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    Bio-prospecting for endophytes of Brassica : 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
    (Massey University, 2019) Roodi, Davood
    Disease and insect pests are major limiting factors for crop production worldwide. Farmers are often heavily reliant on synthetic biochemicals and fertilisers to mitigate the negative impact of pests and disease and to increase crop yield. However, the extensive use of chemicals has led to environmental concerns due to contamination of soil and water, human health issues, disturbance of macro and microorganisms balance and the development of resistance by both insects and fungal pathogens. Use of biological control agents including endophytic microorganisms is an alternative control option to combat these problems. Many endophytes are able to provide their host with beneficial traits such as resistance against insect-pests and pathogens and enhance crop performance under abiotic stresses. Although beneficial microorganisms of brassica crops have been discovered, endophytes of wild brassica’s, particularly those associated with the seed, have been ignored. In this study, we screened seed of various brassica species with a worldwide distribution and isolated 131 bacterial and two fungal species. Molecular identification of bacterial isolates indicates that most seed accessions harboured endophytic bacteria belonging to 17 species. Among these isolates, two species, identified as Methylobacterium fujisawaense and Me. phyllosphaerae were dominant and widespread across the majority of accessions sampled, and originated from different species and locations. The inoculation of oilseed rape (Brassica napus) root with these endophytic bacteria significantly increased the fresh weight of the seedlings. The fungal endophyte species identified were Beauveria bassiana and Geomyces pannorum, isolated from two different accession of a wild brassica species (B. rapa). Inoculation of the seeds of three brassica species, B. napus, B. rapa and B. oleracea with these fungal endophytes resulted in infection of below and above ground tissues of inoculated seedlings but colonisation of the next generation seeds/seedlings did not occur. Seed inoculation, foliar application and soil drenching when the plants were grown on non-sterile soil also did not result in plant colonisation. A dual culture test was performed to study the antagonistic effect of these bacterial and fungal endophytes against Leptosphaeria maculans, the causal agent of phoma stem canker in brassica crops. The highest inhibition rate was recorded for Stenotrophomonas rhizophila, Novosphingobium resinovorum, Pseudomonas azotoformans, Plantibacter flavus, Me. fujisawaense and Me. phyllosphaerae which produced a significant inhibition zone indicating the antagonistic ability of these species. The fungal endophytes also suppressed the growth of the pathogen and created an inhibition zone. In planta tests in which the fungal endophytes were inoculated on to seed of a susceptible oilseed rape cultivar were also undertaken. At the cotyledon leaf stage, the leaf was punctured and spore suspension of L. maculans was placed on the wound site. Inoculated seedlings particularly B. bassiana, significantly decreased phoma stem canker disease symptoms on the cotyledon. To our knowledge, this is the first study that screen the fungal and bacterial endophytes of wild brassica species associated with the seeds and demonstrate their beneficial characteristic when inoculated to brassica crops.
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    TILLING : EMS mutagenesis in Epichloë endophytes and mutation screening using High Resolution Melting analysis and Next Generation Sequencing : 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, 2015) Sidhu, Jaspreet Singh
    Epichloë are fungal endophytes (family Clavicipitaceae) of pasture grasses of the sub family poöideae. These endophytes live in symbiotic association with their hosts and confer resistance to insect and animal herbivory through the production of bioactive secondary metabolites (alkaloids) that are produced in planta. For a number of years endophyte research has been focused at manipulating fungal genes responsible for production of alkaloids which have toxic effects on livestock. However, the techniques used to date involve genetic modification to delete genes responsible for alkaloid production and strict regulations around genetically modified organisms in New Zealand prevent commercialisation of these organisms. Traditional mutagenesis was not practical. To find mutations in secondary metabolite pathways, the mutants had to be inoculated back into plants, which would have been a laborious and time-consuming process. The aim of my research was to develop Targeting Induced Local Lesions In Genomes (TILLING) methodology in Epichloë to disrupt fungal secondary metabolite genes using Ethyl methanesulfonate (EMS) and screen for mutations using high throughput screening techniques such as High Resolution Melting (HRM) analysis and whole genome sequencing, MiSeq. In order to carry out the mutagenesis, uninucleate propagules would be preferred but as most of the filamentous fungi (including