Quality or Quantity? Increasing Legume Yield Using Traditional Inoculants and Rhizobial Nod Factors in the Context of Inter-Strain Competition

Abstract

Rhizobia have been used for decades as biopreparations, successfully replacing synthetic nitrogen fertilizers in legume cultivation. They have a beneficial effect on the growth and yield of these plants when cultivated in soils that are deficient in both nitrogen and indigenous rhizobia. However, such preparations, containing strains that are characterized by high effectiveness in reducing atmospheric dinitrogen, are not universal. Their use is ineffective when plants are grown in soils that are already rich in strains with low effectiveness, because such inoculant strains are unable to effectively compete with native soil populations. This review discusses issues related to the rhizobia–legume symbiosis, with particular emphasis on inter-strain competition occurring in the soil and in the colonized plant tissues. The importance of Nod factors (NFs) in symbiosis and their broad impact on plant physiological and developmental processes are also discussed. Research results on the effects of NF-containing biopreparations on legume growth and yield are summarized. Moreover, this review explains how such preparations can support the growth and yield of legumes growing in soils containing numerous populations of low-effectiveness rhizobia. Finally, the potential for the application of this technology to non-legume plants is presented.

1. Introduction—The Importance of Biological Dinitrogen Reduction

All crops require nitrogen fertilization to achieve satisfactory yields. In countries with well-developed agricultural systems, synthetic nitrogen fertilizers are most commonly used. Unfortunately, this solution can also cause environmental degradation. Moreover, the use of synthetic nitrogen fertilizers to achieve optimal yields often generates the highest cost in plant production [1]. Therefore, other alternative methods and technologies are still necessary and must be developed. One such method is the use of legumes and bacteria, collectively named rhizobia, which establish a symbiotic relationship and participate in the biological reduction of atmospheric dinitrogen.

The amount of nitrogen on Earth is estimated at about 1.6 × 10²³ Tg, of which about 98% is contained in the geosphere and is inaccessible to living organisms. The remaining 2% is present in the atmosphere (about 3 × 10²¹ Tg, mainly N₂), the biosphere (about 10¹⁷ Tg), and the soil (×10¹⁷ Tg: organic compounds undergoing mineralization, NO₃⁻, NO₂⁻, and NH₄⁺) [2]. Microbial denitrification processes lead to the reduction of NO₃⁻ to NO₂⁻ and then to N₂, which is lost to the atmosphere. In turn, biological and abiotic nitrogen reduction processes are responsible for increasing the amount of soil NH₄⁺ at the expense of atmospheric N₂ [3,4,5]. Currently, the biological reduction of atmospheric nitrogen is the main process balancing the denitrification-related loss of nitrogen from the soil to the atmosphere [6,7]. The outcome of total global biological nitrogen fixation is estimated at 100–300 Tg of reduced N per annum, and this value is higher than for overall synthetic N fertilizer production (80–90 Tg N per annum) [8,9,10].

In the process of biological nitrogen fixation, an enzymatic complex called nitrogenase catalyzes the reaction that transforms triple-bond inert atmospheric N₂ into an ammonia molecule. All known nitrogenases are multi-complex prokaryotic enzymes. It is possible that nitrogenase-coding genes have been spread between different species of microorganisms via horizontal gene transfer. Such microorganisms, collectively named diazotrophs, are representatives of both Archaea and Bacteria [11,12]. Diazotrophs can be photoautotrophs, chemolithotrophs, or heterotrophs. The latter are either free-living microorganisms or live on the surface of or inside plant tissues [13].

Due to their potential use in crop production, rhizobia that establish a symbiotic relationship with legumes are the most interesting group. There are estimations suggesting that symbiotically reduced N₂ may supply more than half of the total requirement of the plant [10,14,15,16]. The yield-promoting effect of symbiotic diazotrophs is not limited to one growing season, as about 30% of total plant N in pulse or pasture legumes can be rhizo-deposited and thereafter used by other crops in subsequent growing seasons [10].

