Masters Thesis

Investigating bacterial endophytes in parasitic Cuscuta campestris and its Helianthus annuus host

A diverse community of microorganisms colonize plant surfaces. These microorganisms can colonize the external leaf surface (phyllosphere), the external root surface (rhizosphere), and the plant interior (endosphere). Microorganisms existing internally, called endophytes, can have mutualistic, commensal, or pathogenic relationships with their host plant. Previous work has identified a consistent endophytic community in leaf and petal tissue of the common sunflower (Helianthus annuus), which is dominated by the phyla Proteobacteria and Actinobacteria, and classes α-proteobacteria and β-proteobacteria. The endophyte community in parasitic plants and how it affects the composition of the host plant endophyte community has not been described in detail. Examining the endophyte community composition of H. annuus, the holoparasite Cuscuta campestris (dodder), and their endophytic interactions to build an understanding of microbiota diversity in each species are the primary aims of this study. Moreover, this study intends to describe possible changes in host endophyte microbiota resulting from a dodder infection. The goals are to (i) isolate/identify endophytic bacteria in the parasitic plant C. campestris and a host, H. annuus; and (ii) examine changes in the H. annuus endophyte community resulting from dodder infection. Fifteen samples of dodder tissue, dodder-infected and non-infected H. annuus stem and leaf tissue were field collected in Central California. All tissues were surface washed to minimize phyllospheric bacteria. DNA was extracted by grinding plant tissue in liquid nitrogen and amplified with chloroplast-excluding 16S primers due to the primer’s affinity for non-target DNA. Endophyte PCR products were isolated, sequenced (Illumina platform) and analyzed using the QIIME2 pipeline. The results indicated H. annuus from Population A consistently displayed a significantly greater number of observed OTUs (30 average) compared to C. campestris (16 average). Taxonomic analysis showed the greatest fraction of OTUs were phylum Proteobacteria. These results support previous work as Proteobacteria and Actinobacteria were among the most prevalent phyla identified in H. annuus samples. All H. annuus groups from Population A consistently harbored greater numbers of Proteobacteria OTUs compared to C. campestris groups. The β-proteobacteria class accounted for over 99% of all OTUs identified at the class level in both H. annuus and C. campestris. Findings indicate that the parasite C. campestris and its H. annuus host did not have significant effects on each other’s endophytic communities and maintained significantly different endophyte compositions. The identification and understanding of endophytic community structure in both host and parasite has the potential for improving plant management and parasite prevention strategies.

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