Mycorrhizosphere Interactions to Improve a Sustainable Production of Legumes
The sustainability and productivity of agroecosystems depends exquisitely on the functionality of a framework of plant–soil interactions where microbial populations, including both mutualistic symbionts and saprophytic microorganisms, living at the root–soil interfaces, the rhizosphere, are involved. Among various beneficial and consumable plant species, legumes form useful symbiotic relationships with two types of soil microbiota: N2-fixing bacteria, often called rhizobia, and arbuscular mycorrhizal (AM) fungi. Also, the legume rhizosphere inhabits other valuable microbes such as plant growth-promoting rhizobacteria (PGPR). These microorganisms interact intensely among themselves, and with legume roots, to develop the multifunctional legume mycorrhizosphere, a microcosm environment of variable activities, appropriate for legume productivity. This chapter highlights (1) the types of microorganisms and processes involved in the establishment and functioning of the mycorrhizosphere, (2) the impact of the mycorrhizosphere activities on legume production, and (3) the possibilities to tailor an efficient mycorrhizosphere as a biotechnological tool to improve legume performance in different production systems following efficient rhizobial, PGPR, and AM fungal inoculants.
KeywordsLegume productivity Rhizosphere microorganisms Mycorrhizosphere services Microbial inoculants Mycorrhizosphere tailoring
The great challenge that science and society are currently facing is how to satisfy the increasing demands of healthy foods for the constantly growing human populations. Considering these challenges, there is urgent need to increase agricultural/food production globally. In this regard, intensive agriculture appears, theoretically, fundamental to respond to this challenge. However, high-input agricultural practices are concomitant with the mass consumption of nonrenewable natural resources particularly rock phosphate (RP) reserves (George et al. 2016). Also, the excessive use of agrochemicals in modern farming practices causes stress to the plants and may cause climatic change (Barea 2015). Due to these problems, society and science both are becoming aware on the necessity to follow sustainable agricultural production models which could provide a healthy and nutritious food supply without compromising on yields (Altieri 2004). Similarly, a sustainable management (restoration practices) of natural soil–plant ecosystems is peremptory to preserve the biodiversity and environmental quality (Barea et al. 2011). Concerning quality food production, diverse research approaches have been proposed and practiced to achieve environmental and economical sustainability. One of these approaches, exploitation of soil microbial communities (Barea 2015), has been considered as safe, inexpensive, and environmentally friendly. Indeed, diverse genetic and functional groups of soil microorganisms are known to play decisive roles (microbial services) in agriculture, mainly by propelling nutrient cycling, enhancing plant nutrition, and promoting plant health and soil quality (Lugtenberg 2015).
Microbial activities are particularly relevant at the root–soil interface microhabitats known as the rhizosphere, where microorganisms are stimulated by carbon substrates provided by plant rhizodeposits (Hirsch et al. 2013b). Formation, development, significance, functioning, and managing of the rhizosphere have been reviewed (Barea et al. 2013a). Currently, much attention is given to optimize the functions of root-associated microbiome in enhancing plant nutrient capture and for increasing plant resistance/tolerance to either biotic or abiotic stress factors. Accordingly, several strategies for identifying and utilizing beneficial microbial services have been proposed to promote a sustainable and environmentally friendly agricultural production (Raaijmakers and Lugtenberg 2013; Barea 2015). It is noteworthy to point out that the use of molecular techniques has evidenced that only 1% of microorganisms living in the bulk soil, and 10% of those from the rhizosphere microbiome, are able to grow in standard in vitro culture media and can therefore be isolated and multiplied (Hirsch et al. 2013a). The rest of soil microorganisms are considered as unculturable but can be detected and analyzed for their effectiveness using culture-independent molecular approaches (Barret et al. 2013; Schreiter et al. 2015). Most studies on the plant-associated microbiome focus on bacteria and fungi and both of them can establish either saprophytic or symbiotic relationships with the plant which could either be detrimental or beneficial (Spence and Bais 2013; Lugtenberg et al. 2013a, b). Beneficial plant mutualists are both the N2-fixing bacteria (Olivares et al. 2013) and the multifunctional arbuscular mycorrhizal (AM) fungi (van der Heijden et al. 2015).
The AM fungi play fundamental roles in agro- and ecosystems by driving nutrient and carbon cycles. Essentially, they capture P from soil solution and supply this nutrient to plants. In mycorrhizal symbiosis, the host plant receives mineral nutrients via the fungal mycelium (mycotrophism), while the heterotrophic fungus obtains carbon compounds from the host’s photosynthates. It is universally accepted that mycorrhizal associations, which can be found in almost all agro- and ecosystems worldwide, are fundamental to improve plant fitness and soil quality through key ecological processes (Smith and Read 2008; van der Heijden et al. 2015). The mycorrhizal fungi colonize the root cortex and develop an extraradical mycelium which overgrows the soil surrounding plant roots. This hyphal net is a structure specialized for the acquisition of mineral nutrients from the soil, particularly those whose ionic forms have poor mobility or are present in low concentration in the soil solution, as it is the case with P and N (ammonia). AM colonization changes the chemical composition of root exudates which stimulate to grow rhizosphere microorganisms to generate the so-called mycorrhizosphere, a functional structure which help to improve plant productivity (Azcón-Aguilar and Barea 2015; Barea et al. 2013a).
