Tall Fescue Online Monograph

Click to Expand

Fig. 13-4. Structure of the insect-feeding deterrent peramine.

Pyrrolopyrazine Alkaloid-Peramine

Peramine is the only known pyrrolopyrazine alkaloid in E+ tall fescue (Fig. 13-4). However, it is present in more grass-endophyte symbiota tested than any of the other fungal endophyte-associated alkaloids in grasses (Siegel and Bush, 1997). The pyrrolopyrazine ring structure and the guanidinium group of peramine are rather unusual (Fig. 13-4). Total synthesis was reported by Dumas (1988) and Brimble and Rowan (1988). Peramine concentration generally is around 1 mg/kg in tall fescue and 10- to 30-fold higher in perennial ryegrass (L. perenne L.). Peramine is not active in mammalian bioassays and is known primarily as an insect feeding deterrent. Concentrations in tall fescue often are below the threshold amount reported for insect response to peramine in perennial ryegrass (Rowan, 1993).

Peramine does not have a significant role in tall fescue growth and development in the United States; consequently, understanding of the physiology of the alkaloid to growth and development of tall fescue is lacking. Roylance et al. (1994) reported greatest concentration in the flowering culm (4.2 mg/kg), with lesser amounts in the panicle and leaf blade. There was no apparent seasonal pattern of peramine accumulation in greenhouse-grown plants. Concentrations during the year varied by as much as 132%, with mean values less than 2 mg/kg. Bush and Schmidt (1994) did not measure a significant seasonal variation of peramine in field-grown tall fescue. Keogh and Tapper (1993) and Spiering et al. (2002) reported little difference within plant tissues for accumulation of peramine in perennial ryegrass. However, as leaves aged, greater peramine concentration was found in leaf sheaths and the base of leaf blades. Generally, leaf sheaths and blade bases contained the highest levels of peramine. This suggests that peramine is translocated uniformly and readily throughout the plant from the site of endophyte biosynthesis, the leaf sheaths, to younger leaves. Peramine was not detected in roots of mature plants; however, it was detected in seedling roots, probably as a result of translocation from the seed during germination (Ball et al., 1993). Water deficit stress of greater than -2.0 MPa, compared with less than -1.0 MPa, did not alter peramine accumulation in perennial ryegrass, nor did harvests of tillers of different ages (Barker et al., 1993). Simulated hay making decreased peramine levels in cut tillers by more than 50% in the first 6 d. Dead endophyte tissue at the base of pasture plants contained very low or no peramine (Hume et al., 2007). Results from the more extensive research on peramine in perennial ryegrass was similar to what was known about tall fescue; it is therefore a reasonable assumption that the more detailed results from ryegrass can be ascribed to tall fescue.

In the early work on peramine, results indicated independent relationships among the accumulation of the different alkaloid classes. There appeared also to be differences among host-endophyte genome interactions for accumulation of peramine. More recently, Tanaka et al. (2005) described a gene (perA) for peramine biosynthesis. This is a single multifunctional nonribosomal peptide synthetase (NRPS). Two fungal genes are believed to be required for peramine biosynthesis. The NRPS conjugates amino acids to form short peptides and the second enzyme is a phosphopantetheinyl transferase (PPTase) that supplies the NRPS with an essential cofactor. PerA is distributed widely in Neotyphodium species; however, several mutations have been identified that result in endophyte strains that do not produce peramine (Scott et al., 2007). The possibility of inserting the gene into other fungi or host plants has potential to confer insect resistance to agricultural management protocols, since peramine is an insect feeding deterrent. Pirlo et al. (2007) inserted perA into Penicillium paxilli, which has a functional PPTase gene, and subsequently produced peramine.

Ergot Alkaloids

Ergot alkaloids of tall fescue include many compounds related by an ergoline ring system or a biosynthetic precursor thereof. These alkaloids include clavines, lysergic acid and derivatives, and the ergopeptide and ergopeptine alkaloids (Fig. 13-5).

All are produced by the fungal endophyte N. coenophialum. These alkaloids have a tricyclic peptide and differ only in the amino acids in positions 1 and 2 of the cyclic peptide. All have proline in position 3 (Fig. 13-6).

