Nutrition, clipping, water stress, growth temperature, and utilization affect ergovaline content of the tissue. Nitrate-N and NH4-N fertilization increased ergopeptide alkaloids (measured as ergovaline) in leaf sheath and blade in greenhouse-grown plants by more than 500% over unfertilized plants (Lyons et al., 1986). Similar results were reported by Azevedo et al. (1993). In addition, they reported significantly increased ergovaline in the crown and root tissues with the addition of N. Increasing N fertilizer from 0 to 135 kg/ha increased ergovaline concentrations in leaf blades, stems and seedheads by 88, 103, and 66%, respectively (Rottinghaus et al., 1991). Ergovaline in seedheads increased with added N during seed maturation from mid May to mid June; the increase was from 20 to 135% by time of maturity. Increasing extractable soil P levels from 17 to 50 mg/kg were accompanied by an increase in ergot alkaloids in shoots, but a further increase of extractable P to 96 mg/kg seems to have been related to decreased ergot alkaloid concentrations (Malinowski et al., 1998). Ergot alkaloid concentrations were substantial, 54 mg/kg, in these plants. Roots contained measurable amounts of ergot alkaloids, which increased with P availability at all levels of P.
Based on the seasonal accumulation of ergovaline, temperature would be expected to influence ergot alkaloid accumulation. Endophyte infected tall fescue grown in controlled-environment chambers with a 16-h photoperiod maintained at 15, 21, or 25°C showed that different alkaloids responded independently to temperature. Chanoclavine and ergonovine decreased with time at 15°C, but increased with time at 21 and 25°C (Salminen et al., 2005). Ergovaline content increased with time at 15°C. The maximum temperature used in this experiment is lower than the average daytime temperature in much of the United States tall fescue belt during the summer portion of the growing season. For application to tall fescue toxicity in summer to bovines, accumulation of ergot alkaloids at temperatures of 30 to 33°C would have been more appropriate. Certainly the summer accumulation patterns in Missouri and Kentucky occur at such higher temperatures. It is difficult to separate heat stress and water stress treatments on ergovaline accumulation as they may be confounded with N fertilization and certainly with dry matter accumulation. For example, in a greenhouse experiment a 40-d water deficit increased ergovaline content of tall fescue, especially with high N fertilization (Arechavaleta et al., 1992). At low N fertilization there appeared to be no difference in ergovaline content at low and high water stress. No temperature stress was indicated in this greenhouse experiment, and these data, especially at the low added N fertility, would support the seasonal distribution pattern of ergovaline that suggests water stress in mid summer as well as increased temperature decrease ergovaline content. This is especially true if one assumes tall fescue pasture soils are low or very low in available N. Plant growth and ergovaline biosynthesis decreased with water stress when measured on a per-pot basis (Belesky et al., 1989). However, concentrations of ergovaline in the leaf blade and sheath increased in plants grown in a water deficit regime.
Growth temperature may be confounded with ambient temperature of area of ecotype adaptation (see Chapter 3, Hannaway et al., 2009, this publication). Jensen et al. (2007) collected tall fescue ecotypes from Italy, Spain, and Denmark and grew all populations for 2 wk at 23/18°C (day/night temperatures), followed by 2 wk at 25/20°C with a 16-h photoperiod. Entries from Denmark contained an average of 15.2 mg/kg ergovaline, whereas the entries from Spain averaged only 3.1 mg/kg. The values from Danish populations were extremely high when compared with values measured in the United States and New Zealand. Further investigations are needed on the N. coenophialum strain in these populations and response of other N. coenophialum strains inserted into these tall fescue populations.
Clipping height and defoliation frequency affect ergot alkaloid accumulation. Salminen et al. (2003) measured increased ergonovine and ergocryptine with increased clipping height of 2.5 to 7.5 cm above the soil surface in greenhouse-grown tall fescue. Plants were clipped weekly, and alkaloids were measured monthly. Previously, Belesky and Hill (1997) measured decreased ergovaline concentration with 7-d interval clipping at 5 and 10 cm compared with an uncut control. The authors of this chapter have measured increased ergovaline with repeated harvests at 5 cm when clipped every 21 d compared with an uncut control (unpublished data, 2008).
