Phytochemistry 58 (2001) 395–401 www.elsevier.com/locate/phytochem Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media Jimmy D. Blankenshipa, Martin J. Spieringa, Heather H. Wilkinsona,1, Franklin F. Fanninb, Lowell P. Bushb, Christopher L. Schardla,* aDepartment of Plant Pathology, University of Kentucky, Lexington, KY 40546-0091, USA bDepartment of Agronomy, University of Kentucky, Lexington, KY 40546-0091, USA Received 27 February 2001; received in revised form 18 May 2001 Abstract Lolines (saturated 1-aminopyrrolizidines with an oxygen bridge) are insecticidal alkaloids produced in symbioses of certain Epi- chloe¨ (anamorph-Neotyphodium) species (fungal endophytes) with grasses, particularly of the genera Lolium and Festuca. Prior to the present study, it was unknown whether lolines were of plant or fungal origin. Neotyphodium uncinatum, the common endophyte of meadow fescue (Lolium pratense=Festuca pratensis) produced loline, N-acetylnorloline, and N-formylloline when grown in the defined minimal media at pH 5.0–7.5, with both organic and inorganic nitrogen sources and sugars as carbon sources. In contrast, lolines were not detected in complex medium cultures. GC–MS and 13C NMR spectroscopic analyses confirmed the identity of the alkaloids isolated from the defined medium cultures. Lolines accumulated to ca. 700 mg/l (4 mM) in cultures with 16.7 mM sucrose and 15–30 mM asparagine, ornithine or urea. Kinetics of loline production and fungal growth were assessed in defined medium with 16.7 mM sucrose and 30 mM ornithine. The alkaloid production rate peaked after the onset of stationary phase, as is common for secondary metabolism in other microbes. # 2001Elsevier Science Ltd. All rights reserved. Keywords: Neotyphodium uncinatum; Clavicipitaceae; Epichloe¨ endophyte; Lolium pratense; Festuca pratensis; Poaceae; Meadow fescue; Fungal fermentation; 1-Aminopyrrolizidines; Loline alkaloids 1. Introduction in fermentation cultures (Bacon, 1988; Rowan, 1993, Gurney et al., 1994; Porter, 1994). The lolines are pro- Loline alkaloids (saturated 1-aminopyrrolizidines duced in a number of grass-endophyte symbioses: with an oxygen bridge) (Fig. 1) have been identified in Lolium arundinaceum (=Festuca arundinacea)with some grass-endophyte associations (Siegel et al., 1990), Neotyphodium coenophialum (Siegel et al., 1990), Lolium but are absent in most other organisms. These grasses giganteum (=Festuca gigantea) with Epichloe¨ festucae are naturally infected with symbiotic endophytic fungi, (Leuchtmann et al., 2000), Lolium pratense (=Festuca especially Epichloe¨ spp. and their asexual derivatives pratensis)withNeotyphodium uncinatum (Bush et al., (Neotyphodium spp.), which grow in the intercellular 1993; Justus et al., 1997), L. pratense with Neotyphodium spaces (apoplast) of host plants. In these symbioses, an siegelii (Craven et al., 2001), and endophyte-infected Fes- array of other anti-herbivore alkaloids is produced. Three tuca argentina (Casabuono and Pomilio, 1997), Lolium of the alkaloid classes commonly found in endophyte– temulentum (Dannhardt and Steidt, 1985), Poa autumnalis grass associations—peramine (a pyrrolopyrazine), ergot (Siegel et al., 1990), and Stipa robusta (TePaske et al., alkaloids, and lolitrems (indolediterpenes)—are of known 1993). The plants, Argyreia mollis (Convolvulaceae) and fungal origin because these are reported to accumulate Adenocarpus spp. (Fabaceae) have also been reported to contain loline alkaloids, although endophytes have not been implicated (Hartmann and Witte, 1995; Tofern et * Corresponding author. Tel.: +1-859-257-8758; fax: +1-859-323- al., 1999). 1961. E-mail address: [email protected] (C.L. Schardl). The lolines are secondary metabolites and have an 1 Current address: Department of Plant Pathology and Micro- unusual structure consisting of a saturated necine ring biology, Texas A&M University, College Station, 77843, USA. with an –NRR0 substituent at C1and an oxygen bridge 0031-9422/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00272-2 396 J.D. Blankenship et al. / Phytochemistry 58 (2001) 395–401 Loline alkaloid expression correlates with fungal gen- otype, and in some grass-endophyte symbiota levels can exceed 2% plant dry mass (Craven et al., 2001). Until the present study lolines have been the only grass-endo- phyte associated alkaloid class reported in the literature that was not observed in fungal cultures (Porter, 1994). Knowledge of the origin of lolines would be beneficial for elucidating the biosynthetic pathway and its regula- tion, identifying biosynthesis genes, and expanding bio- technological possibilities for these alkaloids. Since lolines were only detected in endophyte-infected plants, we hypothesized the endophyte was responsible for their production, and that cultures that adequately mimic the internal environment of the plant may sti- mulate loline production. In this paper, it is demon- strated that three of the loline alkaloids found in plant tissue—loline (1), N-acetylnorloline (2), and both rota- mers of N-formylloline (3 and 30)—are produced by N. uncinatum under certain minimal medium conditions. 