2. Legume–Bacterial Symbiosis: Its Importance and the Mechanisms Involved

Rhizobia are bacteria belonging mainly to the alpha-proteobacteria (e.g., the genera Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (Ensifer)) [17,18,19], which are able to develop mutualistic relationships with legumes [20,21]. These symbiotic interactions are of great environmental importance, as it is estimated that rhizobia can establish symbiosis with about 20,000 legume species, including more than 100 agriculturally important legumes spanning all geographical regions [22].

The establishment of symbiosis between rhizobial microsymbionts and legume hosts is a multi-step process involving numerous signaling and regulatory steps. There are many bacterial and symbiotically involved signal molecules like lipochitooligosaccharides (LCOs, also called Nod factors, NFs) [23,24], capsular polysaccharides, cyclic beta-glucans, lipopolysaccharides [25], exopolysaccharides [26,27], and homoserine lactones [28,29]. Plants participate in the symbiosis process by producing flavonoids [23,30], jasmonates [31], aldonic acids [32], xanthones [33], and alkaloids [34]. Although each of these factors may be necessary for the proper development of the symbiotic system, the most important role should be assigned to flavonoids and Nod factors. Each legume species can produce and secrete numerous flavonoid compounds [35,36], which are inducers of the expression of bacterial nod genes into the rhizosphere. The transcriptional activity of these genes results in the synthesis of those proteins responsible for the production and secretion of NFs [20,23,24,30,37].

Rhizobial NFs are composed of an oligosaccharide core containing 3 to 6 N-acetylglucosamine residues linked by beta-1,4-glucosidic bonds, to which fatty acids and other substituents (e.g., acetate, sulfate, fucose, arabinose, etc.) can be attached. The diversity of the nod gene sets present in different species of rhizobia and the presence of different enzymes responsible for introducing substituents into the sugar backbones of Nod factors in a single bacterial strain make each bacterial strain able to produce and secrete a set of structurally different NFs. The profiles of LCO molecules synthesized by different rhizobial species (sometimes even subspecies, called symbiovars) differ, which affects host specificity [20,24,37,38,39,40,41]. There are many factors that may influence symbiotic compatibility (i.e., the ability of a specific rhizobium strain to establish symbiosis with a specific legume species), but interactions between plant flavonoids and rhizobial NodD protein, as well as interactions between rhizobial NFs and plant receptors, are of the highest importance [42,43].

The Nod factors released from bacterial cells are recognized by numerous specific receptors in plant cells [44,45], which leads to the activation of signal transmission pathways and, ultimately, to the activation of numerous transcription factors influencing the expression of plant genes [46,47,48,49]. This, in turn, has an impact on the level and distribution of phytohormones, such as auxins [50,51,52], gibberellins [53], or cytokinins [54,55,56]. Recent transcriptomic studies identified numerous legume genes with double (NF-dependent and phytohormone-dependent) regulation [57]. Moreover, it was demonstrated that not only root [53,55,58,59,60,61] but also foliar [62] transcriptomes can be massively affected by NF treatment. Groups of genes indicated as being NF-affected in their transcriptional activity are associated with very different processes, e.g., nutrient ion absorption, metabolism, cell cytoskeleton remodeling, or the action of plant defense systems [55,58,60,61]. These findings were supported by proteomic studies, which confirmed the large potential of rhizobial NFs to change the functioning of plant cells [63].

Simultaneously with the above-mentioned changes in the plant transcriptome and proteome, plant cells undergo a set of early physiological reactions like intra- and extracellular alkalinization, cell membrane depolymerization, changes in the level of Ca²⁺ ions, and reductions in the salicylic acid level, resulting in the suppression of host defense responses. Bacteria move toward the roots and colonize their surface, whereas the first morphological changes, such as deformation of root hairs and initiation of cortical cell division, can be observed in the plant root system [37,46,47,64,65]. These new meristematic cells that appear in the primary root cortex will give rise to meristems responsible for the growth and development of root nodules. In most cases, root hair curling is a prerequisite for plant tissue colonization. Then, rhizobia penetrate plant tissues using a special structure called an infection thread and participate in the development of symbiotic structures [37,46,47,66,67,68,69,70,71]. Less frequently, a mechanism called crack entry is observed; in such a case, microbes enter the roots through wounds, particularly where lateral or adventitious roots develop [72,73,74]. Finally, regardless of the way in which rhizobia penetrate the plant roots, mature and infected nodules develop. Inside these nodules, the process of N₂ reduction is performed by bacteria that have differentiated into bacteroids living inside plant cells, and plant-derived carbohydrates are exchanged for bacterial-derived nitrogen compounds [48,64,75,76]. Besides bacteroids, saprophytic (non-symbiotic) rhizobia and non-rhizobial strains are also present inside root nodules [77,78,79]. They all live together and multiply during nodule development and return to the soil in increased numbers after disintegration of the nodule.

