Abstract
Fungal endophytes are ubiquitous, phylogenetically diverse microorganisms that form intimate associations with their plant hosts. Chionochloa (Poaceae) tussock grasslands represent a vital ecosystem in New Zealand, supporting rich biodiversity that has been extensively studied and recognized as a critical component of the country’s conservation estate. Despite this, little is known about the diversity of fungal endophytes and the factors influencing these intimate microbial relationships. The high levels of endemism within this indigenous ecosystem also makes it an ideal reservoir for bioprospecting novel microorganisms. This thesis aims to characterize the endophytic fungal communities associated with Chionochloa and identify the abiotic and biotic factors driving their diversity and composition. Both culture-based and culture-independent methods are employed to achieve these objectives, alongside an investigation into the plant growth-promoting benefits of these endophytic fungi.
A culture-based approach was used to characterize the common culturable endophytic fungi associated with Chionochloa rigida (narrow-leaved snow tussock grass) and Chionochloa rubra (red tussock grass) (Chapter 3). A total of 31 common fungal taxa, predominantly belonging to the phylum Ascomycota was identified. Three endophytic species—Beauveria pseudobassiana, Arthrinium sp., and Penicillium glabrum—were consistently detected across all sites, colonizing both roots and leaves, and occasionally from whole spikelets of flowering plants. At the landscape level, the endophytic fungal communities were not significantly different between C. rigida and C. rubra. However, at the level of individual tussocks, significant differences were observed between the roots and the leaves. Roots were significantly more diverse than leaves, and although many of the taxa were shared between the two tissue types, the root endophyte community contained more unique taxa.
To gain a deeper understanding of the fungal endophyte community, a culture-independent approach using ITS2 metabarcoding of fungal DNA from the leaves and roots was employed (Chapter 4). This method revealed a significantly more diverse fungal endophyte community compared to the culture-based approach. The community was predominantly composed of Ascomycota and Basidiomycota fungi. A significant interaction effect between host species and tissue type was observed in the composition and diversity of the endophytic fungal community. In C. rubra, the leaf endophyte community was primarily dominated by Candida and Vishniacozyma, whereas the root community was dominated by Mycena, Lachnum, and Candida. In C. rigida, Candida and Sporisorium were the dominant taxa in the leaves, while Lachnum and Mycena dominated the roots. Although the taxonomic composition between tissue types of both host species largely overlapped, the fungal communities differed significantly in the relative abundances of individual taxa. Indicator species analysis of the culture-independent community revealed that the assemblage of endophytes in the roots of C. rigida and in the leaves and roots of C. rubra was predominantly composed of Basidiomycota fungi.
A comparison of the endophyte communities identified through culturing and ITS2 metabarcoding revealed minimal overlap. The basic local alignment search tool (BLAST) search results showed that four of the cultured taxa, Arthrinium sp., Trichoderma atroviride, Podila humilis, and Paraphaeosphaeria sp., were also identified with ITS2 metabarcoding but was not as prevalent within the endophytic community. Both methods revealed complementary aspects of the total endophyte community in Chionochloa, emphasizing the value of a polyphasic approach for characterizing endophytic microbiomes. FunGuild analysis classified many taxa as saprophytes or plant pathogens, a pattern frequently observed in grass-endophyte studies.
The culturable community revealed endophytic entomopathogenic strains, including B. pseudobassiana SS63, Metarhizium sp. FS, and M. novozealandicum SS99. These strains were subsequently evaluated for their pathogenicity against three major pest species in New Zealand (Chapter 5). All strains demonstrated strong pathogenic activity against the endemic grass grub (Costelytra giveni), the white cabbage caterpillar (Pieris rapae), and the recently introduced fall armyworm (Spodoptera frugiperda). Among these, B. pseudobassiana emerged as the most promising candidate for use as a biopesticide, owing to its rapid growth and sporulation in culture, high virulence, and ability to sporulate and produce infective propagules on infected insect cadavers. Furthermore, the pathogenicity of the entomopathogenic strains against the endemic grass grub represents the first evidence of the potential functional role for specific endophytic fungi in tussock grassland ecosystems.
Fungal endophytes have also been widely recognized for their plant growth-promoting abilities, which elicit physiological responses in host plants that enhance biomass production. To investigate this potential, a subset of common endophytic taxa was inoculated into tomatoes (Solanum lycopersicum cv. Moneymaker) and onions (Allium cepa cv. Pukekohe long keeper). In tomatoes, inoculation with Trichoderma harzianum PBC14 resulted in increased aboveground biomass, while inoculation with Metarhizium sp. FS inoculated in onions led to enhanced root biomass. Additionally, onions treated with Arthrinium sp. and grown in garden beds exhibited increased bulb and leaf weights. Highlighting the biotechnological potential of endophytic fungi associated with New Zealand’s indigenous ecosystems.
Overall, this thesis provides valuable insights into the fungal biodiversity associated with indigenous tussock grasses and offers a deeper understanding of the factors shaping the composition and diversity of fungal endophyte communities within this ecosystem. It emphasizes the importance of integrating both culture-based and culture-independent approaches to form a comprehensive and complementary understanding of microbial systems. The insights from this study advances our understanding of the ecosystem services provided by microbial associations in indigenous tussock grasslands. Additionally, the study underscores both the intrinsic and instrumental value of these grasslands in supporting biodiversity and ecological functions.