Graphania mutans (Walker) and Acremonium lolii (Latch) : the relationship between an insect herbivore and a fungal endophyte of perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Zoology at Massey University

In examining the relationship between Graphania mutans (Walker) (Lepidoptera: Noctuidae) and perennial ryegrass infected with the fungal endophyte Acremonium lolii (Latch in press), the biology of G. mutans was investigated. Two types of larval development are identified: 'fast-track' larvae develop more rapidly through fewer instars and grow much larger than 'slow-track' larvae when reared on both artificial diet and perennial ryegrass. The complexity of Graphania speciation is discussed. The presence of endophyte is shown to confer on perennial ryegrass resistance to G. mutans larvae in the laboratory, with strong antixenosis and possible antibiosis effects exhibited. Feeding preference tests show that neonate and sixth instar fast-track larvae significantly prefer excised endophyte-free ryegrass to endophyte-infected leaves. The effects of endophyte on the development of fast-track and slow-track larvae are to decrease larval weight, head capsule width, and the number of successful pupations. The same methods were used to determine the effects of peramine (an antifeedant compound for Argentine stem weevil extracted from endophyte-infected perennial ryegrass) on fast-track G. mutans larvae. Incorporated into artificial diet at 10ppm, peramine has no affect on neonate and sixth instar larval feeding preference. peramine does affect larval development, causing reduced larval weight, delayed pupation, and, increased mortality. The role of peramine in endophyte-induced resistance, and the possible adaptive significance for perennial ryegrass of endophyte infection is considered. The interactions between G. mutans, endophyte and perennial ryegrass within the pasture ecosystem are discussed, and suggestions and hypotheses presented for future investigation

Chemical control of Poa trivialis on New Zealand racetracks : a thesis presented in partial fulfilment of the requirements for the degree of Master of Applied Science at Massey University

Poa trivialis is a perennial grass weed commonly found in perennial ryegrass (Lolium perenne) racetrack swards throughout New Zealand. Its presence is undesirable as it does not tolerate high wear and is susceptible to dying out over the summer. Two pot trials at Massey University and one field trial carried out at the Awapuni racecourse were conducted during winter and spring 1995 to test the relative susceptibility of Poa trivialis and perennial ryegrass to a wide range of herbicides. A bioassay was also conducted to determine whether herbicide residues from the field trial could affect the germination of perennial ryegrass seed sown soon after treatment. Results showed that none of the chemicals at their chosen respective rates could completely remove Poa trivialis from a racetrack sward in the spring without some damage being caused to perennial ryegrass. Propyzamide and fenoxaprop at rates of 0.2 and 0.15 kg/ha respectively showed the most potential of the chemicals, severely damaging Poa trivialis (causing 50 to 75% reductions) with no adverse effect on perennial ryegrass 8 weeks after spraying. Propyzamide can also provide some control of Poa annua. Fenoxaprop was not improved by increasing the application rate or adding an oil. The performance of fenoxaprop was substantially reduced when applied with either MCPA or a picloram/triclopyr mix. Dalapon and asulam showed good potential to control Poa trivialis but at the high rates tested caused variable or harmful effects to perennial ryegrass. Ethofumesate and chlorpropham applied at rates of 2.0 and 2.5 kg/ha respectively gave inadequate control of Poa trivialis. None of the above herbicides, when used in the field trial resulted in residues which reduced the germination of perennial ryegrass seed sown 3 weeks after spraying. Herbicides tested in the pot trials which showed poor control of Poa trivialis were atrazine, dicamba, isoproturon/diflufenican, linuron, mecoprop, methabenzthiazuron, metsulfuron, pendimethalin, prometryne, thifensulfuron-methyl, triclopyr, and trinexapac-ethyl. Diuron applied at 2.6 kg/ha provided good control of Poa trivialis but caused significant damage to perennial ryegrass. It is concluded that an integrated management approach that incorporates both cultural and chemical techniques will be required to control Poa trivialis on New Zealand racetracks. Future trial work should be carried out on propyzamide applied at rates of 0.2-0.3 kg/ha in autumn to establish the most appropriate time of year to apply this herbicide

Drought tolerance of perennial ryegrass (Lolium perenne L.) and the role of Epichloë endophyte : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand

