Potential for Industrial Application of Microbes in Symbioses that Influence Plant Productivity and Sustainability in Agricultural,Natural,or Restored Ecosystems

Abstract

Microbes in three widespread and evolutionarily stable mutualistic symbioses with plants offer a range of nutritional and other benefits that may be modified or enhanced through selection or genetic manipulation for biotechnological applications. In this review, the biology, ecology, and genetics of arbuscular mycorrhizal fungi partnering with more than 80% of all land plant species; rhizobia bacteria that nodulate legumes and fix atmospheric nitrogen; and Epichloë fungi endophytic on cool-season grasses, are discussed. Research on these symbioses indicates considerable potential to utilize modified or natural microbial strains to stimulate production selectively of desirable compounds in plants or to introduce or over-express genes that can stimulate beneficial effects of the mutualisms on desirable traits in plant hosts, whether in agricultural or more natural ecosystems.

Introduction

No one is truly alone; all creatures, no matter how isolated, interact with others of the same or different species. Therefore, it is no surprise that symbiotic associations are integral to global life processes. Anton de Bary, in an 1879 monograph Die erscheinung der symbiose, first coined the term symbiosis to encompass any interactive partnership between individuals of different species.¹ The association may be of short or long duration; obligatory or discretionary; and beneficial (mutualistic) or harmful (pathogenic) to one or both partners.

Among the vast array of symbioses, three plant-microbe mutualisms are the focus of this paper for the following reasons: the microbe lives partly or entirely inside the plant for part or all of its life cycle; the evolutionary history spans 60–450 million years and is indicative of lengthy coevolution between microbe and plant; and sufficient research has been done to discuss the biology of the mutualisms and how they might contribute to advances in industrial biotechnology.² The arbuscular mycorrhizal mutualism partners a global spanning phylum of fungi (Glomeromycota) with more than 80% of all land plants and is present in most habitats worldwide.^3
–5 Closely related bacterial species collectively known as rhizobia infect roots of many species of legumes, stimulate nodule formation, and reduce atmospheric dinitrogen molecules to compounds that can be utilized by their plant hosts.⁶ Fungi in the genus Epichloë infect cool-season grasses and form a continuum of symbioses that span the full range from mutualism to parasitism.⁷ All of these microbes are associated with major crop plants, and so they have significant impact on human societies through agriculture and/or ecological restoration and management. In that capacity, natural products able to stimulate or optimize these mutualisms or, alternatively, novel compounds produced by the microbes or plant partners that are able to benefit medicine or other industrial applications, warrant investigation and utilization.

The Arbuscular Endomycorrhizal Association

This plant-fungus mutualism has its origin more than 400 million years ago at a time when ancient flora did not form true roots and the fungi likely established a coevolutionary partnership that was both obligate and sustainable.^3,8 Evidence was found in fossils from the Devonian period and in three genes involved in mycorrhizal signaling pathways that were vertically transmitted and highly conserved in all major plant lineages that coevolved with the fungi. ^9,10 The fungal symbiont benefits from a niche free of competition by other soil microbes and a steady supply of carbon for growth and reproduction. The plant host benefits from uptake of soil nutrients otherwise unavailable to roots, especially immobile cations like phosphorus, via an expansive network of hyphae in the rhizosphere.¹¹ Specialized and highly branched arbuscules sandwiched between the cell wall and membrane of root cortical cells provide sites for reciprocal exchange of nutrients between the plant and its fungal endosymbiont–hence the name arbuscular mycorrhizal fungi (AMF) (Fig. 1A-B).¹² Other benefits to the plant host are context dependent (e.g., plant phenology, environmental conditions).¹³ Monoclonal antibodies identified a glycoprotein (named glomalin) coating hyphae and spores that benefits the fungal organism by sealing and protecting the hyphae, and benefits plants by functioning as a chelating agent and contributing significantly to aggregation of soil particles.^14,15 Some plant species have evolved away from the symbiosis, most of which are pioneer weedy species in Brassicaceae, Chenopodiaceae, and other families.¹⁶ The deep evolutionary history of AMF and their intimate association through geologic time may help explain the general absence of plant host specificity and cosmopolitan distribution of many species.⁵ Because of such broad physiological and ecological tolerance, more than 1,200 strains of almost 80 AMF species (35% of known diversity) from a wide range of habitats and all continents except Antarctica have been grown successfully in open-pot cultures using a single mycotrophic host plant sudangrass (Sorghum sudanense) and a standardized sand-soil mix adjusted to pH 6.2–6.5.^17,18

Fig. 1.