3. Rhizobial Populations: Abundance, Diversity, and Competition Between Strains

Studies conducted around the world in places as diverse and distant from each other as Mexico [80], Scandinavia [81], China [82], or Australia [83] have shown that the rhizobial populations inhabiting soils can be very diverse within most of the studied localizations. Such studies have focused on different bacterial taxa, e.g., Rhizobium leguminosarum, which is a symbiont of pea, vetch, or clover [81,84,85,86,87,88,89,90,91], Sinorhizobium (a symbiont of Medicago) [80,91,92], Bradyrhizobium (a symbiont of lupine and serradella) [93,94], or Rhizobium tropici and Rhizobium etli (a symbiotic partner of faba bean) [82,84]. Regardless of the chosen model, in all these cases, high genetic diversity was found between strains belonging to these narrow taxa, which inhabit specific environments [80,81,84,85,87,92,93,94,95]. Other studies showed that the high metabolic and physiological diversity of these local populations is correlated with their high genetic diversity [86,88,89,90,91], which stems from the high variability and evolution of rhizobial genomes [96,97]. Most studies focused on populations inhabiting arable soils, but an analogous high biodiversity of rhizobial populations was also observed in grasslands [85] or soils contaminated with heavy metal ions [86]. It was suggested that the soil type is the primary determinant of the composition of bacterial communities [98], and some agricultural practices, such as liming the soil, could additionally increase rhizobial diversity [84]. Taking these findings together, it can be assumed that the legume hosts in most cases and localizations encounter highly diverse strains of compatible rhizobial microsymbionts, and numerous bacteria with diverse physiological traits compete with each other while colonizing a limited number of nodules developing on plant roots. As shown in some studies, this inter-strain competition occurs not only in the soil [99,100] but also in plant tissues. The presence of different rhizobial strains inside individual infection threads and root nodules [101,102,103,104] and the possibility of colonization of single plants by dozens of different rhizobial strains belonging to the same narrow taxon (e.g., Rhizobium leguminosarum sv. trifolii or sv. viciae) have been reported [89,105].

Research on the biodiversity of rhizobial populations led to another interesting conclusion on the effectiveness of rhizobia–legume symbiosis and the usefulness of inoculant strains. For a long time, it was assumed that soil or seed inoculation should be recommended when the roots of plants grown in a given area had no or only a few root nodules as a result of a very low number of microsymbionts colonizing the soil. However, when abundant root nodulation was observed (especially if the nodules appeared visually effective and contained leghemoglobin, which is synthesized in plant cells colonized by active bacteroids) [106], inoculation was not recommended because it was assumed that symbiotic nitrogen fixation provided the plants with substantial amounts of nitrogen. It is known that rhizobial strains vary in effectiveness, and numerous studies indicate that the strains that make up native populations are mostly of low effectiveness [83,95,107,108,109]. Sometimes, even a tenfold difference in symbiotic efficiency (expressed as a plant mass increase) was observed between the most and least efficient symbionts [109]. It is believed that high symbiotic effectiveness is not beneficial for rhizobia because the presence of multiple strains per plant and the mode of transmission (root-to-root, not seed-borne) will favor rhizobia that invest in their reproduction rather than symbiotic dinitrogen reduction [110,111]. Therefore, the abundant presence of rhizobia in the soil and abundant plant nodulation do not automatically mean that plant requirements for nitrogen are met. In such a case (i.e., the presence of numerous infected but low-effectiveness nodules), it would be necessary to inoculate plants with highly effective strains in order to improve their yield. However, in the case of the presence of numerous autochthonous strains, the success of highly effective inoculants that have been introduced into the environment may be very low; this is because the inoculant strain has to face strong competition and is not able to colonize a sufficiently large part of the nodules to produce noticeable effects [112,113,114].

