High nitrogen supply and carbohydrate content reduce fungal endophyte and alkaloid concentration in Lolium perenne

The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent. Here, we infected two perennial ryegrass (Lolium perenne) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues. Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration. The results stress the need for wider quantification of fungal endophytes in the grassland-foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems.     

Developing a method for laboratory biosafety testing impacts of transgenic plants on non-target insects

A series of laboratory experiments was carried out with the main objective of contributing to the development of biosafety testing methods for transgenic plants or plants produced by other novel technologies. The adult stage of a New Zealand native weevil, Nicaeana cervina (Broun) (Coleoptera: Curculionidae: Entiminae), was used as a test insect as this has become well‐adapted to introduced pasture species, feeding on both ryegrass [Lolium perenne L. (Poaceae)] and white clover [Trifolium repens L. (Fabaceae)]. Ryegrass plants used in experiments were infected with strains of the symbiotic endophytic fungi Neotyphodium lolii (Latch, M.J. Chr. & Samuels) and Epichloe festucae Leuchtm., Schardl & M.R. Siegel and included a transgenic endophyte, which was modified to eliminate production of the insect deterrent, peramine. Nicaeana cervina was exposed to these endophyte‐infected plants along with endophyte‐free ryegrass. Weevils were also exposed to white clover with high or low levels of cyanogens, and to white clover selections bred conventionally to contain high or low levels of crude protein. These bioassays were carried out sometimes over a series of time intervals, and weevil survival, feeding, and reproductive performance variables were measured. Nicaeana cervina survival was generally high, although feeding and oviposition were variable between experiments despite high replication in some cases. A summary of significant differences obtained across all experiments suggested that data on the proportion of weevils feeding and amounts of leaf material consumed were more often significantly different than treatment effects on survival or reproductive performance. A positive relationship between the proportion of weevils feeding and those ovipositing was found, suggesting that a 50% reduction in feeding would result in a 28% reduction in the proportion of weevils ovipositing. Power analysis using the range of values recorded for the variables measured indicated that for proportions of weevils surviving, feeding, or fecund, about 35–42 replicates would be required for a 40% difference in values. For measures of leaf consumption and numbers of eggs per female, fewer replicates were required for 95% power when these values were at the higher end of the range than when they were at the lower end of the observed range. The usefulness of the case study test insect and risk assessment protocols used for biosafety testing are discussed.     

Metabolite analysis of the effects of elevated CO2 and nitrogen fertilization on the association between tall fescue (Schedonorus arundinaceus) and its fungal symbiont Neotyphodium coenophialum

Atmospheric CO₂ is expected to increase to between 550 ppm and 1000 ppm in the next century. CO₂‐induced changes in plant physiology can have ecosystem‐wide implications and may alter plant‐plant, plant‐herbivore and plant‐symbiont interactions. We examined the effects of three concentrations of CO₂ (390, 800 and 1000 ppm) and two concentrations of nitrogen fertilizer (0.004 g N/week versus 0.2 g N/week) on the physiological response of Neotyphodium fungal endophyte‐infected and uninfected tall fescue plants. We used quantitative PCR to estimate the concentration of endophyte under altered CO₂ and N conditions. We found that elevated CO₂ increased the concentration of water‐soluble carbohydrates and decreased the concentration of plant total amino acids in plants. Fungal‐derived alkaloids decreased in response to elevated CO₂ and increased in response to nitrogen fertilization. Endophyte concentration (expressed as the number of copies of an endophyte‐specific gene per total genomic DNA) increased under elevated CO₂ and nitrogen fertilization. The correlation between endophyte concentration and alkaloid production observed at ambient conditions was not observed under elevated CO₂. These results suggest that nutrient exchange dynamics important for maintaining the symbiotic relationship between fungal endophytes and their grass hosts may be altered by changes in environmental variables such as CO₂ and nitrogen fertilization.