Microbial interactions are of paramount importance in the rhizosphere of legumes. Actually, legumes establish beneficial symbiotic relationships with both N2-fixing bacteria and AM fungi (Azcón and Barea 2010). The widespread presence of the AM symbiosis in nodulated legumes and the impact of AM fungi in improving nodulation and N2 fixation are well established (Barea and Azcón-Aguilar 1983). Nodulated and mycorrhizal legumes are fundamental in sustainable agriculture and in natural ecosystems because these symbioses supply major nutrients (N and P) to plants. In addition, legumes live in association with other saprophytic microorganisms which interact with their symbionts in the rhizosphere; the result of that interaction is important for sustainable legume production (Azcón and Barea 2010; Shtark et al. 2011).
The literature related to the influence of the mycorrhizosphere on legume improvement was critically reviewed and presented in the first edition of this book (Azcón and Barea 2010). The present chapter, however, provides recent information on this aspect focusing mainly on the new concepts and approaches, including basic, strategic, and applied insights on this thematic area of mycorrhizosphere formation, functioning, and agro-technological application to benefit legume performance. Here attempts have been made to focus on (1) the types of microorganisms and processes involved in the establishment and functioning of the mycorrhizosphere, (2) the impact of the mycorrhizosphere activities on legume production, and (3) the possibilities to tailor an efficient mycorrhizosphere to be used as a biotechnological tool to improve legumes different agro-production systems.
9.2 Establishment and Functioning of the Mycorrhizosphere
As stated before, both functionally and genetically diverse microbial communities live in close proximity with plants where they interact intensely and perform many activities relevant to the productivity of the soil–plant systems (Barea et al. 2013a). Even though majority of these microbes remain in the rhizospheric soil or rhizoplane, a small subpopulation of such organisms, designated as “endophytes,” is able to penetrate and live within plant tissues (Mercado-Blanco 2015). Some endophytes affect plant growth by influencing pathogens, herbivores, and environmental changes or by producing and making important secondary metabolites available to plants. In this chapter, only rhizosphere microorganisms will be considered. Strictly speaking, other microbial groups, for example, mycorrhizal fungi, rhizobia, and some pathogens, are actually endophytes that colonize plant tissues, but they are considered separately from the core group of “endophytes,” because they are involved in either nutrient transfer from sources outside the root, i.e., soil or atmosphere, or cause disease symptoms in their host plant (Barea 2015). The information reported here mainly focuses on to culturable bacteria and fungi, involved directly or indirectly in enhancing plant nutrition and health. A key microbial group discussed in this chapter is the AM fungi obligate symbionts, which are unculturable in axenic conditions but, in some way, are “culturable” since they can be multiplied when cultivated together with a host plant in the growing substrate.
9.2.1 Beneficial Rhizosphere Bacteria and Fungi in Agroecosystem and Natural Ecosystems
The prokaryotic bacteria and the eukaryotic microscopic fungi have a great variety of trophic/living habits whose saprophytic or symbiotic relationship with the plant could be either detrimental (pathogens) or beneficial (mutualists). The beneficial saprophyte microbes can act as (1) decomposer of organic substances (detritus), (2) plant growth-promoting microorganisms, or (3) antagonists of plant pathogens.
220.127.116.11 Saprophytic Rhizosphere Bacteria and Fungi
Diverse PGPR have been identified as biocontrol agents and used to reduce losses to crops caused by plant pathogens. Of the various PGPR involved in disease management, Pseudomonas spp. have been considered as one of the major groups (Ramos-Solano et al. 2008; Mendes et al. 2011; Lugtenberg et al. 2013a). Three general mechanisms adopted by PGPR for the control of soilborne diseases are (1) the reduction in the saprophytic growth of the pathogens which occurs mainly via antagonistic activities, as mediated by the production of antibiotics, (2) the reduction of the virulence of the pathogen, and (3) the induction of systemic resistance in the host plants (Pieterse et al. 2014). Trichoderma spp. are rhizosphere fungi which promote plant growth and act as pathogen antagonists following the above indicated mechanisms and, additionally, exert as mycoparasitic agent (Hermosa et al. 2012).
Another fundamental activity of PGPR is nutrient cycling, particularly nitrogen fixation (Azcón-Aguilar and Barea 2015) and phosphate mobilization (Khan et al. 2010; Barea and Richardson 2015). The N2 fixation process is the first step in cycling N from the atmosphere to the biosphere, a key N input to plant productivity (Arrese-Igor 2010). Many free-living diazotrophic bacteria are recognized to be able to fix N2 that these microbes use as a source of N for themselves, with a low direct N transfer to the plant, having therefore a limited agronomic significance (Ramos-Solano et al. 2009; Olivares et al. 2013).
