Plants are meta-organisms that are associated with complex microbiomes. Many of the microorganisms that reside on plant surfaces (epiphytes) or within plant tissues (endophytes) do not cause any plant diseases but often contribute significantly to the nutrient supply of their host plant and can help the plant to overcome a variety of biotic or abiotic stresses. The yield potential of any plant depends not only on successful plant traits that improve, for example, the adaptation to low input conditions or other stressful environments but also on the plant microbiome and its potential to promote plant growth under these conditions. There is a growing interest to unravel the mechanisms underlying these beneficial plant microbe interactions because the activities of these microbial communities are of critical importance for plant growth under abiotic and biotic stresses and could lead to the development of novel strategies to improve yields and stress resistances of agronomically important crops. In this chapter, we summarize our current understanding of the beneficial interactions of soybean plants with arbuscular mycorrhizal fungi, nitrogen-fixing rhizobia, and fungal and bacterial endophytes and identify major knowledge gaps that need to be filled to use beneficial microbes to their full potential.
- arbuscular mycorrhizal symbiosis
- biological nitrogen fixation
- tripartite interactions
The plant rhizosphere and phyllosphere is colonized by a wide range of epiphytic and endophytic microorganisms, and these microorganisms can establish beneficial, neutral, or detrimental associations of varying intimacy with their host plant. Recent developments in sequencing technologies have enabled us to study the composition and function of plant microbiomes, but these microbiomes are dynamic and differ among different plant tissues and in response to the environment. The microbiome can also be seen as “the second plant genome” and can consist of 10 times more genes than typical plant genomes . Beneficial microorganisms that are associated with plants hold enormous potential to be developed into microbial fertilizers or microbial pesticides  and new biotechnological tools to improve the nutrient efficiency and stress tolerance of crops, and environmental sustainability of agroecosystems. Specific interactions between microbes and plants, such as the
Soybeans form interactions with nitrogen-fixing rhizobia, and this symbiosis plays a key role not only for the nitrogen (N) nutrition of the plant but also for agricultural productivity since soybean root residues provide N for other plants in crop rotations [3, 4]. Arbuscular mycorrhizal (AM) fungi colonize the root system of the majority of land plants, including soybeans; transfer nutrients such as phosphate (P), N, potassium (K), and other nutrients to their host plants; and improve the resistance of their host plants against abiotic (e.g., drought, salinity, and heavy metals) and biotic stresses . In addition, soybeans are associated with bacterial or fungal endophytes that exhibit a wide range of plant growth promoting capabilities, including the production of phytohormones, an improved N nutrition through biological nitrogen fixation (diazotrophic endophytes), the biosynthesis of ACC (1-aminocyclopropane-1-carboxylate) deaminase, the capability to solubilize phosphate, and also the biosynthesis and release of antimicrobial metabolites or siderophores to inhibit the growth of pathogenic microorganisms .
The plant microbiome is a largely unexplored resource of beneficial microorganisms with diverse properties and a hidden potential to manipulate plant growth and success in stressful environments. However, while the symbiosis of soybeans with rhizobia and AM fungi is well characterized, the functional role of endophytes is only known for a limited number of isolates. Our functional understanding of these interactions is mainly based on experiments with individual symbionts, but there is increasing evidence that individual symbionts can also affect the interactions of the plant with other symbionts [7, 8, 9, 10]. We summarize here the effects of different beneficial microbes on nutrient uptake, yield, and stress resistance of soybeans and identify knowledge gaps that hinder the application of these interactions to their full potential in soybean production systems.
2. Beneficial plant microbe interactions of soybean plants
2.1 Arbuscular mycorrhizal symbiosis
The arbuscular mycorrhizal (AM) symbiosis is arguably the most important symbiosis on earth and is formed by more than 65% of all known land plant species (n > 200,000), including all legumes and many other agronomically important crops, such as wheat, corn, and rice . AM fungi are classified into the fungal subphylum Glomeromycota that consists of less than 350 fungal species . AM fungi co-exist relatively morphologically unaltered with plants for more than 400 million years, and there is evidence that suggests that the AM symbiosis played a critical role for land plant evolution .