Basic features of three widespread and sustainable mutualistic symbioses. (A–C) Arbuscular mycorrhizal associations between fungi in the phylum Glomeromycota and >80% of all plants. (A) Direct blue-stained arbuscules in the cortical cells of a corn root; (B) Mycorrhizal colonization of corn roots, consisting of internal hyphae, arbuscules, and a network of external hyphae bearing abundant asexual spores; (C) Multinucleate cytoplasm within a developing spore (Acaulospora). (D–E) Bacterial associations of rhizobia with legume plants (Leguminoseae). (D) Nodule in a root of Medicago truncatula; (E) Confocal micrograph of a cross-section of mature root nodule showing cells infected with bacteroids expressing green fluorescent protein (photo by C. Wyman). (F–H) Epichloë fungal endophytes associated with cool-season grasses; (F) Sexual stroma of an Epichloë species erupting from inflorescences of its grass host; (G) Close up of an Epichloë stroma (photos F–G by H.L. Barnett); (H) Aniline blue-stained asexual hypha of E. coenophiala growing intercellularly within a pseudostem of tall fescue.

This International Culture Collection of Vesicular-Arbuscular Mycorrhizal Fungi (INVAM) has evolved over the last 24 years as a multiservice repository of living fungal stocks for exploitation by researchers, teachers, students, and industry. INVAM, like most other microbe collections, affords a unique resource because it contains genetically diverse organisms from a wide range of natural habitats; strains are of known identity based on comparisons with reference strains of morphological and 28S rDNA sequences; and public and private institutions seeking to make use of all available biological materials and research knowledge are on equal footing.^19,20 AMF can be unpredictable and difficult to characterize because of clonal reproduction and large genetic population size attributable to a multinucleate thallus (Fig. 1C). ²¹ Considerable genetic diversity can exist within the body of one organism and even within one multinucleate spore of that organism.^22,23 Because of strict asexual reproduction, morphological and genetic polymorphisms become fixed and persist in populations that are evident phenotypically or remain latent.^{21, 24}

While most of the strains in INVAM are well characterized taxonomically, functional diversity is only beginning to be understood.²⁵ Genetic markers are limited to anonymous, beta-tubulin, and rDNA sequences.^26,27 A different approach now being undertaken is to identify a much wider range of expressed molecules as markers. AMF contain abundant lipids with distinct fatty acid profiles, and new technology using laser ablation electrospray ionization linked to a highly sensitive mass spectrometer and statistical software can differentiate hundreds of proteins and fatty acids in spore samples.^28
–30 These data will help achieve a better assessment of phenotypic diversity, link this information to genetic variation, and provide additional criteria for differentiating, selecting, and screening AMF strains.

That greater microbial diversity optimizes and stabilizes diversity in plant communities is a pervasive biological paradigm, and for native communities of AMF it is valid.^31,32 For that reason, all but a few commercial inoculants of AMF list a plethora of species on their labels. In Table 1, only the Pro-Mix product from Premier Tech (Québec, Canada) contains a single fungal strain, Rhizophagus intraradices; all others contain species mixtures, together with other amendments. Single-choice strains are the most suitable approach for biotechnological applications involving a pathway in a specific plant-fungus combination because of the need for control of genotypic/phenotypic purity, monitoring purposes, quality control, and cost containment.

Table 1.

