North Yelm, WA, United States
North Yelm, WA, United States

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Riyaz-Ul-Hassan S.,Indian Institute of Integrative Medicine | Strobel G.,Montana State University | Geary B.,Brigham Young University | Sears J.,Center for Laboratory Services Lee Group
Journal of Microbiology and Biotechnology | Year: 2013

A Nodulisporium sp. (Hypoxylon sp.) has been isolated as an endophyte of Thelypteris angustifolia (Broadleaf Leaf Maiden Fern) in a rainforest region of Central America. It has been identified both on the basis of its morphological characteristics and by scanning electron microscopy as well as ITS sequence analysis. The endophyte produces volatile organic compounds (VOCs) that have both fuel (mycodiesel) and use for biological control of plant disease. When grown on potato dextrose agar, the organism uniquely produces a series of ketones, including acetone; 2-pentanone; 3-hexanone, 4-methyl; 3-hexanone, 2, 4-dimethyl; 2-hexanone, 4-methyl, and 5-hepten, 2-one and these account for about 25% of the total VOCs. The most abundant identified VOC was 1, 8 cineole, which is commonly detected in this group of organisms. Other prominent VOCs produced by this endophyte include 1-butanol, 2-methyl, and phenylethanol alcohol. Moreover, of interest was the presence of cyclohexane, propyl, which is a common ingredient of diesel fuel. Furthermore, the VOCs of this isolate of Nodulisporium sp. were selectively active against a number of plant pathogens, and upon a 24 h exposure caused death to Phytophthora palmivora, Rhizoctonia solani, and Sclerotinia sclerotiorum and 100% inhibition to Phytophthora cinnamomi with only slight to no inhibition of the other pathogens that were tested. From this work, it is becoming increasingly apparent that each isolate of this endophytic Nodulisporium spp., including the Daldina sp. and Hypoxylon spp. teleomorphs, seems to produce its own unique set of VOCs.© the Korean Society for Microbiology and Biotechnology.

Banerjee D.,Montana State University | Banerjee D.,Vidyasagar University | Strobel G.,Montana State University | Geary B.,Brigham Young University | And 4 more authors.
Mycology | Year: 2010

Muscodor albus strain GBA is a newly isolated endophytic fungus from Ginko biloba (family Ginkoaceae) collected in Newport, RI, USA. The cultural characteristics (color, growth pattern) and mycelial/hyphal characteristics resemble many isolates of Muscodor albus. The ITS rDNA sequence of the strain has at least 98% similarity with other isolates of M. albus and M. crispans. This xylariaceaous species effectively inhibits and kills certain test microbes via a mixture of volatile organic compounds (VOCs) that it produces. Some of the target test microbes were totally inhibited by M. albus strain GBA and not by other M. albus isolates, making this isolate unique in its biological activity. The VOCs of this fungus were identified by gas chromatography/mass spectrometry as esters, lipids, alcohols, acids and ketones, including proportionally large quantities of 1-butanol, 3-methyl-, acetate. A terpenoid, not observed in other strains of this fungus, vitrene was tentatively identified in the VOCs of this organism. This is the first record of M. albus in Ginko biloba and is the first report of any M. albus strain from the United States. The organism is normally found in tropical latitudes (16° north/ south) but the plant host M. albus strain GBA is located at 41° north latitude. Most importantly, however, the discovery of M. albus in the USA has enormous implications vis-a.vis governmental regulation of M. albus for use as a biological control agent in agriculture and industry, as this organism naturally occurs in the USA. A discussion on the relationship of this taxon with its host is also included. © 2010 Mycological Society of China.

