Brad Geary

Hypoxylon sp., an Endophyte of Persea indica, Producing 1,8-Cineole and Other Bioactive Volatiles with Fuel Potential

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.

An endophytic/pathogenic Phoma sp. from creosote bush producing biologically active volatile compounds having fuel potential

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.

An Endophytic Nodulisporium sp. Producing Volatile Organic Compounds Having Bioactivity and Fuel Potential

Nodulisporium sp. has been isolated as an endophyte of Myroxylon balsamum found in the upper Napo region of the Ecuadorian Amazon. This organism produces volatile organic compounds (VOCs) that have both fuel and biological potential. Under microaerophilic growth environments, the organism produces 1, 4-cyclohexadiene, 1-methyl- , 1-4 pentadiene and cyclohexene, 1-methyl-4-(1-methylethenyl)- along with some alcohols and terpenoids of interest as potential fuels. The fungus was scaled up in an aerated large fermentation flask, and the VOCs trapped by Carbotrap technology and analyzed by headspace solid –phase microextraction (SPME) fiber-GC/MS. Under these conditions, Nodulisporium sp. produces a series of alkyl alcohols starting with 1-butanol-3-methyl, 1- propanol-2-methyl, 1- pentanol, 1-hexanol, 1-heptanol, 1- octanol, 1-nonanol along with phenylethyl alcohol. The organism also produces secondary alkyl alcohols, esters, ketones, benzene derivatives, a few terpenoids, and some hydrocarbons. It appears that many of the products have fuel potential. Furthermore, the VOCs of Nodulisporium sp. were active against a number of pathogens causing death to both Aspergillus fumigatus and Rhizoctonia solani and severe growth inhibition produced in Phytophthora cinnamomi and Sclerotinia sclerotiorum within 48 hr of exposure. The Carbotrapped materials somewhat mimicked the bioactivities of the culture itself when certain test organisms were exposed to these VOCs. A brief discussion on the relationship of these fungal VOCs to those compounds found in transportation fuels is presented.

Screening Two Lycopersicon peruvianum Collections for Resistance to Tomato spotted wilt virus

The United States Department of Agriculture (USDA) Research Service and the Tomato Genetics Resource Center (TGRC) Lycopersicon peruvianum germplasm collections (16,335 plants from 285 accessions) were screened with the Tomato spotted wilt virus (TSWV) isolates TSWV6 from Hawaii, and Anwa-1 from Western Australia. Using TSWV6 to screen for resistance, 10,634 L. peruvianum plants from 280 accessions were screened for resistance, resulting in 168 (60%) accessions with 1,437 (14%) plants indicating resistance, with all 1,404 89S (Sw-5+/Sw-5+) and 1,456 89R (Sw-5/Sw-5) controls infected. When using Anwa-1 for screening, 864 (15%) of 5,701 L. peruvianum plants were uninfected from 106 of the 181 accessions tested, and 472 (95%) of the 495 89S and 421 (73%) of the 574 89R controls were infected. Of the 172 accessions tested with both isolates, 54 were resistant to one isolate but not the other. Additionally, more accessions from the USDA than from the TGRC collection indicated resistance. TSWV-resistant accessions were somewhat equally distributed throughout the L. peruvianum geographic range, with an observation that northern Chile and southern Peru seemed to have an unusually high portion of accession indicating resistance. The value of Sw-5 is discussed in relationship to potential additional sources of TSWV resistance.

A rapid column technique for trapping and collecting of volatile fungal hydrocarbons and hydrocarbon derivatives

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.

Muscodor albus strain GBA, an endophytic fungus of Ginkgo biloba from United States of America, produces volatile antimicrobials

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.

Functionalized para-substituted benzenes as 1,8-cineole production modulators in an endophytic Nodulisporium species.

A Nodulisporium species (designated Ti-13) was isolated as an endophyte from Cassia fistula. The fungus produces a spectrum of volatile organic compounds (VOCs) that includes ethanol, acetaldehyde and 1,8-cineole as major components. Initial observations of the fungal isolate suggested that reversible attenuation of the organism via removal from the host and successive transfers in pure culture resulted in a 50 % decrease in cineole production unrelated to an overall alteration in fungal growth. A compound (CPM1) was obtained from Betula pendula (silver birch) that increases the production of 1,8-cineole by an attenuated Ti-13 strain to its original level, as measured by a novel bioassay method employing a 1,8-cineole-sensitive fungus (Sclerotinia sclerotiorum). The host plant produces similar compounds possessing this activity. Bioactivity assays with structurally similar compounds such as ferulic acid and gallic acid suggested that the CPM1 does not act as a simple precursor to the biosynthesis of 1,8-cineole. NMR spectroscopy and HPLC-ES-MS indicated that the CPM1 is a para-substituted benzene with alkyl and carboxyl substituents. The VOCs of Ti-13, especially 1,8-cineole, have potential applications in the industrial, fuel and medical fields.