Diverse rhizobacteria and rhizofungi have the capacity to mobilize P from poorly available sources of this element and supply soluble P to plants (Zaidi et al. 2010; Barea and Richardson 2015). The mechanisms whereby P-mobilizing microorganisms release available P from sparingly soluble soil P forms, either inorganic (solubilization) or organic (mineralization), by means of activities largely based on producing specific enzymes and/or chelating organic acids, have recently been discussed (Barea and Richardson 2015). The effect of phosphate-mobilizing microorganisms, mostly PGPR, has been tested under field conditions, but their effectiveness in the soil–plant system is variable (Antoun 2012). One of the reasons for a lack in realizing benefit to the plant is that the P ions made available could be refixed by the soil constituents before they reach the root surface. However, if phosphate ions, as released by the PMB, are taken up by a mycorrhizal mycelium, this would result in a synergistic microbial interaction which improves P acquisition by the plant.
18.104.22.168 N2-Fixing Symbiotic Bacteria
Associative and symbiotic N2-fixing bacteria are fundamental in plant N nutrition (Olivares et al. 2013). The N2-fixing symbiotic bacteria belonging to different genera, collectively termed as “rhizobia,” are able to fix N2 in symbiosis with legume plants (Olivares et al. 2013; de Bruijn 2015). How these bacteria interact with legume roots to form N2-fixing nodules and the molecular aspect determinants of host specificity in the rhizobia–legume symbiosis are described elsewhere in this book. Other bacteria, from the genus Frankia (actinomycetes) are known to form N2-fixing nodules on the roots of the so-called “actinorrhizal” plant species. The associative bacteria, like Azospirillum, colonize root surfaces and establish diazotrophic rhizocenosis with the plant and can even invade intercellular tissues; however N2-fixing structures are not formed (Gutiérrez-Mañero and Ramos-Solano 2010; Bashan et al. 2011; Olivares et al. 2013). Azospirillum enhance N supply to the plant but act mainly by increasing the production of auxin-type phytohormones, which affect the rooting patterns thereby benefiting plant nutrient uptake from soil rather than as N2-fixing bacteria (Dobbelaere et al. 2001).
22.214.171.124 Arbuscular Mycorrhizal (AM) Fungi
Some 50,000 fungal species are recognized to form mycorrhizal symbiosis with about 250,000 plant species and thereby to carry out fundamental roles in terrestrial ecosystems, particularly by propelling nutrient and carbon cycles and other agroecosystem services. Actually, mycorrhizal fungi provide up to 80% of plant N and P (van der Heijden et al. 2015). Several types of mycorrhiza are recognized, but the most widespread and agronomically important type is constituted by the arbuscular mycorrhizal (AM) associations, which colonize approximately 80% of terrestrial plant species growing in almost all terrestrial agroecosystems worldwide (Brundrett 2009).
AM fungi are ubiquitous soilborne microscopic fungi whose SSU rDNA phylogeny revealed that they have a monophyletic origin constituting the phylum Glomeromycota (Schüßler et al. 2001). There are both fossil and phylogenetic evidences that the terrestrial AM fungi are about 460 million years old, suggesting that they existed before the land flora, consisting on bryophyte-like plants, at 450 million years ago (Honrubia 2009; Schüßler and Walker 2011; Barea and Azcón-Aguilar 2013; Selosse et al. 2015). However, molecular clock analyses further revealed its origin of land plant to around 477 million years and that the origin of AM fungi took place 50–200 million years earlier than the land plants (Schüßler and Walker 2011; Shtark et al. 2012; Barea and Azcón-Aguilar 2013). Morphological (fossil records) and phylogenetic (molecular) studies support that AM fungi facilitated plant terrestrialization and that roots coevolved in association with AM fungi in such a way that the majority of the extant vascular plants live associated with AM fungi (Field et al. 2015).
The AM fungal community diversity studies were based initially on the morphological characterization of their large multinucleate spores, until the molecular tools became available (see Robinson-Boyer et al. 2009 for references of pioneering studies). Essentially, molecular-based identification processes include the PCR-amplified rDNA fragments of the spores and/or the mycelia from AM fungi followed by cloning, fingerprinting, and sequencing (Krüger et al. 2011; Brearley et al. 2016). New molecular approaches allow the quantitatively analyses of the effect of environment, geographical location, or management on the AM fungal communities. The Q-PCR technique can be used for simultaneous specific and quantitative investigations of particular taxa of AM fungi in roots and soils colonized by several taxa (König et al. 2010; Redecker et al. 2013). In addition, new techniques of high-throughput sequencing are available which have improved our understanding on the biology, evolution, and diversity of AM fungi (Lumini et al. 2010; Drumbell 2013; Öpik et al. 2013). The morphological characterization of AM fungi is also currently used, as complementary to the molecular methods (Oehl et al. 2011a, b; Redecker et al. 2013).