Kudalkar P.,Montana State University | Strobel G.,Montana State University | Riyaz-Ul-Hassan S.,Montana State University | Geary B.,Brigham Young University | Sears J.,Center for Laboratory Services Lee Group
Mycoscience | Year: 2012

Muscodor sutura is described as a novel species that is also an endophyte of Prestonia trifidi. Uniquely, this fungus produces a reddish pigment, on potato dextrose agar (PDA), when grown in the dark. In addition, the organism makes some volatile organic compounds that have not been previously reported from this genus, namely, thujopsene, chamigrene, isocaryophyllene, and butanoic acid, 2-methyl. These and other volatile compounds in the mixture possess wide-spectrum antifungal activity and no observable antibacterial activity. Most unusually, on PDA, the newly developing hyphae of this fungus grow in a perfect stitching pattern, in and out of the agar surface. The partial ITS-DNA sequence of this organism is identical to that of Muscodor vitigenus but it differs from all other Muscodor spp. Justification for a new species, as Muscodor sutura, is collectively based on morphological, cultural, chemical, and bioactivity properties. © 2011 The Mycological Society of Japan and Springer.

Tomsheck A.R.,Montana State University | Strobel G.A.,Montana State University | Booth E.,Montana State University | Geary B.,Brigham Young University | And 6 more authors.
Microbial Ecology | Year: 2010

An endophytic fungus of Persea indica was identified, on the basis of its anamorphic stage, as Nodulosporium sp. by SEM. Partial sequence analysis of ITS rDNA revealed the identity of the teleomorphic stage of the fungus as Hypoxylon sp. It produces an impressive spectrum of volatile organic compounds (VOCs), most notably 1,8-cineole, 1-methyl-1,4-cyclohexadiene, and tentatively identified (+)-.alpha.-methylene-.alpha.-fenchocamphorone, among many others, most of which are unidentified. Six-day-old cultures of Hypoxylon sp. displayed maximal VOC-antimicrobial activity against Botrytis cinerea, Phytophthora cinnamomi, Cercospora beticola, and Sclerotinia sclerotiorum suggesting that the VOCs may play some role in the biology of the fungus and its survival in its host plant. Media containing starch- or sugar-related substrates best supported VOC production by the fungus. Direct on-line quantification of VOCs was measured by proton transfer mass spectrometry covering a continuous range with optimum VOC production occurred at 6 days at 145 ppmv with a rate of production of 7.65 ppmv/h. This report unequivocally demonstrates that 1,8-cineole (a monoterpene) is produced by a microorganism, which represents a novel and important source of this compound. This monoterpene is an octane derivative and has potential use as a fuel additive as do the other VOCs of this organism. Thus, fungal sourcing of this compound and other VOCs as produced by Hypoxylon sp. greatly expands their potential applications in medicine, industry, and energy production. © 2010 Springer Science+Business Media, LLC.

Singh S.K.,Montana State University | Singh S.K.,MACSAgharkar Research Institute | Strobel G.A.,Montana State University | Knighton B.,Montana State University | And 3 more authors.
Microbial Ecology | Year: 2011

An unusual Phomopsis sp. was isolated as endophyte of Odontoglossum sp. (Orchidaceae), associated with a cloud forest in Northern Ecuador. This fungus produces a unique mixture of volatile organic compounds (VOCs) including sabinene (a monoterpene with a peppery odor) only previously known from higher plants. In addition, some of the other more abundant VOCs recorded by GC/MS in this organism were 1-butanol, 3-methyl; benzeneethanol; 1-propanol, 2-methyl and 2-propanone. The gases of Phomopsis sp. possess antifungal properties and an artificial mixture of the VOCs mimicked the antibiotic effects of this organism with the greatest bioactivity against a wide range of plant pathogenic test fungi including: Pythium, Phytophthora, Sclerotinia, Rhizoctonia, Fusarium, Botrytis, Verticillium, and Colletotrichum. The IC50 values for the artificial gas mixture of Phomopsis sp. varied between 8 and 25.65 μl/mL. Proton transfer reaction-mass spectrometry monitored the concentration of VOCs emitted by Phomopsis sp. and yielded a total VOC concentration of ca. 18 ppmv in the head space at the seventh day of incubation at 23°C on PDA. As with many VOC-producing endophytes, this Phomopsis sp. did survive and grow in the presence of the inhibitory gases of Muscodor albus. A discussion is presented on the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship. © 2011 Springer Science+Business Media, LLC.

