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

Locoweeds are toxic leguminous plants inAstragalusandOxytropisgenera that contain fungal endophytes ofAlternariasectionUndifilumspecies. These fungi produce swainsonine, an alkaloid alpha-mannosidase inhibitor that causes a neurological syndrome, locoism in grazing animals. A SWN gene cluster has been identified in many swainsonine-producing fungi. TheswnKgene, which is an essential component of the swainsonine biosynthetic pathway, encodes a polyketide synthase-nonribosomal peptide synthase (PKS-NRPS). To determine ifswnKwas conserved amongAlternariasectionUndifilumendophytes of locoweed, the sequence of the KS region ofswnKwas 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,andAlternariasp. fromSwainsonaspecies produced distinct clades for all multigene data sets.swnK-KS sequence did not differ among fungi isolated fromAstragalus mollissimusvarieties orA. lentiginosusvarieties. The swnK-KS amino acid sequence was essentially identical among all swainsonine-producingAlternariasp. Two low swainsonine-producing fungi,Alternaria bornmuelleriandA. gansuense, clustered together, as did non-pathogenAlternariaendophytes. TheswnK-KS sequence comparisons were effective in identifying swainsonine production capability and differentiating among swainsonine-producing fungal species.