Urnula sp., an Endophyte of Dicksonia antarctica, Making a Fragrant Mixture of Biologically Active Volatile Organic Compounds.

Urnula 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 50 g of beet pulp waste, using stainless steel carbotraps, yielded over 180 mg of hydrocarbon-based products collected over 6 weeks 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.

Fungi as Architects of the Rimstone Dams in Huanglong, NSD, Sichuan, China.

The Huanglong park area of the Sichuan Province of China is a unique scenic area of the world. It is known for its thousands of aquamarine-colored pools that are formed behind naturally formed rimstone dams of travertine (calcite) along a cold water stream. The travertine, based on its crystalline structural analysis, is of biological origin. This makes sense since the temperature of the waters of Huanglong varies from 5 to 7 °C and thus geochemical crystallization does not occur as it does in other locations around the world possessing thermal pools whose structures are primarily formed through cooling processes. Fungi and bacteria were discovered associated with both leaves associated with the calcite dams as well as in the older parts of well-established dams. Several species of Phytium, a phycomycete and an endophyte, accounted for over 45 % of all of the fungi successfully isolated from the well-established dam samples and at least 85 % in the floating leaf samples. Saprolegnia spp. (Phycomycetes) along with Phoma spp. (Ascomycetes) were noted along with Mortierella sp. as other dam-associated fungi. The fungal hyphae observed on dead leaf material as well as in the calcite dams directly served as nucleation points for the formation of crystalline CaCO3. Eventually, these crystals grow large enough to fuse to make calcite plates which form the main structural feature of all of the travertine dams in this area. Interestingly, each of the individual crystals associated with the dams has an associated hole in its core where a fungal hypha used to reside as observed by scanning electron microscopy. While diatoms were present in the analysis, they too seem to contribute to the structure of the dams but in a minor way. The only bacteria isolated from the older dam of this aquatic environment were Pseudomonas spp. and their role in dam formation is uncertain. Huanglong is a unique and beautiful place, and the water features present in this area can definitely be attributed to those fungal architects that encourage calcite crystal formation.

Postdispersal Infection and Disease Development of Pyrenophora semeniperda in Bromus tectorum Seeds.

The Ascomycete fungus, Pyrenophora semeniperda, attacks a broad range of cool-season grasses. While leaf and predispersal infection of seeds (i.e., florets containing caryopses) have been previously characterized, little is known about the pathogenesis of mature seeds following dispersal. In this study, we examined infection and disease development of P. semeniperda on dormant seeds of Bromus tectorum. Inoculated seeds were hydrated at 20°C for up to 28 days. Disease development was characterized using scanning electron and light microscopy. P. semeniperda conidia germinated on the seed surface within 5 to 8 h. Hyphae grew on the seed surface and produced extracellular mucilage that eventually covered the seed. Appressoria formed on the ends of hyphae and penetrated through the lemma and palea, stomatal openings, and broken trichomes. The fungus then catabolized the endosperm, resulting in a visible cavity by 8 days. Pathogenesis of the embryo was associated with progressive loss of cell integrity and proliferation of mycelium. Beginning at approximately day 11, one to several stromata (approximately 150 µm in diameter and up to 4 mm in length) emerged through the lemma and palea. Degradation of embryo tissue was completed near 14 days. Conidiophores produced conidia between 21 and 28 days and often exhibited "Y-shaped" branching. This characterization of disease development corrects previous reports which concluded that P. semeniperda is only a weak seed pathogen with infection limited to the outermost seed tissues. In addition, the time required for disease development explains why infected dormant or slow-germinating seeds are most likely to experience mortality.

An endophytic Coniochaeta velutina producing broad spectrum antimycotics.

An endophyte (PC27-5) was isolated from stem tissue of Western hemlock (Tsuga heterophylla) in a Pacific Northwest temperate rainforest. Phylogenetic analyses, based on ITS-5.8S rDNA and 18S rDNA sequence data, combined with cultural and morphological analysis showed that endophyte PC27-5 exhibited all characteristics of a fungus identical to Coniochaeta velutina. Furthermore, wide spectrum antimycotics were produced by this endophyte that were active against such plant pathogens as Sclerotinia sclerotiorum, Pythium ultimum, and Verticillium dahliae and lethal to Phythophthora cinnamomi, Pythium ultimum, and Phytophthora palmivora in plate tests. The bioactive components were purified through organic solvent extraction, followed by silica column chromatography, and finally preparative HPLC. The minimum inhibitory concentration of the active fraction to Pythium ultimum, which was gained from preparative HPLC, was 11 µg/ml. UPLC-HRMS analysis showed there were two similar components in the antimycotic fraction. Their molecular formulae were established as C30H22O11 (compound I) and C30H22O10 (compound II) respectively, and preliminary spectral results indicate that they are anthroquinone glycosides. Other non-biologically active compounds were identified in culture fluids of this fungus by spectral means as emodin and chrysophanol--anthroquinone derivatives. This is the first report that Coniochaeta velutina as an endophyte produces bioactive antifungal components.