It is long known that AM fungi can increase the nutrient uptake of their host plant and are able to deliver substantial amounts of P, N, K, sulfur (S), and trace elements, such as copper (Cu) and zinc (Zn) to the plant. Many AM fungi also provide non-nutritional benefits for their host that are critical for plant survival or fitness and improve, for example, the resistance of plants against abiotic (e.g., drought, heavy metal, and salinity) and biotic (pathogens) stresses . In return for these benefits, host plants transfer up to 20–25% of their photosynthetically derived carbohydrates to the fungal symbiont . It was generally believed that carbon is transferred to the fungus in the form of hexoses , but recent evidence suggests that also fatty acids can move across the mycorrhizal interface to the fungal partner (Figure 1) [16, 17, 18].
AM fungi are ubiquitous in soils and can account for up to 50% of the microbial biomass in soils . AM fungi form extensive hyphal networks in soils, and the extraradical mycelium (ERM) of the fungus acts as an extension of the root system and increases the nutrient absorbing surface of the root. The ERM with its mycorrhizosphere (interface between fungal hyphae and the soil) acts as an important conduit between microbial communities and the host plant  and can provide soil microbial communities with plant-derived carbon (C) inputs in large distance from the root. The mycorrhizosphere represents in soils an important ecological niche for diverse microbial communities that are specifically adapted to this mycorrhizosphere. According to estimates, the bacterial density in the mycorrhizosphere is 4–5 times higher than in the plant rhizosphere . However, the presence of AM fungal mycelia does not only lead to quantitative but also to qualitative changes in the microbial community composition in soils . The presence of AM fungal hyphae plays an important role in the bacterial community assembly during decomposition  and affects the access of members of these microbial communities to C sources during decomposition .
Within the host root, the fungus can not only spread intercellularly but also penetrate the root cortex intracellularly, and it forms here highly branched specialized structures called arbuscules that are separated from the plant symplast by the plant periarbuscular membrane . Some AM fungal species also form vesicles, thick-walled, lipid-containing storage organs in the roots. Arbuscules are the site of nutrient exchange between the plant and the fungus, and both the fungal cell membrane and the plant periarbuscular membrane are characterized by the presence of specific transport proteins that play a critical role for the resource exchange between both partners (Figure 1) [15, 25, 26].
The colonization of host roots by AM fungi is based on a molecular dialog between both partners that facilitates partner recognition and triggers responses in both partners that are critical for the establishment of the symbiosis . After fungal spore germination, an extensive hyphal branching in close proximity to host roots can be observed that is triggered by strigolactones and other compounds in root exudates . After attachment to the host root surface and the differentiation of a fungal hyphopodium, the fungus penetrates the root, spreads with the help of a prepenetration apparatus , and forms arbuscules in the cells of the root cortex. This process is initiated by the release of lipochitooligosaccharides, or Myc factors, by the fungus that are perceived by specific receptors on the host root surface and trigger a cascade of molecular responses in the host root. The pathway is called the common symbiotic signaling pathway (CSSP), since similar responses can be observed after the perception of rhizobial Nod factors [27, 30]. A key role for the perception of fungal Myc or Nod factors by the rhizodermis plays the membrane-bound receptor-like kinase SYMRK that activates the mevalonate (MVA) biosynthetic enzyme HMGR1(3-hydroxy-3-methylglutaryl CoA reductase 1). A second set of CSSP proteins is located in the nuclear pore complex and includes the three nucleoporins NUP133, NUP85, and NENA; the ATP-powered Ca2+ pump MCA8; and cation channels encoded by CASTOR and POLLUX involved in the strong Ca2+ oscillations in the nucleus of rhizodermal cells that can be observed shortly after Myc factor perception. Another set of proteins is located in the nucleoplasm and decodes these Ca2+ signals [30, 31]. A Ca2+/calmodulin-dependent protein kinase (CCaMK) phosphorylates with the help of calmodulin CYCLOPS, which then regulates gene expression either directly or through GRAS transcription factors such as NSP1, NSP2, and RAM1 [30, 31, 32]. The elucidation of the CSSP is mainly based on studies in the model legumes
Mycorrhizal plants have two pathways that are involved in the nutrient uptake from the soil: the “plant pathway” via high- and low-affinity transporters in root epidermis and root hairs or the “mycorrhizal pathway” that first involves the uptake of nutrients via the ERM of the fungus, transport to the arbuscules, and then the uptake by the plant from the interfacial apoplast through specialized transporters in the periarbuscular membrane. In response to the colonization with AM fungi, transporters that are involved in the plant pathway are often down-regulated, while mycorrhiza-specific transporters in the periarbuscular membrane are induced , indicating that there is a shift in the nutrient acquisition strategy and that the mycorrhizal pathway can become the dominant pathway for nutrient uptake [34, 35].
