We specifically highlight studies that address assembly processes during seed development and maturation, as these stages are understudied compared to seed dormancy and germination, and they are likely the source of microbes that persist between plant generations . Lastly, we suggest future lines of research to gain a more mechanistic, scale-explicit understanding of seed microbiome assembly.Plant seeds are generally composed of three tissues: a seed coat which provides physical protection , an embryo which is the precursor to the seedling and is made up of an immature root, a stem, and one or more embryonic leaves , and an endosperm which typically consists of carbohydrates and proteins and provides nutrition for the embryo during germination and growth before photosynthesis can occur . Seed development involves three stages . Following fertilization by pollen, the egg cells divide and differentiate into the embryo and endosperm tissues, in a process called histodifferentiation . Next, the cells expand and mature with reduced division, and seed mass increases during this filling stage, as nutrient reserves are deposited into the endosperm . After this, nutrient accumulation declines, and the seed goes into maturation drying and loses about 10%–15% moisture content before it is ready to be dispersed .During seed development, microbes may enter the seed tissues via three distinct routes of transmission: vertical, floral, and horizontal . Vertical transmission involves microbes traveling from other organs of the mother plant to the developing embryo. Such transmission is cited as an ecologically important way for plants to inherit beneficial microbes across generations and for seed associated pathogens to disperse .
Vertical transmission has long been observed in grasses,dutch bucket hydroponic which are hosts to clavicipitaceous fungal endophytes such as Epichloe . Vertical seed transmission has also been observed for non-clavicipitaceous endophytes in Setaria viridis , Triticum , Quercus , and other plants . Floral transmission of microbes into seeds has been studied extensively for pathogens such as Monilinia vaccinii-corymbosi in blueberry and Acidovorax citrulli in watermelon . However, flower-to-seed transmission has also been observed for commensal and beneficial bacteria, for example in Brassica napus . The microbial contributions of the vertical and floral transmission pathways are likely to vary based on a plant species’ pollination mode . Horizontal transmission is the acquisition of seed microbes from the environment, either prior to or after the maturation of the seed as it is still attached to the mother plant or as matured seed disperse and becomes colonized from sources such as air , water , animals , soil , and other seeds in storage . Seed dormancy and germination are likely to represent a very active period of such horizontal transmission, as soil microbes interact with seed exudates and pre-existing microorganisms on and within the seed .The meta community concept was formally described by Leibold et al. , who defined meta communities as sets of local communities that are interconnected by dispersal. This definition arose out of a need to better account for spatio-temporal scales in ecological studies , and also included the impacts of dispersal and habitat heterogeneity on community patterns . Since it was first described, meta community theory has adopted Vellend synthesis that community assembly and composition are driven by four categories of processes: abiotic and host filtering, species interactions, dispersal, and ecological drift .
Categories 1 and 2 represent a deterministic or niche-based process of selection where differences in fitness between taxa, species, or guilds lead to differences in their abundances . Finally, drift is the stochastic fluctuation in species abundances, often due to chance birth, death, and migration events . Framing plant microbiomes as meta communities provides an integrated view of the drivers of their composition, function, and evolution, and of the impacts of these drivers on host health . Traditional meta community ecology states that filtering and species interactions occur at the local scale , while dispersal and drift occur at the regional scale . However, categorizing processes as “local” or “regional” is relative to the community that is being studied, and depends on the scales of interest and on defining the boundaries between a local community and a regional meta community. For plant microbiota, including those associated with seeds, the terms “local” and “regional” are contextual because microbes primarily behave at very small scales , although they can be affected by much larger scale factors . Furthermore, microbes can be ubiquitous across habitats at multiple scales, blurring the boundaries between patches of local communities in the landscape of interest . As we apply the first principles of meta community ecology to plant and seed microbiology below, we will therefore use three categories of spatial scale: macro- , meso- , and micro- scales. Integrating the study of assembly processes across these three scales should give a more complete picture of how microbial communities are assembled, and how emergent community patterns occur at individual scales .