Examples of Commercial Products on the Market Today and Their Uses by Two Major Groups of Fungal Endophytes Partnered with a Wide Range of Plant Species and Rhizobia Bacteria-Forming Nodules in Legumes

ENDOPHYTE	PRODUCT	COMPANY	PURPOSE
Mycorrhizal fungi: Native and introduced strains	MycoApply Diehard Pro-Mix	Mycorrhizal Applications (Grants Pass, OR) Horticultural Alliance (Sarasota, FL) Premier Tech (Rivière-du-Loup, Canada)	Improved phosphorus nutrition, increased growth and fitness of many plant species
Rhizobia: Introduced strains	Nitragin Gold ZHO N-Dure Twin N	Novozymes BioAg (Brookfield, WI) Botanicare (Chandler, AZ) IN TX Microbials, LLC (Kentland, IN) Active Soil (Evesham, UK)	Effective nodulation and nitrogen-fixation of legumes
Epichloë species: Non-toxic genetic variants	MaxQ ArkPlus AR1 or AR37	Pennington Seed Co. (Madison, GA) FFR Cooperative (Lafayette, IN) AgResearch Grasslanz (Palmerton North, NZ)	Biotic and abiotic stress resistance, improved fitness of cool-season grasses

AMF are treated as generalists, but a considerable body of work indicates that not all strains and species are equal.³ The best fungal strain is determined biologically by breadth of tolerance to a wide range of environmental conditions and broadest compatibility across a gamut of herbaceous and woody plant species. Cumulative knowledge of INVAM strains grown in pot cultures for more than two decades and feedback from users of collection strains point to pandemic species in the genus Rhizophagus (in particular, R. clarus and R. intraradices) as having all of the traits that maximize mycorrhizal plant responses at all scales. Anecdotally, the presence of R. intraradices as the dominant fungus in more than 50 commercial inoculants tested during the past decade (regardless of the species listed on the label) support this conclusion. For R. clarus, similarly, all of five strains from INVAM were tolerant to toxic levels of soil aluminum regardless of habitat of origin.³³ In contrast, results were mixed for strains of species in other genera.³³ Rhizophagus species, then, are recommended to increase probability of success when little else is known.

For biotechnology purposes, multiple genes have been identified from AMF that are amenable to exploitation. Some encode novel products that may have industrial potential. One example is glomalin. The gene encoding this glycoprotein has been identified putatively, and it has a high amino acid sequence homology with heat shock protein 60 (hsp 60) expressed in other fungi.³⁴ Transcript profiles from a model plant Medicago truncatula (barrel clover) colonized by a strain of Glomus versiforme identified 67 mycorrhiza-induced genes that included those involved in defense and stress responses and others involved in signaling pathways of the symbiosis.³⁵ Many of these genes are novel and subject to further exploration.

Alternatively, AMF may be used selectively to promote physiological processes that increase production of secondary metabolites, especially active compounds in medicinal plants. In a review by Zeng et al., 47 papers report a general positive effect of the mycorrhizal symbiosis on formation of terpenes, phenolics, alkaloids, aromatic diones, and other compounds.³⁶ However, stimulation of these metabolites varies with fungal strain, host plant genotype, dependency on the mycorrhizal association, and plant growth environment (e.g., fertility, especially phosphorous). Screening becomes essential, and identification of plant-fungus combinations in controlled environments is feasible using whole plant bioassays.^18,37

Lastly, industrial production of diffusible signaling Myc factors may have application in agriculture by stimulating mycorrhizal development and root branching, such as synthetic sulfated and non-sulfated lipochitoligosaccharides.³⁸ Analogous products also serve as nodulation (Nod) factors stimulating infection and nodule development in legumes by nitrogen-fixing bacteria.³⁹

Nitrogen-Fixing Bacteria (Rhizobia)

Even though mycorrhizal associations involve plant-fungus partnerships, some of the same genes and early recognition pathways contributed to later evolution of a mutualistic symbiosis between rhizobia and legumes that results in fixation and uptake of atmospheric dinitrogen molecules.^40,41 This symbiosis is important because sustainable agriculture requires more energetically and environmentally friendly alternatives to the detrimental impacts from heavy fertilization of crop systems with artificial nitrogen.^42,43