Strobel G.,Montana State University | Booth E.,Montana State University | Schaible G.,Montana State University | Mends M.T.,Montana State University | And 2 more authors.
Biotechnology Letters | Year: 2013

The construction and testing of a unique instrument, the Paleobiosphere, which mimics some of the conditions of the ancient earth, is described. The instrument provides an experimental testing system for determining if certain microbes, when provided an adequate environment, can degrade biological materials to produce fuel-like hydrocarbons in a relatively short time frame that become trapped by the shale. The conditions selected for testing included a particulate Montana shale (serving as the "Trap Shale"), plant materials (leaves and stems of three extant species whose origins are in the late Cretaceous), a water-circulating system, sterile air, and a specially designed Carbotrap through which all air was passed as exhaust and volatile were hydrocarbons trapped. The fungus for initial testing was Annulohypoxylon sp., isolated as an endophyte of Citrus aurantifolia. It produces, in solid and liquid media, a series of hydrocarbon-like molecules. Some of these including 1,8-cineole, 2-butanone, propanoic acid, 2-methyl-, methyl ester, benzene (1-methylethyl)-, phenylethyl alcohol, benzophenone and azulene, 1,2,3,5,6,7,8,8a-octahydro-1,4-dimethyl-7-(1-methylethenyl), [1S-(1α,7α,8aβ)]. These were the key signature compounds used in an initial Paleobiosphere test. After 3 weeks, incubation, the volatiles associated with the harvested "Trap Shale" included each of the signature substances as well as other fungal-associated products: some indanes, benzene derivatives, some cyclohexanes, 3-octanone, naphthalenes and others. The fungus thus produced a series of "Trap Shale" products that were representative of each of the major classes of hydrocarbons in diesel fuel (Mycodiesel). Initial tests with the Paleobiosphere offer some evidence for a possible origin of hydrocarbons trapped in bentonite shale. Thus, with modifications, numerous other tests can also be designed for utilization in the Paleobiosphere. © 2012 Springer Science+Business Media Dordrecht.

Booth E.,Montana State University | Strobel G.,Montana State University | Knighton B.,Montana State University | Sears J.,Center for Laboratory Services Lee Group | And 2 more authors.
Biotechnology Letters | Year: 2011

A custom-made stainless steel column was designed to contain various materials that would trap the hydrocarbons and hydrocarbon derivatives during the processes of fungal fermentation ultimately yielding preparative amounts of volatile organic substances (VOCs). Trapping materials tested in the column were Carbotrap materials A and B (Supelco) as well as bentonite-shale from the oil bearing areas of Eastern Montana, the former allowed for the effective and efficient trapping of VOCs from purged cultures of Hypoxylon sp. Trapping efficiencies of various materials were measured by both gravimetric as well as proton transfer reaction mass spectroscopy with the Carbotraps A and B being 99% efficient when tested with known amounts of 1,8-cineole. Trapped fungal VOCs could effectively be removed and recovered via controlled heating of the stainless steel column followed by passage of the gases through a liquid nitrogen trap at a recovery rate of ca 65-70%. This method provides for the recovery of mg quantities of compounds normally present in the gas phase that may be needed for spectroscopy, bioassays and further separation and analysis and may have wide applicability for many other biological systems involving VOCs. Other available Carbotraps could be used for other applications. © 2011 Springer Science+Business Media B.V.

Strobel G.,Montana State University | Singh S.K.,Montana State University | Singh S.K.,MACSAgharkar Research Institute | Riyaz-Ul-Hassan S.,Montana State University | And 3 more authors.
FEMS Microbiology Letters | Year: 2011

A Phoma sp. was isolated and characterized as endophytic and as a pathogen of Larrea tridentata (creosote bush) growing in the desert region of southern Utah, USA. This fungus produces a unique mixture of volatile organic compounds (VOCs), including a series of sesquiterpenoids, some alcohols and several reduced naphthalene derivatives. Trans-caryophyllene, a product in the fungal VOCs, was also noted in the VOCs of this pungent plant. The gases of Phoma sp. possess antifungal properties and is markedly similar to that of a methanolic extract of the host plant. Some of the test organisms with the greatest sensitivity to the Phoma sp. VOCs were Verticillium, Ceratocystis, Cercospora and Sclerotinia while those being the least sensitive were Trichoderma, Colletotrichum and Aspergillus. We discuss the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship with implications for utilization as an energy source. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

