Phylloplane Yeasts Associated with the Japanese Yew and Austrian Pine in Indiana Shannon Newerth, John Cavaletto, M. Catherine Aime
Department of Botany & Plant Pathology, Purdue University, West Lafayette, IN
[email protected]
Abstract
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Little is known about phylloplane yeasts that inhabit conifer needles. The purpose of this experiment was to expand knowledge these in Indiana. Needles were collected from Japanese yew and Austrian pine needles on the Purdue campus and organisms were isolated into pure cultures. DNA was isolated from pure cultures and Polymerase Chain Reaction (PCR) and DNA sequencing were used to help with identifications. As a result, six phylloplane yeasts species were identified. The six yeasts show preferences in host plants, light intensity, and the habitation of one side of a leaf over another. This information along with other studies can broaden our understanding of phylloplane yeast behavior.
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D Fig. 5. Standard Laboratory Protocol. Samples were taken from pure cultures and DNA was isolated using a UltraClean Microbial DNA Isolation Kit (Mo-Bio Laboratories Catalog #12224-50). A Polymerase Chain Reaction (PCR) was used to amplify the ITS region of rDNA locus. Portion of the products were taken and an agarose gel was run to confirm the isolation and amplification of DNA. PCRs were treated with ExoSap and sent for sequencing at Purdue University Genomics Facility.
Fig. 2. Pinus nigra (Austrian pine). Letters refer to sampling sites on map in Fig. 1.
I think that there will be differences in the diversity of epiphytes and endophytes that grow on the leaves of the Austrian pines and Japanese yew because some species of epiphytes and endophytes may prefer a specific host plant over another. I believe that there will be differences in the epiphytes that grow on the top and bottom of the Japanese yes because these types of microbes may have been shown to be more prevalent on the abaxial surface. I think that there will be differences in the diversity of epiphytes and endophytes that grow on the Austrian pines and Japanese yew in lightly shaded areas in comparison to darker shaded areas because they may prefer a specific range of temperature, UV exposure, as well as water availability due to higher evaporation rates.
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Results Table 1. Phylloplane yeast species isolated and identified. More
species were found in low light and on the adaxial leaf surface than in high light and abaxial surfaces.
Methods
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Fig. 3. Taxus cuspidatus (Japanese yew). ). Letters refer to sampling sites on map in Fig. 1.
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Fig. 1. Map Depicting Host Plant Locations at Purdue University. Four host plants (two of each species) were selected for sampling in June 2013. Hosts were selected to represent different light conditions on the day of sampling.
Acknowledgements We thank Rachel Koch for assistance with poster design. We are also grateful to Dr. Merje Toome for assistance with phylogenetic analyses. SN is also grateful to Dr. P:aul Kaiser and Mr. Kevin Banks at Beech Grove Highschool and Dr. Clark Gedney at Purdue for assistance during the initial phase of this project.
Fig. 6. Phylogenetic tree showing placement of new species. Isolate
was from Japanese yew in low light on the abaxial surface. Fig. 4. All samples were placed onto plates of potato dextrose agar (PDA). Epiphyte isolations consisted of attaching leaves to lids of petri dishes with Vaseline. Spores of phylloplane yeasts then shot onto surface of PDA and grew. Yeast cultures were aseptically transferred to fresh plates to get individual species into isolation.
Conclusions This study has shown that the species of host plant does have a effect on the diversity of phylloplane yeasts. It also shows that the amount of light present has an effect on the types and numbers of phylloplane yeasts present. Although results were not presented for the endophytes, this work also showed that these types of organisms live within both the Japanese yew and Austrian pine.
