Theses and Dissertations at Montana State University (MSU)
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Item Identification and cultivation of methyllycaconitine degraders from wild ruminants to protect against larkspur poisoning in range cattle(Montana State University - Bozeman, College of Agriculture, 2021) Grace, Savannah Gray; Chairperson, Graduate Committee: Carl Yeoman and Craig Carr (co-chair); Joanna-Lynn C. Borgogna, Craig A. Carr, Lance B. McNew, Brian Bothner and Carl J. Yeoman were co-authors of the article, 'Degradation of toxic alklaoids in Delphinium occidentale species occurs within the gastrointestinal tract of Montana's wild ruminants' submitted to the journal 'American Society of Animal Science' which is contained within this thesis.; Joanna-Lynn C. Borgogna, Mostafa Elshahed, Lance B. McNew, Brian Bothner, Craig A. Carr and Carl J. Yeoman were co-authors of the article, 'Degradation of the toxic alkaloid, methyllycaconitine by wild ruminant species is predominantly mediated by rumen fungi' submitted to the journal 'American Society of Animal Science' which is contained within this thesis.Tall larkspur (Delphinium spp.) in the western United States present a serious toxicity danger to rangeland cattle. Consumption of Methyllycaconitine (MLA), the toxic alkaloid in larkspur plants, can cause annual losses of 5-15% of range cattle in grazing pastures with sufficient larkspur. With the wide distribution and abundance of larkspur, wild ruminants in Montana likely encounter tall larkspur while foraging; however, no evidence suggests they are negatively affected by MLA's toxic effects. Therefore, we evaluated: i) whether alkaloids in Delphinium spp., and MLA specifically degraded within ruminal specimens collected from Montana's wild ruminant species over 48 h using in vitro incubations; ii) whether observed degradative activities were abiotic, or mediated by either the fungal or non-fungal (mostly bacterial) residents of the ruminal microbiota in wild ruminant specimens; and iii) if representative microbial isolates individually possessed the ability to degrade MLA within in vitro incubations. Rumen samples were collected from wild ruminant species during the 2019 and 2020 hunting seasons using legal methods by volunteer hunters. In all assays, total alkaloid was measured spectrophotometrically, and MLA by High-Performance Liquid Chromatography Mass Spectrometry (HPLC) from initial and final incubations. Our results demonstrated that, with the exception of white-tailed deer, all wild ruminant species exhibited variable degradative abilities in both total alkaloid (P< 0.001) and MLA (P< 0.001) assays and that such degradation was predominantly mediated by ruminal fungi. Additionally, screening of 15 fungal isolates, representing 10 known genera and 2 isolates of unknown taxonomic identity each obtained from herbivorous hosts, determined all were capable of degrading MLA to some extent. Fungal isolates obtained from wild ruminants exhibited greater degradative activity, with Aestipasuomyces R5 isolated from wild sheep degrading 71% of MLA (P<0.001). Overall, our results indicate that degradation of both total alkaloid and MLA-specifically occurs within the gastrointestinal tract of Montana's wild ruminants and that it is largely influenced by fungal activity. Additionally, fungal strains isolated from wild ruminants are capable of degrading MLA and have the potential to be further used as a direct fed microbial to rangeland cattle as an optimal way to mitigate larkspur toxicosis.Item Understanding the Coprophilous fungus Sporormiella as a proxy for megaherbivores(Montana State University - Bozeman, College of Letters & Science, 2020) Ulrich, Barbara Carol; Chairperson, Graduate Committee: Cathy WhitlockIn many studies, the presence of the coprophilous fungus Sporormiella in Quaternary sediments has been interpreted as evidence of past megaherbivore activity. Despite its use as an ecological proxy, little is known about the taxonomy and life history of Sporormiella, and the taphonomic processes that lead to its preservation in lake sediments. This information underlies its utility to interpret past herbivore presence and abundance. Present-day bison (Bison bison) dung from Yellowstone National Park was examined to explore the production, dispersal, transportation, deposition, and preservation of Sporormiella there. While Sporormiella was found in dung samples, sediments from two lakes frequently visited by bison failed to yield Sporormiella spores. Laboratory preparation techniques were modified to increase the likelihood of Sporormiella spore survival, yet no spores were identified with the new treatment. Although the occurrence of spores in lake-sediment samples may indicate herbivore presence, our study suggests that spore absence does not necessarily indicate an absence of herbivores. We