Scholarly Work - Plant Sciences & Plant Pathology
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Item Antiviral Activity of the PropylamylatinTM Formula against the Novel Coronavirus SARS-CoV-2 In Vitro Using Direct Injection and Gas Assays in Virus Suspensions(MDPI, 2021-03) Brown, Ashley N.; Strobel, Gary; Hanrahan, Kaley C.; Sears, JoeSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of novel coronavirus disease 2019 (COVID-19), has become a severe threat to global public health. There are currently no antiviral therapies approved for the treatment or prevention of mild to moderate COVID-19 as remdesivir is only approved for severe COVID-19 cases. Here, we evaluated the antiviral potential of a Propylamylatin formula, which is a mixture of propionic acid and isoamyl hexanoates. The Propylamylatin formula was investigated in gaseous and liquid phases against 1 mL viral suspensions containing 105 PFU of SARS-CoV-2. Viral suspensions were sampled at various times post-exposure and infectious virus was quantified by plaque assay on Vero E6 cells. Propylamylatin formula vapors were effective at inactivating infectious SARS-CoV-2 to undetectable levels at room temperature and body temperature, but the decline in virus was substantially faster at the higher temperature (15 min versus 24 h). The direct injection of liquid Propylamylatin formula into viral suspensions also completely inactivated SARS-CoV-2 and the rapidity of inactivation occurred in an exposure dependent manner. The overall volume that resulted in 90% viral inactivation over the course of the direct injection experiment (EC90) was 4.28 ls. Further investigation revealed that the majority of the antiviral effect was attributed to the propionic acid which yielded an overall EC90 value of 11.50 ls whereas the isoamyl hexanoates provided at most a 10-fold reduction in infectious virus. The combination of propionic acid and isoamyl hexanoates was much more potent than the individual components alone, suggesting synergy between these components. These findings illustrate the therapeutic promise of the Propylamylatin formula as a potential treatment strategy for COVID-19 and future studies are warranted.Item An Endophytic Nodulisporium sp. from Central America Producing Volatile Organic Compounds with Both Biological and Fuel Potential(2013) Hassan, Syed R.-U.; Strobel, Gary A.; Geary, Brad; Sears, JoeA 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.Item Urnula sp., an Endophyte of Dicksonia antarctica, Making a Fragrant Mixture of Biologically Active Volatile Organic Compounds(2017-08) Strobel, Gary A.; Ericksen, Amy; Sears, Joe; Xie, Jie; Geary, Brad; Blatt, BryanUrnula sp. was isolated as an endophyte of Dicksonia antarctica and identified primarily on the basis of its ITS sequence and morphological features. The anamorphic state of the fungus appeared as a hyphomyceteous-like fungus as based on its features in culture and scanning electron microscopy examination of its spores. On potato dextrose agar (PDA), the organism makes a characteristic fragrance resembling peach pie with vanilla overtones. A GC/MS analysis done on the volatile organic compounds (VOCs) of this organism, trapped by carbotrap methodology, revealed over 150 compounds with high MS matching quality being noted for 44 of these. Some of the most abundantly produced compounds included 4-decene, tridecane, 2-decene (E), 2-dodecene, (Z,E)-alpha-farnesene, butanoic acid, pentyl ester, and 1-hexanol,2-ethyl. In addition, vanillin, methyl vanillin, and many other fragrant substances were noted including isomenthol, pyrazine derivatives, and 3-decanone. In split plate bioassay tests on potato dextrose agar (PDA), Botrytis cinerea, Ceratocystis ulmi, Pythium ultimum, Fusarium solani, and Rhizoctonia solani were inhibited at levels of 24 to 50% of their normal growth on this medium. Bioreactors supporting fungal growth on 50g of beet pulp waste, using stainless steel carbotraps, yielded over 180mg of hydrocarbon-based products collected over 6weeks of incubation. Similarly, because this organism is making one of the largest sets of VOCs as any fungus examined to date, producing many compounds of commercial interest, it has enormous biotechnical potential. The role of the VOCs in the biology and ecology of this endophyte may be related to the antimicrobial activities that they possess.Item Volatile antimicrobials from Muscodor crispans, a novel endophytic fungus(2009-01) Mitchell, Angela M.; Strobel, Gary A.; Moore, Emily; Robison, Richard; Sears, JoeMuscodor crispans is a recently described novel endophytic fungus of Ananas ananassoides (wild pineapple) growing in the Bolivian Amazon Basin. The fungus produces a mixture of volatile organic compounds (VOCs); some of the major components of this mixture, as determined by GC/MS, are propanoic acid, 2-methyl-, methyl ester; propanoic acid, 2-methyl-; 1-butanol, 3-methyl-;1-butanol, 3-methyl-, acetate; propanoic acid, 2-methyl-, 2-methylbutyl ester; and ethanol. The fungus does not, however, produce naphthalene or azulene derivatives as has been observed with many other members of the genus Muscodor. The mixture of VOCs produced by M. crispans cultures possesses antibiotic properties, as does an artificial mixture of a majority of the components. The VOCs of the fungus are effective against a wide range of plant pathogens, including