The rhizobia-legume symbiosis begins with release of flavonoid compounds from roots into the soil.⁴⁴ Rhizobia cells move to root surfaces via chemotaxis and are stimulated to release lipochitooligosaccharides (Nod factors) that induce nodule formation from inner cells of the root cortex (Fig. 1D). Root hairs first curl to entrap bacterial cells and form an infection thread that guides the bacteria to the nodule primordia. Bacteria are then encapsulated in plant cell membranes via endocytosis and undergo marked anatomical and physiological transformations to a form, the bacteroid, devoted primarily to symbiotic nitrogen fixation. Microsymbionts lose their cell wall, become pleomorphic to pack together better (Fig. 1E), and induce the expression of the nitrogenase enzyme complex, which catalyzes nitrogen fixation. The bacteroids become highly dependent on the plant host to obtain all compounds necessary for sustainable cell metabolism and symbiotic nitrogen fixation, including oxygen for respiration, carbon skeletons usually in the form of dicarboxylates such as malate and succinate, branched amino acids, minerals, and other inorganic molecules.^45,46

Many legume genes are involved in this process.^47
–49 Scientists once naively thought that introducing only the genes coding for the nitrogenase complex into non-legume plants would lead to nitrogen-fixation capabilities.⁵⁰ However, a cascade of other biochemical pathways is needed to create an optimal environment for bacteroid functionality. Free oxygen molecules inhibit activity of the nitrogenase complex by irreversibly oxidizing a critical FeMo cofactor and so the plant expresses proteins called leghemoglobins in cytoplasm of bacteroid-colonized cells. These molecules act analogously to hemoglobins in vertebrates by binding oxygen to heme groups, thus minimizing contact between oxygen and enzymes. One way to recognize effective nodules visually is by the pink pigments of leghemoglobin concentrated in the core of nodules.

A number of commercial inoculants have been on the market for many years (Table 1), mainly to introduce bacterial populations to areas where legumes have not grown before or to provide more effective strains where native populations are ineffective. A major goal of legume biology research is to gain a better understanding of how the symbiosis occurs and is regulated genetically and physiologically at the molecular and cellular levels to identify signaling and other pathways that can enhance nitrogen-fixation capabilities in new strains and develop more responsive plant genotypes.

Many genes expressed during nodulation and nitrogen fixation are now known, so research is increasingly focused on understanding regulatory networks.^47,51 Regulation of gene expression depends on the synthesis and activity of a suite of transcription factors.^46,49 The molecular function of several key regulatory transfactors during symbiosis establishment, nodule development, and maintenance of nitrogen fixation already have been identified.⁶

Modern molecular biology techniques now allow us to assess expression of virtually all genes in a species. An important intellectual resource of fundamental importance to understanding legume biology has been the development, publication, and maintenance of a Medicago Gene Expression Atlas.^47,52 Each chip used for this analysis encompasses 50,900 probe sets representing most genes in the M. truncatula genome. This service has been constantly updated and currently holds gene-expression information of 254 experiments that were classified according to the treatments they received, had at least three biological replicates, and passed stringent data quality control. From this work, the dynamics of gene expression during symbiotic processes are better understood and key genes can be targeted for functional characterization.

In addition, advanced mathematical and statistical modeling can be used to understand how these genes are interconnected. This is done by creating complex regulatory gene networks in which transcription factors are master regulatory genes coordinating gene transcription in a combinatorial fashion. In this regard, tools such as LegumeGRN utilize algorithms that include sophisticated statistics and machine learning algorithms to create these intricate networks of novel gene interactions.⁴⁹ This type of analysis is at the forefront of molecular genetics sciences and its usefulness relies on the possibility of creating testable hypotheses and/or delineating gene networks that will lead to specific traits of interest.

