While the diversity of the plant microbiome, and its role for plant health, are increasingly recognized, we lack insights into the mechanisms behind the continuity of the microbiome across generations. In the current study, we investigated the transmission of the plant microbiome from seed to seedling, which is a crucial but largely overlooked part of the vertical inheritance process. We first dissected and sequenced individual seeds, which showed that the inherited microbiome is highly distinct within the seed (i.e. embryo vs. pericarp), with a particularly high microbial diversity within the embryo. We then used state-of-the-art culturing devices to grow common oak seedlings in the absence of environmental microbes, which showed that the major part of the inherited microbiome is transmitted from the seed to the seedling. While there was a strong imprint of the embryo’s microbiome on the phyllosphere microbiome, the root microbiome represented a distinct subset of the seed microbiome. Collectively, the results provide clear evidence of microbial inheritance in plants, niche differentiation of the inherited microbiome in both seeds and seedlings, and divergent transmission routes from the acorn to the phyllosphere and roots. These findings shed new light on the source and transmission of plant-associated microbes and increase our understanding of the dispersal and distribution of microbes within natural environments.
The majority of previous studies on the seed microbiome used bulk samples (several pooled seeds) [4, 17, 51] which inflates microbial diversity, especially when evaluating the transmission of the inherited microbiome into individual seeds [12, 21]. The present study demonstrated that individual seeds possessed high microbial diversity, that was higher in the embryo than in the pericarp. These results are in contrast to other recent studies that reported a limited number of bacterial and/or fungal species present in seeds [12, 13].
The composition of the microbial community in embryo and pericarp greatly differed. In particular, the bacterial community of the pericarp was almost completely dominated by the genus Pseudomonas. While this genus was also present in the embryo, it was part of a diverse community that included other bacterial genera. The existence of strong differentiation of the microbial community between different seed parts may be a general pattern, and in this case the lower diversity and evenness in the pericarp may be due to the limited resources present in the pericarp. In this regard, it is interesting to note that no specialists or novel species were identified in the pericarp. Instead, OTUs belonging to a more generalist genus (Pseudomonas), known to thrive in many different habitat types [52, 53], were identified in the pericarp. Although Pseudomonas species are generally considered plant and seed pathogens, they can also induce plant growth and secrete antibiotics into the rhizosphere [54–56]. Notably, despite their high abundance, seeds did not show any disease symptoms. While highly speculative, we suggest that Pseudomonas taxa may have a role in protecting the acorn from soil pathogens, and stimulate the development of the acorn, though direct evidence for this premise will still need to be provided. In contrast to the pericarp, several unidentified fungal and bacterial taxa were identified in the embryo, highlighting the scarcity of studies on seed microbes and the potential for the discovery of novel species that occupy this niche. Although not documented in the present study, it is expected that the inherited microbiome in the acorn primarily originated from the microbiome of the parent tree. A recent study of the maternal effect on the fungal composition of seeds indicated that composition of the tree microbiome had a major influence on the microbial seed community . Other sources of the seed microbiome include floral pathways, via the stigma of flowers, and direct contamination of seeds by microorganisms present in the environment [22, 58].
Contrary to our prediction, the majority of fungal and bacterial taxa (> 95%) of the inherited microbiome were present in the transmitted microbiome in the phyllosphere and roots of germinated seedlings. Although vertical transmission of the microbiome represents a strategy that allows a host to provide their offspring with mutualistic endosymbionts , some of the transmitted microorganisms may have detrimental effects, as has been reported for the plant and insect microbiome [60–63]. Nevertheless, the fact that the transmitted microbiome represented a very large fraction of the seed microbiome further emphasizes the ecological role of seeds as a reservoir and source for community assembly in new seedlings . In contrast to the transmitted microbiome, the transient microbial community consisted of several taxa with very low abundance, including the genera Pseudogymnoascus, Arcobacter, Cystofilobasidium, and Rutstroemia. While it has been argued that the seed represents an end-point for transient microorganisms, the seed may also provide an effective strategy for the dispersal of transient microbes into new environments, or transient microbes may play a role in the decomposition of (parts of) the acorn after dispersal.
Although the fungal and bacterial community in the phyllosphere reflected the community present in the embryo, only part of the microbes present in the embryo and pericarp were identified in the roots of the developing seedling. As a result, the microbial community of the phyllosphere differed significantly from the root community, despite originating from the same source. These differences could be due to a combination of microbial life-history traits and plant regulatory factors that limit the migration of some members of the seed microbiome into the roots. Whether direct or indirect, plant-mediated or microbe-mediated, results of the present study indicate that the distinct microbial communities commonly reported between above- and below-ground plant parts in natural and agricultural studies may originate during the transmission of microbes from seed to seedling, and not, as commonly assumed, due to inherent differences in the microbial communities in the soil and air. Such initial seed-borne differences among the below- and aboveground plant tissues may cause strong priority effects during later development. Our demonstration of partial transmission of the seed microbiome to the roots confirms previous work suggesting that plant seeds are a repository for rhizosphere microbial communities , and the high resemblance of the embryonic and phyllosphere microbiome indicates that seeds may play an even larger role as a repository for the phyllosphere microbial community.