If populations of indigenous rhizobia are numerous, the establishment of symbiotic relationships with the plant host is not solely determined by compatibility, i.e., the ability to correctly exchange molecular signals, which leads to the initiation of nodule formation and the infection of plant tissues. In such cases, competition (the ability of a given strain to occupy root nodules) also comes into play. Competition for nodule colonization occurs primarily between strains that are compatible with a given host and are present in its immediate vicinity, but also occurs with incompatible rhizobia and non-rhizobial microorganisms that live together in the same soil environment. Therefore, in such situations, an increase in plant yield could be achieved if the inoculant strain is (a) compatible, (b) competitive, and (c) highly effective. Screening the environment can reveal the presence of compatible rhizobial microsymbionts for every agriculturally important plant species. Problems arise if the strain is to be highly efficient and highly competitive at the same time because these traits are not correlated; moreover, each trait depends on numerous genetic determinants.

When selecting highly effective strains, simple (though labor-intensive) experiments examining the effect of inoculation on important parameters related to plant physiology, growth, and yield are sufficient.

Unfortunately, despite numerous studies being conducted to date, there are currently no clear conclusions regarding the main traits determining high competitiveness in bacterial strains. There is a fairly obvious relationship between strain competitiveness and bacterial motility [115] or—in some environments—acid pH tolerance [116]. Furthermore, high strain competitiveness may be a result of the ability to produce or resistance to bacteriocins and antibiotic peptides [99,100,117], or the ability to produce biotin and other water-soluble vitamins [118]. Moreover, susceptibility to plant molecular signals (mainly flavonoids) also plays a role in shaping the competitive abilities of rhizobia [119,120]. There are also numerous reports on the relationship between bacterial metabolic characteristics and strain competitiveness, which may be related to the ability to utilize individual substrates (e.g., rhamnose) [121], groups of similar substances (e.g., amino acids) [122], or a whole set of carbon and energy sources [88]. Conversely, some data demonstrate that a significant determinant of whether a given strain will colonize its plant host or not is the distribution of its cells in certain microniches in the soil [123]. This finding sounds likely, considering that legume roots are only susceptible to rhizobial invasion transiently and over a very limited surface area [124]. Moreover, several studies indicated that both the host and microsymbionts affect rhizobial competition, and plants could favor particular bacterial genotypes [125,126,127,128].

Therefore, if strain competitiveness depends on such diverse factors as the overall metabolic potential of bacteria, their responsiveness to plant signals, other specific rhizobial traits, and various environmental factors, then, while finding highly effective strains is feasible, finding highly competitive strains that can be used in more than one specific environment will likely be impossible. On the one hand, this does not seem a favorable situation; on the other hand, it promotes an impulse to search for new solutions, such as the use of preparations containing rhizobial Nod factors.

4. Traditional Rhizobial Inoculants and NF Preparations: Where, When, and How to Use Them to Increase the Yield of Legumes

The potential to exploit biological nitrogen fixation by rhizobia to enhance legume crop yields has long been known. The earliest rhizobial inoculants contained rhizobia isolated from soybean, a crop native to East Asia that was also introduced to other continents in the late 19th century. The first biopreparations containing live cells of these bacteria were produced commercially in the United States over a century ago, and their use has brought farmers significant benefits [129,130]. This is not surprising, considering that the first inoculants contained rhizobia that were symbiotic for soybean, which was originally grown in the eastern region of Asia and spread to other continents at the end of the 19th century [130]. When a new crop is introduced into an area where indigenous legumes are absent, its soils usually lack microsymbionts specific to this plant, and it often takes decades of regular use of biopreparations containing rhizobia for these microorganisms to become a permanent element of the soil environment. Examples of this include the introduction of cultivated legumes to Australia and the introduction of soybeans to Europe and the Americas [130,131,132]. Some rhizobia may also be locally absent due to severely detrimental soil conditions, especially soil pH. For example, most acidic soils are free from Sinorhizobium meliloti (a symbiont of Medicago), and basic soils are often free from Bradyrhizobium lupini (a symbiont of lupines). Other conditions like high temperatures, dryness, and salinity can also significantly reduce the number of or eliminate live rhizobial cells from the soil. Therefore, the use of traditional inoculants in these cases may be a good strategy to ensure a better supply of plants with nitrogen [131,133,134], as introduced strains do not have to compete with autochthonous rhizobia that are compatible with the same plant host.

