Year of Publication

2004

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Agriculture

Department

Plant Pathology

First Advisor

Dr. Christopher Lewis Schardl

Abstract

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.

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