We do note that the microbial communities we found in the offspring phyllosphere and especially the roots are expected to differ from those found in natural systems. First, acorns used in our study were collected directly from the canopy before falling onto the ground. In this regard, the fungal community of acorns has been reported to undergo a shift after falling to the ground and have a greater resemblance to the soil community . The overwintering period is also expected to allow several microbial species to penetrate through the pericarp and establish themselves in both pericarp and embryo. Importantly, the oak seedlings in the present study were grown in sterile chambers and in sterile ultra-pure water, the latter imposing anaerobic condition on the plant roots that may have favored the growth of some anaerobic taxa and limited the growth of other taxa. The fact that we did not add any additional nutrients to the water may also have inhibited the growth of some nutrient-dependent microorganisms [65, 66]. On the other hand, this approach allowed us to eliminate those factors that might have altered the microbial community transmitted to the developing seedling. Despite the expected difference, it is likely that the microbiome that is transmitted from the acorn to the roots would still play a role in plant growth and survival, before being altered or even outcompeted by the soil microbiome .
Some taxa, albeit at a relatively low abundance, were detected in the phyllosphere and roots of the developing seedling but not in seed (embryo and pericarp) tissues. Although it is difficult to pinpoint the exact reason behind this, there are several non-mutually exclusive explanations. A biological-statistical explanation is that there is natural variation between individual acorns. Thus, there is a statistical expectation of slight differences in taxa present in acorns used to characterize the microbial community of the seed vs. the acorns used to germinate seedlings. This would naturally result in some microbes not being present in the set of sequenced embryos and pericarps. A methodological explanation is also plausible in which some taxa that were initially below detection levels proliferated after their transmission to the developing seedling. Lastly, a third explanation is that the experiment had low levels of contamination. Regardless of the reason(s) underlying the presence of unique taxa in the developing seedling, we chose to focus on the core microbiome in an attempt to identify those taxa that have a consistent presence in the acorn, and that were consistently transmitted to the developing seedling. Such core taxa are expected to play a significant functional role in the respective compartments of the holobiont .
In this regard, several taxa we found to be vertically transmitted include taxa with various functions such as production of antimicrobial compounds, detoxification, nutrient uptake, and growth promoting activities. For example, Burkholderia, the most abundant genus in roots, have been reported to have plant-growth-promoting, nitrogen fixing, and plant protection properties [69–72]. In addition to their interactions with plants, some members of Burkholderia have been shown to have an impact on fungal reproduction, and were suggested to be part of the mycorrhizal intracellular microbiome [9, 73]. The genus Cadophora comprises several root endophytes with antimicrobial activity [74, 75]. Cadophora species were also found to produce high levels of plant cell wall-degrading enzymes, as well as small, secreted proteins and aquaporins that typically function in tissue colonization and nutrient acquisition in the intercellular spaces of host plants [76, 77]. The core phyllosphere microbiome included bacteria such as Micrococcus, a genus with widespread occurrence in a variety of different environments. Micrococcus taxa have been previously isolated as endophyte from coffee seeds and banana leaves, and several species of Micrococcus are involved in the detoxification or biodegradation of the toxic carbamate pesticide, carbaryl, and several other environmental pollutants [78–81]. Propionibacterium, a bacterial endophyte previously reported to inhabit apple stems, has been suggested to play a role in solubilizing phosphate into phosphorus via the synthesis of propionic acid . Paracoccus is a genus with high biochemical value due its ability to degrade a wide range of compounds, including denitrification and sulfonate degradation. Paracoccus is also the only known bacterium that possesses all the components of the mitochondrial respiratory chain and oxidative phosphorylation pathway . Species in this genus were reported to be a common endophyte in Phaseolus vulgaris seeds , Ferula sinkiangensis seeds  and Arachis hypogaea seeds and sprouts , as well as several other crops . Delftia species are seed bacterial endophytes known for their antagonistic activity against several plant pathogens, growth-promotion, and nitrogen-fixing properties [87–89]. Rahnella is a bacterium present in oak stems and is suggested to play a role in the pathobiome of acute oak decline [90, 91]. The presence of species of Apiognomonia, the causal agent of oak anthracnose, in acorns and the phyllosphere of developing seedlings may provide useful information on the epidemiology of this disease . Since this fungal pathogen has not yet been reported in Sweden, however, its presence may indicate an endophytic lifestyle or the absence of conditions that are favorable for the development of disease symptoms.