Tall Fescue Online Monograph

Fig. 13-8. Loline alkaloids in grasses with different Neotyphodium species.

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R1 R2 Loline CH3 H N-acetylloline CH3 COCH3 N-formylloline CH3 CHO N-acetylnorloline H COCH3 N-methylloline CH3 CH3 Norloline H H N-formylnorloline HC=O H
Fig. 13-9. Seasonal distribution of N-acetyl plus N-formyl loline in tall fescue in Kentucky. Data are fitted to a spline curve.
Fig. 13-10. Changes in N-acetyl and N-formyl lolines in tall fescue when grown at different temperatures for 4 to 8 wk following an adaptation period of 21/15°C day/night temperatures and 16-h photoperiod.

Pyrrolizidine alkaloids of tall fescue-endophyte symbiotia are saturated in the pyrrolizidine rings. Pyrrolizidine alkaloids of most other plants are unsaturated between carbons 1 and 2 and are generally hepatotoxic and carcinogenic. Pyrrolizidine alkaloids of E+ tall fescue also contain an oxygen bridge between carbons 2 and 7 of the A and B rings (Fig. 13-8). These saturated 1-amino pyrrolizidine alkaloids, loline alkaloids or lolines, are found in much greater abundance than other alkaloids in E+ tall fescue. Levels may exceed 10 g/kg. Tall fescue alkaloids often are referred to as lolines, and they are derivatives of loline. The predominant pyrrolizidine alkaloids produced by the common (wild-type) endophyte strain in KY-31 are N-formylloline (NFL) and N-acetylloline (NAL). Loline, N-formylnorloline, norloline, N-acetylnorloline, and N-methylloline also are found (Blankenship et al., 2005). These compounds differ only by the substituents on the 1-amino group. Lolines have broad-spectrum insecticidal activity (Siegel and Bush, 1996, 1997), but lesser biological activity in vertebrates and often at levels higher than for the other symbiotum alkaloids (Siegel and Bush, 1996; Klotz et al., 2008). Lolines and derivatives found in tall fescue have been synthesized (Tufariello et al., 1986; Petroski et al., 1989; Blakemore et al., 2001). The complete synthesis of loline is arduous and not used to obtain large amounts for bioassay research. Loline may be obtained from plant extracts (Bush et al., 1993), with loline derivatives prepared much more easily (Petroski et al., 1989) than by synthesis.

Pyrrolizidine alkaloids have been found in all tissues of tall fescue. In a plant sampled at flowering, greatest abundance was found in the spikelet and much lesser amounts, in descending order, in rachis, stem, leaf sheath, and leaf blade (Burhan, 1984). Mature seed contains greater amounts of NAL and NFL than the vegetative forage (Bush et al., 1982). Generally, leaf sheath and leaf blade associated with the stem contain less lolines than the pseudostem and leaf blade of vegetative plants. In vegetative plants, the greatest amount of lolines is found in the leaf sheath, with lesser amounts in the leaf blade (Burhan, 1984). In flowering plants, the amount of NAL + NFL in the stem, rachis, and leaf blade had a positive and significantly linear correlation with the amount of endophyte in the respective tissue (Bush et al., 1993). Small amounts of pyrrolizidine alkaloids have been measured in roots from plants grown in soil or sand culture, but not in roots grown in solution culture (Bush et al., 1993). Presence of these alkaloids in all tissues examined strongly suggests translocation within the plant. Koulman et al. (2007) measured N-formylloline in sap from cut leaves and guttation water of tall fescue with wild-occurring strain of endophyte. The observation that lolines were in the guttation water and thus found on the surface of the leaves may be significant for insect deterrence for both surface- and vascular-feeding insects.

Seasonal Accumulation

Seasonal accumulation of pyrrolizidine alkaloids in tall fescue reaches a maximum in late summer in Kentucky and in a similar climatic area of Switzerland (Fig. 13-9) (Bush et al., 1993; Bush and Schmidt, 1994).

The period of accumulation is associated with late summer growth that usually occurs under heat and water stress conditions and low dry weight production. In the southern portion of the tall fescue belt in the United States, lolines have their highest accumulation in July, again when tall fescue is least productive (Putman et al., 1991). Seasonal accumulation pattern for lolines generally is highest during July and August when ergot alkaloid accumulation is lowest (Fig. 13-7 and 13-9).

Environmental Effects

Environment and management parameters influence accumulation of pyrrolizidine alkaloids. Near optimum growth temperatures for tall fescue, 21/15°C day/night, elicited the greatest increase in loline alkaloids during an 8-wk regrowth period (Fig. 13-10). Daytime temperatures of 16°C or less caused a decrease in loline alkaloid concentration, most likely because the dry weight increase was greater than the loline alkaloid biosynthesis. Pyrrolizidine alkaloids increased slightly with daytime temperatures above 30°C. These results are in general agreement with the seasonal accumulation of the loline alkaloids.

Greater water stress from -1.2 to -3.6 MPa over a 12-wk period increased NAL + NFL from 2236 mg/kg to 11,063 mg/kg, a 394% increase (Fig. 13-11). Controls increased from 2439 to 3246 mg/kg, an increase of only 33% (Kennedy and Bush, 1983). Water deficits of only -1.5 MPa caused an increase in these same two alkaloids in most genetic lines tested (Belesky et al., 1989). These observations are in general agreement with the seasonal accumulation pattern for the loline alkaloids.

Concentrations of pyrrolizidine alkaloids do not increase in response to increased soil N as do concentrations of ergot alkaloids. In controlled-environment experiments using container-grown tall fescue, Burhan (1984) and Kennedy and Bush (1983) did not detect a positive relationship between available N and alkaloid accumulation, nor did Belesky et al. (1989) find a consistent response in herbage from fertilized paddocks. The lack of a consistent effect of available N on loline alkaloid accumulation suggests that the effect of N fertilization is minimal.

Concentrations of pyrrolizidine alkaloids increased in regrowth tissue with repeated clipping (Bush et al., 1993). Greatest alkaloid accumulation occurred in the second 3-wk regrowth period, 9 wk after the first harvest (Fig. 13-12). Plant age alone at time of harvest did not influence NAL and NFL concentrations. However, Eichenseer et al. (1991) did measure a decrease in NFL as herbage tissue matured and senesced. Sun-cured hay samples contained about 80% of the starting NAL and NFL, whereas about 90% of the initial pyrrolizidine alkaloids were present after ensiling (Bush et al., 1993). Once the herbage is dry and stored in dry conditions, the loline alkaloids are stable for several years.

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Fig. 13-11. N-acetyl plus N-formyl loline accumulation with water stress achieved during a 12-wk growth period.				Fig. 13-12. N-acetyl plus N-formyl loline accumulation in initial clippings, in 3-wk regrowth following each initial clipping, and in a second 3-wk regrowth clipping following the second clipping.