Keywords

Introduction

The concept of the microbiome was described for the first time as the "ecological community of commensal microorganisms, symbionts or pathogens, which literally occupy a space in our body" [1]. Recently, this term has been used for different environments inhabited by microorganisms [2,3,4]. This term has also been used in the plant context as "an environment, which consists of the plant and all microbes associated with it" [3].

The relationship between plants and their surroundings, especially those plant-microbe interactions with a beneficial output, has been the center of attention of various studies [5]. Traditionally, many researchers have tried to understand these interactions, looking to individual plant-microbe relationships, i.e., a one vs. one approach, but these interactions are much more complex, as they involve a vast diversity of microbes and environmental factors [6].

Plant, soil, soil-borne microbes, and environmental factors together influence the various changes that cooperate to create plant health and productivity. Recent advances in “-omics” research have shed light on microbiome compositions and interactions with the environment [7]. These advances have contributed to the development of novel approaches that seek to improve plant fitness through the artificial selection of microbes with specific effects on host performance. The selection of microbial communities occurs indirectly through host traits that have coevolved together with the microorganisms and influence the microbiomes [8].

In this chapter, firstly, we define each plant compartment, i.e., the rhizosphere, phyllosphere, and endosphere, and within each compartment we describe “who” is there (microbiome structure), “what” they are doing (microbiome functions), and what are the major drivers shaping the assembly of the microbiome. Finally, we discuss the advances in microbiome manipulation and the possibilities of using such manipulation to improve and optimize crop productivity.

The Rhizosphere Ecosystem

The term rhizosphere was coined by the soil bacteriologist Lorenz Hiltner in 1904 [9]. This term is derived from the Greek word rhiza (root) and the Latin word sphaera (sphere), referring to an environment or compartment that encloses the inhabited “microbial world” on the plant roots. The rhizosphere is the narrow zone of soil surrounding the root system where plants and microorganisms interact [10,11,12,13,14] (Fig. 1) and it is characterized by a chemical, biological, and physical gradient that changes radially and longitudinally along the roots [15].

Fig. 1
figure 1

Schematic representation of plant microbiome compartments and frequency of studies describing bacterial phyla in each compartment, i.e., phyllosphere, endosphere, and rhizosphere. Each pie graph shows the frequency of studies reporting bacterial phyla per plant compartment. For example, 18% of 15 studies on the phyllosphere detected Actinobacteria in the bacterial community. Seventy-one studies were surveyed, 15 for the phyllosphere, 29 for the endosphere, and 27 for the rhizosphere. Searches were performed in the Scopus database between February 03, 2016 and March 15, 2016. The search used a combination of words describing plant compartments (“rhizosphere”, “phyllosphere”, “endophytic”, “endosphere”) and investigative techniques (“sequencing”, “metagenomic”, “next-generation sequencing”). Studies using cultivation-dependent approaches were not included in the survey. Phyla cited in only one manuscript were included in the “Others” category

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The idea of microbial colonization of the rhizosphere seems to be supported by the niche theory of species diversity, which is driven by various abiotic and biotic factors, such as plant genotype and soil [5, 13, 16,17,18,19]. Changes in the rhizosphere microbial community begin when the soil microbiota is exposed to rhizodeposits, which are influenced by the plant genotype, including glucose, amino acids, organic acids, polysaccharides, and proteins [10, 13]. Rhizodeposition increases the microbial populations in the rhizosphere, known as the “rhizosphere effect” [11,12,13, 16]. Later, the plant genotype selects and assembles a closely associated microbial community in the rhizoplane and within the plant roots [13, 16, 20]. It has been hypothesized that each plant species selects specific microbial populations as a result of the high degree of host specificity in the coevolution of plants and microbes [5, 13, 21].

Plants release exudates into their direct surroundings to attract, stimulate, or repel microorganisms on the roots. The amount and composition of the rhizodeposits, which structure and modulate the rhizosphere microbial community throughout the plant life cycle, may vary among different plant species [22] and throughout their growth [23], as well as in different stages of root development [5]. Microbial succession starts with the release of carbon from seeds during the germination stage, and microorganisms in the rhizosphere are distributed according to root type and zones, as well as according to their movement through the soil during root growth [13]. In the early stages of plant development (seedlings), alcohol and sugars are released, while in the later stages, amino acids and phenolic compounds predominate [23]. This phenomenon suggests the attraction of a large diversity of microorganisms in the early stages of plant development, while later the release of specific substrates selects certain microorganisms in the rhizosphere [5, 21, 23].

