Duckweed hosts a taxonomically similar bacterial assemblage as the terrestrial leaf microbiome

Abstract

Culture-independent characterization of microbial communities associated with popular plant model systems have increased our understanding of the plant microbiome. However, the integration of other model systems, such as duckweed, could facilitate our understanding of plant microbiota assembly and evolution. Duckweeds are floating aquatic plants with many characteristics, including small size and reduced plant architecture, that suggest their use as a facile model system for plant microbiome studies. Here, we investigated the structure and assembly of the duckweed bacterial microbiome. First, a culture-independent survey of the duckweed bacterial microbiome from different locations in New Jersey revealed similar phylogenetic profiles. These studies showed that Proteobacteria is a dominant phylum in the duckweed bacterial microbiome. To observe the assembly dynamics of the duckweed bacterial community, we inoculated quasi-gnotobiotic duckweed with wastewater effluent from a municipal wastewater treatment plant. Our results revealed that duckweed strongly shapes its bacterial microbiome and forms distinct associations with bacterial community members from the initial inoculum. Additionally, these inoculation studies showed the bacterial communities of different duckweed species were similar in taxa composition and abundance. Analysis across the different duckweed bacterial communities collected in this study identified a set of "core" bacterial taxa consistently present on duckweed irrespective of the locale and context. Furthermore, comparison of the duckweed bacterial community to that of rice and Arabidopsis revealed a conserved taxonomic structure between the duckweed microbiome and the terrestrial leaf microbiome. Our results suggest that duckweeds utilize similar bacterial community assembly principles as those found in terrestrial plants and indicate a highly conserved structuring effect of leaf tissue on the plant microbiome.

Fig 1. Sample collection sites.

(A) Map of New Jersey, USA depicting sample collection sites. Duckweed and ambient water samples were collected from Caldwell House and Passion Puddle sites. Wastewater was collected from United Water Princeton Meadows treatment facility to use as an inoculum for assembly studies. (B) Representative images of duckweed collected from Caldwell House and Passion Puddle sites.

Fig 2. Duckweed hosts a conserved bacterial community that is distinct from the surrounding water bacterial community.

(A) The total number of ASVs observed and assessment of the phylogenetic diversity using Faith’s PD phylogenetic diversity index for DAB and ambient water communities from Caldwell House and Passion Puddle sites. Wilcoxon rank sum test was used for comparison of ASVs and Faith’s PD index (p-value < 0.05 = “*”, p-value < 0.01 = “**”, p-value < 0.001 = “***”, p-value < 0.0001 = “****”). (B) Principal coordinate analysis of DAB and ambient water bacterial communities from Caldwell House and Passion Puddle sites using unweighted UniFrac and generalized UniFrac distances.

Fig 3. Proteobacteria is the major constituent of the duckweed bacterial microbiome.

(A) Phylum, (B) family, and (C) genus composition of DAB and ambient water (AW) bacterial communities from Caldwell House (CH) and Passion Puddle (PP) sites.

Fig 4. A discrete bacterial community steadily assembles onto duckweed.

(A) The number of ASVs and Faith’s PD index for wastewater (WW), ambient wastewater (AWW), and Sp9509 bacterial communities (WWDAB) derived from Princeton Meadows 2015 wastewater inoculum (Wilcoxon rank sum test; p-value < 0.05 = “*”, p-value < 0.01 = “**”, p-value < 0.001 = “***”, p-value < 0.0001 = “****”). (B) Principal coordinate analyses using unweighted (left) and generalized (right) UniFrac distances between WW, AWW, and WWDAB communities.

Fig 5. Bacterial genera are enriched in both the ambient water and DAB community.

ALDEx2 analysis determined 12 significantly enriched bacterial taxa in the ambient water and WWDAB communities compared to the wastewater community (adjusted Welch’s t-test, p-value < 0.05, absolute effect size greater than 1.50). The ALDEx2 distribution for each of these bacterial taxa are displayed. Multiple comparisons were performed using Dunn’s test with command letters displayed. * = bacterial taxa for which bacterial ASVs were found in DAB t0 community, CLR = centered-log ratio.

Fig 6. Different duckweed species host similar bacterial communities.

(A) Principal coordinate analysis of Sp9509 (SpDAB) and Lm5576 (LmDAB) DAB communities using unweighted (left) and generalized (right) UniFrac distances. (B) Scatterplot of bacterial taxa abundance between SpDAB and LmDAB bacterial communities assembled from Princeton Meadows year 2 inoculum. Pairwise Spearman rank correlation coefficient and p-value are displayed. CLR = median centered-log ratio for taxa.

Fig 7. Bacterial taxa in the duckweed core microbiome.

Core taxa were found in at least 6 of the 7 DAB communities analyzed. Displayed is the log2 fold difference between core taxa median centered-log ratio (clr) to non-core community median clr from Caldwell House, Passion Puddle, Princeton Meadows years 1–2, Leishan County (China), Congjiang County (China), and Liping County (China) DAB community studies. Taxa were considered core members if they displayed a 2-fold (log2 > 1) higher abundance in at least 6 studies. Negative values signify taxa abundance was lower than non-core community abundance. Abundance was found to be significantly different (p-value < 0.05) between all core taxa and non-core community comparisons using Dunnett’s test with the non-core community as a control. The median non-core community clr is displayed for each of the 7 DAB communities. No Methylotenera taxa were found in DAB communities from the Princeton Meadows year 1 study.

Fig 8. The duckweed bacterial microbiome resembles the terrestrial leaf microbiome.

The bacterial taxa composition of duckweed, rice, and Arabidopsis bacterial communities were compared. Samples were rarefied to 1000 reads. (A) PCoA using the Bray-Curtis distance for duckweed, rice, and Arabidopsis bacterial communities. (B) The number of bacterial genera from four predominant plant-associated bacterial phyla was calculated for different plant tissues. Multiple pairwise comparison testing was performed using Dunn’s test with Benjamini-Hochberg adjustment and the resultant compact letters are displayed.