months, but declined thereafter. Infections of dense plantings of P. indica seedlings in the plots, approximately 9 months after the organisms had been added, showed that P. dnnamomi had been re-established into the treated plots. The rate of infestation was slower in the treated plots, but eventually disease incidence was as great as in the nontreated plots. After 8.5 months cotton waste and fertilizers were added to the plots; Streptomyces spp. increased after adding the amendments, but M. verrucaria did not.

Fourteen n-alkanols (C1-C12, C14, and C16), 13 α,ω-n-alkanediols (C2-C12, C14, and C16), and 13 ω-chloro-α-alkanols (C2-C12, C14, and C16) were tested against Aspergillus niger, Trichoderma viride, and Myrothecium verrucaria in Sabouraud dextrose agar at pH 4.0 and 5.6. Toxicity to Candida albicans, Trichophyton mentagrophytes, and Mucor mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. The fungitoxicity of these alcohols was influenced by chain length and insignificantly by the pH of the medium and the presence of beef serum. The C10-member of each series was most active; the order of activity of the three groups was chloroalkanols > alkanols > alkanediols. Compared to the fatty acids, the order of fungitoxicity on a weight basis was 2-alkynoic acids > 2-alkenoic acids > ω-chloroalkanols > alkanoic acids > 2-bromoalkanoic acids > 2-fluoroalkanoic acids > n-alkoxyacetic acids > n-alkanols > α,ω-n-alkanediols.

Crude lytic enzymes (predominantly β-(1–3)-glucanases from Oerskovia xanthineolytica, Basidiomycete QM 806 and Rhizopus arrhizus QM 1032 were tested against the walls of log-phase Saccharomyces cerevisiae and germinated spores of Aspergillus aculeatus, Aureobasidium pullulans, Myrothecium verrucaria, Rhizopus arrhizus and Trichoderma viride. Release of reducing sugars and formation of spheroplasts from viable cells were used as criteria to evaluate enzymatic activity. The enzyme preparation from Oerskovia was more active than the other two enzymes for formation of spheroplasts and release of reducing sugars from Au. pullulans, M. verrucaria, S. cerevisiae and T. viride. The basidiomycetous enzyme was also able to attack these viable cells and form spheroplasts, but the rate of release of reducing sugars and the extent of spheroplast formation was lower than for the Oerskovia enzyme. The enzyme from R. arrhizus did not attack viable cells extensively or release spheroplasts from the organisms tested. All three enzyme preparations released reducing sugars from Asp. aculeatus without the formation of spheroplasts. R. arrhizus was resistant to treatment by these enzymes. The crude Oerskovia enzyme exhibited an endo-splitting action pattern against laminarin and lichenin. The ratio of cell lytic activity to laminarinase activity was higher for the Oerskovia preparation than for the preparations from the other two organisms. The basidiomycete enzyme showed a predominantly exo-splitting action pattern against laminarin and did not attack lichenin to a great extent, but was capable of yeast wall lysis; The enzyme from R. arrhizus was predominantly endo-splitting against laminarin and lichenin, and thus similar to the Oerskovia enzyme. However, it showed no action against viable cells. Chitinase assisted the Oerskovia and basidiomycete enzyme preparations in the release of reducing sugars from Asp. aculeatus, M. verrucaria and T. viride. Chitosanase showed the greatest action towards R. arrhizus.

Summary Myrothecium verrucaria , a cellulolytic fungal strain, was tested for cell protein production in presence of different carbon and nitrogen sources. Cellulose powder, followed by paper cellulose, proved to be the best carbon source, while agricultural waste products were comparatively less suitable for fungal growth. Substituting (NH 4 ) 3 PO 4 in the culture medium with organic nitrogen sources improved protein production; corn steep liquor was the most suitable nitrogen source. Ammoniacal-nitrogen was more favourable for protein synthesis than NO 3 −N. Fungal growth and protein yield were markedly affected by the p H value of the culture medium; p H 5.0–5.5 proved to be the optimum p H level. Eight amino acids could be detected in the hydrolysed fungal protein, none of which contained sulphur.

