2022 Volume 10 Pages 344-355
The rhizosphere is the soil that surrounds, and is influenced by, the roots of a plant. It is considered one of the most complex ecosystems on the planet due to the intense interactions that occur between plants and microorganisms, as well as the competition that occurs among the microbial components. Due to this competition and beneficial interactions, a contribution of paramount relevance occurs in terms of chemical, physical, and biological characteristics that allow the plant and crop development. To mitigate ecosystem disturbances, it is necessary to compensate the imbalance of these conditions. Unfortunately, human activities involving strong soil disturbance have significantly affected plant development. Therefore, currently it is a priority to avoid the deterioration of soil ecosystems to remediate the damages that have already occurred. In the case of soil microbiology area, there are many solutions that can be designed and applied with beneficial microorganisms, including both fungi and bacteria, that interact mutualistically with plants or crops. At the same time, a deep understanding of these interactions remains challenging due to their complexity. In this review, biotechnological developments with agricultural or forestry importance are analysed. These include plant growth promoter bacteria, the Azolla-Anabaena symbiotic system, arbuscular mycorrhizal fungi and ectomycorrhizal fungi, as well as their relevance in the production of agricultural and forestry biofertilizers.
The definition of principles and general practices that are appropriate to the current trends and paradigm shift in agriculture are occurring in the present time, among which stand out concepts of soil conservation, such as sustainable land management and conservation agriculture [1]. The use of plant growth-promoting rhizobacteria (PGPR) and Arbuscular mycorrhiza fungi (AMF), as an integrated nutrient management system, can increase the uptake of nutrients by plants and removal from soil [3], as well to increase the efficiency of inorganic fertilizers [4], and the possibility to reduce its application rates for saving costs [5]. In the present time, we face a steep escalation in the cost of agricultural and forestry products because of broken supply chains due to unexpected events in human society; this added to existing problems such as drought, erosion, contamination, or loss of arable land. In this review, we present the microbial symbioses as an alternative to improve agriculture in terms of productivity, viability, and respect to native agroecosystems.
PGPR, which are part of the soil microbiota, help improving the quality of crops [6], providing to plants direct benefits like solubilization of phosphates, production of siderophores that increase the availability of iron, and production of growth regulators such as indoleacetic acid (IAA) [7]. Other indirect benefits provided by PGPR are related to the production of antibiotic compounds, the release of protective enzymes, and the induction of systemic resistance [8]. In return, PGPR are benefited from root exudates that serve them as nutrients as well as carbon and energy sources. These exudates allow some specificity in the recruitment of beneficial organisms [9].
In general, the most widely studied PGPR genera are Bacillus, Pseudomonas, and Azospirillum [8, 10], which are the ones with the most commercial interest due to their ease of cultivation in vitro and the proven beneficial effects on plants. Due to these properties, some PGPR (Bacillus, Pseudomonas, Azotobacter, Azospirillum, Bradyrhizobium, and Mesorhizobium) have been included as part of inoculant formulations, alone or in combination with other rhizobacteria or with AMF [11].
The fixation of atmospheric nitrogen by bacteria plays an important role for the incorporation of this element in available forms, into the ecosystem thus, allowing its assimilation by plants and to fulfill its biogeochemical cycle. Different genera of nitrogen-fixing bacteria have been described, including nodulating symbiotic (Rhizobia and Frankia), associative (Azospirillum, Azoarcus, Herbaspirillum), cyanobacteria (Anabaena, and colony-forming cyanobacteria of the genus Nostoc), and free-living bacteria (Klebsiella and Azotobacter), as well as autotrophic bacteria other than cyanobacteria (Rhodobacter) [12, 13].
The symbiosis of nodule-forming bacteria occurs only in the monophyletic clade referred to as "FaFaCuRo" (Fabales, Fagales, Cucurbitales, and Rosales) [14]. There are also endophytic and extracellular diazotrophic microorganisms, but much less efficient for fixing nitrogen for plants than bacteria that form an obligate symbiosis with plants [15]. Even so, 24% of the nitrogen contained in cereal crops (corn, rice, and wheat) is associated with the physiological activity of non-symbiotic N-fixing microorganisms [16]. Another very useful characteristic of PGPR is their ability to promote mycorrhization (establishment of mycorrhizal symbiosis in roots), which is carried out through the activities described above such as N-fixation, production of plant growth regulators, pathogen control, and solubilization of organic forms of phosphorus into orthophosphates that can be transferred by AMF to plant roots [17].