Characterization of an Endophytic Gloeosporium sp. and Its Novel Bioactivity with "Synergistans".

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.

Identification of the Infection Route of a Fusarium Seed Pathogen into Nondormant Bromus tectorum Seeds.

The genus Fusarium has a wide host range and causes many different forms of plant disease. These include seed rot and seedling blight diseases of cultivated plants. The diseases caused by Fusarium on wild plants are less well-known. In this study, we examined disease development caused by Fusarium sp. n on nondormant seeds of the important rangeland weed Bromus tectorum as part of broader studies of the phenomenon of stand failure or "die-off" in this annual grass. We previously isolated an undescribed species in the F. tricinctum species complex from die-off soils and showed that it is pathogenic on seeds. It can cause high mortality of nondormant B. tectorum seeds, especially under conditions of water stress, but rarely attacks dormant seeds. In this study, we used scanning electron microscopy (SEM) to investigate the mode of attack used by this pathogen. Nondormant B. tectorum seeds (i.e., florets containing caryopses) were inoculated with isolate Skull C1 macroconidia. Seeds were then exposed to water stress conditions (-1.5 MPa) for 7 days and then transferred to free water. Time lapse SEM photographs of healthy versus infected seeds revealed that hyphae under water stress conditions grew toward and culminated their attack at the abscission layer of the floret attachment scar. A prominent infection cushion, apparent macroscopically as a white tuft of mycelium at the radicle end of the seed, developed within 48 h after inoculation. Seeds that lacked an infection cushion completed germination upon transfer to free water, whereas seeds with an infection cushion were almost always killed. In addition, hyphae on seeds that did not initiate germination lacked directional growth and did not develop the infection cushion. This strongly suggests that the fungal attack is triggered by seed exudates released through the floret attachment scar at the initiation of germination. Images of cross sections of infected seeds showed that the fungal hyphae first penetrated the caryposis wall, then entered the embryo, and later ramified throughout the endosperm, completely destroying the seed.

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.

Collophora aceris, a novel antimycotic producing endophyte associated with Douglas Maple.

A novel endophyte designated Collophora aceris, was obtained from stem tissues of Douglas Maple (Acer glabrum var. douglasii) in a Pacific Northwest temperate rainforest. Colonies were slow growing, white, creamy, moist, and translucent to opaque on potato dextrose agar and other media with few aerial hyphae. It also produced solid, dark sclerotia (200-400 µm) on oatmeal agar and no evidence of pseudopycnidia as per other Collophora spp. Conidia were rod-like in the size ranging from 2.2-8.4 × 0.8-1.8 µm and produced holoblastically on conidiogenous cells by budding with no collarette at the budding site. Phylogenetic analyses, based on 18S rDNA sequence data, showed that C. aceris possessed 99 % similarity to other Collophora spp. However, ITS-5.8S rDNA sequence data indicated that the organism was potentially related to Allantophomopsis spp. Finally, combined morphological, physiological, and molecular genetics data indicated that this organism is most like Collophora spp. but it is distinctly unique when compared to all other fungi in this group. It is to be noted that this is the first report of any member of this genus existing as an endophyte. This fungus makes a wide spectrum antimycotic agent (Collophorin) with biological activity against such pathogenic fungi as Pythium ultimum, Phytophthora cinnamomi, Phytophthora palmivora, and Rhizoctonia solani. Collophorin was purified to homogeneity and shown to have a unique mass of 120.0639, an empirical formula of C8H8O1, and UV absorption bands at 260 and 378 nm. This work also indicates that C. aceris possesses the biological potential to provide protection of its host against an array of common plant pathogens.

Developing molecular tools and insights into the Penstemon genome using genomic reduction and next-generation sequencing.

BACKGROUND: Penstemon's unique phenotypic diversity, hardiness, and drought-tolerance give it great potential for the xeric landscaping industry. Molecular markers will accelerate the breeding and domestication of drought tolerant Penstemon cultivars by, creating genetic maps, and clarifying of phylogenetic relationships. Our objectives were to identify and validate interspecific molecular markers from four diverse Penstemon species in order to gain specific insights into the Penstemon genome. RESULTS: We used a 454 pyrosequencing and GR-RSC (genome reduction using restriction site conservation) to identify homologous loci across four Penstemon species (P. cyananthus, P. davidsonii, P. dissectus, and P. fruticosus) representing three diverse subgenera with considerable genome size variation. From these genomic data, we identified 133 unique interspecific markers containing SSRs and INDELs of which 51 produced viable PCR-based markers. These markers produced simple banding patterns in 90% of the species × marker interactions (~84% were polymorphic). Twelve of the markers were tested across 93, mostly xeric, Penstemon taxa (72 species), of which ~98% produced reproducible marker data. Additionally, we identified an average of one SNP per 2,890 bp per species and one per 97 bp between any two apparent homologous sequences from the four source species. We selected 192 homologous sequences, meeting stringent parameters, to create SNP markers. Of these, 75 demonstrated repeatable polymorphic marker functionality across the four sequence source species. Finally, sequence analysis indicated that repetitive elements were approximately 70% more prevalent in the P. cyananthus genome, the largest genome in the study, than in the smallest genome surveyed (P. dissectus). CONCLUSIONS: We demonstrated the utility of GR-RSC to identify homologous loci across related Penstemon taxa. Though PCR primer regions were conserved across a broadly sampled survey of Penstemon species (93 taxa), DNA sequence within these amplicons (12 SSR/INDEL markers) was highly diverse. With the continued decline in next-generation sequencing costs, it will soon be feasible to use genomic reduction techniques to simultaneously sequence thousands of homologous loci across dozens of Penstemon species. Such efforts will greatly facilitate our understanding of the phylogenetic structure within this important drought tolerant genus. In the interim, this study identified thousands of SNPs and over 50 SSRs/INDELs which should provide a foundation for future Penstemon phylogenetic studies and breeding efforts.