The fungicide Dithane M-45 when applied to the soil was regularly and significantly toxic to the total count of fungi at all doses used (4·48, 22·4 and 44·8 mg active ingredient per kg dry soil). The toxicity was regularly increased with dose and persisted till the end of the experiment (15 weeks). When Dithane M-45 was incorporated into the agar medium all test fungi were eliminated by the high dose (24·0 ppm). The low and medium doses were tolerated by some fungi. The decay of filter paper buried in the fungicide-treated soil was significantly reduced by the high dose after 1 and 3 weeks of incubation, while the decay of filter paper treated with this fungicide and buried in untreated soil was significantly retarded by the high dose only after all experimental periods of incubation (up to 15 weeks). The growth and sporulation of nine test fungi were eliminated by the three doses of Dithane M-45 except Chaetomium globosum, Fusarium moniliforme and Trichoderma viride which could grow restrictedly at the low dose only. Production of endo-1,4β- d -glucanase and mycelial dry weight were not significantly affected by the three doses in the case of Aspergillus niger, and by the low dose in the case of Alternaria alternata, Fusarium solani and Myrothecium verrucaria. In the case of other test fungi and other doses, endo-1,4β-glucanase production and mecelial dry weight were significantly inhibited.

Nine macrocyclic trichothecenes, thought to cause toxic effects in herbivors, were isolated from the Brazilian shrub Baccharis coridifolia. The structures of two new components, named miophytocene A and B, are described. Structure analysis was mainly accomplished by application of two-dimensional Fourier transform NMR techniques. The trichothecenes are most likely produced by the soil fungus Myrothecium verrucaria, which was isolated from soil samples around B. coridifolia. It is concluded that the plant is able to absorb and accumulate these mycotoxins. Histopathological studies on rabbits which received roridin E and A per os revealed mainly necrosis and hemorrhages of the mucosa of the colon and in the case of roridin A also of the caecum. Lethality in mice was generally low, intoxication proceeding slowly. It has to be considered, however, that all trichothecenes tested are practically insoluble in aqueous solutions and were applied as emulsions (i.p. and per os application). No trichothecenes or other toxic substances were detected from the Brazilian species Baccharis dracunculifolia, although slight toxic effects have been reported in cattle.

The effect of radiation pretreatment of agricultural cellulosic wastes was investigated through hydrolytic reactions of cellulose. Gamma irradiation significantly increased the acid hydrolysis of rice straw, rice hull and corn husk. The yields of reducing sugar were higher with increasing radiation dose in these materials. The observed radiation effect varied with the cellulosic material but it correlated with neither the cellulose content nor the lignin content. Likewise, the radiation pretreatment accelerated the subsequent enzymatic hydrolysis of rice straw and rice hull by cellulase. The irradiated rice straw appeared to be a better growth medium for the cellulolytic microorganism, Myrothecium verrucaria , than the non-irradiated material. This was attributed to increased digestibility of the cellulose by the microorganism.

A method for the estimation of erythorbic acid in the presence of ascorbic acid utilizing the unique properties of the spore ascorbase of Myrothecium verrucaria has been described. Measurements were made during partially inhibited enzyme oxidations of ratios from 10:1 to 300:1 of the substrate (ascorbic acid) to the enzyme inhibitor (erythorbic acid). Chemical analyses for reduced and oxidized ascorbic acid followed the manometric measurements. Regression lines of the amount of reduced or oxidized ascorbic acid on the logarithm of the corresponding ratio of ascorbic acid to erythorbic acid were developed from the three sets of data and were used satisfactorily for the estimation of the quantity of erythorbic acid present in “unknown” synthetic solutions. Trial measurements were made of erythorbic acid in blood and urine following test loads. Adjustments in the method must be made before definitive results can be obtained with the small amounts of the acids present in plasma. The presence of erythorbic acid was established qualitatively in urine by the method but quantitative relationships were not clearly defined due to limited data and great variation in excretion of the test load.

Cellotriose, cellotetraose, cellopentaose and cellohexaose were prepared by acetolyzing cellulose and fractionating the deacetylated products on a charcoal column. A procedure is described for chromatographic separation and analysis of hydrolyzates of these sugars. Cellobiose and the above sugars, at various initial concentrations, were hydrolyzed at constant enzyme concentration by purified Myrothecium verrucaria cellulase and the hydrolyzates analyzed at various time intervals. For the conditions tested, the turnover numbers of the enzyme were calculated to be approximately as follows: cellobiose, 5–6; cellotriose, increasing directly with initial concentration from 50 to 200; cellotetraose, 400; cellopentaose and cellohexaose, at least 450. The process of chain-splitting appeared to become more random as the chain length increased. The results are discussed.