A process related to the promotion of mycorrhization is mediated by Mycorrhiza Helper Bacteria (MHB), which is the generic name of bacteria that interact with plants and fungi to stimulate mycorrhizal symbiosis [18], receptivity of the root to the mycobiont, root-fungus recognition, fungal growth, modification of rhizospheric soil, the germination of fungal propagules, and supression of host defense response [19]. MHB can be bacteria from rhizosphere, such as Gram-negative Proteobacteria (Agrobacterium, Azospirillum, Azotobacter, Bradyrhizobium, Burkholderia, Enterobacter, Klebsiella, Pseudomonas, and Rhizobium), Gram-positive Firmicutes (Bacillus, Brevibacillus, and Paenibacillus), and Gram-positive actinomycetes (Arthrobacter, Rhodococcus, and Streptomyces) [20]. Biofilms are structures that harbor complex microbial communities on surfaces and interfaces with plants [21] that lead improved access to nutrients and protection against biotic and abiotic stress [22]. Microorganisms that form biofilms consist of a single microbial species or a combination of different species of bacteria, protozoa, archaea, algae, filamentous fungi, and yeast [23]. The synthesis of bacterial exopolysaccharides begins the formation of biofilms on the root surface [24]. Each constituent in biofilm matrix has specific functions, such as bacterial adhesion and cohesion, retention of water, physical barrier against abiotic stress, ion sorption and exportation of cell components, to overcome drought stress and enhance communications between symbionts [25]. The adaptive potential of biofilms is used by beneficial bacteria such as Pseudomonas fluorescens. During colonization/mycorrhization [26], bacteria form a biofilm, with a specific pattern that interacts with the host immune system. This adaptive potential, for the good or the bad, is utilized by both beneficial and pathogenic bacteria to evade the immune system of plants and thus, to promote colonization of symbionts or to cause further infections in the case of pathogens [27]. Knowledge about biofilms formed by pathogens is deeper than the ones formed by beneficial microorganisms. Other mechanisms that promote bacterial colonization are related to quorum sensing and several chemical signals between bacteria and hosts [28]. The functions of these mechanisms are still largely unknown due to the high complexity of the interactions involved. “Omic” sciences and bioinformatics can help understanding these interactions through metabolome, genomic sequencing [29], and “machine learning” approach [30, 31].
Azolla is a genus of mostly tiny floating aquatic ferns, averaging 1 cm in diameter, with water content greater than 90%; this fern grows on static and non-saline water bodies. The symbiosis between this pteridophyte and the N-fixing cyanobacteria Anabaena azollae is permanent. Figure 1 shows the distribution of these ferns when floating on water, a close-up of the fronds, and a micrograph of the cyanobacteria living inside fern fronds. An important characteristic of this aquatic fern is its ability to grow and reproduce rapidly, thus, doubling its biomass in short time.
Figure 1: a) Culture of Azolla filiculoides; b) Fronds of Azolla filiculoides; c) Micrograph of Anabaena azollae cells
This fern may be utilized as a biofertilizer due to its high nitrogen content. In Asian countries, where it is commonly used in traditional rice cultivation [32, 33], its use has been documented since the 1960s [34]. There is a lack of information on the agronomic and environmental effects of replacing N fertilizers with Azolla biomass [35]. An example of a practical application of Azolla as an organic fertilizer for producing either organic tomatoes in greenhouse [36] or corn [37]. Recent studies have reported that this fern also provides several benefits, not only as a biofertilizer for sustainable agriculture but also when cultivated alongside flooded crops [38]. In this regard, the fern biomass not only increases the yield of rice crops [39] increases nitrogen use efficiency [35], reduces NH3 volatilization [40], and reduces rice grain cadmium accumulation [41].
