Bacterial diversity and composition in the fluid of pitcher plants of the genus Nepenthes

Abstract

Pitchers are modified leaves used by carnivorous plants for trapping prey. Their fluids contain digestive enzymes from the plant and they harbor abundant microbes. In this study, the diversity of bacterial communities was assessed in Nepenthes pitcher fluids and the composition of the bacterial community was compared to that in other environments, including the phyllosphere of Arabidopsis, animal guts and another pitcher plant, Sarracenia. Diversity was measured by 454 pyrosequencing of 16S rRNA gene amplicons. A total of 232,823 sequences were obtained after chimera and singleton removal that clustered into 3260 distinct operational taxonomic units (OTUs) (3% dissimilarity), which were taxonomically distributed over 17 phyla, 25 classes, 45 orders, 100 families, and 195 genera. Pyrosequencing and fluorescence in situ hybridization yielded similar estimates of community composition. Most pitchers contained high proportions of unique OTUs, and only 22 OTUs (<0.6%) were shared by ≥14/16 samples, suggesting a unique bacterial assemblage in each pitcher at the OTU level. Diversity analysis at the class level revealed that the bacterial communities of both opened and unopened pitchers were most similar to that of Sarracenia and to that in the phyllosphere. Therefore, the bacterial community in pitchers may be formed by environmental filtering and/or by phyllosphere bacteria.

Comparative Study of Bacterial Communities in Nepenthes Pitchers and Their Correlation to Species and Fluid Acidity

“…However, a significant fraction of Acidobacteria was only present in some highly acidic fluid samples in this study. The low abundance of these bacteria has been found in a few previous studies [14,25]. None of the Nitrospira sp.…”

“…However, whether the contribution of pH and insect-introduced bacteria on shaping the overall community structure is interdependent or separated phenomena is still a question which needs further study. A recent study by Takeuchi et al also suggested that bacterial communities of Nepenthes pitchers may be formed by environmental filtering and/or originated from phyllosphere bacteria [25]. Furthermore, the uniqueness of bacterial communities in NAM could be due to its relatively narrow pH range of the fluids (pH 4.2-4.8) and the evolutionary progression towards a more vegetarian diet of this species.…”

“…Sota et al detected bacteria in unopened pitchers of wild plants of Nepenthes eustachya in Sumatra using acridine orange direct count although they considered their findings might be erroneous [15]. The detection of microbes in closed pitcher fluids was then supported by Chou et al and Takeuchi et al [14,25] who used culture-independent study based on sequencing of 16S rRNA clonal library and 454 pyrosequencing of 16S rRNA gene amplicons, respectively. The presence of native microflora in unopened pitchers was also found in few samples in our study, in which the bacterial communities were dominated by Proteobacteria using tagged 16S rRNA amplicon sequencing by the high-throughput next generation sequencing platform.…”

“…It has been expected that some particular bacteria, e.g., some Acidobacteria genera known to live in acidic environments and Nitrospira recognized to be involved in nitrification processes in nitrogen-rich environments, could represent abundant taxa in the fluid [25]. Acidobacteria are generally discovered in acidic environments, e.g., compost and peat swamp soil [54,55].…”

“…High-throughput sequencing on a 454 pyrosequencing platform was then used to study bacterial communities in pitcher fluids of carnivorous plant genus Sarracenia [17,24]. This method was recently used to explore the bacterial diversity in four Nepenthes species of wild and cultivated origins, and their diversity was found to be related to those in Sarracenia and phyllosphere [25]. The culture-independent approach was also used to isolate novel acidic lipases from a metagenomic library of the microbial community in the pitcher fluid of Nepenthes hybrida [26].…”