2.1.1 Importance of arbuscular mycorrhizal fungi for yield and nutrient uptake of soybeans
Under both greenhouse and field conditions, increases in nutrient content, yield, and overall fitness of soybeans in response to an AM colonization can be observed [36, 37], and soybean yields are significantly correlated to the colonization of the roots with AM fungi . Many reports clearly demonstrate the positive effects of AM fungi on the nutrient uptake of soybeans and here particularly on the uptake of phosphorus (P) and nitrogen (N) [39, 40, 41]. However, the effects can differ greatly among AM fungi. Our own studies demonstrated, for example, that while the AM fungus
Some of the observed differences among these AM fungi seem to be related to the impact of the AM fungus on plant P transporter expression. Fourteen genes of the Pht1 family have been identified in soybeans , and three of these transporters show high expression levels in AM roots . While the colonization of the roots with
2.1.2 Importance of arbuscular mycorrhizal fungi for the stress resistance of soybeans
AM fungi can also increase the resistance of soybeans against other abiotic stresses such as drought, salinity, or soil contaminations. It is known for several decades that the AM colonization can improve the tolerance of soybeans against drought . AM fungi can influence leaf water potential, solute accumulation, and oxidative stress of soybeans under drought stress  and delay nodule senescence triggered by water deprivation . In mycorrhizal soybeans, plasma membrane aquaporins were down-regulated in response to drought stress, and this could reduce the permeability of membranes for water and contribute to water conservation . In addition, both fungal and plant mitogen-activated protein kinases (MAPKs) are upregulated in AM soybean plants under drought stress. MAPK cascades are known to regulate many cellular processes in response to various stimuli, including abiotic and biotic stresses . AM fungi also improve the tolerance of soybeans against salinity. AM plants had a higher biomass and proline concentrations in roots, but reduced proline and Na concentrations in the shoot under salt stress. When the fungus was pretreated with NaCl, the alleviating effects were even stronger, indicating that the acclimation of the fungus to salinity may play a role for the stress response . AM fungi can also improve the tolerance of soybeans against arsenic  and aluminum  by reducing the uptake of these toxic metals.
Soybean yield and productivity is also threatened by many fungal or bacterial diseases and soil inhabiting nematodes. Soybean cyst nematodes (SCNs,
2.2 Nitrogen-fixing symbiosis with rhizobia
Most legume plants are able to interact with N-fixing bacteria called rhizobia that are able to reduce atmospheric dinitrogen (N2) into ammonia (NH3) in specialized root nodules. The symbiosis evolved in legumes between 25 and 50 million years ago [63, 64] and plays an important role for plant nitrogen (N) nutrition. Rhizobia can contribute up to 70% to the total N nutrition, grain legumes can gain up to 300 kg N, and legume trees (e.g.,
Bacteroids express the nitrogenase complex that consists of six protein subunits (two each of NifH, NifD, and NifK), two [4Fe–4S], two (Fe8S7) iron-sulfur clusters, and two iron-molybdenum cofactors (Fe7MoS9N) called FeMoco, which catalyze the N2 reduction to NH3 . The nitrogenase metallocenters are all oxygen-labile and must operate in an environment with a low level of free oxygen, and nodules provide their bacterial symbionts with this oxygen-reduced environment for optimum N fixation . N fixation by bacteroids is a highly energy consuming process, and rapid respiration in the bacteroids is necessary to produce the 16 ATP required for the conversion of each atmospheric N2 into two NH3.
