Recurrent selection in perennial ryegrass (Lolium perenne L.) for reduced levels of ergovaline with particular emphasis on the effect of other ergot alkaloid concentrations

Abstract

Perennial ryegass (Lolium perenne L.) has coevolved with symbiotic Epichloë fungal endophytes (Neotyphodium lolii) to form a mutualistic association. Infection with endophyte imparts unique bioactive properties which increase the host plants tolerance to a range of biotic and abiotic stressors. Different endophyte strains produce different classes of secondary metabolites and concentrations in conjunction with their host. Ergovaline is the end product of the ergot alkaloid pathway and is considered one of the primary secondary metabolites responsible in animal toxicosis. It is desirable when breeding for new ryegrass-endophyte associations to either eliminate, or reduce the level of ergovaline expression, while maintaining adequate levels to provide protection against invertebrate attack. Ergovaline levels have previously been shown to be a highly heritable trait for selection. However the effect of recurrent selection for reduced ergovaline on the regulation of other intermediate ergot alkaloids has not been established. The effect of recurrent selection on associated peramine concentrations and mycelial density and whether environment plays a significant role in the synthesis of the intermediate ergot alkaloid pathway is also largely unknown. An experiment was designed to determine whether ergovaline and other intermediates in the ergot alkaloid pathway are under independent or associated control. An initial base population was developed by interpollinating drought surviving genotypes infected with the novel endophyte strain ‘AR5’. Recurrent selection primarily for reduced ergovaline was then applied for two cycles. Three populations of the recurrent selection cycles (base, first cycle and second cycle generations) were evaluated in a field experiment at Leigh Creek (south-west Victoria, Australia) over 1 year in 2012-13. A split-split plot design allowed the populations to be subjected to different environment (dryland and irrigation) and nitrogen (plus and minus) treatments. Herbage samples were collected for high performance liquid chromatography (HPLC) to determine ergovaline concentration and liquid chromatography-electrospray ionization mass spectrometry (LC-MS/MS) to detect peramine and the intermediate ergot alkaloid pathway concentrations. Enzyme-linked immunosorbent assay (ELISA) was also used to quantify mycelial density. The expected profile of ergot alkaloid pathway alkaloids were detected including chanoclavine, lysergyl alanine, ergine and ergovaline. Ergovaline concentation was inconsistent across the two selection cycles and showed a significant (P<0.05) increase in the first cycle across all environments and treatments, conflicting with the predicted outcome. Ergine and peramine both significantly (P<0.05) increased in concentation following reselection for reduced ergovaline. When the ergot alkaloids are presented as percentage components of the biosyntheic pathway, it did however show that recurrent selection reduced both ergovaline and lysergyl alanine, and simultaneously increased ergine. It appears that there is a biochemical shunt in the pathway from ergovaline towards ergine, rather than a down-regulation of the whole pathway. An alternative ergot alkaloid biosynthetic pathway is proposed. These results support the hypothesis that in this host ryegrass-AR5 population, the ergot alkaloids are under independent host genetic control, and the pathway is inefficient as the intermediates do not convert directly to ergovaline, but accumulate as ergine. Large environmental effects were observed in ergot alkaloid concentations within environments and between nitrogen treatments, with significantly greater (P<0.05) concentrations in the dryland environment. There also appears to be no effect from recurrent selection on mycelial density in this host-endophyte association. Limitations of this particular study are discussed and possibilties for future work are proposed.