An important aspect in AM fungal diversity studies is to consider that AM fungi use three types of propagules for root colonization: spores, fragments of AM roots, and internal mycelium (IRM) and the external AM mycelium (ERM) developing in the root-associated soil (Smith and Read 2008). Most of AM fungal diversity surveys commonly focused on a single propagule compartment, traditionally the spore community in soil. However, with the spread of sequence-based identification methods, many studies have now addressed to the AM fungi colonizing roots, the IRM, or mycorrhizospheric soil samples that include both ERM and spores. The related information on this aspect was reviewed recently by Varela-Cervero et al. (2015, 2016), where the AM fungal diversity was analyzed in the three different propagule types. These studies suggest that AM fungal taxa are differentially allocated among soil mycelium, soil spores, and colonized root propagules in a natural environment. Obviously, these results are relevant for exploiting AM fungal diversity and designing vegetation restoration programs employing AM inoculation. The analysis of the genome of the AM fungi has been addressed in several studies based on functional molecular approaches (Gianinazzi-Pearson et al. 2012). The complete genome of the model AM fungus Rhizophagus irregularis (formerly Glomus intraradices) has been sequenced (Tisserant et al. 2013; Lin et al. 2014).
The most significant biological characteristic of AM fungi is their ability to establish association with members of almost all phyla of land plants, regardless of their taxonomic position, life form, or geographical distribution (Smith and Read 2008; Brundrett 2009). In this context, it is relevant to note that AM fungi exhibit little host specificity, while a single plant root can be colonized by many different AM fungi. However, a certain degree of host preference (functional compatibility) has been demonstrated, a fact having ecological and agronomical consequences. From the ecological point of view, a diverse AM fungal population is needed for maintaining the diversity, stability, and productivity of natural ecosystems, while an appropriate selection of AM fungal inocula is fundamental for the effectiveness of AM symbioses in plant production in agricultural systems (Barea and Azcón-Aguilar 2013).
9.2.2 The AM Symbiosis: Characteristic, Establishment, and Functions
This review will focus only on AM symbiosis and fungi, but a brief reference to the other mycorrhizal types is given. There are two main types of mycorrhiza: ecto- and endomycorrhiza (Smith and Read 2008; van der Heijden et al. 2015). In ectomycorrhizas, the fungus forms a mantle of hyphae around the feeder roots. The mycelium penetrates the root and grows between the cortical cells forming the so-called Hartig net where nutrient exchange between partners takes place. About 2% of higher plant species, mainly forest trees in the Fagaceae, Betulaceae, Pinaceae, and some woody legumes form ectomycorrhiza. The fungi involved belong mostly to class Basidiomycetes and Ascomycetes (mushrooms and truffles). In endomycorrhizas, the fungi colonize the root cortex both intercellularly and intracellularly and develop an extraradical mycelium growing in soil, a network of hyphae without extructuring a mantle around the root. There are three types of endomycorrhizal fungi: “ericoid,” “orchid,” and “arbuscular.” Of these, arbuscular fungi are the most common type and widely distributed throughout the plant kingdom. The widespread and ubiquitous AM symbiosis is characterized by the treelike symbiotic structures, termed “arbuscules” that the fungus develops within the root cortical cells and where most of the nutrient exchange between the fungus and the plant occurs. An intermediate mycorrhizal type, the ectendomycorrhiza, is formed by plants in families in the Ericales and in the Monotropaceae and Cistaceae. In these mycorrhizal associations, the fungi form both a hyphal mantle and intracellular penetrations (Smith and Read 2008). The AM symbiosis is that established by plant of agronomic interest and by herbaceous, arbustive, and some trees in natural ecosystems (Barea and Azcón-Aguilar 2013; van der Heijden et al. 2015). The AM associations are known to benefit plant fitness and soil quality, mainly by improving nutrient acquisition and plant health (Barea 1991). As the AM symbiosis is the mycorrhizal type formed by legume plants (all but Lupinus spp.), this review will focus only on the AM symbiosis and their role at improving these species, the target of this chapter, both in agricultural and natural systems.
The cellular and developmental programs controlling the processes of AM formation, from propagule activation until the intracellular accommodation of the fungal symbiont, have recently been reviewed (Gutjahr and Parniske 2013; Bonfante and Desirò 2015). This molecular cross talk prior to physical contact is the recognition by the fungus of plant signaling molecules, the strigolactones, which stimulate the fungus to ramify (López-Ráez et al. 2011). On the other side, plants perceive diffusible fungal signals, called “Myc factors” (lipochitooligosaccharides), analogous to the nodulation (NOD) factor of nitrogen-fixing rhizobia, which induce symbiosis-specific responses in the host root (Genre and Bonfante 2010; Maillet et al. 2011; Bonfante and Desirò 2015). After contacting the root epidermal cells, the fungal hyphae form an appressorium from which the fungus penetrates the epidermal cells to develop an intraradical mycelium until the formation of the tree-shaped structures, the arbuscules. Each fungal branch within a plant cell is surrounded by a plant-derived periarbuscular membrane and an apoplastic interface between the plant and fungal plasma membranes. The resulting structure is fundamental for the exchange of symbiotic signals and nutrient between symbionts (Gutjahr and Parniske 2013; Bonfante and Desirò 2015).