The successful use of inoculants, resulting in a significant increase in crop yields, depends in part on proper preparation, storage, and application in the field [129,130,131,132]. However, there is often a gap between the conditions under which the research was conducted and the conditions under which such biopreparations are commercially applied or used by unskilled growers. Additionally, applying these biopreparations on a large scale may be difficult [129,135]. Enhancing farmers’ knowledge and skills and developing media/inoculum fluids containing substances that increase the survival of bacterial cells can improve the results in many cases, but the compatibility of inoculants with fungicides and insecticides is low, especially when they are used for seed treatment, and this topic still requires intensive research [136].

Another obstacle that may result in ineffective inoculant use, which is much more difficult to overcome, is the competition from indigenous soil-inhabiting rhizobia. This problem affects plants when they have been cultivated for thousands of years in their areas of likely origin and when related species occur nearby in the wild—for example, peas in Europe and Southwest Asia, soybean in East Asia, or beans in Mesoamerica. In such cases, the long presence of the plant host in the environment promotes the easy recruitment of compatible microsymbionts from the environment. Moreover, the regular multiplication of bacteria in host tissues in each growing season results in (a) a high number of compatible rhizobia, reaching up to 10⁵ cells/g of soil, and (b) bacterial abundance in most soils in a given area [84,134]. This is obviously not a rule that applies to all environments in a given area, but it can be expected with high probability that, for example, the use of traditional inoculants for peas, vetches, broad beans, or lentils in the Mediterranean basin will encounter significant difficulties due to competition from autochthonous rhizobia (Figure 1).

As previously mentioned, preparations containing live rhizobial cells have been used widely in the field for over a century because they are relatively cheap and easy to produce. This has increased the area of those soils colonized by rhizobia. As a result, even in areas formerly devoid of legume microsymbionts (such as Australia), diverse rhizobial populations can be found nowadays. These populations often include strains that are likely to have arisen as a result of horizontal gene transfer between non-symbiotic native saprophytic strains and symbiotic strains introduced as inoculants [138]. Thus, several decades ago, traditional inoculant technology yielded good results in such areas, but now this approach needs to change because “no competition” conditions have changed into “high competition” conditions. Sometimes losing this competition in the colonization of nodules does not have to mean that the inoculants are absolutely useless. This may be the case if such rhizobial inoculants should possess additional traits like the production of phytohormones, solubilization of precipitated phosphorus, production of siderophores, etc. [139]. These traits may have a positive effect on plant growth and yield, but failure to colonize the nodules deprives such inoculants of their most important feature, which is increasing the supply of available nitrogen for plants.

Legumes control many developmental processes in root nodules [69,140,141]. Furthermore, these plants have evolved varied mechanisms of control over microsymbionts [142], which can influence, among other things, whether low-performing nodules receive those substances required for their further development. However, this does not change the fact that the presence of diverse rhizobial populations in the soil causes individual plants to be infected by particularly numerous strains. In such conditions, none of these strains alone can significantly impact plant growth—they act as a combination, and their effectiveness is the result of the symbiotic efficiency of all these components [143]. Therefore, under high competition, it seems reasonable to abandon efforts to colonize the largest possible pool of developing nodules with those strains supplied as highly effective inoculants and to focus on increasing the number of effective root nodules instead. In such a situation, the differences in the symbiotic efficiency of the strains that colonize these nodules should be ignored, assuming that the plants will employ mechanisms that support the growth of nodules with higher symbiotic efficiency at the expense of nodules with lower symbiotic efficiency.