attribute the absence of spores in sediments to local climatic and seasonal factors that may affect production and transport in the watershed, sedimentary processes that may destroy spores after deposition, and harsh laboratory processing techniques that may damage or destroy spores. More research remains to be done to evaluate the importance of these factors before using Sporormiella as a reliable proxy of herbivore activity.Item The characterization of fungicide resistance, population structure, and aggressiveness of fungal species associated with ascochyta blight of pulse crops in Montana(Montana State University - Bozeman, College of Agriculture, 2019) Owati, Ayodeji Stephen; Chairperson, Graduate Committee: Mary Burrows and Bright Agindotan (co-chair); Bright Agindotan, Julie Pasche and Mary Burrows were co-authors of the article, 'The detection and characterization of QOI-resistant Didymella rabiei causing ascochyta blight of chickpea in Montana' in the journal 'Frontiers in plant science' which is contained within this dissertation.; Bright Agindotan and Mary Burrows were co-authors of the article, 'First microsatellite markers developed and applied for the genetic diversity study and population structure of Didymella pisi associated with ascochyta blight of dry pea in Montana' in the journal 'Fungal biology' which is contained within this dissertation.; Bright Agindotan and Mary Burrows were co-authors of the article, 'Characterization and detection of fungal species associated with ascochyta blight of dry pea in Montana' submitted to the journal 'Plant disease' which is contained within this dissertation.; Bright Agindotan and Mary Burrows were co-authors of the article, 'The development and application of real-time and convention SSR-PCR assays for rapid and sensitive detection of Didymella pisi associated with ascochyta blight of dry pea' submitted to the journal 'Plant disease' which is contained within this dissertation.Ascochyta blight (AB) of pulse crops causes yield loss in Montana, where 1.24 million acres were planted to pulses in 2018. Pyraclostrobin and azoxystrobin, quinone outside inhibitor (QoI) fungicides, have been the choice of farmers for the management of AB in pulses. QoI-fungicide-resistant Didymella rabiei isolates were found in one chickpea seed lot each received from Daniels, McCone and Valley Counties, MT, from seed produced in 2015 and 2016. Multiple alignment analysis of amino acid sequences showed a mutation that replaced the codon for amino acid 143 from GGT to GCT, introducing an amino acid change from glycine to alanine (G143A), which is reported to be associated with QoI resistance. Under greenhouse conditions, disease severity was significantly higher on pyraclostrobin-treated chickpea plants inoculated with QoI-resistant isolates of D. rabiei (QoI-R) than sensitive isolates (QoI-S) (p-value = 0.001). D. rabiei-specific PCR primer pair and probes were developed to discriminate QoI-R and QoI-S isolates. In North America, AB of dry pea is caused by a complex of fungal pathogens (Didymella pisi, Peyronellaea pinodes, and Peyronellaea pinodella). D. pisi is the predominant causal pathogen of AB of dry pea in Montana resulting in yield losses. Thirty-three microsatellite markers (SSR) were developed and used to analyze the genetic diversity and population structure of 205 D. pisi isolates from four geographical regions of Montana. Unweighted Neighbor-joining, principal coordinate, and population structure analyses grouped these 205 isolates into two major sub-groups. The clusters did not match the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 15% among populations. There was moderate genetic variation in the total populations (PhiPT = 0.153). Recently, a shift in pathogen composition has been observed in Montana from D. pisi to P. pinodes and P. pinodella. Also, a Phoma sp. was found associated with AB contaminated dry pea seeds and included in this study. Mycelial growth and sporulation were evaluated at different temperatures. Also, the pathogenicity of Phoma sp. and the difference in aggressiveness among the fungal pathogens was evaluated. At all temperatures, Phoma sp. had the highest growth rate (p-value = < 0.001) and produced more spores than the other species (p-value = < 0.001). P. pinodes caused greater disease severity than the other species when inoculated on pea plants (cv. Carousel, p-value < or = 0.001). The Phoma sp. was not pathogenic. Peameal agar was used to visually discriminate between fungal species. Diagnosis of AB of dry pea is challenging because of the complex of pathogens involved. Also, they have slow growth rate and different morphotypes. Currently, there are no PCR-based assays developed for D. pisi or any of the fungal pathogens associated with the AB complex of dry pea. D. pisi specific SYBR green SSR-qPCR and conventional SSR-PCR