the fungi Pythium ultimum, Phytophthora cinnamomi, Sclerotinia sclerotiorum and Mycosphaerella fijiensis (the black sigatoka pathogen of bananas), and the serious bacterial pathogen of citrus, Xanthomonas axonopodis pv. citri. In addition, the VOCs of M. crispans killed several human pathogens, including Yersinia pestis, Mycobacterium tuberculosis and Staphylococcus aureus. Artificial mixtures of the fungal VOCs were both inhibitory and lethal to a number of human and plant pathogens, including three drug-resistant strains of Mycobacterium tuberculosis. The gaseous products of Muscodor crispans potentially could prove to be beneficial in the fields of medicine, agriculture, and industry.Item Muscodor albus strain GBA, an endophytic fungus of Ginkgo biloba from United States of America, produces volatile antimicrobials(2010-09) Banerjee, Debdulal; Strobel, Gary A.; Geary, Brad; Sears, Joe; Ezra, David; Liarzi, Orna; Coombs, JamesMuscodor 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.Item Geobacillus sp., a Thermophilic Soil Bacterium Producing Volatile Antibiotics(2010-01) Ren, Yuhao; Strobel, Gary A.; Sears, Joe; Park, MelinaGeobacillus, 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.Item An endophytic Myrothecium inundatum producing volatile organic compounds(2010) Banerjee, Debdulal; Strobel, Gary A.; Booth, B.; Sears, Joe; Spakowicz, Daniel; Busse, S.Myrothecium inunduatum was isolated as an endophyte from a euphorbeacean herb, Acalypha indica in NE India. This fungus when grown in shake culture produced an abundance of foam. Contained in the foam was a mixture of volatile organic compounds (VOCs) some of which were hydrocarbons and hydrocarbon derivatives. The most prevalent compounds were 3-octanone, 3-octanol, and 7-octen-4-ol. Numerous other volatile organic compounds were also produced including many terpenes, organic acids, ketones, and alcohols. The VOCs of this fungus demonstrated growth inhibitory activity against a number of plant pathogenic fungi including Pythium ultimum and Sclerotinia sclerotiorum. However, when grown in microaerophilic conditions, the organism produced a number of hydrocarbons of interest as fuel related hydrocarbons including octane and tentatively identified- 1,4- cyclohexadiene, 1-methyl- and cyclohexane, (1-ethylpropyl) and others. An NMR method was used to measure VOC production which peaked at day 15 in a time course experiment. Numerous substrates can serve to support the production of VOCs by this fungus including potato broth and beet pulp extracts.Item The production of myco-diesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum (NRRL 50072)(2010-12) Strobel, Gary A.; Knighton, W. Berk; Kluck, Katreena; Ren, Yuhao; Livinghouse, Tom; Griffin, Meghan; Spakowicz, Daniel; Sears, JoeAn endophytic fungus, Gliocladiun 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.Item An endophytic/pathogenic Phoma sp. from creosote bush producing biologically active volatile compounds having fuel potential(2011-05) Strobel, Gary A.; Singh, Sanjay K.; Ul-Hassan, Syed Riyaz; Mitchell, Angela M.; Geary, Brad; Sears, JoeA 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.Item Modulation of volatile organic compound formation in the Mycodiesel-producing endophyte Hypoxylon sp. CI-4(2012-11) Ul-Hassan, Syed Riyaz; Strobel, Gary A.; Booth, Eric; Knighton, W. Berk; Floerchinger, Cody; Sears, JoeAn endophytic Hypoxylon sp. (strain CI-4) producing a wide spectrum of volatile organic compounds (VOCs), including 1,8-cineole, 1-methyl-1,4-cyclohexadiene and cyclohexane, 1,2,4-tris(methylene), was selected as a candidate for the modulation of VOC production. This was done in order to learn if the production of these and other VOCs can be affected by using agents that may modulate the epigenetics of the fungus. Many of the VOCs made by this organism are of interest because of their high energy densities and thus the potential they might have as Mycodiesel fuels. Strain CI-4 was exposed to the epigenetic modulators suberoylanilide hydroxamic acid (SAHA, a histone deacetylase) and 5-azacytidine (AZA, a DNA methyltransferase inhibitor). After these treatments the organism displayed striking cultural changes, including variations in pigmentation, growth rates and odour, in addition to significant differences in the bioactivities of the VOCs. The resulting variants were designated CI4-B, CI4-AZA and CI4-SAHA. GC/MS analyses of the VOCs produced by the variants showed considerable variation, with the emergence of several compounds not previously observed in the wild-type, particularly an array of tentatively identified terpenes such as α-thujene, sabinene, γ-terpinene, α-terpinolene and β-selinene, in addition to several primary and secondary alkanes, alkenes, organic acids and derivatives of benzene. Proton transfer reaction mass spectroscopic analyses showed a marked increase in the ratio of ethanol (mass 47) to the total mass of all other ionizable VOCs, from ~0.6 in the untreated strain CI-4 to ~0.8 in CI-4 grown in the presence of AZA. Strain CI4-B was created by exposure of the fungus to 100 µM SAHA; upon removal of the epigenetic modulator from the culture medium, it did not revert to the wild-type phenotype. Results of this study have implications for understanding why there may be a wide range of VOCs found in various isolates of this fungus in nature.