The number of microorganisms in the rhizosphere is higher than that in bulk soil, due to the carbon availability in the rhizosphere. Generally, gram-negative bacteria are stimulated by rhizodeposition, whereas gram-positive bacteria are inhibited [10]. Proteobacteria (α, β, γ), Firmicutes, Actinobacteria, Bacteroidetes, Crenarchaeota, Acidobacteria, Ascomycota, and Glomeromycota, and also unclassified bacteria, represent relatively large groups detected in the rhizosphere [5, 12, 13] (Fig. 1).

The microorganisms found in the rhizosphere can have beneficial or deleterious effects on the growth and health of the plant [13]. The beneficial microbes, among others, include mycorrhizal fungi and rhizobia, which provide phosphorus and nitrogen; siderophore-producing bacteria, which facilitate iron acquisition; and plant-growth-promoting rhizobacteria (PGPR), which promote plant growth [12, 14, 24]. PGPR can suppress disease by mechanisms such as competition for nutrients and microsites, parasitism and antibiosis, or by inducing systemic resistance to pathogens in the plant [13]. There are some examples of microorganisms that promote plant adaptation to abiotic stresses such as drought, flooding, saline stress, temperature or pH extremes, and high concentrations of toxic compounds, and these cases reveal complex associations of microorganisms with plants as a result of coevolution in their native habitats [13, 25]. Biotic stress includes the presence of phytopathogenic microorganisms such as nematodes, fungi, and oomycetes, which have agronomic importance because they reduce the yields of food, feed, fiber, and fuel crops [12].

Given that root exudates are strongly linked to the recruitment of the microorganisms that comprise the rhizosphere microbial community, it can be seen that the rhizosphere is closely involved with plant health and growth; therefore, the understanding of rhizosphere functioning and ecology is key to increasing crop yield.

The Phyllosphere

The second compartment of the plant microbiome is the phyllosphere, or aerial plant surface, which is characterized as being nutrient poor when compared with the rhizosphere [26]. The phyllosphere is composed of microbial cells that are able to colonize the aerial plant surfaces [27, 28] that are dominated by the leaves, although the term phyllosphere can be used for any aerial part of the plant [29] (Fig. 1).

The microbial habitat on the surfaces of leaves may be one of the largest microbial habitats on earth, with the terrestrial leaf surface area estimated to exceed 108 km2 globally [30]. The phyllosphere microbiome is composed of viruses, bacteria, filamentous fungi, yeasts, algae, and, occasionally, protozoa and nematodes [26]. Bacteria are the most abundant of the cellular organisms in the phyllosphere community, present in numbers between 106 and 107 cells cm−2 of leaf tissue [26, 29]. Fungi and archaea are apparently less abundant; however, their population has not been estimated yet [26, 30, 31].

Overall, species richness in phyllosphere communities is high [32]; however, the bacterial community diversity is lower than the diversity of the communities in the rhizosphere or bulk soil [31, 33]. Advances in sequencing technologies have vastly expanded our understanding of plant microbiome structure, including that in the phyllosphere [34]. At the phylum level, the phyllosphere bacterial communities are composed mainly of Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria [35], with a predominance of the classes Alphaproteobacteria and Gammaproteobacteria [36, 37] (Fig. 1). Further analysis of community composition at the genus level suggests that Pseudomonas, Sphingomonas, Methylobacterium, Bacillus, Massilia, Arthrobacter, and Pantoea are consistently found as part of the phyllosphere microbiome across a wide range of plant species [35].

The colonization of plant leaf surfaces, in large part, occurs through the immigration of bacteria, fungi, and other microorganisms from air, soil, water, seeds, or through animal sources [29]. Furthermore, studies have shown that some of these microorganisms of the foliar microbiome can be transferred not only through environmental exchange, but also vertically, through generations of plants [38]. Neighboring environmental ecosystems can also randomly contribute to the assembly of the foliar microbiome [39]. Even after the stabilization of phyllosphere microbial communities, variations may occur, caused by nutritional heterogeneity in different regions on the leaf surface, where the carbon sources (e.g., glucose, fructose, and sucrose) are spatially heterogeneous, leading to distinct microbial assemblages on the leaf veins, which are regions near the stomata and surface appendages [26, 29]. Large fluxes in temperature, moisture, and radiation throughout the day and night also cause changes in the phyllosphere microbiome structure [26, 29, 40]. In some cases, this spatial heterogeneity is promoted by the organization of microbial cells into biofilms, which are a common feature of organisms in the phyllosphere, acting as aggregators and protectors of the microbial cells under the frequently inhospitable conditions [26, 41].