Extracts from spores and mycelium of Myrothecium verrucaria contain an oxalic acid decarboxylase similar to the one Shimazono found in a wood-rotting fungus. The enzyme is highly specific for oxalic acid having no activity on 13 other carboxylic acids including formic acid. The stoichiometry approached 1 mole of oxalic acid per mole of formic acid and carbon dioxide. However, the carbon dioxide yield was about 10% low and there was an average of 0.85 μmole of oxygen consumed per 20 μmoles of oxalic acid decomposed particularly in the later stages of the reaction. The enzyme was sensitive to several heavy metal and sulfhydryl inhibitors. It was inactive under anaerobic conditions and was inhibited by certain reducing agents. The inhibition was reversed by oxygen but not by other electron acceptors. It is suggested that reductive cleavage of certain disulfide bonds inactivates the enzyme and that this process is reversible.

Muconomycin A (M-A) and muconomycin B (M-B), two nonnitrogenous antibiotics derived from culture broths of Myrothecium verrucaria, possess unique structural features and high mammalian toxicity. The ld 50 in mice (i.p.) was found to be 0.50 to 0.75 mg/kg for both antibiotics. M-A induced a severe creatinuria in rats, and this effect was attenuated by pre-administration of vitamin E. Both antibiotics were found to inhibit creatine phosphokinase (CPK) in vitro; I50 values were 29.8 × 10−4 M for M-A and 7.0 × 10−4 M for M-B. CPK was then assayed, at various concentrations of substrate, with M-A. A Hunter-Downs plot of the data showed that M-A was a noncompetitive inhibitor of CPK; the Ki was 3.5 × 10−4 M. It is suggested that the high toxicity of these antibiotics, as well as their ability to cause creatinuria, may be related to their inhibitory effect on CPK.

Summary 1. Initially the growth of Myrothecium verrucaria was found to be inhibited through cyanamide. A concentration of 10-4 M of cyanamide caused a slight inhibition. 2. The addition of cyanamide to the culture medium brought about an induction of a cyanamide hydratase. This enabled the fungi to grow, with cyanamide as the only source of nitrogen. Urea was formed first, and then decomposed into ammonia and carbon dioxide. 3. Cyanamide hydratase was isolated from the fungi mycelium and concentrated approximately seventy times. 4. The characteristics of cyanamide hydratase were examined, i. e., to show that the reaction NH = C = NH + H2O → (NH2)2CO could be proved quantitatively, and that there was an absence of inhibitors in the preparations. In addition, it was found that the temperature optimum ranged between 40–55° C, that the pH optimum was pH 7.7 and that the Km of the reaction was 3,7 · 10-6 Mol/l. The cyanamide hydratase seemed to be crucial for the reaction. The velocity of the reaction was greatly enhanced through additional Mn2+. The enzyme was classified as one of the E. C. group 4.2.1. 5. The question of an active secretion of the enzyme into the culture medium was discussed.

Activities of several enzymes associated with cellulolysis were compared using as substrates cell-walls of Lolium multiflorum and cotton cellulose. Purified enzymes C 1 (see Ref. 1 for definition), C. x (CM-cellulase) and β-glucosidase were employed as well as culture filtrates containing C x . Activities were determined by ability to digest the substrates and to release H 2 O-soluble phenolic compounds from the grass cell-walls. The culture filtrates most active on cotton cellulose were obtained using the fungi Trichoderma viride and Fusarium solani ; with grass cell-walls the most active were from T. viride , Gliocladium roseum , a species of Basidiomycetes, and one strain of Myrothecium verrucaria (IMI Strain 25 291). For the crude enzyme preparations tested, there were highly significant correlations between the digestibility of grass cell-walls and the UV-absorption of the filtrate at λ max 290 nm and at λ max 324 nm but there was no significant correlation between the digestibility of grass cell-walls and that of cotton cellulose. Partially purified C 1 and C x from two different fungal sources showed activity on both substrates. Differences in MW of the H 2 O-soluble phenolic compounds obtained by treatment of grass cell-walls with C 1 and C x components suggest that these enzymes could have different modes of action. Synergism between C 1 and C x from T. koningii occurred with both substrates but with C 1 and C x from F. solani synergism only occurred with cotton cellulose.