Specifically, the ergopeptine, ergovaline, has been the center of much research attention because of associated vasoconstrictive properties. Lyons et al. (1986) reported the presence of five ergopeptine alkaloids-ergovaline, ergosine, ergonine, ergoptine, and ergocornine-in pasture samples of E+ tall fescue (Fig. 13-6). Ergovaline accounted for 84 to 97% of the total ergopeptine alkaloids present. Total ergopeptine alkaloids, as ergovaline equivalents, were as high as 2.8 mg/kg in leaf sheaths and 0.3 mg/kg in leaf blades. The presumption is that the epimers ergovalinine, ergosinine, ergoninine, ergoptinine, and ergocorninine would also be found (Smith and Shappell, 2002). Small amounts of ergotamine have been reported in E+ tall fescue (Yates et al., 1985; Porter, 1995). Many simpler ergot alkaloids may be present in greater quantities than ergovaline (ergopeptine) alkaloids, and these may be significant in tall fescue toxicosis, although this is yet to be determined (Hill et al., 2001; Klotz et al., 2006, 2007a, 2007b). In the sample above, with 2.8 mg/kg ergovaline, the total ergot alkaloids concentration measured as ergonovine was 4.8 mg/kg in the leaf sheath, whereas the leaf blade contained only 1.0 mg/kg total ergot alkaloids. The additional ergot alkaloids were most likely clavines and lysergic acid derivatives. Total synthesis of ergovaline has been reported (Stadler et al., 1964).

Click to Expand

Fig. 13-5. A generalized scheme for ergot alkaloid biosynthesis in tall fescue/Neotyphodium coenophialum symbiotum. DMAPP, dimethylallyl-diphosphate; Trp, tryptophan; DMATrp, dimethylallyl-tryptophan; draW, gene encoding for DMATrp synthase; LPS1 and LPS 2, two subunits of d-lysergyl peptide synthetase; and lpsA and lpsB, encode for the LPS enzymes.

Amino acid 1 R1 Amino acid 2 R2 Ergocornine L-valine i-Pr L-valine i-Pr Ergonine L-2-aminobutyric acid Et L-valine i-Pr Ergoptine L-2-aminobutyric acid Et L-leucine i-Bu Ergosine L-alanine CH3 L-leucine i-Bu Ergovaline L-alanine CH3 L-valine i-Pr Ergotamine L-alanine CH3 L-phenylalanine CH2Ph Fig. 13-6. Structures and amino acids in the common ergopeptine alkaloids of the tall fescue/Neotyphodium coenophialum symbiotum. Proline is always the amino acid in position 3.

Distribution Within the Plant

Much higher levels of ergot alkaloids are found in the leaf sheaths than in the leaf blades of tall fescue (Lyons et al., 1986), suggesting limited translocation from the site of synthesis to the leaf blade. This conclusion is supported by the lack of correlation between fungal protein quantity and ergot alkaloid concentration in herbage (Hiatt and Hill, 1997). However, ergot alkaloid concentration should be a function of duration of biosynthetic activity, which would not have to correlate with total fungal protein. Ergovaline was found in cut leaf exudates or guttation water of tall fescue infected with wild-type endophyte in New Zealand, supporting the concept of translocation (Koulman et al., 2007). Seedheads and seed contain the greatest ergot alkaloid levels. Rottinghaus et al. (1991) reported almost 5.0 mg/kg in seedheads, much higher than in the stems and leaves, which had levels less than 0.5 mg/kg. In tall fescue the concentration from greatest to least is generally seed, crown, stems, leaves, and roots (Rottinghaus et al., 1991; Azevedo and Welty, 1995). The absolute amount will depend on the growth environment and the host-endophyte genetic combination.

Seasonal Distribution

In Kentucky, in the northern portion of the tall fescue belt, accumulation of ergovaline generally is low during winter and increases during spring growth to a high level in mid May near the time of seed fill (Fig. 13-7). During the hotter, drier growth conditions of summer, the vegetative tissue has less ergovaline than during spring and fall. A second peak in ergovaline concentration occurs during fall. Rottinghaus et al. (1991) in Missouri reported a similar accumulation pattern of increased levels during spring growth with maximum levels in seedheads. Regrowth in June, July, and August contained much lower levels of ergovaline, but concentration began to increase again in September and reached a fall maximum in early October, decreasing into the winter months. In the southern reaches of the tall fescue belt in the southeastern United States, maximum ergovaline accumulation occurred in May, with the March, April, and June concentrations at about 30 to 50% of those occurring in May (Agee and Hill, 1994; Belesky et al., 1988). Ergovaline concentrations in May were about 1 mg/kg and were from plants with seedheads emerged. Because of high summer temperatures, tall fescue mainly grows only during these months in Georgia, and very low levels were measured during other times of the year.