9.2.3 Mycorrhizosphere Establishment
9.3 Tripartite Symbiosis in Legumes: Establishment and Functioning
The establishment of a functional and effective mycorrhizosphere is a key issue for legume productivity improvement (Azcón and Barea 2010; Muleta 2010). Here, the interaction between rhizobial bacteria and AM fungi and their interaction with the roots of their common legume host is discussed considering (1) a shared signaling pathway for AM fungi and root nodule symbiosis establishment, (2) physiological interactions related to the formation and functioning of the tripartite symbiosis, and (3) the use of 15N to ascertain the role of AM fungi in N2 fixation.
9.3.1 Establishment of Arbuscular Mycorrhiza and Root Nodule Symbiosis: A Shared Signaling Pathway
Root colonization by AM fungi depends on a number of genes termed symbiosis (SYM) genes (Gutjahr and Parniske 2013; Bonfante and Desirò 2015). Such a plant–microbe symbiotic toolkit evolved with the AM symbiosis from basal land plants to extant flora (Delaux et al. 2013). Even more, ancestors of land plants, the green algae in the Charophytes, appeared preadapted for symbiosis as they possessed SYM genes, a symbiotic toolkit which was later recruited and further developed alongside AM fungi and plant coevolution (Delaux et al. 2015). In addition, during plant evolution, the SYM genes were again recruited for other plant root symbioses, like the N2-fixing rhizobial root nodules; thus a similar plant gene toolkit can modulate both types of legume symbioses. Actually, the legume–rhizobia symbiosis evolved much later than the AM symbiosis from a set of preadaptations during coevolution with AM fungi; thus the legume root symbioses may be considered as a component of an “evolutionary plant–microbial continuum” (Shtark et al. 2012). The genes required for AM establishment were identified first in legumes but were then found in many AM host plants (Parniske 2008; Bonfante and Genre 2010). The SYM genes, common to both symbioses, are known to encode proteins involved in a signaling transduction pathway starting with the perception of microbial signals at the plant plasma membrane, by means of a receptor kinase, and ending with the intracellular accommodation of both symbionts, AM fungi and rhizobial bacteria, into the host cell (Genre et al. 2013; Lagunas et al. 2015; Bonfante and Desirò 2015; Sun et al. 2015).
9.3.2 Formation and Functioning of the Tripartite Symbioses: Physiological Interactions
The AM associations are recognized as an adaptive strategy for P acquisition in soils with low P availability which is an important nutrient for legumes, required growth, nodulation, and N2 fixation (Olivares et al. 2013; Azcón-Aguilar and Barea 2015). Since the mycorrhizosphere interactions in legumes are important for N and P cycling, the tripartite symbiosis becomes special in sustainable agriculture in order to improve the productivity of both, woody and herbaceous, legumes (Courty et al. 2015).
Numerous experiments have been conducted to study the physiological and biochemical basis of AM fungal x rhizobia interactions. The information, reviewed by Azcón and Barea (2010), reinforce the idea that the main cause of such interactions is the supply of P by the AM fungi to satisfy the high P demand for nodule formation and N2 fixation, leading to an increased fixation rates.
A topic of research interest is whether AM fungi and rhizobia compete for photosynthates. Since legumes use up to 4–16% of photosynthesis products to satisfy the demands of their heterotrophic rhizobial and AM fungal symbionts, a reduction in productivity, therefore, results under photosynthate limitation. The main conclusions of former studies, as reviewed by Ha and Gray (2008), are that when host photosynthesis is limited, AM fungi usually show a competitive advantage for carbohydrates over the rhizobia, but under normal situations, the photosynthetic capacity of plants exceeds the carbon demand of the tripartite symbiosis. Pioneering results show that AM plants have developed a mechanism for enhancing photosynthesis to compensate for the C cost of the symbioses, as further corroborated (Mortimer et al. 2008). Accordingly, a comprehensive meta-analysis of potential photosynthate limitation of the symbiotic responses of legumes to rhizobia and AM fungi was carried out (Kaschuk et al. 2010). These authors analyzed 348 data points from published studies with 12 legume species using response variables plant yield, harvest index, and seed protein and certain lipid production. They found an increase in the target parameters supporting that legumes are not C limited under symbiotic conditions.
9.3.3 Evaluation of N2 Fixation and Role of AM Fungi in This Process: A 15N Approach
Plant-available N occurs in six isotopic forms; but only two of them are stable: 14N with a natural abundance of 99.634% and 15N with a natural abundance of 0.366%. The ratio of 15N/14N remains almost constant in the atmosphere, plants, and soils (Zapata 1990). However, addition of a small amount of 15N-enriched inorganic fertilizer to soil increases the 15N/14N ratio and consequently the soil N pool. In this context, a basic concept in agronomy must be considered: “when a plant is confronted with two or more sources of a nutrient, the nutrient uptake from each source is proportional to the amounts available in each source” (Zapata 1990). Consequently, plants, after growing in a 15N-labeled soil, will take N from soil at the isotopic proportion provided by the new 15N/14N ratio after the 15N-enriched inorganic fertilizer addition, according to the known amount and richness of the added 15N. Simple calculations allow us to quantify the amount of N in plant derived from soil or from the fertilizer.
