Plant gnotobiology: Epiphytic microbes and sustainable agriculture

ABSTRACT

In 1963, a monograph by Thomas D. Luckey entitled Germfree Life and Gnotobiology was published, with a focus on animals treated with microbes and reference to the work of Louis Pasteur (1822–1895). Here, we review the history and current status of plant gnotobiology, which can be traced back to the experiments of Jean-Baptiste Boussingault (1801–1887) published in 1838. Since the outer surfaces of typical land plants are much larger than their internal areas, embryophytes “wear their guts on the outside.” We describe the principles of gnotobiological analyses, with reference to epiphytic metylobacteria, and sunflower (Helianthus annuus) as well as Arabidopsis as model dicots. Finally, a Californian field experiment aiming to improve crop yield in strawberries (Fragaria ananassa) is described to document the practical value of this novel research agenda.

Introduction

Recently, the concept of a “microbe-human superorganism” has been introduced, and the general idea of bacterial symbioses in animals and plants is discussed extensively in the biomedical literature.¹ However, the key term in this area of research, gnotobiology, is only occasionally used in the context of botanical sciences, despite the fact that Julius Sachs (1832–1897) had integrated bacteria into the emerging discipline of experimental plant physiologiy.² In this Addendum we describe the origin and significance of gnotobiology, with a focus on plants, and outline possible practical applications of this innovative research program.

Biologists have long known that all eukaryotic organisms, animals, plants, humans, have evolved and are adapted to life in a microbial world.³ Based on this insight, the first experimental protocol describing how laboratory animals could be reared in the absence of bacteria (i.e., under germ-free conditions) was published in 1896 (see ref. 4 a monograph of T. D. Luckey 1963, entitled Germfree Life and Gnotobiology).

During the 1950s, this experimental strategy for analyzing the relationships between bacteria and their hosts, notably the addition of specific microbes to germ-free multicellular organisms, developed into a new scientific discipline, termed gnotobiology (from the Greek “gnosis”, i.e., knowledge, and “bios”, i.e., life).⁵

Historically, the basic idea of gnotobiotic experimental analysis is credited to the work of Louis Pasteur (1822–1895). In 1885, the French chemist speculated that, upon the complete removal of microbial associates, animal life may become impaired or even impossible⁴ (see ref. 6 for recent examples of animal gnotobiotic research, such as the analysis of the mutualistic insect [aphid]-bacteria [Buchnera sp.]-symbiosis).

However, one of the most important recent discoveries of animal gnotobiologists is the finding that the large internal surface of the gut of mammals, such as mice (Mus musculus) and humans (Homo sapiens) contains numerous beneficial bacteria of known taxonomic status.¹ These prokaryotic “gut microbes” are co-evolved microbial partners, or intestinal symbionts, that drastically improve the “quality of life” of their corresponding host organism.^6,7 Hence, Pasteur's hypothesis has been confirmed, at least with respect to aphids and mammals.^1,6

Origin of plant gnotobiology

In the shadow of this anthropocentric research that is significant for human health, plant gnotobiology developed. This branch of the botanical sciences can be traced back to the work of the French chemist and agricultural scientist Jean-Baptiste Boussingault (1801–1887). In 1838, he performed a set of experiments by transplanting legumes in sand that had been sterilized, but did not contain any nitrogen. Boussingault observed that the legumes continued to grow under germ (and N)-free conditions (Fig. 1A). Accordingly, he concluded that these plants fix atmospheric nitrogen via an unknown mechanism. Based on this finding, the French chemist discovered that, following the growth of legume crops such as peas (Pisum sativum) (Fig. 1B), an increase in soil nitrogen (N) occurrs.⁸ Decades later, it was discovered that root modules that contain N₂-fixing bacteria (Rhizobium sp) are responsible for this positive effect on the nutrition of crop plants.^8,9

Figure 1.

Scheme illustrating the key experiment of J.-B. Boussingault of 1838, performed with pea (Pisum sativum) plants that were raised in sterile sand (A). O = sand without mineral salts; KP = sand with potassium and phosphate; KPS = sand with potassium, phosphate and nitrate. The root system of a pea plant (B) is characterized by numerous nodules that contain nitrogen-fixing bacteria (adapted from ref. 9).

