Genetic Relationships in the Toxin-Producing Fungal Endophyte, Alternaria oxytropis Using Polyketide Synthase and Non-Ribosomal Peptide Synthase Genes

The legume Oxytropis sericea hosts a fungal endophyte, Alternaria oxytropis, which produces secondary metabolites (SM), including the toxin swainsonine. Polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) enzymes are associated with biosynthesis of fungal SM. To better understand the origins of the SM, an unannotated genome of A. oxytropis was assessed for protein sequences similar to known PKS and NRPS enzymes of fungi. Contigs exhibiting identity with known genes were analyzed at nucleotide and protein levels using available databases. Software were used to identify PKS and NRPS domains and predict identity and function. Confirmation of sequence for selected gene sequences was accomplished using PCR. Thirteen PKS, 5 NRPS, and 4 PKS-NRPS hybrids were identified and characterized with functions including swainsonine and melanin biosynthesis. Phylogenetic relationships among closest amino acid matches with Alternaria spp. were identified for seven highly conserved PKS and NRPS, including melanin synthesis. Three PKS and NRPS were most closely related to other fungi within the Pleosporaceae family, while five PKS and PKS-NRPS were closely related to fungi in the Pleosporales order. However, seven PKS and PKS-NRPS showed no identity with fungi in the Pleosporales or the class Dothideomycetes, suggesting a different evolutionary origin for those genes.

Control of Silky Crazyweed (Oxytropis sericea) with Aminopyralid + 2,4-D and Picloram + 2,4-D on Native Rangeland

Techniques for preventing crazyweed toxicity in livestock have generally fallen into two categories: excluding livestock access to infested ranges during early spring and fall, and controlling crazyweed populations through herbicide application. Although picloram has been used to control crazyweed effectively in the past, aminopyralid has shown efficacy at lower application rates, exhibits less potential off-target movement, and has been classified as a reduced-risk product. Differences in the response of silky crazyweed and nontarget grasses and forbs to picloram + 2,4-D and aminopyralid + 2,4-D were investigated. Picloram + 2,4-D was applied at a rate of 0.3 kg ae ha−1picloram + 1.1 kg ae ha−12,4-D, and aminopyralid + 2,4-D was applied at a rate of 0.1 kg ae ha−1aminopyralid + 1.2 kg ae ha−12,4-D. Silky crazyweed canopy cover, number of flowering stalks, plant size, and biomass decreased 15 mo after herbicide treatments (MAT) with average percentage of relative reductions of 92, 95, 90, and 99%, respectively. Crazyweed density decreased by 1.5 ± 0.2 SE plants m−2and 1.3 ± 0.2 plants m−2, a relative reduction of 95 and 80%, 15 MAT in aminopyralid + 2,4-D– and picloram + 2,4-D–treated plots, respectively. Plots treated with aminopyralid + 2,4-D had 4% lower nontarget forb canopy cover than did picloram + 2,4-D plots 15 MAT. Grass biomass remained similar within treatments over time for control, aminopyralid + 2,4-D and picloram +2,4-D plots, and was similar in all plots 15 MAT. Plots treated with herbicides had, on average, 11% greater grass cover than did control plots 15 MAT (aminopyralid + 2,4-D: 89%; picloram + 2,4-D: 85%; control: 76%).

Detection and localization of the endophyte Undifilum oxytropis in locoweed tissues

Poisoning of livestock owing to grazing on locoweeds results in significant economic losses in the western United States. Some Oxytropis spp. locoweeds contain a seed-transmitted endophytic fungus, Undifilum oxytropis, which produces the toxic alkaloid swainsonine. We sought to localize and characterize growth patterns of the fungus within leaves and petioles of Oxytropis lambertii Pursh and Oxytropis sericea Nutt. to help define the types of interactions between the fungus and its hosts. Vegetative hyphae were observed within locoweed tissues using integrated imaging. Topographical images from scanning electron microscopy revealed the presence of the endophyte in the pith tissue of petioles. The fungus was identified between plant cells but did not appear to penetrate host cells. Transmission electron microscopy images of thin sections revealed that hyphae were closely associated with host cell walls. Oxytropis sericea was innoculated with green fluorescent protein-transformed U. oxytropis and observed by confocal microscopy, confirming the presence of the endophyte hyphae in leaves and petioles. The fungus was identified only in the pith of petioles using fluorescence and in the vascular bundle throughout extracellular spaces in leaves. These results revealed no signs of a pathogenic interaction between plant and fungus and support the hypothesis of a mutualistic or commensal relationship.

Localization of endophytic Undifilum fungi in locoweed seed and influence of environmental parameters on a locoweed in vitro culture system

Endophytic Undifilum oxytropis (Q. Wang, Nagao & Kakish) Pryor, Creamer, Shoemaker, McLain-Romero, & Hambleton found within toxic locoweeds ( Astragalus and Oxytropis spp.) produces the alkaloid swainsonine, which is responsible for locoism in grazing animals. We sought to determine the location of U. oxytropis within locoweed seed, develop endophyte free plants, and assess the influence of environmental stresses on locoweed and endophyte cultures. Undifilum was identified within the parenchyma layers in seeds of Astragalus lentiginosus M.E. Jones using light microscopy and polymerase chain reaction. Astragalus lentiginosus and Oxytropis sericea Nutt. seedlings produced in embryo culture without seed coats did not contain swainsonine or fungus. Plants produced from whole seed contained U. oxytropis in both foliage and root tissues. When the in-vitro cultured plants of O. sericea and U. oxytropis cultures were subjected to environmental stresses including high temperature, low and high pH media, nutrient deficient media, and polyethylene glycol (PEG) amended media to simulate water deficit, both dry mass and swainsonine levels were affected. Swainsonine levels were greatest for O. sericea and Undifilum cultures in PEG or hydrochloric acid amended media. Plants grown in PEG-amended media had significantly greater dry mass, while Undifilum grown in PEG-amended media had lower dry mass than other treatments.