These approaches and concepts form the basis to measure N2 fixation rates of nodulated legumes. For quantitative measurements, an appropriate “non-fixing” reference crop is needed (Danso 1986). Furthermore, both N2-fixing and non-fixing plants are grown on a 15N-labeled soil, and both type of plants will take up 15N and 14N at a similar rate. However, since the N2-fixing plants use atmospheric N as an additional source of available N, the ratio 15N/14N will be lower in the N2-fixing plant (Danso 1986). The technique can be used to select the more efficient rhizobial strain that shows the highest reduction in 15N/14N ratio. These methodologies are used to measure N2 fixation by rhizobia–legume symbioses under field conditions (Azcón-Aguilar and Barea 2015).
These techniques have also been applied to ascertain the role AM fungi in N2 fixation by nodulated legumes, as reported in the first edition of this book (Azcón and Barea 2010), later discussed thoroughly (Azcón-Aguilar and Barea 2015). Briefly, the effect of the co-inoculation of rhizobia and AM fungi on N2 fixation by legumes growing under field conditions using 15N-aided methodologies was investigated first time by Barea et al. (1987). A lower 15N/14N ratio was recorded for the shoots of rhizobia-inoculated AM plants compared to those obtained in non-mycorrhizal plants. This finding indicated an enhancement of the N2 fixation rates which was induced by the AM activity on the rhizobium–legumes symbiosis. Other studies further corroborated the contribution of the AM symbiosis to N2 fixation by rhizobia-inoculated legumes both under greenhouse and field conditions (Toro et al. 1998; Barea et al. 1989a, b; Chalk et al. 2006).
9.4 Mycorrhizosphere Managing for Improving Legume Productivity
Managing mycorrhizosphere interactions (mycorrhizosphere tailoring) is a feasible biotechnological tool to improve plant growth and health and soil quality (Azcón-Aguilar and Barea 2015). The impact of mycorrhizosphere tailoring on legume performance has been tested under field conditions and involves the common host AM plant, N2-fixing nodulating rhizobia, and phosphate-mobilizing bacteria (Azcón and Barea 2010; Shtark et al. 2012, 2015a; Zhukov et al. 2013; Larimer et al. 2014). However, the success of these organisms under field environment has been variable. The inoculum production technologies, as a basis for tailoring legume mycorrhizosphere, are discussed in the following section.
9.4.1 Field Strategies for Testing a Managed Legume Mycorrhizosphere
Only few studies involving AM fungi–rhizobia interactions have been carried out under field conditions for a sustainable agricultural production. Since the pioneering work on dual inoculation of Medicago sativa grown in normal cultivation systems in an arable soil (Azcón-Aguilar et al. 1979), some experiments aimed at evaluating the role of AM fungi in improving N2 fixation, either in controlled or in real field conditions, have been carried out all over the world. The accumulating data, however, suggest that several factors must be considered so that the inoculation effects of microbial symbionts on legumes become successful: (1) selection of appropriate rhizobial strain/AM fungus combination (Azcón et al. 1991; Ahmad 1995) and (2) fertility level of soils. In this context, a beneficial impact of AM fungi on legume symbiotic performance was corroborated mainly under low soil P levels (Chalk et al. 2006; Uyanoz et al. 2007; Pagano et al. 2008). Conversely, the AM symbiosis was not effective and did not promote N2 fixation in soils with high levels of available P (Zaidi and Khan 2007; Lesueur and Sarr 2008).