The product of biological N fixation (BNF) is ammonia, which diffuses out of the bacteroids into the acidic symbiosome space and is here protonated to ammonium. The symbiosome membrane is energized by an H+-ATPase, which pumps protons into the symbiosome space and thereby promotes the uptake of NH3/NH4+ into the plant cytosol, where NH4+ is rapidly assimilated into amino acids, and the ureides, allantoin and allantoic acid . A candidate for the uptake of NH4+ from the symbiosome space is
BNF is an energy expensive process, which requires 16 ATP to fuel the reduction of one N2. Plants allocate up to 30% of their photosynthetically fixed C to rhizobia , which is oxidized in the bacteroids to ATP. The N2 fixation rate of rhizobia is higher when the nodules receive more C, suggesting that the allocation of C to nodules is a limiting factor for BNF. Transgenic
2.2.1 Significance of rhizobia for soybean agriculture
According to estimates, soybeans with their rhizobia populations fix around 20 million tons of N each year, and this has an enormous influence on agricultural productivity, not only on soybeans, but also on other crops in crop rotation systems [3, 4]. Soybean residues in the soil enrich the soil with N, improve soil organic matter, and can lead to yield increases in non-legume crops that follow soybeans. Crop rotations or intercropping systems of cereals with legumes can result in higher crop yields without fertilizer additions . However, conventional agricultural management practices and other anthropogenic factors can have a negative impact on rhizobial function. In addition, excessive tillage, applications of higher N fertilizer dosages, and extended fallow periods can also have detrimental effects on rhizobia populations in soils. As a consequence, integrating this symbiosis more efficiently in modern agricultural practices is crucial to limit the amount of fertilizers used and make agriculture more environmentally sustainable. Exploring ecologically best fitted ecoregions for soybeans and best adapted soybean cultivars will help farmers to produce more yield with reduced inputs. Rhizobial strains differ in their efficacy in symbiosis with different soybean cultivars, and the input of N into agricultural systems can be increased by the inoculation of legumes with optimized rhizobia for different environments . The development of better inoculation strategies and specifically adapted rhizobia for different soybean cultivars could reduce the dependency of farmers on agrochemicals and enhance food security .
2.3 Tripartite symbiosis with arbuscular mycorrhizal fungi and rhizobia
In natural environments, legume roots form tripartite interactions and are simultaneously colonized by both AM fungi and rhizobia [7, 79]. Tripartite interactions have been shown to improve plant productivity, seed yield, P and N acquisition, and photosynthetic rates [10, 80, 81]. The rhizobial nitrogenase complex requires at least 16 ATP to reduce one N2 molecule into two NH3. Consequently, nodules act as strong P sinks in legume root systems to provide sufficient P resources to the bacteroids for optimum BNF [79, 82]. Since AM fungi are able to improve the P nutrition of legume plants, AM fungi can increase the BNF by root nodules by at least 50% . Nonmycorrhizal soybean plants have lower nodule numbers and weights and particularly under low P supply lower N fixation rates [7, 83]. AM fungi can also provide their hosts with microelements that are essential for N2 fixation, including zinc, iron, manganese, and molybdenum [84, 85].
AM fungi and rhizobial bacteria can act synergistically and can improve plant productivity, seed yield, and grain quality [7, 10, 81]. However, the prior inoculation by either rhizobia or AM fungi can also reduce the subsequent colonization by the other symbiont . Plants control the extent of root colonization by both symbionts by an autoregulatory mechanism, possibly to limit the high C costs associated with these interactions [83, 87]. Whether AM fungi and rhizobia interact antagonistically or synergistically depends on the environmental context  and the compatibility between symbiotic partners [10, 88]. For example, the rhizobial strain STM 7183 is more compatible with the AM fungus
Both interactions are costly, and the host plant allocates up to 20% of its photosynthetically fixed C to its fungal [14, 90] and up to 30% to its N-fixing symbionts (Figure 2) . C acts as an important trigger for symbiotic functioning, and a reduction in the C fluxes to the symbionts decreases BNF by rhizobia , and P and N uptake and transport by AM fungi [92, 93, 94]. Considering the high C costs of these symbioses for the host, plants are under a selective pressure to strongly regulate the C fluxes to both root symbionts, but these control mechanisms are currently poorly understood. Resource exchange between host and AM fungi is controlled by a reciprocal reward mechanism that is driven by biological market dynamics . Our own results recently demonstrated that similar mechanisms may also control the resource to C exchange in tripartite interactions;