Perennial ryegrass is the most important grass species in New Zealand. Due to climate change, drought will become more severe and frequent in New Zealand, which makes it increasingly important to improve drought tolerance of perennial ryegrass. There are many ryegrass cultivars in the seed market; however, very limited information is available about drought tolerance of these cultivars. Therefore, the first aim of this thesis was to compare drought tolerance of several market-leading perennial or long-rotation ryegrass cultivars in order to provide cultivar information for pastoral industry. Epichloë festucae var. lolii fungal endophyte naturally colonises perennial ryegrass. Reported effects of endophyte on drought tolerance of the host perennial ryegrass are multifarious. Therefore, the second aim of this thesis was to investigate effects of endophyte on drought tolerance of perennial ryegrass comprehensively. Two main experiments were conducted in this PhD project. In the first experiment, endophyte-free (E–) and endophyte-infected (E+) cloned plants of seven perennial or long-rotation ryegrass cultivars (Grasslands Commando, Ceres One50, Banquet II, Alto, Bealey, Trojan and Avalon), an un-released elite perennial ryegrass line (URL) and one Mediterranean tall fescue cultivar (Grasslands Flecha) were subjected to a cycle of drought and rehydration from December 2012 to May 2013 while other clones of the same plants were irrigated. In the second experiment, two perennial ryegrass cultivars One50 and Commando infected with and without the AR37 endophyte were subjected to a glasshouse experiment. Eight genotypes of each cultivar with and without endophyte infection were either under irrigation or withheld irrigation for two weeks and then rehydrated for one month. A series of plant morphological and physiological responses were measured in each experiment. In the rainout shelter experiment, it was found that Flecha tall fescue was more tolerant to drought than ryegrass cultivars, but this was attributed to its small plant size induced by the partial summer dormancy. Introducing germplasm from Mediterranean areas would be an option to improve drought tolerance of perennial ryegrass in New Zealand. Among evaluated ryegrass cultivars, Banquet II was relatively more drought tolerant than other cultivars, which was also mainly due to its small plant size. In the glasshouse experiment, it was found that Spanish germplasm based One50 was more drought tolerant than „Mangere? ecotype based Commando, suggesting that Spanish germplasm has conferred enhanced drought tolerance to perennial ryegrass in New Zealand. Under both irrigated and non-irrigated conditions, endophyte infection reduced the herbage yield, decreased the relative water content, osmotic potential and stomatal conductance (as indicated by carbon isotope discrimination) and increased the proline concentration of the host compared to E– plants. Also, a majority of these effects were more pronounced in the URL (infected with AR37) and One50 (infected with AR1). It was concluded that E+ plants are at a disadvantage compared to E- plants when insect pressure is artificially controlled, no matter whether the water availability is high or low

Fungal alkaloid occurrence during seedling establishment and early growth in Lolium perenne seedlings infected with Epichloë festucae var. lolii and the influence of adult Argentine stem weevil (Listronotus bonariensis) feeding on alkaloid concentrations : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Agriculture Science at Massey University, Palmerston North, New Zealand

Fungal endophytes of the genus Epichloë often form stable, symbiotic, and mutualistic relationships with grasses of the Pooideae, including perennial ryegrass (Lolium perenne L.). The endophyte provides various benefits to its grass host, through the production of secondary metabolites, which are exploited in New Zealands pastoral agriculture systems. The endophyte can give its host grass an ecological advantage in certain challenging environments, such as during seedling establishment, where young plants are especially vulnerable to insect predation, such as feeding by adult Argentine stem weevil (ASW, Listronotus bonariensis). This thesis focuses on understanding the alkaloid concentrations that occur in endophyte-infected perennial ryegrass seedlings during the early establishment phase. A glasshouse experiment was conducted in which fungal alkaloid concentrations (peramine, lolitrem B, ergovaline, and epoxy-janthitrems) were measured in perennial ryegrass seedlings infected with Epichloë festucae var. lolii strains AR1, AR37, NEA2, and NZCT for 69 days after sowing. From the data it is inferred that an initial translocation of alkaloids stored in seed during maturation into the developing shoot of the germinating seedling occurs, followed by a period of alkaloid dilution due to seedling expansion, and finally production of newly metabolised alkaloids in the plant. Alkaloid concentration were found to peak in 8–10 day old seedlings, giving the seedling a “kick start” in protection of the emerging seedling from adult ASW feeding during the first 11 days after sowing. The influence of adult ASW feeding on alkaloid concentrations in endophyte-infected perennial ryegrass seedlings was also tested. The study demonstrated that adult ASW feeding can influence alkaloid production, although peramine, the main alkaloid responsible for ASW deterrence was not significantly affected. Findings from this thesis improve understanding of the role of fungal alkaloids in endophyte-infected perennial ryegrass seedlings during establishment, and help explain results from earlier studies describing seedling susceptibility to adult ASW

