Early studies with different host lines indicated that presence or absence of the endophyte was associated with presence or absence of the loline alkaloids (Bush et al., 1982; Belesky et al., 1988; Hiatt and Hill, 1997). In addition, the level of alkaloid accumulation varied in the presence of the endophyte. Recently, endophyte strain has been shown to alter loline alkaloid accumulation quantitatively and qualitatively (Ball et al., 2006). Different endophyte strains inserted into KY-31 caused loline alkaloid accumulations ranging from none to 1983 mg/kg. Tall fescue containing endophyte strain AR542 did not accumulate NAL and NFL but did accumulate significant amounts of N-acetyl norloline. It must be noted that virtually all insect bioassay data have been obtained with NAL and NFL. As different pyrrolizidine alkaloids accumulate in different host-endophyte combinations, the biological fitness of the symbiotum may change accordingly. A symbiotum with primarily N-acetylnorloline did not deter fall armyworm feeding as the wild-type symbiotum (Ball et al., 2006). Other endophyte strains were associated with accumulation of all three of these pyrrolizidine alkaloids. Blankenship et al. (2001) demonstrated that the loline alkaloids were of fungal origin, thus complementing similar findings for peramine and ergot alkaloids. Perennial ryegrass with its common strain of N. lolii accumulates no or very low levels of loline alkaloids. Insertion of endophyte strains from tall fescue into perennial ryegrass caused the accumulation of NFL in all lines tested and of N-acetyl norloline in one line; no NAL was found from any of the lines tested (Easton et al., 2007). These results demonstrate the significance of both the endophyte and the host for pyrrolizidine alkaloid accumulation.
Subsequent research by Schardl and associates has described the biosynthetic pathway and the genetic control of pyrrolizidine alkaloid accumulation in tall fescue (Schardl et al., 2007). Proline (Fig. 13-8) contributes C atoms 5 to 8 and the N atom at position 4 (Blankenship et al., 2005). Homoserine contributes C atoms at 1-3 and the 1-amino group and methionine the carbon atoms of the N-formyl and N-methyl substituents of NFL (Faulkner et al., 2006). The sequence of formation is that the proline and homoserine moieties form the pyrrolizidine ring structure and then the ether bridge is added between C-1 and C-7. A genetic cross of haploid Epichloë festucae Leuchtm., Schardl, and Siegel from Festuca gigantea (L.) Vill., a loline producer and from Festuca rubra L., a loline nonproducer, produced loline-expressing and nonexpressing phenotypes in a 1:1 ratio. In a haploid system this ratio would be expected if the factor were at a single locus. Subsequently, the group found in N. uncinatum (Gams, Petrini & Schmidt) Glenn, Bacon, Price & Hanlin, the endophyte of L. pratense and a diploid fungus, two gene clusters of 8 or 9 genes associated with the loline phenotype (Spiering et al., 2005). These clusters have been designated LOL1 and LOL2. Continuing efforts are underway to establish the enzyme activities of the gene products in these clusters and thus determine the biosynthesis pathway and mechanisms of the complete biosynthesis. Schardl et al. (2007) reviewed in detail the genetics and biosynthesis of these 1-amino pyrrolizidine alkaloids. Understanding the genetics of loline alkaloid biosynthesis is important to attempts to express or overexpress these alkaloids in other grasses or other agriculturally important plants.
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