assays were developed for rapid detection and quantification of D. pisi both in-planta and in-vitro.Item Restoration of whitebark pine on a burn site utilizing native Ectomycorrhizal suilloid fungi(Montana State University - Bozeman, College of Agriculture, 2017) Jenkins, Martha Lee; Chairperson, Graduate Committee: Cathy L. CrippsThe compilation of threats both natural and anthropogenic and the resulting loss of whitebark pine has led scientists and land managers to actively pursue a strategy for restoration of this keystone species. A range-wide strategy for restoration has been developed by leading managers in the field and focuses on promoting rust resistance, conserving genetic diversity, saving seed sources, and employing restoration treatments (Keane et al. 2012). These strategies are applied across the range of whitebark pine and rely on the collaboration of land managers, scientists, and academics. Seed Source The most promising strategy for restoration of whitebark pine is the out-planting of blister rust resistant seedlings (Keane et al. 2012). Due to the continuous loss of mature cone-bearing whitebark pine, it is necessary to collect seed for blister rust resistance screening, genetic conservation, and out-planting... For the large-scale planting of 36,000 whitebark pine seedlings on the Eureka Basin Burn in the Beaverhead-Deerlodge National Forest, the first year survival of the 800 seedling subsample was high overall (94%). A method for examining how seedling-level planting variables such as colonization by suilloid ectomycorrhizal fungi, microsite type and position, slope, and potential soil moisture (TWI) affect seedling health and survival was developed and seedlings were monitored 3 and 14 months after planting. Further monitoring will continue to examine how long term seedling success is affected by these variables.Item Physiological characteristics of fungi associated with antarctic environments(Montana State University - Bozeman, College of Agriculture, 2016) Kudalkar, Priyanka Sushil; Chairperson, Graduate Committee: John C. PriscuThe permanent ice covers on the lakes of Antarctica's McMurdo Dry Valleys region harbor a diverse group of phototrophic and heterotrophic microorganisms that metabolize during the short summer months when solar radiation produces melt inclusions within the ice and provides energy to drive photosynthesis. Laboratory cultures of fungi were obtained from ice cores taken from Lakes Bonney (east lobe) and Chad, and sediments collected from Subglacial Lake Whillans (West Antarctica). Using molecular techniques, the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) was sequenced to identify fungal types and to determine whether they may be unique to this region. Four axenic fungal cultures, Tetracladium ellipsoideum, Lecythophora hoffmannii, Mucor sp., and an unidentified Ascomycota were successfully isolated. These isolates are closely related to organisms that have been previously reported in Antarctica and other cold habitats. The isolates were tested for growth characteristics under various temperature and nutrient regimes. Temperature response experiments revealed that all the isolated fungi were psychrotolerant and growth rates were greatest at 25°C. Of major significance in evaluating the potential of Antarctic fungi as a bioresource is their ability to produce bioactive compounds. Two out of four isolated organisms exhibited antimicrobial activity against several plant pathogens. The metabolic potential and preferred substrate utilization was examined by exposing fungal isolates to a variety of substrates in a 96 well "Biolog" plate. A strong correlation was found among substrate utilization, isolates, temperature and the different carbon substrates. This experiment revealed that the isolated fungi have preferences for different labile carbon substrates at 4°C and 24°C which may imply different physiologies at different times of year in the lake ice-covers. Results from my studies will help understand the role of fungi in lake ice and subglacial lake sediment ecosystems, and the physiology of fungi living in cold environments.Item Candida albicans mitochondrial dynamics during a yeast to hyphal transition(Montana State University - Bozeman, College of Agriculture, 1998) Kobayashi, Scott D.Item A study of a-aminobutyronitrile in Rhizoctonia solani(Montana State University - Bozeman, College of Letters & Science, 1972) Liu, Francis Hing SoonItem Systematics of Rocky Mountain alpine Laccaria (basidiomycota, agaricales, tricholomataceae) and ecology of Beartooth Plateau alpine macromycetes(Montana State University - Bozeman, College of Agriculture, 2003) Osmundson, Todd WilliamItem Ectomycorrhizal fungi of whitebark pine (Pinus albicaulis) in the Northern Greater Yellowstone Ecosystem(Montana State University - Bozeman, College of Agriculture, 2006) Mohatt, Katherine