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Polyclonal antibodies were produced in sheep for the development of a competitive enzyme-linked immunoassay for use in quantifying loline alkaloids in pasture samples. Lolines are aminopyrrolizidine secondary metabolites produced by fungal endophytes present in tall fescue and meadow fescue grasses, and confer increased resistance to a range of grass pests. Immunizing and plate coating antigens were prepared with derivatized loline dihydrochloride. Cross-reactivity studies indicated that the assay developed has broad specificity and antibody binding to loline analogues is affected by structural changes in the side chain of the loline molecule. Results obtained using the optimized immunoassay were compared with those determined by gas chromatography. The assay provides a sensitive and rapid analytical method that detects the loline analogues of interest and has been applied in pasture breeding programmes. The assay limit of quantitation for lolines in pasture is 3 µg/g in dried herbage. [ABSTRACT FROM PUBLISHER]

Endophytes of the genusEpichloë(Clavicipitaceae, Ascomycota) frequently occur within cool-season grasses and form interactions with their hosts that range from mutualistic to antagonistic. ManyEpichloëspecies have arisen via interspecific hybridization, resulting in species with two or three subgenomes that retain all or nearly all of their original parental genomes, a process termed allopolyploidization. Here, we characterizeEpichloë hybrida, sp. nov., a mutualistic species that has increasingly become a model system for investigating allopolyploidy in fungi. TheEpichloëspecies so far identified as the closest known relatives of the two progenitors ofE. hybridaareE. festucaevar.loliiandE. typhina. We confirm that the nuclear genome ofE. hybridacontains two homeologs of most protein-coding genes fromE. festucaeandE. typhina, with genome-wide gene expression analysis indicating a slight bias in overall gene expression from theE. typhinasubgenome. Mitochondrial DNA is detectable only fromE. festucae, whereas ribosomal DNA is detectable only fromE. typhina. Inheriting ribosomal DNA from just one parent might be expected to preferentially favor interactions with ribosomal proteins from the same parent, but we find that ribosomal protein genes from both parental subgenomes are nearly all expressed equally inE. hybrida. Finally, we provide a comprehensive set of resources for this model system that are intended to facilitate further study of fungal hybridization by other researchers. [ABSTRACT FROM AUTHOR]

Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxyfluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor white M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fnngal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fuoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed. [ABSTRACT FROM AUTHOR]