Comparing the fungal community in pitcher plant fluid of Sarracenia purpurea and Nepenthes robcantleyi individuals




Shicheng Zu

Introduction

The pitcher plant is a carnivorous plant that thrives in low-nutrient areas. In order to supplement nutrients not found in the surrounding soil, the pitcher plant lures insects into the sticky liquid inside its pitfall trap (Koopman, et al., 2010). When the insects die, the plant protease nepenthesin dissolves the prey remain to release critical nutrition.

Fungi belong to a large group of eukaryotic organism that distinguish themselves from other kingdoms by chitin cell walls and lack of chloroplast. Playing a significant role in ecosystem, fungi contribute to the organic material turnover and food chain maintenance. This project is to characterize the fungal community makeup of two pitcher plant species: Sarracenia purpurea and Nepenthes robcantleyi. Sarracenia purpurea originates in North America. It differentiates itself from other pitcher plant species by its leaves directly growing from the root. Nepenthes robcantleyi, the notorious tropical species is famous for its colorful trumpet-shape pitchers hanging down from the vines. DNA barcode technique allows us to characterize fungal species in its native environment directly. Advance in molecular biology confers each species with its own DNA barcode. DNA barcode technique uses a short DNA segment as a biomarker to characterize the unknown species by comparing the amplified barcode gene sequence to the sequence data of existing species. This short DNA fragment should cover all taxa of interests and contain enough SNP to provide discriminatory power. This project used the Internal Transcribed Spacer (ITS) locus and Large Subunit (LSU) locus to characterize fungal species in pitcher plant fluid.

Methods
1)Sarracenia purpurea pitcher fluid was gift from Paul Smith's College VIC. Nepenthes robcantleyi pitcher fluid came from Native Exotics Inc.
2)DNA isolation was performed by FastDNA® Spin isolation kit (MP Biomedicals).
3)ITS (Internal Transcribed Spacer) and LSU (Large Subunit) locus PCR amplification.
4)PCR product purification, pGEM-T® vector ligation and transformation.
5)Blue/white screening.
6)Colonies were directly sequenced to check species.

Results and Discussion
1) ITS PCR amplification showed a band of 1kb for each sample that was in accordance with the ITS band size in reference (Schoch. et al. PNAS 2012).
2) LSU PCR amplification showed a band of 0.6kb for Sarracenia purpurea and a 0.5kb band for Nepenthes robcantleyi which suggested that different fungal species may exist in those two pitcher plant species. ITS identified more fungal species than LSU in this project because amplified ITS gene size was much larger and contained more variations than LSU to provide discrimination. Therefore when using the DNA barcode technique to characterize species in an unknown community, it is preferential to use combination of different DNA barcode genes.
3) The common discovery of Candida species by ITS and LSU suggested that Candida species probably existed and predominated in Sarracenia purpurea pitcher fluid. However, their description and the reason why candida species are so adapted to Sarracenia purpurea are still largely unknown. C. palmioleophila was reported in 1991 as an opportunistic pathogen causing intravenous catheter-associated fungemia (Rasmus H. Jensen, et al., 2011).
4) ITS-based phylogenetic tree implied that the three identified fungal species in Sarracenia purpurea pitcher fluid were closely related; The 30 uncultured fungus in Nepenthes robcantleyi were closely related. However, the uncultured fungal species in Nepenthes robcantleyi were not related to the identified fungal species in Sarracenia purpurea. Further culture-dependent method characterization would be required.

Figures


Figure 1-ITS gene PCR amplification. Lane1 and lane2 were two pitcher fluid individuals from Sarracenia purpurea. Lane3 to Lane8 were six pitcher fluid individuals from Nepenthes robcantleyi. Sample 2 and sample 8 are used to do ligation.


Figure 2-LSU gene PCR amplification. Amplification of LSU gene showed a 0.6kb band for Sarracenia purpurea and 0.5kb bands for Nepenthes robcantleyi. Sample1 and sample3 were used to do ligation.


Figure 3-For LSU in Sarracenia purpurea, 24 colonies confirmed the existence of Candida palmiolephia. For ITS in Sarracenia purpurea, 20 colonies confirmed the existence of Candida palmiolephia; 3 colonies identified Phoma herbarum; 3 colonies identified Lambertella advenula. For LSU in Nepenthes robcantleyi, all colonies identified uncultured fungus. For ITS in Nepenthes, 29 colonies identified uncultured fungus.


Figure 4-ITS phylogenetic tree implied that the three identified fungal species in Sarracenia purpurea pitcher fluid were closely related; the 30 uncultured fungus in Nepenthes robcantleyi were closely related. However, the uncultured fungal species in Nepenthes robcantleyi were not related to the identified fungal species in Sarracenia purpurea. Further culture-dependent method characterization would be required.


Summary
The microbial makeup of pitcher plant fluid is remarkably diverse. This microorganism-enrich fluid provides an ideal platform to characterize fungal community by using DNA barcode technique. This project employed two DNA barcode gene, ITS and LSU, to characterize fungal communities in two pitcher plant species: Sarracenia purpurea and Nepenthes robcantleyi. DNA sequence analysis confirmed Candida palmiolephia and Candida pseudoglaebosa existence in Sarracenia purpurea pitcher fluid individuals. However, their description and the role in symbiotic relationship with the Sarracenia purpurea need further investigation. The ITS phylogenetic tree suggested that the three identified fungal species in Sarracenia purpurea were closely related; However, the uncultured fungal species in Nepenthes robcantleyi were not related to the identified fungal species in Sarracenia purpurea. To further characterize those uncultured fungus, culture-dependent method would be required.
Reference
1) Conrad L. Schoch, Keith A. Seifert, Sabine Huhndorf, Fungal Barcoding Consortium, PNAS, 2012
2) Rasmus H. Jensen and Maiken C. Arendrup, Candida palmioleophila: Characterization of a previously overlooked pathogen and Its unique susceptibility profile in comparison with Five Related Species, Journal of clinical microbiology, Feb. 2011.
3) Koopman, Fuselier,The carnivorous pale pitcher plant harbors diverse, distinct, and time--dependent bacterial communities. Applied and Environmental Microbiology,76(6), 1851-1860.

Full Paper

Acknowledgments

I would like to thank Catharina Grubaugh, Katherine Reid and Dr. Berish Rubin for their guidance in designing and support to make this project possible.


This document was last modified 05/16/2014.
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