4d.Progress report.
This report serves to document research conducted under a Trust Fund Cooperative Agreement with the National Research Initiative and ARS. Additional details of research can be found in the report for the parent project 1907-22410-003-00D "Microbial and genetic resources for biological control and high-value uses".

This project was directed toward defining the role of destruxins, a family of biologically active peptides, in the insect disease process of Metarhizium anisopliae. Destruxins are cyclic depsipeptides with chemical features suggesting synthesis by a nonribosomal peptide synthetase (NRPS). Knockout (KO) mutants were created by disrupting a NRPS gene fragment, ma267 predicted to be involved in destruxin production via Agrobacterium tumefaciens-mediated transformation. All KO strains were mitotically stable and phenotypically similar to wild type, but all strains produced comparable levels of destruxins. Based on these results, we concluded that gene ma267 does not encode the destruxin NRPS. There were no detectable differences in pathogenicity among the KO mutants and wild type strains in multiple bioassays with beet armyworm and Colorado potato beetle. We conclude that the unknown NRPS gene ma267 does not encode a virulence factor for insect disease. Efforts are underway to identify the natural product synthesized by ma267 since it is appears to be expressed early in the infection cycle. Quantitative estimates of destruxin levels in mycosed larvae from HPLC MS/MS analyses ranged from 1 microgram-10 picograms/insect. Destruxin was not detectable in all cadavers and destruxin estimates for insects killed by KO strains did not differ from those for insects killed by WT strains. A fourth KO transformant has an additional uncharacterized mutation evidenced by a marked increase in the production of yellow pigment. We identified two major components, NG-391 and NG-393, previously reported from Fusarium sp. as stimulants of nerve-cell growth; these compounds have not been previously reported from a Metarhizium. These compounds are closely related to the fusarins, mutagenic Fusarium mycotoxins that contaminate corn. Detectable levels of these compounds were also found in control strains; levels in two KO mutants were 60- and 20-fold higher, respectively, than the other strains analyzed. Several new NRPS gene fragments cloned from cDNA of DTX-producing cultures will serve as targets for the next gene disruption studies. Neither destruxins nor the fusarin-like compounds appear to be required for virulence of the fungus, suggesting that they are dispensable for infection. Although further proof will be needed, this information is relevant to strain selection and development since toxin-deficient strains will be perceived as safer for insect biological control than those with the genetic potential to make such compounds.