Genes and gene products that might enhance nitrogen uptake in cereals and other non-legume crops have not yet been identified.^53,54 However, rhizobia have been demonstrated to infect and colonize roots of transgenic rice.⁵⁵ The next step is to identify the minimum suite of genes necessary for the successful transformation of selected crops so they fix some nitrogen. The publication of genome sequences of model legumes such as barrel clover (M. truncatula), Lotus japonicus, and soybean (Glycine max) is accelerating our capacity to accomplish that.^41,56,57 Differences and similarities across a range of root-microbe symbioses that encompass legumes/rhizobial, angiosperm/non-rhizobial, and plant/mycorrhizal associations are being revealed.⁵⁸ Comparative proteomics studies are divulging common and novel protein networks associated with nodulation.⁵⁹ Comprehensive collections of plant mutants with tagged insertions are providing biological tools to explore the effects of gene deletions on nitrogen-fixation processes as well as on legume development and biochemistry.^60,61

Endophytes of Grasses (Epichloë)

Many agriculturally important forage and turf grasses have symbiotic relationships not only with mycorrhizal fungi, but also with fungi in the genus Epichloë. These fungi spend most or all of their life cycles as endophytes living asymptomatically between cells of leaves and stems. Benefits of endophyte infection include improved drought tolerance, shoot growth and tillering, seed production and germination, enhanced resistance to insects and endoparasitic nematodes, and enhanced mineral nutrition.^62
–64 These benefits are offset to varying degrees by toxicity of the endophyte to herbivores after ingestion of infected grasses. For example, cattle are afflicted with maladies such as weight loss or poor weight gain, low fertility, poor milk production, inability to control body temperature, lameness progressing to a dry gangrene of the limbs and tail, and tremors and other nervous system effects.^65,66 Basic knowledge of endophyte biology and the genetics of alkaloid biosynthesis may allow modification of alkaloid profiles of endophytes to reduce these toxic effects.

Epichloë species differ in their relationships with their grass hosts, ranging from mutualistic to antagonist depending on the frequency of sexual reproduction.⁶⁷ During sexual reproduction of Eplichloë, a fungal mass forms on a developing inflorescence (Fig. 1F-G) so that no grass seed is produced. The fungus then spreads horizontally to other plants via spores from the stroma. Some Epichloë species are strictly asexual and never form stroma. Hyphae grow intercellularly within leaves or stems of the plant throughout the life cycle of the fungal endophytes (Fig. 1H), and so they are transmitted vertically only by seed. Some Epichloë species fall between these two extremes and balance vertical (seed) transmission and horizontal (sexual stroma) transmission. The asexual seed-borne endophytes have the greatest impact on agriculture and offer a unique opportunity for industrial applications because they produce the most highly active alkaloids and do not form stroma on plant inflorescences. Two widely planted hosts of these Epichloë species are tall fescue (Lolium arundinaceum) and perennial ryegrass (Lolium perenne).

Collectively, Epichloë species produce four classes of bioactive secondary metabolites: ergot alkaloids (against vertebrates and insects); indole-diterpenes (against vertebrates and some insects); lolines (against insects); and peramine (a deterrent to some insects).^68,69 Species produce metabolites from one to three of these classes.^64,69 For example, Epichloë coenophiala commonly infecting tall fescue produces ergot alkaloids, lolines, and peramine. Epichloë festucae var. lolii is found in perennial ryegrass and produces ergot alkaloids, indole diterpenes, and peramine. The biosynthesis of each of these families of compounds has been studied in detail and reviewed recently.^66,70
–72

The ergot alkaloids present an interesting dilemma because their anti-insect activities are beneficial, but their anti-mammalian activities are detrimental. These secondary metabolites are derived from tryptophan, and compounds interact differentially with combinations of receptors for serotonin, dopamine, and adrenaline such that responses may be stimulatory or inhibitory.^71
–73 The complex nature of these interactions results in a wide range of activities that include vasoconstriction, smooth muscle contraction, and nerve or reproductive disturbance.

