Effect of abiotic stress on the success of the vertical transmission and survival during seed storage of two novel endophytes in perennial ryegrass : A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Lincoln University

Abstract

Epichloë fungal endophytes, which live in symbiosis with perennial ryegrass (Lolium perenne L.) are vertically transmitted only, totally depending on the success of the host plant for their dissemination and survival. New Zealand (NZ) leads the world in terms of adoption of endophyte technology. These novel associations are very successful in farming practices, as the endophytes provide bio-control through the production of secondary metabolites. Alkaloids like ergovaline, lolines, peramine and epoxy-janthitrems control at least six major pasture pests that decrease the quality and persistence of NZ grasslands. Both endophyte and host plant grow in synchronization via a unique intercalary hyphal extension mechanism. However this relationship is far from being perfect as the endophyte can be lost at any stage of the host plant life cycle, either during the pre or post zygotic stage. This research aimed to identify abiotic factors affecting transmission during the development of the perennial ryegrass seed crop and survival of the endophytes in seed during storage, and used two commercial novel endophytes strains, AR1 and AR37 and three perennial ryegrass cultivars, ‘Prospect’, ‘One -50’ and ‘Samson’. Waterlogging (WL) was imposed during three plant development stages (GS 30, GS59 and GS 70) to investigate any effect on the vertical transmission of the endophyte. WL had no significant effect on ryegrass seed germination percentage, or on endophyte transmission. In both cultivars and with both endophytes, the transmission exceeded 70%. Germination did not differ between the cultivars or between endophyte strains. For the growth parameters measured, WL had no significant effect on dry matter (DM) production, number of reproductive tillers per plant, seed yield per plant or thousand seed weight (TSW). However, TSW differed significantly between cultivars and between the endophyte strains independently of the WL treatments of the experiment. The effects of combinations of three rates of nitrogen (0, 150, 250 kg ha⁻¹yr⁻¹) with three rates of potassium (0, 200, 400 kg ha⁻¹yr⁻¹) applied during crop development on the transmission of AR1 and AR37 endophyte strains and the effect on plant growth parameters were investigated. The grand mean transmission was 87%. There were no significance differences among treatments for transmission rate, or between the two cultivars or endophyte strains. However there was a significant linear response (p = 0.05) for transmission between endophyte strains to the applied N only. With no N, strain AR37 transmitted 8.7% more than AR1 in cultivar ‘Prospect’. At 150 and 250 kg N ha⁻¹yr⁻¹, there were no differences either between cultivars (cv) or endophyte strains. N and K application rates had no significant effect on seed germination between endophyte strains. However, there was a significant difference in germination between cultivars, with ‘Prospect’ having a 5.6% higher germination than ‘One-50’. The grand mean of germination was very low (73%), indicating poor quality. K significantly increased DM production, but there was no significant difference between the biomass produced when 200 kg or 400 kg ha⁻¹yr⁻¹ of K were used. The production of DM between endophyte strains did not differ. Both fertilisers increased the number of reproductive tillers produced for both cultivars, but not between endophyte strains. Cultivar ‘Prospect’ produced 47 more reproductive tillers than ‘One-50’. Both fertilisers significantly increased seed yield. Furthermore, the effect of temperature and relative humidity during seed storage on the survival of these endophytic associations was determined. Combinations of four temperatures and four relative humidity levels, created and controlled using saturated salts solutions in sealed containers, were used to determine the endophyte survival in perennial ryegrass infected with strain AR37 during 418 days of storage. The accumulated thermal unit approach (ATU) was used to identify the “best before time” to maintain viable endophyte ≥ 70%. At 4°C endophyte viability remained above 70% for 1800 ATUs at 24%, 40% and 50% RH (relative humidity). At 20°C, endophyte viability at all four % RH remained above the industry threshold for 2500 ATU. At 30°C, by 1500 ATU endophyte viability in seeds stored at 40%, 50% and 70% RH had fallen below the 70% threshold and for those seeds stored at 24% RH, this point was reached at 2500 ATU. Maintaining endophyte viability above 70% was possible at three different temperatures (4°C, 15°C and 20°C) and relative humidity levels of both 24% and 40% when the accumulated thermal units were below 2000. At 70% RH, the endophyte maintained its viability for only up to 1000 ATUs. At 30°C and 24% RH, the endophyte maintained its viability for 45 days, being the only safe storage combination at that high temperature (2250 ATUs). Ambient temperature with higher relative humidity levels increased SMC and as a result negatively affecting endophyte viability when SMC >10 %. For all storage conditions, endophyte viability was always lost before seed viability. In addition, the effect of different concentrations of carbon dioxide (CO₂) during storage on seed and endophyte viability was investigated. Controlled environments were created by capturing seed respiration at 5°C, 20°C and 30°C in sealed containers and by the use of ascorbic acid dust (AnaeroGen™) which caused anoxia and elevated the CO₂ levels in sealed containers at 20°C. Seeds from cultivar Samson containing the novel AR37 endophyte strain and the same cultivar endophyte free (E-) were used in this experiment. The endophyte viability and the seed germination were assessed after short periods of storage (1, 2, 4, 8, 16 d) and up to 32 days; in airtight glass vials. Seed respiration rate increased with temperature and seeds containing endophyte (E+) had a higher respiration rate than seeds without endophyte (E-). Seeds stored at higher temperatures (30°C), with higher accumulative CO₂ concentrations due to respiration of seeds, lost the endophytes at a faster rate than seed stored at lower temperatures (5°C). The time of storage had a significant negative effect on endophyte survival. The effect of temperature was significant on endophyte survival between seed stored at 30°C compared with the ones at 5°C or 20°C. At equal temperature regimes there was no significant difference found between seed lots E+ or E-. Overall, there was no significant difference between seeds infected with AR37 and endophyte free seeds after 32 days of storage. Where anoxic conditions with high concentrations of CO₂ were created, both time of storage and high concentrations of CO₂ in an airtight container negatively affected endophyte viability. Overall high CO₂ concentrations and lack of O₂ did not reduce the germination rate of either E+ or E- seeds stored for 32 days