Several studies based on managing the mycorrhizosphere of legumes for the revegetation of degraded areas suffering disturbance of their plant cover have been carried out (Azcón and Barea 2010). The information on the interactions of AM fungi and rhizobia, obtained from restoration (by revegetation) under field experiments, most of them concerning with Mediterranean desertification-threatened areas, was further analyzed (Barea et al. 2011). Two model experiments carried out in semiarid Mediterranean ecosystems of southeast Spain are briefly discussed. In one of them six species of woody legumes, adapted to the drought and nutrient-deficient condition of the target environment, were inoculated with both N2-fixing bacteria and AM fungi (Herrera et al. 1993). The target shrub legumes included both native (Anthyllis cytisoides and Spartium junceum) and allochthonous (Robinia pseudoacacia, Acacia caven, Prosopis chilensis, and Medicago arborea) species. After 4 years of field grown, only the native shrub legumes were able to survive and thrive under the experimental conditions. It was also shown that the tailored mycorrhizosphere improved plant survival, outplanting performance, and biomass production. Anthyllis cytisoides, a highly mycotrophic legume species from the natural succession, adapted to drought and nutrient-deficient soils, was selected for further revegetation studies. The idea was to promote an integral restoration of the target degraded ecosystem. For that, seedlings with an optimized mycorrhizosphere were transplanted to facilitate plant establishment and nutrient acquisition and to improve physicochemical properties of soil. The established mycorrhizosphere-tailored plants acted as “resource islands,” while inoculum supplied nutrient to the surrounding vegetation (Barea et al. 2011). In a follow-up study, Requena et al. (2001) carried out a time course (3 years) field experiment. During this study, A. cytisoides seedlings were inoculated with a mixture of five taxa of AM fungi representing the natural abundance and diversity and rhizobial symbionts, and the inoculated plants were transplanted into the target degraded area in semiarid Mediterranean ecosystem of southeast Spain. Control seedlings only got AM fungal and rhizobial inocula from the field soil along the time course trial. Tailoring A. cytisoides mycorrhizosphere at pre-transplanting assisted the establishment and nutrient acquisition by the test plant and also increased N content and organic matter accumulation in soil and the formation of hydrostable soil aggregates around plants. Lavandula seedlings (a small shrub species from the natural succession of the site) were also transplanted in some plots in the same experimental area, to be grown either alone or near A. cytisoides plants. The soil in these plots was labeled with 15N to measure N2 fixation and N transfer from the fixing A. cytisoides to the non-fixing Lavandula plants (Requena et al. 2001). It was demonstrated that AM inoculation improved N2 fixation and N transfer.
The role of AM fungi to promote an integral restoration of target degraded ecosystems based on using nodulated legumes from the natural succession of the site was tested in other experiments (Medina et al. 2004; Alguacil et al. 2005, 2011). These authors found that AM inoculation enhanced plant establishment and growth and improved physico-biochemical properties of soil, including enzymatic activities related to C, N, and P cycles and hydrostable soil water-stable aggregates. An increase in the amount and diversity of AM propagules was also found (Alguacil et al. 2011; Martínez-García et al. 2011). These findings supported the hypothesis that the AM management strategy are extremely important in improving both plant development and soil quality and can be considered as a successful biotechnological tool to aid the restoration of self-sustaining ecosystems.
9.4.2 Interactions Between AM Fungi and Phosphate-Solubilizing Microbes: Importance in Legume Improvement
Multitrophic interactions involving AM fungi and phosphate -solubilizing bacteria (PSB) and their consequential impact on legumes (Azcón and Barea 2010; Zaidi et al. 2010) are discussed in the following section.
During the interactions between AM fungi and PSB, PSB solubilize phosphate ions that AM fungi can capture and transport to the plant (Zaidi et al. 2010; Azcón-Aguilar and Barea 2015). The interactions between these two groups of microorganisms concerning on how they affect the development of each other have been analyzed recently (Ordoñez et al. 2016). The mechanisms whereby P-mobilizing microorganisms release available P from sparingly soluble soil P forms involve solubilization (inorganic P) or mineralization (organic P) (which are activated by specific enzymes and/or chelating organic acids, respectively, released by PSB into the surrounding environment (Richardson et al. 2009; Zaidi et al. 2009, 2010; Barea and Richardson 2015)). However, the Pi made available by PSB acting on sparingly soluble P sources may not reach to the root surface due to limited diffusion of this ion in soil solution (Barea and Richardson 2015). This connects with the well-known fact that the external mycelium of the AM fungi is involved in plant uptake of solubilized P (Smith and Smith 2011, 2012). Therefore, it was proposed that if P is solubilized by PSB, AM fungi can tap these Pi ions and translocate them to plants suggesting a microbial interaction, which could improve P supply to the host plants synergistically or additively, as first suggested by Azcón et al. (1976). The 32P-based methodologies have been applied to assess how the interaction among AM fungi with PSB contributed to plant P nutrition, using RP as a source of sparingly available P, and different plant species, mainly legumes (Azcón-Aguilar and Barea 2015). The interactions between AM fungi and PSB are important for P acquisition by the legume plants, and several experiments have investigated this mycorrhizosphere activity. In general, these studies found that dual inoculation produced an increased biomass and P content of plants co-inoculated with AM and PSB and reduced the specific activity (SA = 32P/31P quotient) of the host plant. This lowering of SA indicates that the plant used an extra amount of 31P, solubilized by the PSB from either endogenous or added as RP and that solubilized P can be captured by the AM mycelium from the soil microhabitat where PSB demonstrated P solubilizing activity (Barea et al. 2007; Barea 2010). Conclusively, plants dually inoculated with both AM fungi and PSB appear to be more efficient in terms of P supply compared to non-inoculated or singly inoculated plants.
9.4.3 Implementing Inoculum Production Technology: Tailoring Legume Mycorrhizosphere
The technology for production of rhizobial, free-living PGPR, and AM fungal inoculants was described in the first edition of this book (Azcón and Barea 2010; Patil and Alagawady 2010). Since then some recent advances in this area have been made which are discussed in the following section.