The use of Azolla has been associated not only with an increase in rice yields (8–14%) but also with a more efficient N-cycling. Under such cultivation, N-fixation can reach up to 52 kg N ha-1 in the presence of Azolla, while the N-volatilization can decrease by 12 to 42% [35]. Another important characteristic of this fern is its high protein content (200-400g kg-1 of biomass on a dry basis) [42], and the essential amino acid composition, being leucine, lysine, arginine, and phenylanine (and/ or tyrosine) predominant [43].
An essential aspect of Azolla ferns is the production of chlorophyll as well as carotenoids and anthocyanins [44]. Anthocyanins are synthesized by some Azolla species during adaptation under stressful conditions such as high light intensity and low temperature. These adaptations are reflected in a decrease in the size of fronds and in the chlorophyll a/b ratio, as well as in increased concentrations of carotenoids and anthocyanins that result in color changes of these ferns from green to reddish [45]. The production of carotenoids in Azolla species has relevant potential applications as part of poultry diets [46, 47] or for the industrial production of carotenoids [48].
Azolla biomass have been used as a food source or as a nutritional supplement for animals (mainly in poultry farming) and for human consumption, with favorable results [49]. However, one problem related to the consumption of Azolla biomass is the presence of antinutritional factors, which may discourage their use if the biomass is not properly processed. Antinutritional factors are inescapable in most plants intended for human consumption such as cereals and legumes [50, 51]. In the case of Azolla species, these factors include phytic acid, cyanide [52] and tannins that are associated with proteins, which difficult its application as supplement [53] due to potential inhibitions on the digestive enzyme trypsin [54].
Although antinutritional factors should be eliminated before consumption [55], between these apparently undesirable molecules exist some others that possess some useful biological properties. For instance, polyphenols produced by Azolla [56] may present certain chemical homogeneity [57] and have beneficial antioxidant activities based on in vivo hepatotoxicity tests [58] and anti-inflammatory, antiapoptotic activities [59], and antimicrobial properties [60]. In addition, extracts obtained from Azolla have potential effects as bioinsecticides against mosquito larvae of the Aedes genus [61, 62].
Azolla ferns also have environmental applications since they may absorb and accumulate potentially toxic elements from water bodies, thus, directing them for water bioremediation projects [63, 64, 65]. Overall, these aquatic ferns have important biological benefits that make them suitable for industrial processes as well as for developing biotechnologies directed to the agri-food sector.
Arbuscular mycorrhiza is a symbiosis between microscopic fungi (Glomeromycotina) colonizing the cortical cells of the roots of most terrestrial plants. In this regard, the absence of mycorrhizae is considered abnormal for most plant species, with negative consequences at ecosystemic level [66]. AMF are considered primary biotic components of the soil, and their symbiosis provides beneficial effects on plant growth by favoring nutrient assimilation and increasing resistance to abiotic and biotic stress [67, 68]. Therefore, AMF play a significant role on enhancing crop production and yield and on maintaining balanced microbial communities that contribute on improving both quality and health of soil [69]. The most common AMF genera found as part of inoculant formulations are Glomus, Funneliformis, Rhizophagus, and Septoglomus. The most used species in commercial inoculants are Rhizophagus irregularis and Funneliformis mosseae which are known for their ubiquity, their generalist habits, and their ability to colonize a wide variety of plants [70]. Most of AMF inoculants are well proven based on inducing growth promotion, improving nutritional status, and protecting against biotic and/or abiotic factors for important horticultural, forestry, and agricultural plant species [71, 72, 73].
In soil ecosystems, AMF coexist successfully with rhizobacteria [74]. Therefore, some inoculant formulations include not only propagules (spores, hyphae or root fragments) of one or more species of AMF, but viable biomass of some rhizobacteria whose efficacy has been previously evaluated. Developing combined inoculants is a complicated biotechnological challenge involving the assessment of different metabolic needs and growth rates of microorganisms [75]. The number of patents based on AMF has increased significantly worldwide in the last two decades. Around 696 patents were granted mainly from private companies, research centers and universities [76]. This fact shows that there is a real need for using beneficial microorganisms in agricultural production.