Schaible G.A.,Montana State University | Strobel G.A.,Montana State University | Mends M.T.,Montana State University | Geary B.,Brigham Young University | Sears J.,Center for Laboratory Services Lee Group
Microbial Ecology | Year: 2015

Gloeosporium sp. (OR-10) was isolated as an endophyte of Tsuga heterophylla (Western hemlock). Both ITS and 18S sequence analyses indicated that the organism best fits either Hypocrea spp. or Trichoderma spp., but neither of these organisms possess conidiophores associated with acervuli, in which case the endophytic isolate OR-10 does. Therefore, the preferred taxonomic assignment was primarily based on the morphological features of the organism as one belonging to the genus Gloeosporium sp. These taxonomic observations clearly point out that limited ITS and 18S sequence information can be misleading when solely used in making taxonomic assignments. The volatile phase of this endophyte was active against a number of plant pathogenic fungi including Phytophthora palmivora, Rhizoctonia solani, Ceratocystis ulmi, Botrytis cinerea, and Verticillium dahliae. Among several terpenes and furans, the most abundantly produced compound in the volatile phase was 6-pentyl-2H-pyran-2-one, a compound possessing antimicrobial activities. When used in conjunction with microliter amounts of any in a series of esters or isobutyric acid, an enhanced inhibitory response occurred with each test fungus that was greater than that exhibited by Gloeosporium sp. or the compounds tested individually. Compounds behaving in this manner are hereby designated “synergistans.” An expression of the “median synergistic effect,” under prescribed conditions, has been termed the mSE50. This value describes the amount of a potential synergistan that is required to yield an additional median 50 % inhibition of a target organism. In this report, the mSE50s are reported for a series of esters and isobutyric acid. The results indicated that isoamyl acetate, allyl acetate, and isobutyric acid generally possessed the lowest mSE50 values. The value and potential importance of these microbial synergistic effects to the microbial environment are also discussed. © 2014, Springer Science+Business Media New York.

Ren Y.,Montana State University | Strobel G.,Montana State University | Sears J.,Center for Laboratory Services Lee Group | Park M.,Montana State University
Microbial Ecology | Year: 2010

Geobacillus, a bacterial genus, is represented by over 25 species of Gram-positive isolates from various man-made and natural thermophilic areas around the world. An isolate of this genus (M-7) has been acquired from a thermal area near Yellowstone National Park, MT and partially characterized. The cells of this organism are globose (ca. 0.5 μ diameter), and they are covered in a matrix capsule which gives rise to elongate multicelled bacilliform structures (ranging from 3 to 12 μm) as seen by light and atomic force microscopy, respectively. The organism produces unique petal-shaped colonies (undulating margins) on nutrient agar, and it has an optimum pH of 7.0 and an optimum temperature range of 55-65°C. The partial 16S rRNA sequence of this organism has 97% similarity with Geobacillus stearothermophilus, one of its closest relatives genetically. However, uniquely among all members of this genus, Geobacillus sp. (M-7) produces volatile organic substances (VOCs) that possess potent antibiotic activities. Some of the more notable components of the VOCs are benzaldehyde, acetic acid, butanal, 3-methyl-butanoic acid, 2-methyl-butanoic acid, propanoic acid, 2-methyl-, and benzeneacetaldehyde. An exposure of test organisms such as Aspergillus fumigatus, Botrytis cinerea, Verticillium dahliae, and Geotrichum candidum produced total inhibition of growth on a 48-h exposure to Geobacillus sp.(M-7) cells (ca. 107) and killing at a 72-h exposure at higher bacterial cell concentrations. A synthetic mixture of those available volatile compounds, at the ratios occurring in Geobacillus sp. (M-7), mimicked the bioactivity of this organism. © 2010 Springer Science+Business Media, LLC.

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