The mycotoxin zearalenone (ZEN) can be degraded by a lactone hydrolase ZHD, which was derived from Gliocladium roseum. Here, based on the native ZHD encoding gene zhd101, a codon optimized zhd gene ...was synthesized, which was used for high expression of ZHD in Pichia pastoris GS115. Meanwhile, to further improve the expression of recombinant ZHD, the plasmids containing 1 to 4 copies of the zhd expression cassette were constructed, respectively, using the biobrick method. The protein expression in the recombinant P. pastoris X3c, which was transformed with the plasmid containing 3 copies of zhd expression cassette, was the highest. In addition, the enzymatic activity of ZHD against ZEN was defined for the first time based on a standard curve of peak area vs ZEN concentration. The ZEN degradation activity of ZHD from shake flask fermentation was calculated as 22.5U/mL with the specific activity of 4976.5U/mg. Furthermore, the high-density fermentation of P. pastoris X3c strain was also performed in 5L fermenter. The maximum enzyme activity of the supernatant was 150.1U/mL, which were 6.7-fold higher than that of the shake flask fermentation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
1 Department of Plant Sciences, Montana State University, Bozeman, MT 59717, USA
2 Department of Chemistry, Montana State University, Bozeman, MT 59717, USA
3 Department of Molecular Biophysics and ...Biochemistry, Yale University, New Haven, CT 06520, USA
4 Center for Lab Services/RJ Lee Group, 2710 North 20th Ave, Pasco, WA 99301, USA
Correspondence Gary Strobel uplgs{at}montana.edu
An endophytic fungus, Gliocladium roseum (NRRL 50072), produced a series of volatile hydrocarbons and hydrocarbon derivatives on an oatmeal-based agar under microaerophilic conditions as analysed by solid-phase micro-extraction (SPME)-GC/MS. As an example, this organism produced an extensive series of the acetic acid esters of straight-chained alkanes including those of pentyl, hexyl, heptyl, octyl, sec-octyl and decyl alcohols. Other hydrocarbons were also produced by this organism, including undecane, 2,6-dimethyl; decane, 3,3,5-trimethyl; cyclohexene, 4-methyl; decane, 3,3,6-trimethyl; and undecane, 4,4-dimethyl. Volatile hydrocarbons were also produced on a cellulose-based medium, including heptane, octane, benzene, and some branched hydrocarbons. An extract of the host plant, Eucryphia cordifolia (ulmo), supported the growth and hydrocarbon production of this fungus. Quantification of volatile organic compounds, as measured by proton transfer mass spectrometry (PTR-MS), indicated a level of organic substances in the order of 80 p.p.m.v. (parts per million by volume) in the air space above the oatmeal agar medium in an 18 day old culture. Scaling the PTR-MS profile the acetic acid heptyl ester was quantified (at 500 p.p.b.v.) and subsequently the amount of each compound in the GC/MS profile could be estimated; all yielded a total value of about 4.0 p.p.m.v. The hydrocarbon profile of G. roseum contains a number of compounds normally associated with diesel fuel and so the volatiles of this fungus have been dubbed myco-diesel. Extraction of liquid cultures of the fungus revealed the presence of numerous fatty acids and other lipids. All of these findings have implications in energy production and utilization.
Abbreviations: PTR-MS, proton transfer mass spectrometry; SPME, solid-phase micro-extraction
Biotransformation of Echinocystic acid (EA,1) using G. roseum CGMCC 3.3657 has been investigated, which leads to the isolation and identification of two novel Echinocystic acid derivatives, 4, ...16α-dihydroxy-3,4-seco-olean-12-en-3,28-dioic acid (2) and 16α-hydroxy, A-homo-3α-oxa-olean-12-en-3-one-28-oic acid (3). Their structures have been elucidated by analysis of spectroscopic data. This biocatalysis could serve as an efficient tool complementary to classical chemical methods for the transformation of EA.