The simplest way to increase the number of root nodules is to interfere with the signaling system that functions at the initial stages of symbiosis. Increasing the quantity of flavonoids that induce the production of NFs and/or NFs that induce the formation of root nodule meristems can increase the number of root nodules, thus increasing the volume of plant tissue that is available for rhizobial infection. Legumes have mechanisms that limit the formation of excessive nodules or even inhibit nodulation under conditions of good nitrogen supply [69,140,141]. However, NFs secreted into the soil by rhizosphere rhizobia are often decomposed, excessively diluted in the soil solution, or degraded by soil microorganisms [144] or plant host enzymes [145], which results in reduced root nodulation. There are two potential ways to increase the level of rhizobial NFs in the rhizosphere: indirectly, i.e., by using flavonoid compounds that increase the expression of nod genes in rhizobial cells, resulting in increased synthesis and the secretion of LCOs into the rhizosphere, or directly, i.e., by isolating NFs from bacterial cultures and using them as a preparation stimulating the nodulation of plant roots. The application of specific flavonoids like quercitin, luteolin, hespertin, or naringenin (used for the activation of inoculant strains or as seed-coating solutions) increased the nodulation of bean and alfalfa plants [146,147] or the nodulation and plant dry mass of pea and lentil plants [148] under laboratory conditions. Pretreatment of Bradyrhizobium japonicum with genistein increased the nodulation, total protein yield, and grain yield of soybean under laboratory and field conditions [149,150]. Seed exudates, which are mixtures of flavonoids, can also be used for the enhancement of plant–rhizobia symbiosis [23,30] and might even be economically more justifiable due to their lower production cost. The beneficial effect of such exudates, resulting in increased nodulation and the production of a greater mass of vegetative organs, was reported for clover and pea specimens grown in greenhouse experiments [120,151]. Nowadays, flavonoids are used commercially to promote Bradyrhizobium–soybean symbiosis by increasing nodulation and N₂ fixation in agricultural practice [119,152].

The first experiments testing the effect of preparations containing rhizobial LCOs were conducted in the laboratory with the use of sterile or nonsterile media under strictly controlled plant cultivation conditions (e.g., growth in phytotrons) and brought promising results. The treatment of seeds with NFs improved germination and increased root growth in the seedlings of the legumes (clover, vetch, pea, bean, soybean, and black gram) [153,154,155,156,157]. In the later stages of plant development, LCO application contributed to enhanced lateral root formation (in alfalfa) [158], along with significantly better nodulation (in alfalfa, clover, vetch, and pea) [155,156,159], photosynthetic activity and/or chlorophyll content (in pea and soybean) [160,161], and plant nitrogen content (in pea) [161]. All these changes resulted in significantly better growth of clover, vetch, pea, and soybean [155,156,160] and an increased yield from pea plants [161].

Similar results were obtained in experiments conducted in greenhouses or vegetation halls with uncontrolled temperature and humidity. In the case of pea plants, both physiological (nitrogenase activity and photosynthetic efficiency) [162,163] and morphological (number of root nodules) parameters [56,164] were increased after the application of preparations containing NFs, which finally resulted in improved plant growth and yield [162,163,164,165].

The effects of preparations with NFs were also tested under field conditions. It was reported that LCO treatment enhanced nitrogenase activity and plant nitrogen content [166], as well as the nodulation, growth, and yield of pea [167] and soybean [168,169]. Unfortunately, the beneficial impact of LCOs was not observed in all cases. For example, the application of NFs increased soybean yield under conventional tillage but not under no-tillage practices [168]. It was also reported that severe drought during the reproductive period [168] or a long wet period during the early stages of plant growth [167] resulted in a lack of positive effects after the application of the NFs, which should not be surprising. It is known that severe drought can reduce crop yields, regardless of all the positive yield-forming factors. However, the application of LCOs can increase yield at a medium water-stress level, which is most commonly observed under field conditions [168]. In turn, temporary water excess reduces the ability of plants to form nodules [170] and can reduce the concentration of NFs in the soil solution in the immediate vicinity of developing plants. However, numerous experiments have shown that such biostimulants as LCOs might be a good tool for increasing yields in non-extreme conditions. There are also reports about the beneficial effect of foliar application of LCOs on plant growth [160,161], which makes these preparations more versatile and convenient to use.