Endophyte Strain NRRL 50072 producing volatile organics is a species of Ascocoryne

A fungal endophyte (NRRL 50072) isolated from Eucryphia cordifolia from Patagonia that produces hydrocarbon derivatives is identified as an Ascocoryne sp. It was classified as Clonostachys rosea (Gliocaldium roseum) strain C-13 = NRRL 50072 primarily based upon its morphological characteristics. The fungus produced slimy clumped conidia on verticillated conidiophores and red- pigmented particles in culture. However, DNA sequence analysis (ITS rDNA) suggested a 99% identity to Ascocoryne sarcoides rather than to fungi assigned to the group of Gliocladium-like anamorphs. Comparative genetic, biological and morphological studies confirmed that the anamorphic stage of an authenticated field-collected culture of Ascocoryne sarcoides AV-70 shares many of the same morphological, and genetic features as NRRL 50072, but the latter is unable to produce synnematal masses unless it is grown on a proper substratum. These data suggest that NRRL 50072 is most closely related to the asexual stage of A. sarcoides, namely a Coryne sp. Serial transfer of the A. sarcoides AV-70 resulted in major cultural changes in the fungus especially in the production of aerial hyphae, pigment production and the number of synnemata being formed. This report appropriately sets the taxonomic framework for further molecular biological and biochemical work on NRRL 50072 and related fungi.

Development and use of microsatellite markers for genetic diversity analysis of cañahua (Chenopodium pallidicaule Aellen)

Cañahua (Chenopodium pallidicaule Aellen) is a poorly studied, annual subsistence crop of the high Andes of South America. Its nutritional value (high in protein and mineral content) and ability to thrive in harsh climates make it an important regional food crop throughout the Andean region. The objectives of this study were to develop genetic markers and to quantify genetic diversity within cañahua. A set of 43 wild and cultivated cañahua genotypes and two related species (Chenopodium quinoa Willd. and Chenopodium petiolare Kunth) were evaluated for polymorphism using 192 microsatellite markers derived from random genomic cañahua sequences produced by 454 pyrosequencing of cañahua genomic DNA. Another 424 microsatellite markers from C. quinoa were also evaluated for cross-species amplification and polymorphism in cañahua. A total of 34 polymorphic microsatellite marker loci were identified which detected a total of 154 alleles with an average of 4.5 alleles per marker locus and an average heterozygosity value of 0.49. A cluster analysis, based on Nei genetic distance, clearly separated from wild cañahua genotypes from the cultivated genotypes. Within the cultivated genotypes, subclades were partitioned by AMOVA analysis into six model-based clusters, including a subclade consisting sole of erect morphotypes. The isolation by distance test displayed no significant correlation between geographic collection origin and genotypic data, suggesting that cañahua populations have moved extensively, presumably via ancient food exchange strategies among native peoples of the Andean region. The molecular markers reported here are a significant resource for ongoing efforts to characterize the extensive Bolivian and Peruvian cañahua germplasm banks, including the development of core germplasm collections needed to support emerging breeding programs.

Potato silver scurf affected by tuber seed treatments and locations, and occurrence of fungicide resistant isolates of Helminthosporium solani

The effectiveness of various seed-tuber treatments was evaluated for control of silver scurf on potato (Solanum tuberosum), caused by Helminthosporium solani, at four locations in Washing-ton and Oregon using seed-tubers from the same source. Disease incidence was determined at harvest and following storage, and differed significantly among locations. The highest incidence of disease was observed at Redmond, OR and the lowest was at Hermiston, OR. Significantly less silver scurf occurred on progeny-tubers, regardless of location, when seed was treated with the fungicide treatments fludioxonil, fludioxonil + quintozene, azoxystrobin, or thiophanate-methyl + mancozeb compared with the nontreated control. A sample of H. solani from seed planted in the Columbia Basin of Washington and Oregon was assessed for resistance to thia-bendazole (TBZ) and thiophanate-methyl (TPM). Sensitivity of 20 isolates of H. solani to TBZ was determined on V8 media amended with TBZ. Four isolates, selected as a subset from the 20 isolates tested for TBZ sensitivity, were tested for resistance to TPM, mancozeb, and TPM + mancozeb on amended V8 media. Isolates differed significantly in sensitivity to both chemicals. Thirteen isolates out of the 20 tested were resistant to TBZ. One out of the four subsets of isolates was resistant to both TBZ and TPM. TBZ sensitivity was not related to geographic origin of the isolates. Variation in resistance was evident on a small scale as seen with one resistant and one sensitive isolate collected from the same tuber. This is the first report that silver scurf incidence is affected by location despite the same tuber-seed source and also the first report of TBZ and TPM resistant isolates of H. solani from the Columbia Basin (Oregon and Washington) production area.