Epichloë) are multinucleate in nature, spores were thought to be an ideal alternative for mutagenesis. However, many of the commercially used Epichloë strains, such as AR1 and AR37, do not readily produce spores. Therefore an alternative mutagenesis system using fungal protoplasts was investigated and employed. EMS mutagenesis showed that the number of colonies derived from protoplasts after mutagenesis declined steadily at a reproducible rate as measured by time-course of 0, 15, 30, 45 and 60 minutes to give LD50 values. At 60 minutes there was decline in the number of colonies to the levels of 10% of the initial number. To determine the effectiveness of EMS as a mutagen positive selection, using 5-Fluoroorotic acid (5-FOA), was also performed on the mutagenized protoplasts to derive the mutation frequency of 6 mutations per 1000 mutants compared to 0.002 mutations per 1000 for non-mutagenized protoplasts. This suggests a 3000-fold EMS-induced increase in the frequency of mutations. Having established mutation frequency from the 5-FOA, positive selection and steady decline in number of colonies from EMS mutagenesis, an EMS mutant library was screened using next generation sequencing. . However, high throughput whole genome sequencing (MiSeq) led to the detection of only three verifiable point mutations (1 in 10Mb). Microscopic observations revealed that while individual protoplasts were largely (85%) uninucleate, protoplasts typically formed clumps containing 15-30 protoplasts. In theory, multiple nuclei would lead to an overestimation of the number of mutations since each nucleus would accumulate different SNPs. However, MiSeq sequencing did not detect this, probably due to being filtered out during bioinformatics processing. Thus if methods can be devised for plating single protoplasts, EMS mutagenesis should be applicable to this system. TILLING technology can be used to reduce the time for endophyte discovery and improvement. My research demonstrated that this procedure, although very promising in terms of benefit to fungal improvement, carries certain difficulties with it that we had to address such as mutagenesis using protoplasts and subsequent mutation discovery. I succeeded in establishing TILLING methodology for mutagenesis of E. festucae strain Fl1 as well as optimising protocols to screen mutants.
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    Distribution of Neotyphodium lolii-endophyte metabolic activity in perennial ryegrass (Lolium perenne, L.) and its implications for alkaloid distribution and photosynthesis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2000) Spiering, Martin Jürgen
    Neotyphodium lolii is a fungal endophyte of perennial ryegrass {Lolium perenne). In this symbiosis, alkaloids are produced that significantly impact on the performance of farmed animals. Little was previously known about the physiological conditions for the endophyte in the plant leading to their production. A strain of N. lolii, previously transformed with the β-D-glucuronidase (GUS) gene of E. coli under the control of a constitutive fungal promoter, was used for investigations into the in planta metabolic activity of the endophyte; distribution of the alkaloids ergovaline, peramine, and lolitrem B; and photosynthesis. In vitro studies with this transformed N. lolii strain demonstrated the utility of constitutive GUS expression for assessing the metabolic state of the endophyte. By using constitutive GUS expression and a method for quantitation of the in planta biomass of the endophyte, the endophyte metabolic state (EMS) in the grass plant was determined. The EMS was high and uniform in plant tissues and genotypes differing in endophyte concentration, indicating that proliferation of the endophyte in the plant is not controlled via the EMS. Ergovaline, peramine, and lolitrem B exhibited each a characteristic within-tiller distribution maintained across different plant genotypes. None of the alkaloids was distributed in exact proportion to the distribution of metabolically active endophyte mycelium. Differences in the accumulation of the alkaloids per mycelium were observed between plant tissues and plant genotypes, suggesting differential rates of synthesis and/or degradation of the alkaloids in the mycelium and translocation within the grass tiller. Rates of net photosynthesis at high light intensities were lower in plants infected by N. lolii, indicating for an effect on photosynthetic capacity. However this effect was plant-age dependent. Plant growth was not strongly affected by the endophyte, but infected plants had consistently lower leaf elongation rates. The changes in leaf elongation and photosynthetic capacity in infected plants might signal for an effect of N. lolii on the nitrogen metabolism of its host. The experiments concerning the in planta EMS, alkaloid distribution, plant growth, and photosynthesis were conducted in a controlled environment the establishment of which was part of this study. In addition, for the detailed mapping of ergovaline within the grass tiller a method for quantitative extraction of this alkaloid was developed and optimised.
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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.