The microbial communities found in the phyllosphere may perform key processes related to plant development; for example, nitrogen fixation [42, 43], protection from invading pathogens [44], modification of metabolites, and the biosynthesis of phytohormones [45]. Metagenomic and metaproteomic studies showed that microbes in the phyllosphere could produce proteins that promote substrate uptake, via porins and ABC transporters; resistance to stresses, including reactive oxygen species (ROS); and nutrient cycling [31]. Methylobacteria are involved in methanotrophy and are often detected in phyllosphere communities [46, 47].

The interactions between the plant and the phyllosphere microbial communities, and the variations in their distinct environmental factors, modulate the assemblage of these microbial communities in the phyllosphere and contribute to the heterogeneity in their abundance and structure in distinct plant species. New molecular technologies have shown the importance of microbial functions in the phyllosphere and have provided new insights into the major drivers of microbial community composition. The combination of multiple “omics” technologies will lead us to a system-level understanding of the phyllosphere microbial communities and their physiological potential.

The Endosphere

The endosphere consists of the inner plant tissues, inhabited by microorganisms intimately interacting with the host plant [28, 48, 49]. This compartment is composed of the internal root tissue (endorhizosphere), internal shoot and leaf tissue (endophyllosphere), internal plant reproductive tissue, and the internal seed tissue [50,51,52,53,54,55]. Endophytic microorganisms are organisms that reside internally in plant tissues for at least part of their life cycle [48] without causing visible disease symptoms [56] and they can be accessed from the plant after surface disinfection by cultivation-dependent and/or molecular approaches [57,58,59] (Fig. 1). Although this concept is one of the most commonly accepted ones and is currently applied, it is important to note that there are niches on the surfaces of aerial parts and roots where microorganisms may remain protected from the action of the chemical products usually used for surface disinfection. Recent studies have used sonication to remove surface layers of the plant tissue and to access the endophytic microorganisms on the remaining tissue [17, 20].

Endophytes are beneficial or commensal, and they can shift between parasitic and mutualistic life strategies [60, 61]. Their beneficial role in plant development and health can be mediated and is characterized by metabolic interactions, including the production of plant growth hormones [62,63,64], antibiotics, and toxicants [65, 66]; the improvement of nutrient uptake; and/or increasing the plant tolerance to biotic and abiotic stresses [62, 67, 68]. In addition to these characteristics, the lifestyle of endophytes can also involve altering/inducing the gene expression of plants’ defense and metabolic pathways [66, 69, 70], and, depending on the type of interaction, members of the endosphere microbiome can induce both local and systemic alterations in the host [71]. As an example of these alterations, genome analysis of Bacillus pumilus INR7, an endophytic bacterium that promotes plant growth and induces systemic resistance against several plant patogens, revealed the presence of non-ribosomal peptide synthetase gene clusters for the production of antibacterial compounds such as surfactin, bacillibactin, and bacilysin, as well as genes for the biosynthesis of growth promoters such as indole-3-acetaldehyde and 2,3-butanediol [72].

The endosphere microbiome structure is driven by soil type, host phylogeny, and/or microbes. The soil traits that affect microbial recruitment from bulk soil are soil type [20, 53, 73], soil pH [53, 74], local edaphic conditions [75], and anthropogenic management factors, such as fertilizer and pesticide application and soil preparation [76, 77] The endosphere microbiome structure is also variously affected by plant species [78], plant life stage [77, 79], and plant health, as a result of the differences in root architecture and types of exudates [16]. Finally, the capacity of microbes to reach inner plant tissues and establish themselves there also affects the microbial composition of the endosphere. Endophytes need to have the capacity to reach the root surface, and to express genes for the invasion of plant tissue and the colonization of a niche within the plant tissue [80]. Studies have shown that the endosphere is mainly composed of bacterial phyla, such as Proteobacteria, Actinobacteria, Bacteriodetes, and Firmicutes [17, 20, 77, 81], and fungi, including Ascomycota and Basidiomycota [35, 82,83,84] (Fig. 1).

Endophytes are classified as systemic/true and transient/nonsystemic [56] or as obligate and facultative [48]. Systemic or obligate endophytes are dependent on the plant metabolism, and are disseminated among plants by vertical transmission or by vector activity [48]. In addition, systemic endophytes do not produce any visible symptoms of disease in the host at any life stage [56]. Because they live in a low-competition and low-predation environment, obligate endophytes have evolved to produce specific metabolites that support their interaction with the host [85]. In contrast, facultative or transient endophytes live inside plant tissues for at least part of their life cycle, without producing any apparent disease symptoms in the plants, but they become pathogenic when the host plant faces resource-limited conditions [86]. Transient endophytes vary both in diversity and abundance, depending on changes in the environment [83] and they face high levels of competition in the rhizosphere before entering the plant [80], therefore producing many metabolites that are involved in both their defense and in interactions with the plant [85].

