Endophytic Microbiome Variation Among Single Plant Seeds

Abstract

Like other plant compartments, the seed harbors a microbiome. Seed microbiome members are the first to colonize a germinating seedling, and they may initiate the trajectory of microbiome assembly for the next plant generation. Therefore, the members of the seed microbiome are important for the dynamics of plant microbiome assembly and the vertical transmission of potentially beneficial symbionts. However, it remains challenging to assess the microbiome at the individual seed level (and, therefore, for the future individual plants) due to low endophytic microbial biomass, seed exudates that can select for particular members, and high plant and plastid contamination of resulting reads. Here, we report a protocol for extracting microbial DNA from an individual seed (common bean, Phaseolus vulgaris L.) with minimal disruption of host tissue, which we expect to be generalizable to other medium- and large-seed plant species. We applied this protocol to determine the 16S rRNA V4 and rRNA ITS2 amplicon composition and examine the variability of individual seeds harvested from replicate common bean plants grown under standard, controlled conditions to maintain health. Using DNA extractions from individual seeds, we compared seed-to-seed, pod-to-pod, and plant-to-plant microbiomes, and found highest microbiome variability at the plant level. This suggests that several seeds from the same plant could be pooled for microbiome assessment, given experimental designs that apply treatments at the parent plant level. This study adds protocols and insights to the growing toolkit of approaches to understand the plant-microbiome engagements that support the health of agricultural and environmental ecosystems.

Seed microbiota revealed by a large‐scale meta‐analysis including 50 plant species

“…Additionally, this meta‐analysis included mainly seed microbiota characterization from multiple seeds (> 3 seeds in sample) with > 1000 reads. More work is needed at the single‐seed level (Abdelfattah et al ., 2021; Bintarti et al ., 2021; Chesneau et al ., 2021) to assess seed‐to‐seed variation and the proportion of seeds that do not have detectable microbiota (i.e. sterile seeds) (Newcombe et al ., 2018; Ridout et al ., 2019).…”

Abiotic Treatment to Common Bean Plants Results in an Altered Endophytic Seed Microbiome

“…Because we do not expect microbiome members to be actively doubling inside the seed (and therefore relativized abundances inside the seed to reflect fitness differences as an outcome of growth therein; see reference 33 for a discussion of this), we used a presence-absence assessment (Jaccard index) of β-diversity. There was a statistically supported difference in bacterial/archaeal microbiome composition between treated seeds and control seeds ( Fig.…”

“…Together, these results suggest that any perturbations that affect the host plant may also affect its microbial communities, and these alterations may result in specific outcomes for plant stress tolerance and resilience. Since previous seed microbiome studies have found that seed microbiomes contain relatively simple communities of tens to dozens of taxa ( 21 , 33 , 40 ), even alterations in the composition or abundances of a few taxa may have consequences for microbiome assembly of the next plant generation.…”

“…There could be a microbial shift toward a state associated with positive effects on plant health, as opposed to dysbiosis [ 44 ]). However, the expectation of high variability should be taken into consideration for future studies, as sufficient replication will be needed to power statistical tests ( 33 ).…”

“…Twenty seeds from each plant were collected for DNA extraction following the protocol of a previous study ( 21 ) with minor modifications to include surface sterilization ( 33 ). The seeds were surfaced sterilized in 10% bleach, rinsed five times with sterile DI water, and placed in sterile 50-mL centrifuge tubes with 30 mL of sterile 1× phosphate-buffered saline (PBS) with 0.05% Tween 20 and shaken at 140 rpm at room temperature for 4 h. After shaking, the tubes were centrifuged at 500 × g for 15 min, and the supernatant and seeds were discarded.…”