The enzyme of Myrothecium verrucaria spores has an indicated absolute specificity for l -ascorbic acid and is irreversibly inactivated by d -isoascorbic acid, oxygen being necessary for this reaction. It is postulated that a stable ternary complex is formed between enzyme, isoascorbate, and oxygen. Studies of the inactivation show it to be markedly affected by pH and to have a positive temperature coefficient. Results of further studies on certain characteristics of the enzyme are reported.

4-Fluoroisoleucine was produced by ammonolysis of 2-bromo-4-fluoro-3-methylpentanoic acid, which resulted from the bromofluorination of 4-methyl-2-pentenoic acid. It did not inhibit Plasmodium berghei in mice at 640 mg/kg and was not toxic to the animals. The fluoroamino acid inhibited Aspergillus niger, Trichoderma viride, Myrothecium verrucaria, Trichophyton mentagrophytes, and Mucor mucedo in Czapek solution agar at a concentration between 10(4) and 10(3) microgram/ml. Growth of Escherichia coli was inhibited 25% at 900 microgram/ml in a defined medium.

Eleven n-alkoxyacetic acids and their methyl esters, in which the alkyl group was C1–C9, C11, or C13, were tested against Aspergillus niger, Trichoderma uiride, and Myrothecium verrucaria in Sabouraud dextrose agar at pH 4.0 and 5.6. Toxicity to Candida albicans, Trichophyton mentagrophytes, and Mucor mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. The fungitoxicity of the acids was influenced by chain length, pH of the medium, and absence or presence of adsorbents. The toxicity of the esters was influenced primarily by chain length and to a lesser extent by the medium pH and the presence of beef serum. The order of activity of the n-alkoxyacetic acids was according to the number of linear atoms in the chain: 11 > 12 > 10 > 9 > 8 > 7 > 6 > 14 > 16. T. mentagrophytes was the organism most strongly affected by these compounds, with the esters being slightly more active than the acids. Compared to other fatty acid analogs, the order of fungitoxicity, on a weight basis, was 2-alkynoic acids > 2-alkenoic acids > alkanoic acids > 2-bromoalkanoic acids > 2-fluoroalkanoic acids > n-alkoxyacetic acids.

Fourteen 2-n-alkynols (C3-C14, C16, and C18) were tested against Aspergillus oryzae, Aspergillus niger, Trichoderma viride, and Myrothecium verrucaria in Sabouraud dextrose agar at pH 5.6 and 7.0. Toxicity to Candida albicans, Candida tropicalis, Trichophyton mentagrophytes, and Mucor mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. Fungitoxicity was strongly influenced by chain length, slightly by the pH of the medium, and significantly by the presence of beef serum. 2-n-Undecyn-1-ol was the most active member of the series, and there was marked synergism between it and ketoconazole.

Twelve 3-n-alkyn-1-ols (C4-C12, C14, C16, and C18) were tested against Aspergillus oryzae, Aspergillus niger, Trichoderma viride, and Myrothecium verrucaria in Sabouraud dextrose agar at pH 5.6 and 7.0. Toxicity to Candida albicans, Candida tropicalis, Trichophyton mentagrophytes, and Mucor mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. Fungitoxicity was strongly influenced by chain length, slightly by pH of the medium, and significantly but not strongly by the presence of beef serum. 3-n-Decyn-1-ol, 3-n-undecyn-1-ol, and 3-n-dodecyn-1-ol were the most active members of the series. Synergism toward C. albicans and C. tropicalis was observed between 3-n-undecyn-1-ol and ketoconazole, and a mixture of 3-n-undecyn-1-ol, 2-n-undecyn-1-ol, and ketoconazole in Sabouraud dextrose agar at pH 7.0 in the presence of 10% human serum.

Disposal of paper mill waste by bioconversion is a novel approach which allows a direct conversion of cellulosic solid waste into fungal biomass. A cellulolytic microorganism Myrothecium verrucaria has been used for protein production under batch conditions in flasks. Biomass yield of 0.375 g per g of substrate consumed with 50–60% solid waste usage was observed. The nutritional quality of the biomass in terms of fat and protein contents shows promise for feed supplementation of monogastric animals.