The use of microbial inoculants can be beneficial for crops, but the ecology of soil microorganisms can be severely affected by introducing non-native and invasive species [77]. Therefore, it is important to ensure that native microbial species are not displaced when using commercial products containing AMF [78]. This potential ecological problem has stimulated the interest in developing microbiome engineering in situ [79] to improve the application of inoculants. The application of inoculants represents a low environmental risk at some conditions such as closed horticulture systems that use artificial soil (inert substrates) or protected agriculture. However, it is preferable to use inoculum derived from the native microbiota instead of commercial products with non-native microbial isolates. The application of AMF well-adapted to specific edaphoclimatic conditions for cultivating field-crops, thus, favoring greater probability of success. The use of non-native AMF may be effective for the rehabilitation and recovery of severely degraded soils, or for the remediation of soils contaminated with organic and inorganic compounds.
Fungi are heterotrophic macroscopic or microscopic microorganisms that are distributed among different genera; some macroscopic fungi are relevant to human nutrition since they are an excellent source of food. These macroscopic fungi are either collected at their natural habitat or grown by using industrial agricultural methods.
Saprophytic fungi play an ecological role by degrading soil organic matter and allowing it to reenter the food chain. In addition, some of these fungi are suitable for industrial production, and have been mass-consumed for decades, as happened for Agaricus bisporus, Lentinula edodes or Pleurotus ostreatus. Due to the saprophytic nature of these fungi, some species are recognized as a source of extracellular enzymes of industrial relevance like peroxidases (lignin degradation), pectinases, amylases, xylanases (hemicellulose degradation), cellulases, and chitinases [80]. They can also be an alternative source of packaging material to replace expanded polystyrene (styrofoam) with fungal mycelium [81].
Some fungi do not depend on a saprophytic lifestyle as a means of survival, but instead form ectomycorrhizae, a symbiosis with the roots of around 5,000 plant species, including mainly gymnosperms and angiosperms, and more than 20,000 species of fungi, mainly Ascomycetes and Basidiomycetes. This symbiosis is a mutualistic symbiotic relationship. Being heterotrophic organisms, ECMF are unable to make their own food and depend on carbon sources derived from plant symbionts, technically known as phytobionts. In return, ECMF, called mycobionts, mobilize water, macro- and micronutrients to their plant hosts [82]. From a morphoanatomical perspective, this symbiotic relationship is characterized by three structures: i) Hartig’s network, made up of hyphae that penetrate the interstitial spaces of the cortical cells of the roots; ii) The mantle, a fungal layer covering the roots; and iii) the external mycelium, one of the most fascinating biological structures, connecting the soil system with the plant component of natural ecosystems or forest plantations, where ectomycorrhizae thrive [83, 84].
It can be said that without the ectomycorrhizal symbiosis there would be no forests on the planet. A recent study that analyzed 1.1 million forest plots throughout the world estimated that 60% of all trees on the planet are associated with ectomycorrhizae [85]. In addition, ECMF form living bonds connecting trees through common mycelial networks. In this way, forest ecosystems can be considered entities connected by ECMF. Water, nutrients, and signal molecules flow through these fungal networks for protection against pathogens. Thus, it is possible to affirm that without the ectomycorrhizal symbiosis there would be no forests on the planet [86].
The biotechnological potential of ectomycorrhizal fungi can be exploited through the production of bioinoculants for forest trees. These bioinoculants can be obtained from two sources: mycelial cultures or spores. Each of these sources has certain advantages and disadvantages. ECMF have the advantage that once the fungal strains are isolated and their effectiveness proven, it is possible to carry out propagation at an industrial scale using large fermenters. Furthermore, the preparation of mycelial inoculants can be carried out at any time of the year and in any required quantity. The disadvantage is that, due to their specific nutritional requirements, only a small part of the ECMF have been isolated and an even smaller number has been successfully produced at industrial scale. Production plantations of some of these species can be found around the world, including United States of America, Chile, and New Zealand. In Chile, Suillus luteus is known as “pine callampa” and constitutes an important source of economic income while waiting for wood-producing trees to grow [87]. In New Zealand, plantations of Lactarius deliciosus, known as “milk cap”, provide a relevant ingredient for the gourmet cuisine of that country [88]. Another disadvantage of the use of mycelial inoculants is that they have a short shelf life. During growth, mycelial strains can undergo phenotypic modifications, including changes in their physiological characteristics. In the most complex scenario, their growth can stop. Moreover, the production of forest bioinoculants based on mycelium requires a complex and expensive infrastructure, while the production of inoculants requires costly inputs such as culture media, specific reagents, and expensive laboratory materials.