Post-harvest management can affect alkaloid concentrations in herbage. Field drying of tall fescue hay reduced ergovaline about 50% (Garner et al., 1993). The amount of alkaloid loss seemed to depend on the curing conditions, as Hume et al. (2007) measured a difference in loss between spring and summer conditions when considering alkaloid concentrations in perennial ryegrass. A much more rapid loss was observed during summer than in spring. They measured very little loss if the natural drying occurred within 24 h with the cut herbage left on the sward. If the herbage was dried artificially during 24 h at 55°C, about 30% of the ergovaline was lost. Ammoniation of ground tall fescue seed with 35% moisture significantly reduced (54%) ergovaline and the pyrrolizidine alkaloids 24% during a 7-d treatment period (Simeone et al., 1998). This alkaloid reduction was associated with an improved animal response. Ensiling tall fescue did not decrease ergovaline content of the harvested herbage (Turner et al., 1993).
During investigations on plant management to alter ergot alkaloid content, many authors have suggested that both the endophyte and the plant genotype influenced their results. Hill and coworkers (Adcock et al., 1997; Agee and Hill, 1994; Hiatt and Hill, 1997; Hill et al., 1991; Roylance et al., 1994) concluded that the host plant genome was important in the accumulation in the symbiota of ergopeptide alkaloids produced by the endophytic fungus. Schmid et al. (2000) further concluded that the host controlled intensity of endophyte infection and alkaloid production within a symbiotum. Hill et al. (2002) selected five tall fescue populations with a common endophyte strain that accumulated different ergot alkaloid concentrations. One low-alkaloid line, with approximately 50% of the ergot alkaloid content of the E+ control, was evaluated for lamb weight gain. Gains from the low alkaloid line were significantly lower than those obtained from the E+, but higher than those from the E- control lambs. Bouton et al. (2002) demonstrated the feasibility of infecting tall fescue cultivars lacking N. coenophialum with a non-ergot producing endophyte as a useful strategy for maintaining the positive agronomic characteristics of tall fescue while lowering the toxicity (see Chapter 20, Bouton, 2009, this publication). More recent research has confirmed these observations and provided symbiota to alter the accumulation of ergot alkaloids in planta.
Investigations into ergot alkaloid biosynthesis in Claviceps purpurea (Fr.:Fr.) Tul. and subsequently in Neotyphodium spp. have elucidated much of the ergopeptine biosynthetic pathway (Schardl et al., 2006). The significant beginning and ending steps are illustrated in Fig. 13-5. Addition of the dimethylallyl moiety from dimethylallyl-diphosphate (DMAPP) onto tryptophan by the enzyme dimethylallyl tryptophan synthase (dimethylallyl tryptophan [DMATrp] synthase) is the determinant step toward ergot alkaloid biosynthesis. The gene dmaW codes for this enzyme and disruption of dmaW essentially inhibits biosynthesis of ergot alkaloids. This first step and subsequent steps for synthesis of the clavines to lysergic acid amide have been well detailed by Schardl et al. (2006). The final step in ergopeptine biosynthesis is the addition of the tripeptide chain to lysergic acid amide. Two subunits of d-lysergyl peptide synthetase, LPS 1 and LPS 2, are responsible for the addition of the amino acids. LPS 2 is responsible for activation of lysergic acid and may act alone or in concert with LPS 1. LPS 1 is responsible for activation of the amino acids and participates with LPS 2 to yield the ergopeptide lactam. Further hydroxylation and oxidation yield the ergopeptide alkaloids. Gene lpsA encodes for LPS 1 and lpsB gene encodes for LPS 2. Disruption of lpsB in C. purpurea did not produce ergopeptine alkaloids (Correia et al., 2003) but did produce intermediates in the pathway.
Modified strains of Neotyphodium have been developed to inhibit completely or greatly reduce the amount of ergot alkaloids produced in tall fescue symbiota (Florea et al., 2007). Functionality of different endophyte strains is dependent on the tall fescue genotype/population, and research is ongoing to find improved combinations for maximum positive animal response (Agee and Hill, 1994; Adcock et al., 1997; Hill et al., 2002). Several different tall fescue endophyte strains (Latch et al., 2000) have been patented (see Chapter 25). Ball et al. (2006) used several different Neotyphodium strains from tall fescue with divergent alkaloid profiles to study fall armyworm [Spodoptera frugiperda (J.E. Smith)] feeding on tall fescue. KY-31 was artificially inoculated with the selected strains and expression of alkaloid accumulation measured. Only the wild-type strain had measurable ergovaline accumulation, but the pyrrolizidine alkaloids differed qualitatively and quantitatively. Utility of artificial combinations will be dependent on finding the best combinations for optimum forage and animal production in different environments. A potential resource for determination of significance of intermediates in ergopeptide biosynthesis to toxicosis would be symbiota where the endophyte has the lspA and lpsB genes disrupted to stop ergopeptide formation, but with the earlier portion of the pathway functional and/or shunted (Correia et al., 2003; Panaccione et al., 2003).
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