Several studies have shown that seed microbial communities differ significantly across geographic locations, i.e., at the macroscale, for example in B. napus , Elymus nutans , Phelipanche ramosa and Pseudotsuga menziesii . For most of these studies, the abiotic factors that are important for structuring these seed microbial communities remain to be identified. However, we can assume that these factors are similar to the ones that drive macro-scale differences in the microbial communities on/in other parts of the plant. In communities associated with leaves, roots, and fruits, such factors include temperature , precipitation , humidity , and soil conditions . In a study of above ground microbial communities in Vitis vinifera, Bokulich et al. found that fungal communities of seeded fruit were associated with net precipitation, relative humidity, and temperature. During dormancy in the soil, the bacterial communities of Noccaea caerulescens seeds were correlated with soil pH and cation composition . Not much is known either about variation in seed microbial community as a function of abiotic factors at the meso-and microscales, although again, much can be learned from studies on other above ground plant tissues. At the meso-scale of an individual plant, microbial communities can vary with tissue location such as canopy height in trees. Unterseher et al. cultured fungi from leaves at different canopy heights in several tree species. They found that species richness was greater in the lower canopy. Harrison et al. went on to use next-generation sequencing in a survey of the needle fungi of Sequoia sempervirens at different height positions, and found that there were distinct communities present at each height across trees. While they did not measure microclimate variables within the trees sampled, they suggested that the observed variation could be attributed to the amount of sunlight . At the micro-scale , factors such as exposure to ultraviolet radiation and water availability can also be important. Hayes et al. described variation in the bacterial communities and UV radiation along individual flower petals in two sunflower species. They found that while there was no significant difference in community composition along petals, there was variation in UV tolerance in association with source petal position . Another potentially important factor may be water availability, which has been shown to affect bacterial survival, growth,dutch buckets system and movement on leaf surfaces . For many macro-scale studies, a major limitation is the use of location as a proxy for environmental conditions, which precludes linking variation in microbial communities to specific environmental factors. Because site effects are impacted by environmental, spatial, and temporal factors, it can be difficult to parse out how location and environment influence seed microbiota . Also, most of these studies do not explore if and how environmental conditions actually select for microbial traits and taxa. In vitro experiments suggest that there is potential for environmental filtering, as demonstrated by thermotolerance in fungal endophytes of desert plants , salt stress tolerance in fungal root endophytes , water stress tolerance in bacterial endophytes , and oxidative stress tolerance in the fungal endophyte Epichloë festucae .
Similar characterization of seed microbial tolerance and survival when challenged with different environmental conditions could provide a more mechanistic understanding of abiotic filtering. Such studies would be particularly insightful at the micro-and meso-scales.Variation in plant microbial communities is often studied and interpreted as a result of plant genetics, which represents filtering through host selection. Studies at the macro-and meso-scales have revealed that plant genetics can significantly impact microbial community composition in different parts of the plant, although seeds are clearly underrepresented in the body of literature on this topic. Microbial community variation has been associated with specific genes in leaves and roots of various plants , an approach that has not yet been applied to seeds, as far as we know. Seed line has been weakly associated with microbial community variation in Zea mays and B. napus . Seed accessions of Oryza were also associated with variation in bacterial and fungal community composition, with significant compositional shifts between wild and domesticated accessions . In a study of the bacterial and fungal communities associated with grapes, Singh et al. found that host genotype had an impact particularly within individual sites, whereas abiotic conditions better explained microbial community variation between sites. This is consistent with the notion that host effects are difficult to reveal without carefully controlling for environmental factors, which would suggest, by extension, that environmental factors may have a greater relative impact on seed microbiota than plant genotype. A recent study showed however that the fungal community composition of Quercus petraea internal seed tissue was largely influenced by the mother plant, with only weak significant environmental influences . Studying the roles of plant functional traits in seed microbiome assembly and dynamics provides the mechanistic framework to understand host filtering. Some of the clearest examples of these mechanisms come from the field of plant pathology, where plant traits can be used to predict disease outcomes . One obvious suite of traits to study are plant defenses. As agents of plant regeneration, seeds are one of the most defended plant organs, protected by both chemical and physical defenses . Some of these defenses come from the mother plant, such as through innate floral defenses in angiosperms . Many studies on plant defense traits are obviously focused on protection against pests and pathogens , but can be extended to other members of the microbial community . Using microscopy, the microbial communities within seeds of Citrullus lanatusand Q. petraea were found to differ in abundance and composition depending on seed sub-structure. Since Q. petraea is a wind-pollinated species, the variation in seed sub-structure colonization observed by Fort et al. suggests physical filtering of microbes during vertical and horizontal transmission. Although few studies have explored the role of micromorphology of developing seeds in microbial community acquisition , there are plenty examples of such micro-scale studies come from work on the floral microbiome. Spinelli et al. used microscopy and fluorescent tagging to study the growth and movement of the bacteria Erwinia amylovora and Pantoea agglomerans on flowers of apple and pear . They found that the bacteria migrate from the stigma to the nectaries along a stylar groove in both species, indicating topographical effects on survival, population growth, and dispersal . Similarly, Steven et al. characterized at the high spatial resolution the floral bacterial communities on apple using next-generation sequencing and found that different flower parts were enriched with different bacterial families . It is intriguing to think that variation in microtopography on flowers and stigmas may contribute to host filtering during the process of flower-to-seed horizontal transmission of microorganisms.The role of species interactions in meta community dynamics is important, but often overlooked in meta community ecology studies . In plant microbiota research in general, much focus has been on pathogen antagonism interactions, for example with an eye toward applications in disease control . However, there is much interest and opportunity to better understand interactions between and among non-pathogens in plant and also seed microbial communities. As with traditional ecology studies, much of the work on species interactions in seed microbial communities focuses on competition and antagonism. For example, Raghavendra et al. inoculated Centaurea stoebe flowers with pairs of fungi and then cultured those fungi out of mature seeds. They always isolated the same single fungus from each pairing out of seeds across parent genotypes, and proposed that competition was the primary driver of selection .