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Gliocladium roseum 67-1 selected by the authors has been demonstrated to be a highly effective biocontrol agent of the diseases caused by Sclerotinia sclerotiorum and Rhizocotonia solani. A rapid and ...accurate quantification of the agent is urgently needed for understanding its interaction with disease pathogens in soil and improving its effectiveness for disease management. Primers and probes for quantification in soil by real-time PCR were designed from ITS regions of the fungi belonging to several taxon. Specificity tests of the primers and probes using genomic DNA indicated that they could specifically amplify ITS rDNA of the biocontrol agent. Standard curve developed by using the plasmid containing the ITS rDNA of the agent gave rise to 0.9989 as linear correlation coefficient between DNA concentrations and Ct values, and 94.9% as amplification efficiency. DNA at 10 super(-9) g/ml could be detected. Quantification of the agent added in natural soil by amplifying soil DNA obtained with Mobio UltraClean soil DNA kit showed that Ct value was highly correlated with population density of the fungus (coefficient 0.984). Primary tests on the interaction between R. solani AG1 and the biocontrol agent indicated that population of the pathogen could be suppressed by 75% after co-incubation in natural soil for 2 weeks under 20C and 15% water content although population of the isolate 67-1 declined a lot at the same time.
Rhizoctonia solani AG-3 forms sclerotia on potato tubers, reducing quality and marketability. Potato discs with sclerotia were exposed to saturated soil, and following toothpick baiting, hyphae of ...R. solani parasitised with mycelia that either coiled around or penetrated cell walls were observed. Similar parasitic behaviour was observed in dual cultures. Clonostachys rosea was identified as the mycoparasite by morphology and ITS sequencing. When co-inoculated onto stems grown from disease-free seed potatoes, tubers of the C. rosea + R. solani inoculated plants had significantly lower black scurf (P < 0.0001) and higher yield (P = 0.0002) than R. solani inoculated controls.
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Harmanecká Cave is located in the Harmanec Valley to the northwest of Banská Bystrica city, in the southern part of the Great Fatra Mountains, Slovakia. This cave is the most important underground ...locality of bat occurrence in Slovakia (population of 1000 to 1500 individuals). The study aimed at mycological evaluation of the air, the water, and the rock surface of Harmanecká Cave in Slovakia. The samples were taken on 24 July 2014. To examine the air, the Air Ideal 3P sampler was used. Microbiological evaluation of the rock surface was performed using swab sampling and the water by using the serial dilution technique. The authors observed a relationship between air temperature and the concentration of fungi. The concentration of airborne fungi increased with the increase in the air temperature and decreased with distance from the entrance to the cave. The density of airborne fungi isolated from the outdoor air samples was 810.5 colony-forming units (CFU) per 1 m³ of air and from 27.4 to 128.5 CFU for the indoor air samples. From the rock surface inside the cave, 45.0 to 106.6 CFU per 1 cm² were isolated, whereas from the water, 29.9 CFU per 1 ml were isolated. Seven species of filamentous fungi were isolated from the external air samples and 12 species of filamentous fungi and 3 species of yeast-like fungi from the internal air samples. From the surface of the rocks inside the cave, 5 species of filamentous fungi and 1 species of yeast-like fungi were cultured, whereas from the water samples, 6 species of filamentous fungi were cultured. Cladosporium spp. were the fungi most frequently isolated from the external air; from the internal air, Penicillium urticae was most frequently isolated; from the rock surface, it was Gliocladium roseum; and from the water, it was P. chrysogenum. The species found in the cave can be pathogenic for humans and animals, especially for immunocompromised persons, and they can also cause biodegradation of the rocks. However, the concentration of airborne fungi inside the cave did not exceed official limits and norms stated as dangerous for the health of tourists.
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BFBNIB, EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
In the present study, spherical, crystalline, monodispersed selenium nanoparticles were biosynthesized by an economical, environment friendly, easy, sustainable and green methodology using fungi
...Gliocladium roseum
. The biosynthesized selenium nanoparticles were characterized by using UV Spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), X-ray diffraction (XRD) spectroscopy and scanning electron microscopy with energy dispersive X-ray (SEM-EDX). The size of biosynthesized selenium nanoparticles obtained by TEM was in the range of 20–80 nm. There were some large particles of more than 100 nm but less than 150 nm also seen. XRD spectroscopy analyses revealed that biosynthesized selenium nanoparticles were hexagonal crystalline in nature. The FTIR spectroscopy study confirms presence of functional groups which were associated with proteins and biomolecules excreted extracellularly by fungi. These proteins and biomolecules believe to serve as template for reduction and stabilization of selenium nanoparticles. Moreover, these biomolecules were may also help in controlling size and aggregation.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Zearalenone is a mycotoxin produced by several species of Fusarium genus, most notably Fusarium graminearum and Fusarium culmorum. This resorcylic acid lactone is one of the most important toxins ...causing serious animal and human diseases. For over two decades it has been known that the mycoparasitic fungus Clonostachys rosea (synonym: Gliocladium roseum, teleomorph: Bionectria ochroleuca) can detoxify zearalenone, however no such attributes have been described within the Trichoderma genus.
