Investigating Epichloë endophyte transmission in Poaceae hosts : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatū, New Zealand
Vertically-transmitted Epichloë endophytes are agriculturally important fungi that colonise the aerial plant tissues of cool-season grasses within the Poaceae. Plants colonised by selected strains of Epichloë have superior protection from herbivores, thus affirming the important role of these endophytes in New Zealand farming systems. However, the development and marketability of endophyte-based products is often hindered by failures of endophyte transmission. This research investigated: (1) the developmental timing of endophyte colonisation of the seed embryo; (2) the identity of soluble sugars related to endophyte aging during seed storage; (3) the comparative endophyte hyphal density in the shoot apex and florets of high- and low-transmission genotypes; and (4) the molecular mechanisms for endophyte transmission from the inflorescence primordia to the unfertilised ovary.
Through a detailed investigation, utilising confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Festuca arundinacea), the endophyte hyphal colonisation in the ovary (pre-fertilisation) through to the fully mature seed stage was tracked. Confocal microscopy images revealed that endophytes have colonised the embryo sac before host grass fertilisation.
Tall fescue seeds, either endophyte-free or infected with one of three endophyte strains (AR584, AR605 or common-toxic) were subjected to a 2x2 factorial combination of two factors (accelerated aging or not, and seeds imbibed or not) and the sugar profiles in the seeds were investigated. Trehalose was the sugar that correlated most closely with the loss of endophyte during seed aging. After imbibition, the concentrations of trehalose significantly declined in the endophyte-infected seed tissues, suggesting that the endophyte-oriented trehalose was utilised during imbibition. In addition, the sugar alcohols mannitol and ribitol were found in high concentrations in endophyte-infected embryo and endosperm tissues. These two sugars, therefore, could be potentially used as indexes to estimate endophyte biomass.
Two experiments were performed to investigate the endophyte hyphal density in the vegetative and reproductive tissues of perennial ryegrass (Lolium perenne): namely quantification of the endophyte density in the shoot apex tissues using real-time PCR, and analysis of immunoblot colour intensities of laterally bisected florets from six endophyte-grass genotypes (high-transmission [HT]: genotypes 11, 103, 107; low-transmission [LT]: genotypes 13, 79 and 83) and from three positions (bottom, middle and top) of the spike. The florets were collected at three growth stages (Stage I [unfertilised], Stage II [ten days after Stage I] and Stage III [twenty days after Stage I]). Real-time PCR analysis showed that the HT genotypes generally had higher endophyte densities in the shoot apex tissues compared with the LT genotypes. The immunoblot analysis showed that the immunoblot intensities in genotypes 11, 103, 107 and 13 were significantly higher than the other genotypes at Stage I, while the immunoblot intensities in the three HT genotypes were significantly higher than the LT genotypes at Stage II. However, there were no significant differences in the intensities between any of the genotypes at Stage III. Microscopy confirmed that HT genotypes carried a higher density of endophyte hyphae in the shoot apex tissues and ovaries (Stage I) than the LT genotypes. The data indicated that increased endophyte biomass is one factor that enhances endophyte transmission from the parent plant to mature seeds in the HT genotypes.
RNA-Seq was used to measure the transcriptional response in two types of tissues (inflorescence primordia and the ovary) in the HT and LT genotypes. This study showed that 102 genes were commonly or exclusively differentially-expressed between the HT and LT genotypes in the inflorescence primordia and/or the ovary. Functional enrichment analyses by agriGO showed that the highly enriched gene ontology (GO) terms between the HT and LT genotypes were involved in serine family amino acid metabolic processes (GO:0009069) and cytoplasmic membrane-bounded vesicle function (GO:0016023) in both the inflorescence primordia and the ovary. More differentially-expressed genes (DEGs) coding for trehalose-6-phosphate phosphatase were induced during development from the inflorescence primordia to the ovary in the HT than the LT genotypes, demonstrating the higher demand for trehalose in the HT than the LT genotypes during endophyte transmission. More genes regulating salicylic acid were significantly repressed while more genes related to jasmonic acid metabolism were significantly induced during development from the inflorescence primordia to the ovary in the HT than the LT genotypes. It is proposed that the lower salicylic acid metabolism and higher jasmonic acid metabolism during development from the inflorescence primordia to the ovary in the HT genotypes might be related to increased endophyte transmission frequencies.
Chapter 3 was removed for copyright reasons but was published as: Zhang, W., Card, S.D., Mace, W.J., Christensen, M.J., McGill, C.R., & Matthew, C. (2017). Defining the pathways of symbiotic Epichloë colonization in grass embryos with confocal microscopy. Mycologia 109(1): 153-161. https://doi.org/10.1080/00275514.2016.1277469
Chapter 4 was accepted for publication as: Zhang, W., Mace, W.J., Matthew, C., & Card, S.D. (2019). The impact of endophyte infection, seed aging, and imbibition on selected sugar metabolite concentrations in seed. Journal of Agricultural and Food Chemistry, 67(25), 6921-6929. https://doi.org/10.1021/acs.jafc.9b01618