Diversity and Effects of Fungicides on the Pecan Phyllosphere Microbiome, and Fungal Interactions with a Novel Yeast

Abstract

The phyllosphere microbiome is one of the largest habitats on earth, hosting any organism that can adapt to it. Phyllosphere yeasts in particular can dominate this habitat, but to this date have been chronically understudied with much novelty left to discover and classify. In the presence of fungicides, phyllosphere yeast taxa have been shown to have altered abundance. Pecans are native to the southeastern US, and are grown for nutrients like fiber, copper, and zinc. Fungal pathogens like powdery mildew and scab can affect the fitness of nuts. These pathogens are controlled by fungicide applications, up to and over 10 times a growing season. Under these high fungicide usage conditions, it is hypothesized that these chemical applications may alter phyllosphere yeast taxa abundance. Chapter 1 reviews previous literature discussing phyllosphere microbiomes and effects of fungicides on natural community members, as well as current knowledge of phyllosphere yeasts. In chapter 2, we seek to classify the pecan phyllosphere microbiome from a two-year experiment using molecular and bioinformatic tools for community profiling and analysis. We hypothesize that fungicide applications will have a non-targeted effect on the phyllosphere microbiome, specifically regarding yeast species. We also predict that fungicide-treated leaves will have a disrupted network structure compared to non-treated control leaves. Thus, 12 pecan trees were sampled before and after fungicide applications over the course of two years, 2021 and 2022, to provide community profiling and determine effects of fungicides on community members. Findings from this study support the hypothesis that fungicides have a non-targeted effect on the pecan phyllosphere microbiome, decreasing richness of fungicide-treated leaves and causing a significant difference in community composition in 2021. In addition, findings show that network hubs change between non-treated leaves and treated leaves. In chapter 3, we explore fungal-fungal interactions between a selection of environmental yeast species and a group of fungi to support the hypothesis that these environmental isolates can produce compounds that inhibit fungi. After this, we hone in on a novel Dothideomycete yeast, EMM_F3, to explore metabolic properties that may be causing inhibition of fungi—specifically Fusarium graminearum PH-1—, and taxonomically place it using comparative genomics techniques to other high quality Dothideomycete species. Initial findings support the hypothesis that environmental yeasts can have an inhibitory effect on various fungi. When focusing on EMM_F3, we determined that inhibition to PH-1 was due to metabolites, and we attempted to classify these metabolites through both genome sequencing and gas chromatography-mass spectrometry. Metabolites of interest included clavaric acid, tridecane, and nonadecane. Genome comparison placed EMM_F3 closely to relatives Delphinella strobiligena (a pine pathogen), and Aureobasidium pullulans (an extremophile yeast that has known biocontrol properties). In Chapter 4 we discuss the impacts of these experiments. Thus, this thesis promotes ideas for balancing pest management and preservation of biodiversity in agricultural systems and encourages exploration of the phyllosphere—specifically yeast species—to help understand species diversity and microbial interactions, as well as elucidate new biocontrol directions or new biologically useful metabolites or proteins.