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.
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.
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.
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.
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.
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