We screened for the presence of zearalenone lactonohydrolase homologs in isolates of Clonostachys and Trichoderma genera. We report first finding of expressed zearalenone lactonohydrolase in Trichoderma aggressivum. For three isolates (T. aggressivum, C. rosea and Clonostachys catenulatum isolates), we were able to reconstruct full coding sequence and verify the biotransformation ability potential. Additionally, we assessed progression of the detoxification process (in terms of transcript accumulation and mycotoxin decomposition in vitro).In silico, search for origins of zearalenone lactonohydrolase activity in model fungal and bacterial genomes has shown that zearalenone lactonohydrolase homologs form a monophyletic fungal clade among the a/b hydrolase superfamily representatives. We corroborated the finding of functional enzyme homologs by investigating the functional sites (active site pocket with postulated, noncanonical Ser-Glu-His catalytic triad) conserved in both multiple sequence alignment and in homology-based structural models.
Our research shows the first finding of a functional zearalenone lactonohydrolase in mycoparasitic Trichoderma aggressivum (an activity earlier characterised in the Clonostachys rosea strains). The supporting evidence for presence and activity of functional enzyme homologs is based on the chemical analyses, gene expression patterns, homology models showing conservation of key structural features and marked reduction of zearalenone content in cultured samples (containing both medium and mycelium). Our findings also show divergent strategies of zearalenone biotransformation ability (rapid induced expression and detoxification vs. gradual detoxification) present in several members of Hypocreales order (Trichoderma and Clonostachys genera). The potential for lactonhydrolase activity directed towards zearalenone and/or similar compounds is likely ancient, with homologs present in several divergent filamentous fungi among both Sordariomycetes (Bionectria sp., Trichoderma sp., Apiospora montagnei) and Leotiomycetes (Marssonina brunnea f. sp. 'multigermtubi').
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Clonostachys rosea on strawberry leaf discs exposed to doses of UV-B radiation and challenged by Botrytis cinerea, 3, 7, and 10days after inoculation (dai). A. Incidence of C. rosea with different ...inoculum concentrations at several doses of UV-B radiation; B. Incidence of B. cinerea on leaf discs challenged by C. rosea; C. Leaf disc area with C. rosea conidiophores; D. Leaf disc area with B. cinerea conidiophores. Display omitted
► The UV-B radiation caused a strong deleterious effect to Clonostachys rosea conidia. ► UV-B radiation significantly reduced the growth of Clonostachys rosea on leaf discs. ► The UV-B radiation caused reduction on antagonized ability of Clonostachys rosea to control Botrytis cinerea.
Clonostachys rosea is effective to control of Botrytis cinerea on strawberry, although is highly susceptible to ultraviolet radiation and has reduced ability to antagonize a pathogen in solar radiation conditions. The objective of this work was to evaluate the ability of an isolate of C. rosea, previously selected for its tolerant to UV-B radiation, to control B. cinerea on strawberry leaves in controlled experiments. Leaf discs of 1cm diameter were placed on Petri dishes and each received 20μL of a C. rosea LQC 62 concentrations (104, 105, and 106conidiamL−1). They were then exposed to UV-B irradiance 600mWm−2 (0, 2.1, 4.2, and 6.3kJm−2), and after radiation, half of the discs were inoculated with an aliquot of 10μL B. cinerea (105conidiamL−1). The colonization of fungi on the leaf disc was measured with diagrammatic scale formation of conidiophores. The presence and sporulation of C. rosea on leaf disc was influenced by the dose of UV-B radiation and the conidial concentration of antagonist. The incidence and severity of B. cinerea on leaf discs were inversely correlated to presence and sporulation of C. rosea. The growth of the pathogen was higher in the lower C. rosea concentration. The highest concentration of C. rosea (106conidiamL−1) reduced the incidence and severity by 91% and 98% of B. cinerea on strawberry leaf discs. The UV-B radiation reduced the ability of C. rosea to control B. cinerea. The higher dose of UV-B reduced the presence and sporulation of C. rosea by 20% and 42%, respectively. Consequently, the incidence of B. cinerea increased twice and the severity was three-folder higher. Taken together this data means that, for the development of biological control agents based products, the effect of UV-B should be considered on the efficacy studies.
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