Responses of perennial ryegrass populations to grazing method and endophytic relationships : A thesis submitted in partial fulfilment of the requirements for the Degree of Master of Agricultural Science at Lincoln University

New Zealand dairy farmers rely primarily on swards based on perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) for feeding dairy cows. The direct-grazed, low-cost production system that these pastures enable provides the New Zealand dairy industry with a competitive advantage for products in the international market. As a result, the industry contributes significantly to New Zealand’s export earnings and national gross domestic product. However, farmers in the upper North Island have expressed concern about the poor productivity of their perennial ryegrass pastures beyond three years post-sowing and have linked this to more-frequent occurrence of drier and warmer conditions for pasture growth during summer and autumn. Recent research has shown that even pastures sown with new ryegrass cultivars inoculated with the most-advanced endophyte strains cannot sustain high productivity under the severe climatic conditions and increased insect pest burdens being experienced in many parts of the Waikato, Bay of Plenty and Northland. In some cases, this is due to the death of ryegrass plants and ingress of lower-producing weed species, while in other cases reasonable ryegrass plant density can be sustained, but growth rates are poor. There is limited information on the plant population and phenological factors associated with these changes, making it difficult for farmers, agronomists and plant breeders to develop effective solutions. The study described in this thesis investigated the differences in phenotypic trait expression of perennial ryegrass plants removed from 10-year old pastures grazed by dairy cows in the Waikato (‘survivor’ plants) versus plants grown from the original seed lines that were used to sow the pastures and had, therefore, not been subject to any environmental selection pressure (‘reference’ plants). The study aimed to help inform future plant breeding efforts to utilise phenotypic changes and reselect for traits that are associated with better plant survival. Three populations were compared: reference plants; survivors from pastures rotationally grazed by dairy cows since they were established in autumn 2011 (‘control’); and survivors from pastures managed as per the control treatment, but where grazing was deferred for ~ 120 days from mid spring to mid-summer to promote new seedling recruitment two years before plants were collected from the field (‘deferred’). Four perennial ryegrass cultivars were included in the design: Nui standard endophyte (SE); Commando AR37, a mid-season heading diploid; Alto AR37 a late season heading diploid; and Halo AR37 a very late heading tetraploid. Thus, 12 populations were compared. Initially, there were 60 plants per population giving a total of 720 plants which were grown individually in pots at Lincoln University. The first phase was a pilot study during the early acclimatisation period where progress towards equalisation of plant growth across the populations was tracked. Biomass accumulation and reproductive development were assessed from late February to late April. Substantial flowering activity was recorded in the control and deferred populations for all cultivars, but not in the reference population, illustrating the expected carry-over effects related to differences in plant source (clonal fragments from the field versus seed-derived for the reference population). Biomass accumulation was initially lower in the reference population but there were no differences between populations by day 105 when plants had tillered-out to a similar degree. All plants were then analysed for the presence or absence of endophyte and, where endophyte was present, its genetic identity (strain). Plants with no endophyte, or with an endophyte strain that was not AR37 (or not SE, for Nui), were discarded and plant numbers rebalanced to 30 plants per population that were confirmed as ‘true-to-type’ for the original host genotype – endophyte strain combination sown in 2011. Four further populations of control plants (one for each cultivar; total 240 plants) were available for endophyte analysis from a site in Canterbury where the same experimental design and seed lines as used in the Waikato was established at the same time. The grazing deferral treatment was not implemented at this site. For Waikato, the percentage of plants that were true-to-type was significantly higher in the reference population (82%) than the control and deferred populations (69-71%). Overall, Commando had significantly lower percentage true-to-type than the other cultivars (60% versus 78-82%), with a high presence of standard endophyte (22%) indicating contamination by volunteer ryegrass plants and poorer persistence of the original population. For the Canterbury control populations, mean true-to-type percentages were 85-98% across the four cultivars, i.e. generally higher than for the original seed lines. The second phase was a detailed phenological study, where plant, leaf and tiller characteristics were measured on the true-to-type plants only, and flowering observations were conducted for 70 days starting in mid-October. Phenotypic measurements included: leaf weight, length, width, area, specific leaf area and thickness; pseudostem weight, length and width; and visual scores of rust, tillering and growth habit. Control plants had significantly lower leaf dry weight, shorter leaves and smaller lamina area than the reference plants, with no interaction between population source and cultivar. Deferred populations did not differ from reference populations in these traits, indicating grazing deferral ‘re- set’ the plant phenotype back from the control state to its original state, at least for the first 18 months after deferral. There were no differences between populations in pseudostem characteristics, growth habit or tiller density scores, but reference populations showed a trend towards higher incidence of rust compared with control and deferred. A significant shift toward later flowering in the control population compared with the reference population was found for Nui, Commando and Alto, but not for Halo. In summary, differentiation toward smaller leaves, greater disease resistance, and later flowering was observed after nearly ten years under grazing in the Waikato populations when potential contaminants were excluded and any effects due to endophyte presence/absence or variant strains were removed. It appears that the shift towards longer leaves can be reversed by deferring grazing to allow recruitment of new seedlings into the population. In contrast, shifts in rust resistance and flowering in control populations were not reversed as a result of spring/summer grazing deferral. Further investigation of the genetic factors controlling the reversibility of leaf traits could help identify breeding objectives for maintaining the persistence of yield advantages in ryegrass pastures. Future studies should also examine possible long-term benefits of this management practice to bring about phenotypic change for yield improvements and consider the duration of possible carry-over effects