Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi.

Swainsonine-a cytotoxic fungal alkaloid and a potential cancer therapy drug-is produced by the insect pathogen and plant symbiont Metarhizium robertsii, the clover pathogen Slafractonia leguminicola, locoweed symbionts belonging to Alternaria sect. Undifilum, and a recently discovered morning glory symbiont belonging to order Chaetothyriales. Genome sequence analyses revealed that these fungi share orthologous gene clusters, designated "SWN," which included a multifunctional swnK gene comprising predicted adenylylation and acyltransferase domains with their associated thiolation domains, a ß-ketoacyl synthase domain, and two reductase domains. The role of swnK was demonstrated by inactivating it in M. robertsii through homologous gene replacement to give a ∆swnK mutant that produced no detectable swainsonine, then complementing the mutant with the wild-type gene to restore swainsonine biosynthesis. Other SWN cluster genes were predicted to encode two putative hydroxylases and two reductases, as expected to complete biosynthesis of swainsonine from the predicted SwnK product. SWN gene clusters were identified in six out of seven sequenced genomes of Metarhzium species, and in all 15 sequenced genomes of Arthrodermataceae, a family of fungi that cause athlete's foot and ringworm diseases in humans and other mammals. Representative isolates of all of these species were cultured, and all Metarhizium spp. with SWN clusters, as well as all but one of the Arthrodermataceae, produced swainsonine. These results suggest a new biosynthetic hypothesis for this alkaloid, extending the known taxonomic breadth of swainsonine producers to at least four orders of Ascomycota, and suggest that swainsonine has roles in mutualistic symbioses and diseases of plants and animals.

A search for the phylogenetic relationship of the ascomycete Rhizoctonia leguminicola using genetic analysis.

Rhizoctonia leguminicola, which causes fungal blackpatch disease of legumes and other plants, produces slaframine and swainsonine that are largely responsible for causing salivation, lacrimation, frequent urination, and diarrhea in grazing animals including cattle, sheep, and horses. The original identification of R. leguminicola was based only on morphological characters of the fungal mycelia in cultures because of the lack of fungal genetic markers. Recent investigations suggested that R. leguminicola does not belong to genus Rhizoctonia and is instead a member of the ascomycetes, necessitating an accurate reclassification. The objective of this study was to use both genetic and morphological characters of R. leguminicola to find taxonomic placement of this pathogen within ascomycetes. Internal transcribed spacer region (ITS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) encoding gene were amplified from R. leguminicola isolates by PCR using universal primers and sequencing. Rhizoctonia leguminicola ITS and gpd sequences were aligned with other fungal sequences of close relatives, and phylogenetic trees were constructed using neighbor-joining and parsimony analyses. Rhizoctonia leguminicola isolates were clustered within a clade that contains several genera of ascomycetes belonging to the class dothideomycetes. We suggest that the fungus is misidentified in the genus Rhizoctonia and propose its reclassification in a new genus within the phylum Ascomycota.

Production of the alkaloid swainsonine by a fungal endophyte in the host Swainsona canescens.

Legumes belonging to the Astragalus, Oxytropis, and Swainsona genera have been noted by ranchers in the Americas, Asia, and Australia to cause a neurologic disease often referred to as locoism or peastruck. The toxin in these legumes is swainsonine, an α-mannosidase and mannosidase II inhibitor. Recent research has shown that in Astragalus and Oxytropis species swainsonine is produced by a fungal endophyte belonging to the Undifilum genus. Here Swainsona canescens is shown to harbor an endophyte that is closely related to Undifilum species previously cultured from locoweeds of North America and Asia. The endophyte produces swainsonine in vitro and was detected by PCR and culturing in S. canescens. The endophyte isolated from S. canescens was characterized as an Undifilum species using morphological and phylogenetic analyses.

Two new species of Undifilum, fungal endophytes of Astragalus (locoweeds) in the United States.

New species of Undifilum, from locoweeds Astragalus lentiginosus Vitman and Astragalus mollissimus Torr., are described using morphological characteristics and molecular phylogenetic analyses as Undifilum fulvum Baucom & Creamer sp. nov. and Undifilum cinereum Baucom & Creamer sp. nov. Fungi were isolated from dried plants of A. lentiginosus var. araneosus, diphysus, lentiginosus, and wahweapensis collected from Arizona, Oregon, and Utah, USA, and A. mollissimus var. biglovii, earleii, and mollissimus collected from New Mexico, Oklahoma, and Texas, USA. Endophytic fungi from Astragalus locoweeds were compared to Undifilum oxytropis isolates obtained from dried plant material of Oxytropis lamberteii from New Mexico and Oxytropis sericea from Arizona, Colorado, New Mexico, Utah, and Wyoming. Extremely slow growth in vitro was observed for all, and conidia, if present, were ellipsoid with transverse septa. However, in vitro color, growth on four different media, and conidium size differed between fungi from Astragalus spp. and U. oxytropis. Neighbor-joining analyses of internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase (GPD) gene sequences revealed that U. fulvum and U. cinereum formed a clade distinct from U. oxytropis. This was supported by neighbor-joining analyses of results generated from random amplified polymorphic DNA (RAPD) fragments using two different primers.