Thesis (M.S.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 59 p. : ill. Vita. Includes abstract. Includes bibliographical references.

No description available.

Ingestion of endophyte infected (E+) fescue by pregnant mares can cause significant reproductive problems. Plateau® and Cimarron® herbicides suppress fescue while leaving desired forages unharmed. To determine if these herbicides are harmful to pregnant mares, they were allowed to graze pastures treated with Plateau®, Cimarron®, or vehicle carrier. Pregnancies were monitored via ultrasonography, blood chemistry, and hematology. Of the components measured only creatinine differed among treatments over time (P=0.0003) and that increase was only significant in one of four studies. Two additional experiments were conducted to determine the effect of the herbicides on alkaloids within E+ fescue. A greenhouse experiment utilizing 52 pots of E+ fescue treated with Plateau®, Cimarron®, or nothing was inconclusive, as some alkaloids increased while others decreased. These results indicated that UV light may be required for normal plant death. In a field experiment 12 plots of mixed vegetation were sprayed with the same treatments, and herbicides decreased ergovaline, N-formylloline, and lysergic acid content (P=0.0460, P=0.0324, P=0.0093 respectively). In conclusion, the herbicides did not alter blood components outside physiological norms, but the alkaloids were still present in dying E+ fescue. It may be safest to remove late gestation mares until E+ fescue is completely decayed.

Neotyphodium are fungal endosymbionts of grasses that reproduce asexually by infecting the hosts seed. This relationship has traditionally been considered mutualistic, with the fungus improving host fitness by alleviating important stresses. To determine the importance of biotic and abiotic stresses in mediating the endophyte-grass interaction, I investigated the relationship between grazing pressure by collared pikas and Neotyphodium sp. infection frequency in the grass Festuca altaica in an alpine meadow. I conducted a factorial design experiment combining endophyte infection, grazing history, fungicide and fertilizer. Leaf demography and herbivory damage were monitored every two weeks. In areas with chronic grazing history, infected plants were significantly less productive than uninfected tussocks, but there was no difference at low grazing history. There was no effect of infection on the likelihood of herbivory. Contrary to predictions of the mutualism theory, the Neotyphodium sp. / F. altaica symbiotum varied from parasitic to neutral across our gradient of interest. / Ecology

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains x, 149 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 130-149).

Some endophytes in mutualistic associations with Festuca, Lolium and other grass species produce insecticidal loline alkaloids (1-aminopyrrolizidines; LA). These loline alkaloids have a saturated pyrrolizidine ring system (two-rings sharing a carbon and nitrogen atom), a 1-amine substituted with methyl, acetyl, or formyl groups, and an oxygen bridge between C-2 and C-7. The development of a reliable system of production of LA in cultures of the Lolium pratense (meadow fescue) endophyte, Neotyphodium uncinatum, facilitated work on the LA biosynthetic pathway. N. uncinatum produced norloline, loline, methylloline, N-acetylnorloline (NANL), N-formylloline (NFL), and N-acetylloline as detected in culture filtrates. The total production of the two most abundant alkaloids, NANL and NFL, approached 1000 g ml-1 of fungal filtrate. 1H and 13C chemical shifts were previously reported for this group of alkaloids. Extraction and synthesis of sufficient quantities of the alkaloids allowed determination of previously unknown 15N chemical shifts of some LA. Knowledge of 13C and 15N chemical shifts allowed identification of precursors by feeding stable-isotope-labeled compounds. Initially, due to structural similarity to other plant pyrrolizidines, this study examined putrescine and spermidine as possible precursors to LA. Feeding of 14C putrescine to the fungal cultures failed to demonstrate any enrichment in the LA, but enriched spermidine. In contrast, cultures fed with positionally labeled 2H, 13C and 15N amino acids namely, L-ornithine, L-proline, L-aspartate, L-homoserine, and L-methionine demonstrated specific isotopic enrichment in NFL. Determination of the enrichment from the labeled amino acids utilized 13C and 15 N NMR (nuclear magnetic resonance) and gas chromatography-mass spectroscopy (GC-MS). This study allowed the biosynthetic origins of all carbons and nitrogens of NFL to be determined. NFL incorporated L-proline into the B-ring and L-homoserine into the A-ring and 1-amine. The results strongly indicated that polyamines are not precursors of LA and implicated a novel biochemical pathway for the synthesis of LA.

