Molecular Characterization of a Fungal Ketide Synthase Gene Among Swainsonine-Producing Alternaria Species in the USA

Locoweeds are toxic leguminous plants in Astragalus and Oxytropis genera that contain fungal endophytes of Alternaria section Undifilum species. These fungi produce swainsonine, an alkaloid α-mannosidase inhibitor that causes a neurological syndrome, locoism in grazing animals. A SWN gene cluster has been identified in many swainsonine-producing fungi. The swnK gene, which is an essential component of the swainsonine biosynthetic pathway, encodes a polyketide synthase-nonribosomal peptide synthase (PKS-NRPS). To determine if swnK was conserved among Alternaria section Undifilum endophytes of locoweed, the sequence of the KS region of swnK was compared between various swainsonine-producing fungi. The internal transcribed spacer (ITS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) regions from the same fungi were also assessed. Sequences were examined at the nucleotide and protein levels. Alternaria oxytropis , A. fulva , A. cinerea, and Alternaria sp. from Swainsona species produced distinct clades for all multigene data sets. swnK –KS sequence did not differ among fungi isolated from Astragalus mollissimus varieties or A. lentiginosus varieties. The swnK-KS amino acid sequence was essentially identical among all swainsonine-producing Alternaria sp. Two low swainsonine-producing fungi, Alternaria bornmuelleri and A. gansuense , clustered together, as did non-pathogen Alternaria endophytes. The swnK -KS sequence comparisons were effective in identifying swainsonine production capability and differentiating among swainsonine-producing fungal species.