Understanding the life cycle of the floral microbiota of Leptospermum scoparium (mānuka) : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

Leptospermum scoparium is a shrub native to New Zealand and holds high cultural value (taonga = a treasure) and economic value due to the antimicrobial mānuka honeys produced from its nectar. Previous studies demonstrated a high frequency of association between microorganisms and the leaves, stems and roots of L. scoparium, with some modifying the secondary metabolite profile of L. scoparium leaves and improving growth. Flowers are an important niche for microorganisms; however, no research has focused on the L. scoparium anthosphere (flower), despite its potential impact on the honey industry. The aim of this research was 1) to characterize the endophytic and epiphytic microbial communities associated with L. scoparium flowers, 2) to characterize the effect of floral development on the anthosphere community of L. scoparium, 3) to determine the origin of the microbial communities associated with L. scoparium flowers, and 4) to determine the effects of soil on the bacterial communities associated with L. scoparium flowers. To answer these objectives, bacterial and fungal communities were characterized using 16S rRNA and ITS1-based community profiling (or cultivation independent approach) by high throughput sequencing. Host contamination (plastid and mitochondrial sequences) in 16S rRNA amplicon sequencing from plant samples impedes plant microbiota studies, thus I first optimised the 16S-sequencing method for L. scoparium bacterial microbiota studies (overall increase of bacterial sequences by 30%) based on modifications to the published Cas9-16S seq method. I then characterized for the first time which bacteria and fungi that live in (endophytic community) and on (epiphytic community) L. scoparium flowers. The results showed that nectar-consuming yeasts from Aureobasidium and Vishniacozyma genera, functionally diverse filamentous fungi from the Cladosporium genus, and bacteria from Candidatus Phytoplasma, Pseudomonas and Pantoea dominated the L. scoparium anthosphere. The L. scoparium epiphytic community was richer (5.6 times and 2.5 times more bacterial and fungal OTUs) and differed in composition relative to the endophytic community (p < 0.001), demonstrating filtering by the plant. This study demonstrated that a wide diversity of microbial taxa inhabit the L. scoparium anthosphere. As flower morphology and biology change rapidly over time, dynamic niches for microorganisms are formed and lost. Floral physiology at each life stage can therefore influence arrival, persistence, and loss of microbial species; however, this remains poorly understood despite its potential consequences for host reproductive success. Thus, I characterized the effect of transition through five floral stages on the flower-inhabiting bacterial and fungal communities, from immature bud to spent flower, and subsequent allocation to seed. The results showed a core microbiota (representing >80% of all fungal reads and between 47.5% and 94.0% of all bacterial reads at each floral stage) persisted across this dynamic niche despite high microbial turnover, as observed in shifts in community composition (p = 0.001) and diversity (increase in alpha diversity between closed and open floral stages, p<0.001) as flowers matured and senesced. The results demonstrated that floral stages are strong drivers of the L. scoparium anthosphere community assembly and dynamics. One of the challenges for research seeking to understand the origin and dynamics of floral microbial communities is that every year floral microbiota must begin anew, and, unlike other vegetative organs (roots, stems, and leaves), develop and exist for a limited time. In this study, I collected flowers from L. scoparium plants over two consecutive flowering seasons to understand the effect of seasonality on shaping the microbial community. I also characterized the microbiota within different niches of L. scoparium; flowers, bagged flowers (to exclude animal visitation), leaves, underlying flower stems and lateral roots, and surrounding the plants; soil and bee surfaces to understand the origin of flower-inhabiting microorganisms. The results showed that the L. scoparium anthosphere community was recruited from more than one reservoir. Local reservoirs (leaves, stems) were an important source of microorganisms for the L. scoparium anthosphere (41% of the bacterial and 81% of the fungal community potentially originated from leaves), however pollinators were also important for bacterial community assembly (>25% bacterial community potentially originated from bee visitations). The selection or specialisation of microorganisms was demonstrated across flowering season with a few highly prevalent and abundant taxa (representing >90% of the reads for fungal and bacterial communities in both years). However, most of the taxa retrieved were not prevalent in flower samples indicating stochasticity in the anthosphere assembly process that may depend on environmental conditions, vectors and the microbial community surrounding the flower at the time. Finally, as the rhizosphere is an interface for plant microbial acquisition, soil is an important factor for microbial colonisation of plants. To date there has been little research conducted on the contribution of soil bacteria to the anthosphere bacterial community. Thus, seedlings of L. scoparium were planted in pasteurized potting mix and in soils collected from three contrasting sites. Once the first flowers appeared (~1.5 years), flowers and soil were collected for 16S rRNA gene community profiling. I found that soil played a relatively small role in bacterial community composition of L. scoparium flower, as the anthosphere bacterial community for plants grown in only one soil, ‘home soil’, was different compared to the anthosphere bacterial community for the plants grown in the other soils or pasteurize potting mix (p <0.05). In this research, new understanding of the spatial and temporal floral microbiota of L. scoparium was produced, demonstrating its likely origin and recruitment pathways and the impacts of floral development, organ/tissue filtering and soil on community assembly and dynamics. Finally, this research demonstrated that interaction between microorganisms is likely to play an important role in shaping L. scoparium anthosphere. This study builds fundamental knowledge in microbial ecology of healthy flowers as well as producing a foundation for further research on enhancing the production of mānuka honey by manipulating floral microorganisms.