Browsing by Author "Simpson, Wayne Roydon"

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    A high frequency change, which is both inducible and reversible, results in altered colony morphology of a fungal symbiont (Neotyphodium lolii) and dwarfing of its grass host (Lolium perenne) : this thesis is presented in partial fulfilment of the requirements for the degree of Master of Science (MSc) in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2009) Simpson, Wayne Roydon
    Fungal endophytes of the genus Neotyphodium form stable symbiotic associations, with grasses, that are symptomless and generally considered to be mutualistic. The benefits that these fungi confer to their grass hosts are exploited in pastoral agriculture systems. The production of a range of secondary metabolites, specifically alkaloids including peramine and ergovaline can give their host plants an ecological advantage in certain environments. Neotyphodium endophytes are asexual and have lost the ability to transfer horizontally between hosts making seed transmission a vital feature of the association. This thesis reports the occurrence of phenotypically different perennial ryegrass plants (Lolium perenne) in a population infected with Neotyphodium lolii. Here we show that the change in the plants is directly attributable to a variant endophyte that they host. Isolation of the variant endophyte reveals a change in colony growth compared to the wild-type resident endophyte in the population, which has a white and cottony phenotype. Colonies of the variant endophyte are smaller than wild-type colonies and mucoid, with hyphal filaments forming aggregates. Evidence shows that the switch between colony morphologies occurs at a very high frequency, is reversible, and appears to be environmentally induced. This suggests that the switching phenomenon involves gene regulation rather than mutation. When endophyte-free plants are infected, with either white and cottony (wild-type) or mucoid (variant) fungal colonies, they assume a morphology consistent with the state of the fungus at the time of inoculation, that is normal or dwarfed, respectively. In addition, re-isolation of endophyte from either normal or dwarfed plants always yields white and cottony or mucoid colonies, respectively, suggesting that the host environment stabilizes the state of the fungus. Proteomic profiling revealed differences in protein expression between plants infected with either the wild-type or mucoid fungus. Furthermore, host plants containing the mucoid fungus have never flowered or produced seed. Thus, if this change in the fungal symbiont occurs in a competitive natural environment the mucoid fungus and its host plant may not persist beyond the first generation. This thesis provides insights into the plastic nature of fungal endophyte/grass symbiota and discusses possible mechanisms for the observed morphological switching in culture and host dwarfing.
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    Hordeeae Epichloë endophytes and the formation of synthetic symbioses with cereal grasses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Simpson, Wayne Roydon
    This thesis examined two classes of organism that live in symbiosis, grasses and fungi. Specifically it dealt with grasses of the tribe Hordeeae (Triticeae) in the subfamily Pooideae and Epichloë (Epichloë / Neotyphodium) fungi of family Clavicipitaceae. Epichloë endophytes, particularly asexual forms, have important roles in pastoral agricultural systems in the Americas, Australia and New Zealand. Selected strains add value to grass-based forage systems by providing both biotic and abiotic stress resistance. Cereal grasses such as wheat, barley and rye are important to human and animal nutrition and indeed to the foundation and maintenance of Western civilisation. Modern Hordeeae cereal grasses such as wheat, barley and rye do not host Epichloë endophytes, although grasses of some genera within the tribe, such as Elymus and Hordeum, do so. Both organism classes, Epichloë endophytes and cereal grasses, are of great importance in their own contexts; this research examined the possibility of bringing them together in symbiosis with the ultimate goal of improving cereal production systems. In this study, a screen of wild Elymus and Hordeum grasses in Gansu Province, China showed high levels of Epichloë infection. A diverse range of fungal genotypes was identified using SSR markers, and chemical screening revealed the production of alkaloid metabolites consistent with the range seen in Epichloë-infected pasture grasses of tribe Poae. Importantly, strains were identified that did not produce the mammalian toxins ergovaline or Lolitrem B, although less toxic intermediates such as the indole diterpene paspaline and ergot clavine alkaloids were identified. In addition, strains were identified that produced the insect deterrents/toxins peramine and loline. Inoculation studies performed in this study demonstrated that cereal grasses could be successfully infected by artificial means using cultured Epichloë fungus, although II infected plants generally had poor morphological phenotypes. While alkaloid production of synthetic associations was qualitatively the same as that of native associations, relative quantitative differences were observed between native Elymus and synthetic rye. Differences in infection frequencies and host phenotypes were observed between Epichloë strains. The choice of Epichloë strain used for inoculation profoundly affected the outcome of the symbiosis, ranging from no infection to stunted plants that died prematurely, infected dwarf plants through to normal phenotype plants. Host genotype was also observed to impact infection frequency and phenotype. Family differences in infection phenotype in outcrossing rye suggested a host genetic basis for the observed variation, while population differences in selfing rye indicated that genetics may not have been the sole driver. Consistent phenotypes were observed from the self-fertilizing cereals wheat and barley but, unlike rye, these were not amenable to recurrent selection. Finally, the infection of wheat alien addition/substitution lines showed that there is potential to select wheat-based germplasm with improved phenotypes. Thus, both Epichloë genotype and host genotype underpinned successful compatible symbiosis. This work demonstrated that cereal grasses could be synthetically infected with Epichloë and that agriculturally useful metabolites were produced by these symbioses. The manifestation of infection phenotypes highlighted the necessity for careful selection of germplasm for inoculation and a need for selection and breeding of cereal grasses after infection.