A comprehensive review on the formulation and practical perspectives of inoculant technology for Rhizobium and PGPR has recently been published (Bashan et al. 2014). The authors pointed out a number of top priorities of research to implement the production steps and delivery systems for the bacterial inocula. Special emphasis must be given to the evaluation of carriers and to improve the survival of microorganisms in the inoculants and their shelf life. They encourage the implementation of polymeric/encapsulated formulations. Several companies are producing PGPR inoculum products worldwide (Ravensberg 2015; Kamilova et al. 2015; Borriss 2015).
Concerning AM fungi-based inocula, the difficulty to culture these obligate symbionts fungi in the absence of their host plant is a major obstacle to produce AM inoculants and for the development of inoculation techniques. Despite these problems, several procedures have been developed to multiply AM fungi and to produce high-quality inocula either on-farm, ex vitro in greenhouses, or in vitro monoxenic root organ cultures (Ijdo et al. 2011; Rouphael et al. 2015). The resulting materials (spores, hyphae, root fragments, etc.), from “culturing” AM fungi, are incorporated into different carriers to produce several formulations, including encapsulation, to be applied at an agronomical scale using different techniques. Inoculation of nursery-produced seedlings is a recommendable method for establishing selected fungi in the roots before planting out into the field, as is the case with horticulture and plantation crops. Mixed microbial inoculants, including PGPR, are recommended (Rouphael et al. 2015). This is particularly relevant in the case of legumes because they associate with rhizobia, PGPR, and AM fungi (Azcón and Barea 2010). In this context, Shtark et al. (2015a, b) suggested the development of multicomponent microbial inoculants for legume improvement and to decrease the use of mineral fertilizers and pesticides. However, these authors point out constraints for the certification of these multicomponent inoculants due to the current procedures imposed by governmental registration of inoculants. Several companies worldwide are producing plant-based AM inoculum products which are now commercially available to be applied in forestry, agriculture, and horticulture (Vosátka et al. 2008; Gianinazzi et al. 2010; Singh et al. 2014). A key point is, however, to develop appropriate methodologies for assaying the establishment, persistence, and effectiveness of AM fungal inoculants in the field (Verbruggen et al. 2013). In this context, Pellegrino et al. (2012) used molecular tracing techniques for such purposes, using alfalfa as a test plant. The results revealed the success of AM fungal inoculum establishment and its effectiveness.
Apart from microbial inoculations, other opportunities to exploit the beneficial activities of soil microorganisms are now emerging. Diverse research approaches are currently challenged to ascertain whether plant rhizosphere can be engineered to encourage beneficial organisms while preventing the presence/emergence of pathogens (Achouak and Haichar 2013; Spence and Bais 2013). To address these issues, some approaches are used which are based on combining molecular microbial ecology with ecophysiology and plant genetics which will allow a better understanding of plant–microbiome interactions in the rhizosphere (Zancarini et al. 2013). This “biased rhizosphere” concept/action is a challenge for the future, and possible approaches have recently been discussed (Savka et al. 2013). According to Bakker et al. (2012), one strategy to manipulate the plant to recruit beneficial microorganisms in its rhizosphere relies on developing plants able to shape their microbiome by targeting particular taxa for specific functions such as N2 fixation, P mobilization, biocontrol, etc. Undoubtedly application of biased rhizosphere to foster beneficial microbial services opens new opportunities for future agricultural developments based on exploiting the beneficial microbial services to reduce the agrochemicals inputs thereby achieving environmental sustainability and economic objectives.
Both plant mutualistic symbionts and saprophytic microorganisms living at the root–soil interfaces, the rhizosphere, are essential for plant nutrition and health. Legumes are plant species of great agricultural/environmental importance, known to establish beneficial symbiotic relationships with N2-fixing bacteria and AM fungi, associated with many saprophytic microorganisms developing the so-called mycorrhizosphere. Managing the microbial symbionts and saprobes, including PGPR, involved in legume mycorrhizosphere has a great relevance to improve legume productivity either in sustainable agriculture or in the maintenance of natural plant communities. Consolidated information supports that opportunity exists to exploit the interactive effects of phosphate-mobilizing PGPR, N2-fixing rhizobia, and AM fungi, through tailored management of the mycorrhizosphere, thereby benefiting P and N cycling and plant nutrition. In addition, the interactions among AM fungi and certain PGPR help plants to tolerate the negative impact of biotic (pathogens, insect, parasitic plants) or abiotic (salinity, drought, contaminants) stressors. The technologies for the production of efficient rhizobial, PGPR, and AM fungal inoculants nowadays are commercially available and are used in the field of agriculture, horticulture, and revegetation of degraded ecosystems. The production of legumes employing selected microbial inoculants is likely to become even more important in future due to the agroecological threats of agrochemicals, which urgently requires to be reduced, and even avoided, to increase food quality, sustainable food production, and environmental protection. Therefore, to popularize and improve the use of tailored mycorrhizospheres in legume plants is a major challenge for the scientists, farming communities, and industry.
This research was supported by the Andalusian Research Programme (Project CVI-7640) and the Spanish National Research Programme (R & D)-European Union (Feder) (Project CGL2015-69118-C2-2-P).
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