Stochastic Inoculum, Biotic Filtering and Species-Specific Seed Transmission Shape the Rare Microbiome of Plants

Abstract

A plant's health and productivity is influenced by its associated microbes. Although the common/core microbiome is often thought to be the most influential, significant numbers of rare or uncommon microbes (e.g., specialized endosymbionts) may also play an important role in the health and productivity of certain plants in certain environments. To help identify rare/specialized bacteria and fungi in the most important angiosperm plants, we contrasted microbiomes of the seeds, spermospheres, shoots, roots and rhizospheres of Arabidopsis, Brachypodium, maize, wheat, sugarcane, rice, tomato, coffee, common bean, cassava, soybean, switchgrass, sunflower, Brachiaria, barley, sorghum and pea. Plants were grown inside sealed jars on sterile sand or farm soil. Seeds and spermospheres contained some uncommon bacteria and many fungi, suggesting at least some of the rare microbiome is vertically transmitted. About 95% and 86% of fungal and bacterial diversity inside plants was uncommon; however, judging by read abundance, uncommon fungal cells are about half of the mycobiome, while uncommon bacterial cells make up less than 11% of the microbiome. Uncommon-seed-transmitted microbiomes consisted mostly of Proteobacteria, Firmicutes, Bacteriodetes, Ascomycetes and Basidiomycetes, which most heavily colonized shoots, to a lesser extent roots, and least of all, rhizospheres. Soil served as a more diverse source of rare microbes than seeds, replacing or excluding the majority of the uncommon-seed-transmitted microbiome. With the rarest microbes, their colonization pattern could either be the result of stringent biotic filtering by most plants, or uneven/stochastic inoculum distribution in seeds or soil. Several strong plant-microbe associations were observed, such as seed transmission to shoots, roots and/or rhizospheres of Sarocladium zeae (maize), Penicillium (pea and Phaseolus), and Curvularia (sugarcane), while robust bacterial colonization from cassava field soil occurred with the cyanobacteria Leptolyngbya into Arabidopsis and Panicum roots, and Streptomyces into cassava roots. Some abundant microbes such as Sakaguchia in rice shoots or Vermispora in Arabidopsis roots appeared in no other samples, suggesting that they were infrequent, stochastically deposited propagules from either soil or seed (impossible to know based on the available data). Future experiments with culturing and cross-inoculation of these microbes between plants may help us better understand host preferences and their role in plant productivity, perhaps leading to their use in crop microbiome engineering and enhancement of agricultural production.

Keywords: endophyte; phyllosphere; plant microbiome; plant mycobiome; rare microbiome; rhizosphere; seed microbiome; soil microbiology; vertical transmission.

Figure 1

Uncommon and abundant (with a relative proportion of greater than 10% in at least one sample) OTUs of bacterial 16S and fungal ITS in (A) seeds and (B) spermospheres. Uncommon OTUs were defined as having an occupancy of less than 53% across seeds or spermospheres of the 17 different plant species. Bacterial OTUs are shown in the top blocks, fungal OTUs on the bottom blocks. Next to each OTU ID# is the predicted genus of that sequence. OTU read proportion is represented by color as shown in the legend, with red squares also showing the proportion as a number.

Figure 2

Statistical analysis of total microbial diversity in 17 different plant species grown on sterile sand or field soil. Juvenile cassava (A) and sorghum (B) plants in sterile sand on the left, field soil mixed with sand on the right. PCA of binary-transformed bacterial 16S (C) and fungal ITS (D) OTU counts. Scatterplots of the number of different bacterial 16S (E) and fungal ITS (F) OTUs observed in each sample. Sand-grown samples are displayed as circles, while soil-grown samples are displayed as triangles. Mean values of OTUs observed are indicated by horizontal black bars.

Figure 3

Phylum-level classification of uncommon OTU diversity and read abundance for fungi (A,B) and bacteria (C,D) in shoots, roots and rhizospheres. Uncommon OTUs were those that were found in 9 or fewer of the 17 plant species and were calculated by addition across all plant species per sample type. Percentage of uncommon versus total OTUs in each sample type is indicated above each bar. Read proportion per phylum was the result of averaging the number of reads in uncommon OTUs across all plant species. OTU and read taxonomy are indicated by coloring according to the legends at the right.

Figure 4

The 40 most abundant yet uncommon OTUs of fungal ITS and bacterial 16S in shoots, roots and rhizospheres of 17 different plant species grown in sealed jars on either field soil or sterile sand. Reads were summed across repetitions and then transformed into relative percentages. Sand-grown plants are labelled in white, while soil-grown samples have a brown label. Samples were organized by Bray–Curtis dissimilarity and those that grouped by plant species are highlighted by a purple block atop the column. Square shading is by percentage value, with dark blue being 0%, up to 0.1% being light blue, between 0.1–0.25% being green, 0.25–0.5% being light yellow, 0.5–1% being dark yellow, 1–5% being orange and greater than 5% being red.

Figure 5

The 17 most abundant fungal ITS OTUs occurring in less than 53% of shoot, root and rhizosphere samples fro m 17 different plant species grown in sealed jars on sterile sand or farm soil. Uncommon reads were added across repetitions and transformed into relative percentages, and the most abundant OTU for each plant is shown. Rows with an asterisk are represented in more than 53% of sampl es; however, they appeared to show a large increase in abundance in one specific plant. Cells are shaded by percentage value, with 0% being dark blue, up to 0.15% being light blue, between 0.1–0.25% being green, 0.25–0.5% being light yellow, 0.5–1% being dark yellow, 1–5% being orange and greater than 5% being red with white numbers inside. Predicted inoculum source in soil-grown plants is shown as gray (unknown), green (seed) or brown (soil) colored bars, while pink dots indicate plants selectively filtering for the fungus and black dots indicate a stochastic supply of inoculum.

Figure 6

List of the 17 most abundant yet uncommon bacterial 16S OTUs (occurring at a level of more than 0.15% in 9 or fewer different plant species) in shoots, roots and rhizospheres of 17 different plant species raised inside sealed jars on either farm soil or sterile sand. Reads from soil-grown samples were added across repetitions and transformed into relative percentages, and the most abundant OTU for each plant sample is shown. Cells are shaded by percentage value with 0% being dark blue, up to 0.15% being light blue, between 0.1–0.25% being green, 0.25–0.5% being light yellow, 0.5–1% being dark yellow, 1–5% being orange and greater than 5% being red with white numbers inside.