Breeding Morphogenetic Traits to Match Genotypes to Their Utilization

A divergent selection for lamina length was done from natural populations of perennial ryegrass. Tested in a multi-site experiment in France, the short-leaved perennial ryegrass was more productive under simulated grazing and less productive under infrequent cutting than the long-leaved perennial ryegrass. Matching cultivars to their management is possible by selecting for morphogenetic traits. However, our results suggest the range of adaptation to different managements of perennial ryegrass cultivars could be extended by increasing their phenotypic plasticity

Exploration of flowering control in Lolium perenne L.

End of project reportFlowering or heading in Lolium perenne (perennial ryegrass) is induced by a period of vernalization, followed by long days at higher temperatures. When heading occurs there is a reduction in the feed quality of the forage and therefore extending the period of vegetative growth or eliminating heading during the growing season will improve the potential of perennial ryegrass in agriculture. Conversely, a better control of flowering time and increased heading will lead to higher seed yield for commercial producers. The aim of this project was to investigate the underlying genetic control of flowering time in perennial ryegrass. An F1 population was created by crosspollinating two lines with different heading dates and a genetic linkage map was constructed using Simple Sequence Repeat (SSR) markers. The population and genetic linkage map was then used to identify Quantitative Trait Loci (QTL) associated with heading date, spike length and spikelets per spike. A number of QTL were identified for all traits, some of which had not previously been identified in perennial ryegrass. A Suppression Subtractive Hybridization (SSH) study was also employed to identify genes differentially expressed between an extremely late flowering line and earlier flowering sibling line. Expression analysis of a number of identified genes through floral induction was performed using real time RT-PCR. This revealed a number of transcripts with expression profiles indicative of a role to play in floral induction

Performance and quality of tall fescue (Festuca arundinacea Schreb.) and perennial ryegrass (Lolium perenne L.) and mixtures of both species grown with or without white clover (Trifolium repens L.) under cutting management

There is increasing interest in tall fescue (Festuca arundinacea Schreb.) in Western Europe and elsewhere, mainly because of its better drought resistance and yield potential compared with perennial ryegrass (Lolium perenne L.). Important drawbacks of tall fescue, compared with perennial ryegrass, are its lower digestibility and voluntary intake. Mixtures of both species might combine the advantages of each, and species interactions may eventually lead to transgressive overyielding. We compared the agronomic performance of tall fescue, perennial ryegrass and tall fescue-perennial ryegrass mixtures, as pure-grass swards or in association with white clover (Trifolium repens L.). Tall fescue-perennial ryegrass mixtures differed in the proportion and ploidy of the perennial ryegrass component. Yield, feed quality and botanical composition were measured in the 3years after the sowing year. We found significant effects of ploidy of the ryegrass variety and of the proportion of ryegrass in the initial seed mixture on the botanical composition of the swards. Nevertheless, all swards were dominated by tall fescue at the end of the experiment. No overyielding of the mixtures compared with that of single-species swards was found, but feed quality was intermediate between that of the single-species swards. Mixed swards had better drought resistance than L.perenne and higher feeding quality than F.arundinacea swards

N2-fixation and residual N effect of four legume species and four companion grass species

