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Applied and Environmental Microbiology, April 2009, p. 2200-2211, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.00953-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Indole-Diterpene Biosynthetic Capability of Epichloë Endophytes as Predicted by ltm Gene Analysis{triangledown}

Carolyn A. Young,1,2,3* Brian A. Tapper,4 Kimberley May,1,2 Christina D. Moon,5,{dagger} Christopher L. Schardl,5 and Barry Scott1,2

Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand,1 National Centre for BioProtection, Massey University, Private Bag 11 222, Palmerston North, New Zealand,2 The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73401,3 AgResearch Grasslands, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand,4 Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546-03125

Received 25 April 2008/ Accepted 20 January 2009

Bioprotective alkaloids produced by Epichloë and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. The LTM locus of Neotyphodium lolii (Lp19) and Epichloë festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm 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 10 ltm genes were present in only three epichloë endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. lolii containing the entire LTM 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 in ltm 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.

* Corresponding author. Mailing address: The Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401. Phone: (580) 224-6860. Fax: (580) 224-6802. E-mail: cayoung{at}noble.org

{triangledown} Published ahead of print on 30 January 2009.

{dagger} Present address: AgResearch Grasslands, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand.

Applied and Environmental Microbiology, April 2009, p. 2200-2211, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.00953-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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