Indole-Diterpene Biosynthetic Capability ofEpichloëEndophytes as Predicted byltmGene Analysis

Bioprotective alkaloids produced byEpichloë and closely related asexualNeotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. TheLTM locus ofNeotyphodium lolii (Lp19) andEpichloë festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10ltm genes were present in only three epichloë endophytes. A predominance of the asexualNeotyphodium spp. examined contained 8 of the 10ltm genes, with only oneN. lolii containing the entireLTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present inltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.