An endophytic Nodulisporium sp. from Central America producing volatile organic compounds with both biological and fuel potential.

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 Paleobiosphere: a novel device for the in vivo testing of hydrocarbon producing-utilizing microorganisms.

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,8aß)]. 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.

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.

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 Phomopsis sp. possessing bioactivity and fuel potential with its volatile organic compounds.

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 IC(50) 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.

A survey of Penstemon's genome size.

Penstemon is the largest genus in North America with more than 270 reported species. However, little is known about its genome size. This information may be useful in developing hybrids for landscape use and for gaining insight into its current taxonomy. Using flow cytometry, we estimated the genome size of approximately 40% of the genus (115 accessions from 105 different species). Genome sizes for both reported and probable diploids range from P. dissectus 2C = 0.94 pg (1C = 462 Mbp) to P. pachyphyllus var. mucronatus 2C = 1.88 pg (1C = 919 Mbp), and the polyploids range from P. attenuatus var. attenuatus 2C = 2.35 pg (1C = 1148 Mbp) to P. digitalis 2C = 6.45 pg (1C = 3152 Mbp). Chromosome counts were done for ten previously published and four previously unreported Penstemon species (P. dissectus, P. navajoa, P. caespitosus var. desertipicti, and P. ramaleyi). These counts were compiled with all previously published chromosome data and compared with the flow cytometry results. Ploidy within this study ranged from diploid to dodecaploid. These data were compared and contrasted with the current taxonomy of Penstemon and previously published internal transcribed spacer and chloroplast DNA phylogenetic work. Based on genome size and previous studies, reassigning P. montanus to the subgenus Penstemon and P. personatus to the subgenus Dasanthera, would better reflect the phylogeny of the genus. Furthermore, our data concur with previous studies suggesting that the subgenus Habroanthus be included in the subgenus Penstemon. The DNA content of subgenus Penstemon exhibits high plasticity and spans a sixfold increase from the smallest to the largest genome (P. linarioides subsp. sileri and P. digitalis, respectively). Our study found flow cytometry to be useful in species identification and verification.

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.

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.

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.

Postinfection Activity of Selected Late Blight Fungicides.

Two sets of experiments were done in the greenhouse with 'Russet Burbank' potatoes infected with Phytophthora infestans. The first determined the effectiveness of selected late blight fungicides in restricting lesion length and spore production when applied after stem lesions had occurred on potted plants. The second quantified the curative activity of selected fungicides applied 12 to 48 h after inoculation of excised leaves with P. infestans. Such information may be beneficial in selecting an effective fungicide for late blight management when symptoms of the disease are present in a field. Expansion of established stem lesions was consistently restricted by one application of either dimethomorph plus mancozeb or metiram plus triphenyltin hydroxide. Sporulation on established stem lesions was consistently inhibited by either one application of dimethomorph plus mancozeb or cymoxanil plus mancozeb. Two applications of propamocarb hydrochloride plus chlorothalonil were required to restrict lesion expansion and inhibit sporulation. Size and number of lesions and sporulation on leaflets were reduced when propamocarb hydrochloride plus chlorothalonil was applied up to 48 h after inoculation.

Fungicide Application for Late Blight Management: A Boom Attached to a Center Pivot Irrigation System.

Chlorothalonil was applied to commercial potato fields of cvs. Russet Norkotah and Russert Burbank in replicated trials through either a center-pivot irrigation system or a spray-boom-attached-to-the-pivot system. Water rates were 26,192 and 675 liters/ha for the center pivot and attached boom, respectively. Three crop canopy levels were sampled within 2 h and again after 6 days following fungicide applications made at 7-day intervals. Efficacy of the two application methods was tested by inoculation of individual excised leaves with an isolate of strain US-1 or US-8 of Phytophthora infestans and by chemical residue analysis. Severity of late blight did not differ between the two fungicide application methods 2 h after application but, after 6 days, disease severity was significantly less on leaves where chlorothalonil was applied by the attached boom. Fungicide residues were three times higher on leaves where chlorothalonil was applied by the attached boom than with the center-pivot system. Significantly more disease occurred on fungicide-treated and non-treated leaves inoculated with the US-8 than the US-1 strain. Severity of late blight on inoculated leaves was inversely related to the amount of chlorothalonil residue on leaves.