Components of successful pest management programs must be complementary and not antagonistic. This project examined interactions between natural enemies of the black cutworm, Agrotis ipsilon (Hufnagel), an important turfgrass pest, and host plant resistance by endophytic grass. Agrotis ipsilon nucleopolyhedrovirus (AgipMNPV) was examined as a bio-insecticide for controlling A. ipsilon in turfgrass. Fresh (1-week-old) AgipMNPV residues killed 76−86% of neonates hatching from eggs on golf course tees, however, residual control of implanted larvae lasted no more than a few weeks. Combinations of AgipMNPV with adjuvants, such as optical brightener and lignin, failed to accelerate or extend efficacy of the virus. AgipMNPV seems better suited for targeted control of early instars than for season-long control. Several applications per growing season would likely be needed to maintain high enough titers on turfgrass to effectively control cutworms. The addition of a chitin synthesis inhibiting turfgrass fungicide failed to synergize AgipMNPV infectivity to A. ipsilon. Choice tests revealed the fungicide residues to be a mild feeding deterrent, the likely cause of slightly reduced mortality from virus infection seen in field trials. Combination applications in turfgrass might interfere with larval ingestion of a lethal virus dose, resulting in prolonged feeding in the field. I examined how feeding on perennial ryegrass (Lolium perenne) with or without Neotyphodium lolii, its alkaloid-producing fungal endophyte, affects susceptibility of A. ipsilon to AgipMNPV. Feeding on endophytic grass neither compromises nor synergizes infectivity of AgipMNPV in the cutworm midgut. However, reduced consumption or avoidance of less-palatable endophytic grass could decrease ingestion of virus and rates of subsequent mortality in the field. Host feeding on endophytic grass had differing effects on the tachinid fly, Linnaemya comta, a fast-developing solitary parasitoid, and the encyrtid wasp, Copidosoma bakeri, a slow-developing gregarious parasitoid. L. comta development did not appear to be affected when its host fed on endophytic grass; in contrast, C. bakeri suffered negative fitness effects. These results suggest that parasitoid life strategy and taxonomy play a role in endophyte mediated tritrophic interactions.

Content removed due to copyright: Young, C. A., Bryant, M. K., Christensen, M. J., Tapper, B. A., Bryan, G. T., & Scott, B. (2005). Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass. Molecular Genetics and Genomics, 274(1), 13-29. / Lolitrems are indole-diterpene alkaloids produced by Epichloë and Neotyphodium endophytes in association with their host grass Lolium perenne. Some indole-diterpene (ID) alkaloids are proposed to have insecticidal properties, but lolitrem B is known as the causative agent of the animal syndrome ryegrass staggers. Lolitrems are preferentially synthesised in planta. which suggests that the genes required for lolitrem biosynthesis are symbiotically expressed. The lolitrem biosynthesis pathway has been proposed as a metabolic grid based on the identification of likely intermediates from endophyte-infected ryegrass. Closely related ID compounds are expected to serve as substrates for the same enzyme, but until recently these steps had not been validated. The identification and characterisation of a Petticillium paxilli gene cluster required for the synthesis of the ID paxilline has identified key enzymes required for the production of the ID backbone. Based on the similarity of lolitrem B to paxilline it was proposed that these two biosynthesis pathways would share orthologous early steps but later steps to convert paxilline to the more complex lolitrem B would require additional enzymes. The lolitrem biosynthesis genes (ltm) were isolated using degenerate PCR and from candidate genes identified as ESTs in cDNA libraries. Ten ltm genes were identified that had functions consistent with those required for lolitrem B biosynthesis. The 10 ltm genes were contained on three gene clusters that are separated by repetitive AT-rich sequences that contain remnants of retrotransposons. The ltm clusters 1 and 2 contain eight genes, seven of which are orthologues of the characterised P. paxilli paxilline biosynthesis gene cluster (pax). Functional characterisation of ltmM an FAD-dependent monooxygenase and ltmC a prenyl transferase confirmed these two genes were required for ID biosynthesis and were orthologues of paxM and paxC, respectively. All 10 ltm genes have similar expression profiles and were highly expressed in planta where the production of lolitrem B is most prevalent. The taxonomic distribution of the ltm genes has established which endophyte strains are likely to produce ID compounds. This work provides the basis for elucidation of the lolitrem biochemical pathway and opens the way for determining how the plant regulates the synthesis of this important group of bioprotective molecules.

LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.

LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.