2. Results Fig. 1. Structures of loline alkaloids. The common endophyte of L. pratense, N. uncinatum, between C2 and C7 (Yates et al., 1990; Powell and Pet- was examined for potential production of loline alkaloids roski, 1992). Lolines thus differ from most plant pyrroli- in culture because grasses symbiotic with this species zidines, which are synthesized from homospermidine accumulate lolines at high levels. In a minimal medium (Bo¨ ttcher et al., 1993) and have a –CH2OR group at C1. modified from Chung and Schardl (1997), with mannitol Nothing is known about the biosynthesis of the endo- as the carbon source and asparagine as the nitrogen phyte-associated lolines, but based on this difference, the source, N. uncinatum produced 1, 2, 3,and30. The iden- loline alkaloids have been suggested to be derived from tities of these alkaloids were confirmed by GC–MS and spermidine or spermine (Bush et al., 1993). The alkaloids 13C NMR spectroscopic analyses. Another loline alka- are found throughout the plant in these associations and loid, N-acetylloline, is (together with 3 and 30) one of the have protective effects for the plant due to their anti- most abundant alkaloids in L. pratense–N. uncinatum invertebrate and feeding deterrent activities (Patterson et symbiota (Siegel et al., 1990), but was not detected in al., 1991; Justus et al., 1997; Dougherty et al., 1998; the culture. No loline alkaloids were detected from the Wilkinson et al., 2000). Lolines are broad-spectrum cultures grown in potato dextrose broth (PDB). insecticides (Siegel and Bush, 1997); and in grasses sym- Various carbon sources typically present in plant biotic with strains of E. festucae segregating for loline apoplasts were examined. Initially, mannitol was chosen alkaloid expression, accumulation of these alkaloids because it is a good carbon source for growth of the was clearly associated with activity against certain aphid related endophyte, N. coenophialum (Kulkarni and species (Wilkinson et al., 2000). Thus, the loline alka- Nielsen, 1986). Fructose, glucose, and sucrose were then loids are of great interest as natural plant protectants. tested as alternative carbon sources because sucrose is a Although studies do indicate immunosuppressive effects major transport sugar in the apoplast, while glucose and in mice (Dew et al., 1990) and depressed feed intake has fructose are produced from sucrose by the action of been observed in rats fed loline alkaloids (Jackson et al., invertase secreted by endophytes (Lam et al., 1994). As 1996), it has not been conclusively shown that the loline shown in Fig. 2, sucrose was the best of these carbon alkaloids at in planta levels are toxic to mammalian sources for loline production, giving a range of 500–750 herbivores (Siegel and Bush, 1997). Lolines do not share mgmlÀ1 (up to 4 mM) total of 2, 3,and30. This was a the same potent anti-mammalian hepatotoxicity and 12-fold increase of production over cultures with manni- carcinogenicity as plant pyrrolizidines, 1-hydroxy- tol. Glucose or fructose also tended to increase produc- methylpyrrolizidines, which contain C-1,2 unsaturation tion only slightly compared to mannitol cultures, but in the necine ring (Hincks et al., 1991); cytochrome P450 not to the level obtained using sucrose. converts the 1-hydroxymethylpyrrolizidines to more Nitrogen sources tested were asparagine, arginine, bioactive forms, which to varying degrees are capable of ornithine, glutamine, ammonium sulfate, and urea, in crosslinking of DNA (Kim et al., 1999). cultures at pH 5.5 with sucrose as carbon source. J.D. Blankenship et al. / Phytochemistry 58 (2001) 395–401 397 Nitrogen concentration was maintained at 30 mM total also gave much greater levels than expressed in cultures nitrogen atoms. In this experiment, cultures with urea as with asparagine or arginine. The level of loline alkaloid the sole nitrogen source yielded the greatest overall level production at day 20 was 5-fold greater when ornithine of loline alkaloid production, ca. 425 mgmlÀ1 of culture rather than asparagine was the nitrogen source (Fig. 3). filtrate (Fig. 3). Ornithine as the sole nitrogen source Altering concentrations of phosphate between 6.8 and 68 mM, MgSO4 between 0.2 and 4.0 mM, mannitol between 8.3 and 16.7 mM, or asparagine between 15 and 30 mM had only slight effects on