The biosynthetic pathway of ergot alkaloids leads to multiple products that differ in their benefits to the fungus or its host plant.^71,74 Some intermediates or branch end products can accumulate to concentrations exceeding that of the main end product. For example, perennial ryegrass containing the endophyte E. festucae var. lolii x E. typhina isolate Lp1 accumulates early pathway ergot alkaloids (clavines) to 7.2 nmol/g of plant material, whereas the pathway end products derived from lysergic acid (ergovaline and ergine) accumulate to only 3 nmol/g.⁷⁵ Inefficiency in converting one product to another may be a favorable trait because accumulation of intermediates and end products afford different benefits to the fungus or host plant.⁷⁴ For example, mutants accumulating early pathway clavines but lacking late pathway lysergic acid derivatives effectively deter rabbit feeding but are less able to deter feeding by insects.^75,76 This example of metabolite partitioning is less favorable to commercial application because of retention of anti-mammalian activity. However, further research on the pathway, where ratios of intermediates and end products are altered, may lead to genetically transformed endophytes with a combination of anti-insect properties and reduced toxicities to grazing animals.

Epichloë fungi provide an opportunity for strategic intervention to improve animal health. The presence of endophytes in forage and turf grasses increases grass-host fitness and stress tolerance, but animal toxicity and performance currently limit utility and value of endophytes. Establishing endophyte-free pastures by planting endophyte-free seeds has not been sustainable because the endophyte-free plants are not competitive with encroaching natural, endophyte-infected plants. Tall fescue and ryegrass seed containing endophyte variants lacking ergot alkaloids or indole diterpenes are now on the market (Table 1). These products have worked well in some environments, and toxicity to foraging animals has been lowered.^77
–79

One question is whether pastures containing these plants will be sustainable with competition from ingress of native grasses containing toxic alkaloids that confer additional benefits to increase fitness.⁸⁰ For example, some tall fescue varieties infected with non-toxic endophytes do not have the lesion nematode resistance conferred by more common toxic E. coenophiala isolates.⁸¹ Intentional modification of endophyte alkaloid pathways could provide alkaloid profiles (or chemotypes) that retain many of the native benefits and also minimize toxicity to animals. This approach is technologically feasible since the Epichloë endophytes of tall fescue and perennial ryegrass are vertically transmitted only through seed. As a consequence, the endophyte is an inherent component of the host plant and is transmitted faithfully and exclusively from one plant generation to the next. Genetically modified endophytes producing lower concentrations or fewer types of alkaloids may be able to control toxicoses while benefiting pasture productivity and sustainability.

Interactions Between Symbioses

All of the symbioses discussed in this paper have, for the most part, been studied separately from each other. However, these partnerships rarely occur alone in nature and can interact in ways that benefit or detract from each other. The almost ubiquitous mycorrhizal association on a range of plants, including legumes and grasses, will interact with, and be affected by, any other biotic or abiotic influences on growth and reproduction of plants.^3,11 AMF and rhizobia mutualisms are often interdependent, with the fungal associates providing phosphorous nutrition critical to the activity of enzymes involved in nitrogen fixation.^82
–84 Some of the same genes and early recognition pathways associated with development of the mycorrhizal association (triggered by Myc factors) are also expressed by rhizobia during nodulation (triggered by Nod factors).³⁸ Epichloë endophytes of grasses interact with mycorrhizal fungi by inhibiting colonization and subsequent mycorrhiza-mediated plant benefits, possibly because of either translocation of toxic metabolites or competition for available photosynthetic carbon.⁸⁵ These examples of interplay between associations emphasize the need to begin taking a systems approach when defining plant outcomes or benefits in the field following application of bacteria, fungi, genetic transformants, or other products marketed by industry. Naturally, the target symbiosis takes center focus, but secondary influences by co-occurring plant-microbe interactions require attention as well if any application is to achieve optimal impact and sustainability.

Footnotes

Acknowledgments

Research on the mutualisms discussed in this review is supported by the Davis College of Agriculture, West Virginia University (Morgantown), the National Science Foundation (DBI-1349308 to Joseph B. Morton) and the United States Department of Agriculture-National Institute of Food and Agriculture (2012-67013-19384 to Daniel G. Panaccione and 2010-65115-20384 to Vagner A. Benedito).

Author Disclosure Statement

No competing financial interests exist.

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