Some products containing rhizobial NFs are available on the market. Many of them are simple preparations intended for seed dressing and contain only LCOs [171,172,173,174]. Their manufacturers indicate that it is also possible to use some of these formulations in post-emergence applications [173] or propose products that work with the preparations to increase the stability of NFs used for seed dressing over 200 days [174], which increases flexibility and comfort of use. More complex products containing rhizobial NFs, fungi (Penicillium bilaiae and Trichoderma virens), and bacteria (Rhizobium leguminosarum and Bacillus amyloliquefaciens) [175] can also be found. According to the manufacturer’s recommendations, such multifunctional products are intended for peas and lentils and could increase root nodulation and support the supply of nitrogen, phosphorus, and potassium to plants.

The above examples show that NFs can be successfully used in various conditions (Figure 2). This technology seems to be more versatile than traditional inoculants, ensuring good results in soils that do not contain legume-compatible microsymbionts.

In the absence of indigenous microsymbionts or inoculation with highly effective rhizobia, the effect of NFs on plant growth cannot be significant, as those structures associated with biological nitrogen reduction are not formed. However, under such conditions, by stimulating lateral root meristems, LCOs can positively influence root system development and, thus, indirectly improve water and micronutrient uptake by plants. Significant beneficial effects of NF application can be observed in any case of contact between legumes and compatible microsymbionts: with inoculants alone, with indigenous bacteria alone, and in the case of simultaneous contact with both indigenous bacteria and a selected inoculant strain. In each of these cases, the use of NFs increases the number of nodules, and the presence of microsymbionts attempting to colonize these structures is a signal allowing plants to continue their development and growth.

Studies on the effect of NF preparations on legumes have yielded several interesting observations that may be useful in the further development of this technology. Phytohormones play a central role in the abiotic stress response [176], and rhizobial LCOs may influence the action of phytohormones or act together with them on the expression of plant genes. Therefore, attempts have been made to use NFs as stress relievers, and these attempts were successful in several cases. It was demonstrated that treatment with NFs can alleviate water stress in soybean [56,177] and mitigate the adverse effect of soil compaction on the nitrogenase activity, nitrogen content, plant mass, and yield of pea plants [162]. It was also reported that NF treatment can overcome the adverse impact of low temperature and low pH, but not salt stress, on legume root hairs [178]. Moreover, increased resistance of soybean to powdery mildew was reported after LCOs application [179]. There are studies reporting the suppression of innate responses after LCO treatment in legumes [180] and the NF-dependent activation of plant defense-related genes [181]; therefore, the picture is still unclear. Taking into account the complexity of the interactions between rhizobia–legume symbiosis and plant defense processes [182], a full description of the effect of NF treatment on the modification of plant defense processes will require more research.

5. Future Prospects—Extending NF Technology to Non-Legume Plants

The best results of the use of NF preparations are achieved when they can influence processes involving rhizobia and those related to biological dinitrogen reduction. However, laboratory studies conducted with NFs alone (without microorganisms) have shown that they can support such processes as root hair curling, the reinitiation of cell division, and the stimulation of lateral root formation in different legume species [145,158,183,184].

Moreover, it was reported that NF treatment resulted in better colonization of the legume plant Lablab purpurea by the mycorrhizal fungus Glomus mussae [185]. This last observation, together with the information that LCOs with structural similarity to rhizobial NFs are produced by arbuscular mycorrhizal fungi [186] and ectomycorrhizal fungi [187], opens up completely new possibilities for preparations containing such biostimulants. New data suggest that the production of LCOs is common among fungi, and these LCOs (called Myc-LCOs) may function as signals regulating fungal growth and development [188]. Rhizobial LCOs and Myc-LCOs not only share structural similarities; they can also activate a signaling pathway called the common symbiosis pathway, which is essential for root nodulation as well as root colonization by arbuscular fungi [189,190,191]. As a result, together, they can control the same plant developmental processes, e.g., root branching [192].