The use of spores as a source of inoculum has been successful in the cultivation of one of the most expensive mushrooms in the world, the black Perigord truffle (Tuber melanosporum). The price of 1 kg of this truffle is 170 US dollars on average, while the average price of 1 kg of the Italian white truffle (Tuber mangnatum) is 8,000 US dollars. The biotechnological production of ectomycorrhizal plants with truffles is a growing industry worldwide, and some companies produce hundreds of thousands of ectomycorrhizal trees each year, mainly oaks (Quercus ilex), chestnuts (Castanea sativa), and pines (Pinus radiata). The establishment of plantations of mycorrhizal trees with various species of truffles is an industry worth billions of dollars annually, mainly in Spain, France, Italy, New Zealand, and Australia. In the production of these plants, the spores of the truffles are used as an inoculum source. The advantage of using spores as a source of ectomycorrhizal inoculum is that it is a cheap technology that does not require an expensive infrastructure or special reagents [89]. In addition, many species of ectomycorrhizal fungi can be cultivated using this technology. The fungi that have been cultivated with this biotechnology belong mainly to the Gasteromycetes group of fungi in Basidiomycetes or to the Pezizomycetes group in Ascomycetes. These fungi contain enormous amounts of spores in usually globose or subglobose sporomes. The shelf life of spore-based bioinoculants is much longer than that of mycelium and can reach one or two years depending on the species. The disadvantage of this biotechnology is that it is seasonal, that is, it depends on the time of natural production of the fungi, which with climate change has become increasingly difficult to predict accurately. A modification of the use of Gasteromycetes spores has been developed at Colegio de Postgraduados during the last three decades using pilei (hats) or hymenia (fertile part of the fungus) for Agaricales and Boletales, which include most ECMF. Beneficial effects of these fungi have been demonstrated in terms of growth, physiology, and nutritional translocation of inoculated trees. The survival rate in the field has also increased a consequence of ectomycorrhizal inoculation. To date, 132 combinations of fungi and trees (pines and oaks) native to Mexico have been successfully tested with this biotechnology, which is efficient, cost-effective, simple, and is now ready for industrial-scale production [90] (Figure 2).
Figure 2: a) Ectomycorrhiza of Pinus greggii with Laccaria bicolor; b) Ectomycorrhiza of Pinus pseudostrobus with Hebeloma alpinum; c) Ectomycorrhiza of Pinus greggi with Laccaria laccata; d) Sporome of Lacaria laccata in Pinus montezumae; e) Suillus luteus in Medellin, Colombia; f) Tuber melanosporum; g) Effect on the growth of Pinus greggii inoculated with edible fungi; h) Pinus montezumae inoculated with Hebeloma leucosarx; i) Effect of ectomycorrhizal inoculation on Pinus montezumae; j) Production of inoculants based on pilei of Laccaria laccata
Symbiotic interactions in the rhizosphere occurred naturally in all ecosystems. The basic knowledge about such interactions constitutes a useful sustainable alternative to improve plant growth and crop production as well as to favor healthy and innocuous food for human purposes. In addition, symbiotic or non-symbiotic microbial interactions also represent a natural alternative for alleviating and recovering disturbed soils and environments derived from chronic human activities. However, in general terms, despite the extensive knowledge on this biological subject, some aspects of these interactions between plants and microorganisms are still not well understood due to their enormous complexity. The latter is necessary for establishing and implementing effective biotechnological techniques and inputs to be directed for improving both plant production systems and ecological restoration processes.
Authors thank M.Sc. Jorge Valdez-Carrasco for his valuable help photo shooting and scaling Azolla micrographs.