The microbiomes associated with above-ground (phyllosphere), below-ground (rhizosphere), and internal (endosphere) tissues are distinct, especially considering that the endosphere is where specific metabolic capacities are required to survive. Endophytes have a significant effect on the host plant by modulating its health, growth, and development. Naveed et al. [87] observed that Enterobacter sp. strain FD17 promoted the growth and health of maize grown under natural conditions, increasing grain yield by 42% and reducing the time until flowering. Mendes et al. [62] reported that the endophytic Burkholderia spp. showed ability to control the growth of the sugarcane pathogen Fusarium moniliforme. Khan et al. [88] have shown that tomato plants inoculated with endophytic Sphingomonas sp. LK11 showed increases in shoot length, chlorophyll content, and shoot and root dry weights, indicating that the phyto-hormones produced by this strain may help in increasing crop growth. Although there are still gaps in our knowledge of endophytes, the investigation of these microbes as a bioresource for plant growth-promoting regulators and as biocontrol agents for disease and pest management represents opportunities for improving crop yield and health in a sustainable way.

Manipulation of the Plant Microbiome Toward Improved Plant Health

According to the latest United Nations projections, the world population will exceed ten billion by 2100 [89]. In order to meet the demand for food, both the land area used by agriculture and productivity must increase in the near future. In this scenario, intentional manipulation of the plant microbiome may be an alternative way to improve agriculture sustainability. This would be done by exploiting rhizosphere microorganisms with beneficial traits to, for example, make nutrients more available for plants or increase plant tolerance to biotic and abiotic stresses, consequently decreasing the dependence on chemical input in agriculture.

Manipulating the plant microbiome can be achieved simply by promoting good management of soil. Crop rotations increase the diversity of microorganisms in soil, promoting high resilience to plant pathogens [90]. Bakker et al. [91] showed that where resource changes altered the bulk soil microbial community, the effects were observed in the rhizosphere of two different cultivars of corn, suggesting that rhizosphere microbial communities are altered depending on the site history and selective events.

The stimulation of certain microorganisms or the introduction of inoculants is another strategy for plant microbiome manipulation. This approach aims to establish a beneficial community that competitively excludes plant pathogens. Reducing the time of niche exploration is crucial for enhancing microbial root-colonizing capacity [80, 92]; this can be achieved by the co-inoculation of several beneficial strains, including endophytes. The inoculation of a bacterial consortium might also promote the release of antimicrobial compounds [93] that improve the suppression of soil-borne pathogens [94].

The inoculation of microorganisms also has the potential to improve plant nutritional status. Rhizobium spp. are some of the most common microorganisms used as inoculants in legumes and their use can supply almost all of the nitrogen required by legume crops [95]. Phosphorus-solubilizing microorganisms can also be applied as inoculants, either alone or in association with rock phosphate [96]. A limitation in the use of inoculants is that the densities of the inoculated microorganisms are subject to decline over time, and the inoculants have to be able to survive under different field conditions. It is also important to consider that inoculants must be free of metabolites that are hazardous for humans, animals, and plants [97].

The plant genotype, in interaction with environmental conditions, is responsible for regulating the release of exudates in the rhizosphere soil, and this exudate release is one of the main drivers of the microbiome structure. In this context, the microbiome may be manipulated by changing the amount and quality of root exudates through plant breeding or genetic modification [98,99,100]. However, it is important to note that this strategy is limited in many ways: (a) the methods are very time-consuming and are restricted to the target species/cultivar; (b) traditionally, breeding programs do not consider the interaction among plants and microorganisms when new cultivars are being developed [101]; and (c) the quantity and quality of the exudates vary tremendously among soil types and physiological conditions of plants, making the exudates difficult to manipulate [102].

Although less commonly studied, manipulation of the microbiome of aerial plant parts can also be a strategy for improving plant growth and health. Falk et al. [103] suggested that it is possible to reduce the severity of powdery mildew infections caused by Uncinula necator on grapevines by releasing the conidia of the mycoparasite fungus Ampelomyces quisqualis. Several pesticides applied in agriculture have the potential to affect the natural occurrence of a microbial community [104, 105], while it has already been shown that the natural leaf microbiome is beneficial to the plant. Perazzolli et al. [106] showed that the naturally occurring microbiomes of grapevine leaves could reduce signs of powdery mildew on the surfaces of the leaves under controlled conditions.

Optimizing plant-microbiome interactions through microbiome manipulation has the potential to improve crop sustainability, reducing the impacts of traditional agricultural practices. Although many efforts have been made to understand the factors controlling microbiome assemblage, manipulating the microbiome is still a challenge to be addressed.