Some studies indicate that legumes and non-legumes may, in some cases, respond to rhizobial NF factors in a similar manner. For example, it was reported that NFs could have developmental effects on non-leguminous plants, such as carrot, Norway spruce, or Arabidopsis [193,194,195]. In Arabidopsis, rhizobial NFs were able to induce numerous morphogenetic changes, such as an increase in the number of root tips and an increase in the root and leaf surface area [195]. After LCO treatment, similar Ca²⁺ fluxes and the inhibition of reactive oxygen species production were observed in Arabidopsis, tomato, corn, and soybean [196]. Proteomic analyses revealed that NFs-treated Arabidopsis grown under salt stress [197], as well as soybean [63], increased the production of a similar set of enzymes. Studies of maize transcriptome changes after LCO treatment revealed the induction of genes involved in root growth promotion and the repression of stress-related genes [198]. Large changes in plant hormone levels after NF treatment were also reported in Arabidopsis [199] and Brachypodium, where the stimulation of lateral root formation was also recorded [200].

This information is in agreement with reports about improved germination and increased root growth in seedlings of corn, rice, cotton [154], and barley [201] after NF treatment. The application of rhizobial LCOs enhanced the photosynthesis and growth of corn [160], growth of canola roots [202], flowering of tomato plants [203], or flavonoid production in buckwheat [204]. It was also reported that NF treatment mitigated salt stress in maize [205]. There are products on the market containing rhizobial NFs that are recommended for use in both soybean and corn cultivation [172], but there are still not many such formulations. It is quite possible that this offer will be expanded in the future. Patent databases contain information about preparations with rhizobial NFs that increase the fresh and dry weight of corn [206,207], the yield of tomatoes, or the number of buds in bedding plants such as impatiens or marigolds [206]. At the same time, work is underway on technical improvements; for example, numerous solutions have been developed to increase the solubility of NFs in aqueous solutions using anionic and non-ionic detergents [208]. A genetically modified strain of Cupriavidus necator (formerly Ralstonia eutropha) that is capable of synthesizing rhizobial NFs has also been produced [209]. This is an interesting solution because this engineered microorganism can produce various organic compounds using CO₂ as a sole carbon source and H₂ as a sole energy source [210]. Therefore, its use in the industrial production of NFs seems to be more economically justified and environmentally friendly than the use of saprophytic rhizobia.

Taking these developments together, it can be speculated that the use of NFs (rhizobial and/or Myc-LCOs) may increase as part of sustainable agriculture practices. It seems that NFs potentially have a broader range of applications than traditional inoculants containing live rhizobial cells, as they can affect a much broader set of plants, including monocotyledons. It is possible that new products containing such metabolites will soon appear on the market and will be used in many different environmental conditions, which will be the best test of the usefulness of such compounds in modern agriculture.

6. Final Remarks

Humans have long been cultivating legumes, and new cultivation methods have been employed to increase their yield over the centuries. One significant innovation was the discovery of symbiotic interactions between these plants and rhizobial bacteria, as this allowed the development of biopreparations containing live bacterial cells to replace synthetic nitrogen fertilization. Their use increased plant yields while reducing the cost of cultivation.

As the knowledge of microbiomes inhabiting soils and the complexity of the functioning of rhizobia–legume symbiotic systems increased, it became clear that the use of “simple” inoculants (live rhizobial cells with a carrier) is often insufficient and will not bring the expected beneficial yield effects because such inoculants are unable to penetrate the barrier created by autochthonous, less effective, but highly competitive rhizobia. In such a situation, the use of preparations containing rhizobial LCOs seems to be a solution that effectively circumvents the problem. Since it is difficult to replace less effective indigenous strains with highly effective strains used for inoculation, the use of NFs can simply increase the number of nodules, i.e., they increase the volume and mass of the plant tissue in which rhizobia conduct symbiotic dinitrogen reduction. In such a case, the effectiveness of the strains becomes less important, and quantity can be transformed into quality. As shown by the experimental results, this leads to increased yields using only indigenous soil populations.

Additional arguments in favor of using such biostimulants as rhizobial NFs are as follows: (a) they favorably influence not only the number of nodules but also other morphological and physiological characteristics of plants, (b) they can also be used in non-legume crops, (c) they can be used as a sole biopreparation or together with traditional rhizobial inoculants, and (d) they can be successfully used not only for seed dressing but also as foliar sprays.