Rose; Chairperson, Graduate Committee: Cathy L. Cripps.Whitebark pine (Pinus albicaulis) is an integral component of subalpine ecosystems in the Western United States where it is considered a keystone species. The mature forests which colonize harsh treeline terrain provide habitat for flora and fauna an essential food source to grizzly bears, and are also important in watershed dynamics. Threats which have led to the decline of this tree species, up to 40-90% in parts of its range, include blister rust, mountain pine beetle, fire suppression, and climate change. Pines are obligate ectomycorrhizal symbionts, and host mutualistic fungi on their roots beneficial to tree establishment and sustainability; however, little is known of the ectomycorrhizal (ECM) fungi crucial to the survival of P. albicaulis. The goal of this study was to discover the species of ECM fungi associated with P. albicaulis in the Northern Greater Yellowstone Ecosystem. A survey of fruiting structures of ECM fungi in mature P. albicaulis forests from five mountain ranges revealed at least 44 species. ECM fungi in the Boletales and Cortinariales comprised 50% of the species, 24% were hypogeous, and one Chroogomphus species is new to science. An examination of the roots of P. albicaulis seedlings using morphotyping and sequencing of the ITS region revealed 19 species of ECM fungi, 11 of which were not previously revealed by sporocarps, including 2 Tomentelloid types. Cenococcum geophilum was the most frequent (64% of seedlings) and abundant (49% of root tips) ECM fungus on roots. Pinus albicaulis seeds are primarily distributed by Clark's nutcrackers which cache seed on open slopes at a distance from mature forests. A comparison of the ECM fungi on seedlings in avalanche paths and adjacent mature forests on Scotch Bonnet Mountain revealed a similar species richness, however species composition only partially overlapped . Of necessity, some "seedling clusters" were sampled instead of single seedlings, mostly from paths and they appeared to host more ECM fungi, which likely skewed results. Current efforts to restore this tree, especially by the out-planting of rust-resistant seedlings, can benefit from this research as a knowledge of the ECM fungi could help reestablish this tree in peril.Item Impact of severe fire on ectomycorrhizal fungi of whitebark pine seedlings(Montana State University - Bozeman, College of Agriculture, 2009) Trusty, Paul Evan; Chairperson, Graduate Committee: Cathy L. Cripps.Whitebark pine (Pinus albicaulis) is a threatened keystone species in subalpine zones of Western North America critical to watersheds and maintenance of high elevation biodiversity. Pine nuts are an important food for wildlife including grizzly bears. Whitebark pine stands have experienced losses up to 90% due to white pine blister rust, mountain pine beetles and replacement due to fire suppression. Active management strategies include letting natural fires burn or applying prescribed fires to clear understory fir, stimulate seedling regeneration and provide openings for nutcrackers to plant seeds. However, post-fire plantings of rust-resistant seedlings have low survival rates. This study evaluated the impact of fire on the mycorrhizal fungi which are obligate mutualists with whitebark pine and to address management concerns. The 2001 Fridley fire burned a portion of a mature whitebark pine forest and a year later 20,000 seedlings were planted. After four years, natural and planted seedlings, on the burn and controls in the adjacent unburned forest were well colonized by mycorrhizal fungi (>90%) although a portion may be nursery E-strain. The severe burn reduced mycorrhizal diversity 27% on natural and planted seedlings and caused a significant shift in mycorrhizal species (determined by ITS sequencing, principal component analysis and multidimensional scaling). Seedlings in the burn (natural and planted) were dominated by Pseudotomentella nigra, Wilcoxina species and Amphinema byssoides while natural seedlings in unburned forest hosted mainly Cenococcum geophilum and Piloderma byssinum. Differences were minimal between planted and natural seedlings in the burn, but roots of planted pines retained the container shape. The functional significance of a species shift to seedling survival is not yet known. Seedlings in all treatments hosted suilloid fungi (Rhizopogon, Suillus) important in pine establishment. A greenhouse bioassay of burned and unburned soils using nursery seedlings did not reflect the full diversity found in the field study, but did reveal suilloid fungi indicating that bioassays can be used as a pre-planting assessment tool for this group. Despite high mycorrhization and availability of suilloids, seedling survival was low (22-42%) suggesting the timing/type of mycorrhization and/or other biotic/abiotic factors are a concern.