AM fungi have stronger effects on plant gene expression than rhizobia , but our current understanding of the molecular mechanisms involved in the C allocation to individual root symbionts is limiting. An overexpression of a leaf sucrose phosphate synthase of
2.4 Symbiosis with endophytic bacteria or fungi
Endophytes are defined as organisms that live inside plant hosts for at least part of their lives, without causing apparent disease symptoms in the host as a result of this colonization . Fungal and bacterial endophytes are nearly ubiquitous across all groups of vascular plants , but there is a large biological diversity among endophytes, and it is not rare for some plant species to host hundreds of different endophytic species . Fungal endophytes have been shown to enhance growth and seed production or protect against environmental stresses such as drought or P deficiency or provide defense against herbivory through the synthesis of various biologically active metabolites, such as alkaloids. In soybeans diverse communities of fungal endophytes can be found, and several of these endophytes have plant growth-promoting capabilities and enhance, for example, soybean growth in nickel- or copper-contaminated soils by reducing the levels of stress-related phytohormones such as abscisic acid and jasmonic acid ; or increase glutathione activities and thereby reduce oxidative stress . The inoculation of soybean plants with fungal endophytes can also lead to higher shoot biomasses, chlorophyll contents, and photosynthetic rates compared to noninoculated soybeans under salt stress and decrease the abundances of SCN in soils .
Soybeans host also a diverse group of bacterial endophytes, and many endophytic bacteria have plant growth-promoting capabilities , such as the ability to produce plant growth hormones, or ACC (1-aminocyclopropane-1-carboxylate) deaminase, solubilize phosphate, or release antimicrobial metabolites or siderophores that can inhibit the growth of pathogenic microorganisms. ACC deaminase reduces the levels of ethylene, an important stress hormone in plants. Several endophytic bacteria are also diazotrophs and have like rhizobia bacteria the ability to fix N. Bacterial endophytes also interact with rhizobia bacteria and can enhance root nodulation and activity, and as a consequence, the N content of soybean plants . The dual inoculation with rhizobia and a salt-tolerant bacterial endophyte led to synergistic responses and promoted the fitness of soybean plants under salt stress .
3. Important research gaps and future challenges
Beneficial plant microbe interactions with AM fungi, rhizobia, or bacterial and fungal endophytes have enormous potential to improve plant growth and nutrient uptake in stressful environments and to increase the environmental sustainability of soybean agriculture. However, while the beneficial effects of AM fungi and rhizobia on soybean productivity are long known, the effect of only a small number of endophytes is currently known. The plant microbiome is a still unexplored resource of microorganisms with a so far hidden potential to promote plant growth and success under abiotic or biotic stress conditions, and with unknown effects on the plant phenotype.
The obligate lifestyle of AM fungi has made for a long time the production of fungal inoculum in large quantities difficult, but the development of sterile transgenic root organ cultures has led to an increased commercialization of AM fungal inocula for the utilization in agroecosystems . Although increases in yield and biomass have been reported in different crops after inoculation with these inocula [36, 110], in other studies, inconsistent or neutral effects were observed . AM fungi differ in the benefit that they provide for their host plant , and mycorrhizal growth responses are highly context dependent. Several factors can alter the success of AM fungal inoculation in agroecosystems, including plant/fungal compatibility, the degree of competition with the native microbial population, or timing of inoculation . All these aspects need to be taken into consideration to find the most adapted and specific conditions for an efficient use of AM fungal inocula in a given field or for a certain crop. Our current understanding of the effect of beneficial plant microbes on soybeans is mainly based on studies with single symbiont, but plant productivity and stress resistance in agroecosystems depend on diverse microbial communities and the interactions among the different microorganisms in these communities. Identifying and characterizing the molecular mechanisms responsible for the functioning of different plant microbe interactions is crucial to harness these symbiotic microorganisms in agroecosystems. Currently, most knowledge is gathered on model legumes, such as
We wish to acknowledge funding from the USDA (2017-67014-26530), the SD Soybean Research and Promotion Council, the Agricultural Experiment Station at SDSU, and the funding from the North Carolina Agricultural Research Service (NCARS) for K.G.
Conflict of interest
The authors have no conflict of interest.