Endophytes of Pseudowintera colorata (horopito) : A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Lincoln University

Abstract

Pseudowintera colorata (horopito) commonly known as New Zealand pepper tree is a native medicinal plant, known for its antimicrobial properties. International studies have demonstrated that endophytes of medicinal plants play key roles in maintaining plant health, tolerance to biotic and abiotic stresses and production of secondary metabolites. However, there have been no studies on the endophytes of P. colorata. The objectives of this study were to i) investigate the community structure of the endophytic bacteria, Actinobacteria and fungi in the tissues of P. colorata and identify members of the core endomicrobiome, ii) investigate the bioactive potential of the culturable endophytic bacteria, Actinobacteria and fungi, iii) investigate the influence of selected members on the growth and chemistry of P. colorata in vitro and in vivo. The endophytic communities in P. colorata were characterized by denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq. Plants from ten sites across New Zealand were analysed by DGGE and it was revealed that tissue type was the main factor influencing the endophytic communities in P. colorata (PERMANOVA, P=0.001). Richness of Actinobacteria, Betaproteobacteria and fungi was higher in stems compared to leaves and roots. For a subset of three sites, the interaction of plant location with maturity influenced the microbial communities across all groups analysed except for Alphaproteobacteria and total fungi (PERMANOVA, P=0.226 and P=0.164 respectively). Using Illumina MiSeq for analysing the bacterial communities from ten sites, it was confirmed that tissue type affected the bacterial communities in P. colorata. Illumina data revealed that Gammaproteobacteria was the most abundant class (89.1%) followed by Alphaproteobacteria (10%). In addition, two OTUs belonging to Pseudomonas were identified as members of P. colorata core endomicrobiome. A total of 350 endophytic bacteria, 200 endophytic fungi and nine endophytic Actinobacteria were recovered from P. colorata plants from ten sites across New Zealand. The majority of endophytic bacteria were isolated from the stem (57.1%, n=200), followed by roots (37.1%, n=130) and leaves (5.7%, n=20). Eleven endophytic bacteria showed strong antagonistic activity against four phytopathogenic fungi Neofusicoccum luteum, N. parvum, Neonectria ditissima, Ilyonectria liriodendri and four endophytic bacteria were active against bacterial pathogens Pectobacterium atrosepticum, P. brasiliensis, Staphylococcus aureus, Escherichia coli. In addition, endophytic bacteria also produced siderophores on chrom-azurol S agar (CAS). Based on 16S rRNA gene, the endophytic bacteria were identified as members of genera Bacillus, Pseudomonas, and Pantoea. The endophytic fungi Pezicula sp. PRY2BA2, Metarhizium sp. PR1SB1 were active against all the phytopathogenic fungi tested. Six endophytic fungi showed high activity against Candida albicans in vitro. A total of nine endophytic Actinobacteria were recovered onto selective agar. Sequencing the 16S rRNA gene revealed that the culturable members belonged to genera Streptomyces, Micromonospora, Nocardia, Nakamurella and Microlunatus. Major bands (n=20) from DGGE gels were sequenced and were identified as uncultured bacteria, Streptomyces sp. and Angustibacter peucedani. Nocardia sp. TP1BA1B and Streptomyces sp. UKCW/B solubilized phosphate in tricalcium phosphate agar (TCP) and secreted siderophores. This is the first study to identify Actinobacteria communities in P. colorata and to examine the functional traits of cultured representatives. The effect of endophytes displaying in vitro activity was examined by reintroduction as a soil drench. Bacillus sp. TP1LA1B and Nocardia sp. TP1BA1B in addition to increasing the shoot height, also significantly increased the shoot, root biomass and the number of internodes of P. colorata seedlings compared to the control (P=0.016, P <0.001, P=0.007 and P <0.001 respectively). This is the first study to investigate the influence of endophytes on the growth of P. colorata. Overall, this study revealed the community structure of endophytic bacteria, Actinobacteria and fungi in P. colorata for the first time and is one of the only two studies on native plants in New Zealand. Members of the endomicrobiome displayed in vitro activity as measured by antimicrobial and nutrient mobilisation assays. The endophytes were able to influence host plant growth when applied as soil drenches and some were able to recolonize the host endophytically demonstrating a route from soil to root. This study indicated that P. colorata harbours unique endophytes which have a key role in the ecology of the plant.