Inclusion of forage legumes in low-input forage mixtures improves herbage production and soil fertility through addition of nitrogen (N) from N2-fixation. The impact of different grass-legume mixtures on the N contribution of the forage mixture has rarely been investigated under comparable soil and climatic conditions. We conducted a field experiment on a sandy soil at two nitrogen levels with seven two-species forage mixtures: alfalfa, bird’s-foot trefoil, red clover, or white clover in mixture with perennial ryegrass, and white clover in mixture with meadow fescue, timothy, or hybrid ryegrass. We found high N2-fixation of more than 300 kg N ha-1 from both red clover and alfalfa even when the two mixtures received 300 kg total-N ha-1 in cattle slurry. The addition of cattle slurry N fertilizer lowered N2-fixation for white clover and red clover as expected, but for bird’s-foot trefoil and alfalfa no changes in the proportion of N derived from N2-fixation was observed. We conclude that the competition for available soil N from perennial ryegrass in mixture was an important factor for the proportion of N in alfalfa, white clover, and bird’s-foot trefoil obtained from N2-fixation. White clover had a high proportion of N derived from atmosphere for all companion grasses despite significant differences in white clover proportion. Although the perennial ryegrass-alfalfa mixture in the grass phase yielded more than twice the N from N2-fixation compared to white clover in the perennial ryegrass mixture, this did not in the following year lead to higher residual N effects of alfalfa. Both in terms of N yield in the grass phase and N yield in the subsequent spring barley red clover contributed most to the improvement of soil N fertility

Effects of cutting frequency on plant production, N-uptake and N2 fixation in harvested and below-harvest plant biomass of grass clover

Nitrogen (N) accumulating in stubble, stolons and roots is an important component in N balances in perennial ryegrass-white clover swards, and the effects of cutting frequency on the biomass of above- and below-harvest height were studied during two consecutive years. Total dry matter (DM) and total N production, and N2 fixation, were measured at two cutting frequencies imposed in the summers of two years either by cutting infrequently at monthly intervals to simulate mowing or by frequent cutting at weekly intervals to simulate grazing. Total DM production harvested was in the range 3000 - 7000 kg DM ha-1 with lower DM production associated with the frequent cutting treatment, and it was significantly affected by the different weather conditions in the two years. The higher cutting frequency also reduced the biomass below harvest height, but the different weather conditions between years had less effect on stubble and, in particular, root biomass. The biomass of white clover roots was significantly lower than that of perennial ryegrass roots, and remained at a relatively constant level (200-500 kg DM ha-1) throughout the experiment, whereas the biomass of perennial ryegrass roots increased from 2 400 kg DM ha-1 in the year of establishment to 10 200 kg DM ha-1 in the infrequent cutting treatment and 6 650 kg DM ha-1 in the frequent cutting treatment by the end of the experiment, giving shoot: root ratios of 4.7 – 16.6 and 0.5 – 1.6 for white clover and perennial ryegrass, respectively. Annual N2 fixation was in the range 28 – 214 kg N ha-1, and the proportion of N fixed in stolons and roots was on average 0.28. However, since the weather conditions affect the harvested DM production and the shoot: root ratio, care must be taken when estimating total N2 fixation based on an assumed or fixed shoot: root rati

Micro-mineral profile in different grassland species

The aim was to investigate the micro-mineral profile of herbage as affected by grassland species, cutting time and seasonality and in relation to dairy cow requirements. The different grassland species were grown and harvested in mixtures with one grass and one legume for two growing seasons. The species turned out to have very individual mineral profiles, not influenced by year. Among the legumes red clover (Trifolium pratense) had high concentrations of Co, Cu and Zn, white clover (Trifolium repens) of Mn and Fe, lucerne (Medicago sativa) of Se and lotus (Lotus corniculatus) of Co, Mn, Zn and Fe. Among the grasses perennial ryegrass (Lolium perenne) generally had the highest concentrations of all the micro-minerals. Hybrid ryegrass (Lolium hybridum) had slightly lower concentrations than perennial ryegrass for all minerals. Meadow fescue (Festuca pratensis) had lower concentrations still, and timothy (Phleum pratense) had, with the exception of Zn, the lowest concentrations. In general, the mineral concentrations were higher in summer than in spring growth. During the growth Cu, Zn and Fe concentrations decreased, whereas Co, Se and Mg were unaffected. It was concluded that a mixture of red clover and perennial ryegrass had the best profile of micro-minerals for cattle feeding