As mentioned above, Julius Sachs, who referred to the work of Boussingault, was the first to consider bacteria as plant-associated microbes.^2,10 Since the outer leaf surface of most land plants (embryophytes) is large, whereas the internal areas, such as the lumen of the vascular bundles and the intercellular spaces, are small, green, sessile organisms “wear their guts on the outside.” The aerial parts of green plants, notably the leaves, are colonized by bacteria at densities of up to 10 million microbes per cm² (refs. 11, 12). Recent studies with germ-free (gnotobiotic) land plants (bryophytes, angiosperms etc.) have shown that many of these epiphytic prokaryotes, such as methylobacteria, secrete hormones (auxin, cytokinines), and hence are beneficial to their sessile host. Accordingly, they have been classified as growth-promoting phytosymbionts^7,13 Hence, not only animals, but also plants are superorganisms or chimeras composed of pro- and eukaryotic cells. Green plants benefit from their outer coat of “hardworking” microbial partners and are, like humans, characterized by a meta-genome. In addition to epiphytic microbes, endophytes may also be importance as symbionts of green plants. This topic is beyond the scope of the present article.

Experimental analysis of plant-associated bacteria

In order to explore the interconnected networks of microbes that inhabit the outer surface of land plants (embryophytes), 2 experimental approaches have been employed. First, root-, stem- and leaf-associated bacteria are isolated from crop plants, such as seedlings of sunflower (Helianthus annuus) (Fig. 2A), a species in which no endophytic bacteria were detected (L. Doerges and U. Kutschera 2014; unpublished). In the next step, the microbes are grown on agar plates and characterized, based on their morphology, metabolic activities, and taxon-specific rRNA-sequences.^2,7,11,13 However, this cultivation-dependent analysis cannot provide a complete picture of the surface-associated microbiome.¹⁴

Figure 2.

Principle of plant gnotobiology. Sunflower (Helianthus annuus) seedlings emerge from the soil under real-world (non-sterile) conditions (A). Within a few days, the germ-free embryo develops into a juvenile plant. All organs (root, shoot) are colonized, via the seed coat, by epiphytic microbes (B, C). Using sterile sunflower seeds (i.e., achenes) raised in a germ-free environment, a gnotobiotic reconstitution system can be established to investigate the role of specific bacterial strains on organ growth in vitro (cultivation in a germ-free glass jar). Ba = epiphytic bacteria, Co = cotyledon, Ep = epidermis, Sc = seed coat, St = stomatum.

In order to gain a deeper insight, high-throughput genomic sequencing has been employed to explore the total gene content of the heterogeneous epiphytic microbial community.¹⁵ Using Thale cress Arabidopsis thaliana as model dicot,¹⁴ Bai et al. have identified 7943 “colony forming units” (i.e., bacterial isolates) from the leaves and roots obtained from plants grown in soil. Using a gnotobiotic Arabidopsis-system (i.e., colonization of germ-free plants with synthetic microbial assemblages), Bai et al. were able to characterize the prokaryotic communities that established themselves on sterile plant surfaces.¹⁵

Three major conclusions emerged from this seminal study.¹⁵ First, in contrast to microbes that live in the soil or aquatic environments, most plant-associated bacteria are readily cultivated. This culturability may be due to the fact that roots and leaves are oxygen-rich habitats that provide nutrients for their prokaryotic epiphytes. Second, Bai et al.¹⁵ documented an extensive taxonomic overlap between the microbiota of leaves vs. roots, a finding that is consistent with a previous study on field-grown sunflower plants.¹⁶ Third, the gnotobiotic reconstitution system, using germ-free host plants that were inoculated with known microbes, yielded the surprising result that microbiome assembly in vitro was similar to that found on plants grown in the wild. Taken together, these novel insights form the basis for a rational approach to understand the role of the microbiome for plant health, organ growth and agricultural productivity of entire communities.

Agricultural experiments: Basic and applied gnotobiology

Practical applications of plant physiology, with respect to crop yield, were envisioned by Sachs in the 19th century.^2,10 More recently, the principle of gnotobiology has been employed in field experiments. In 2015, I-Cultiver, a consortium of agricultural and food science experts, conducted a 9-month long time-course analysis with commercial strawberries (Fragaria ananassa) grown in California. The strawberry beds (Fig. 3A) were treated with a liquid product composed of a broad spectrum of microbes and soluble carbon (“Vesta”). As a good source of nutrients, strawberries are a valued crop for their antioxidant and anti-inflammatory properties. Strawberries also provide healthy doses of Dietary Fiber, Vitamin C, Manganese, Folate and Potassium. It was hypothesized that microbial activities in the soil and on roots mobilize nutrients, and make them available for uptake by the plant. The investigation revealed that the treatment significantly and dynamically modified the microbial community structure. Surprisingly, the treatment reduced the soil microbiome diversity (D. Coleman-Derr 2016; unpublished), possibly through competition for nutrients and resources, effectively replacing the potential for establishment of disease-causing pathogens with the beneficial biota. There was an enhancement in root growth, increased water content in roots over time, and enhanced nitrogen and carbon (per dry weight) assimilation in the strawberry leaves (T. Raab 2016; unpublished). These results (Fig. 3B) indicate that plant-associated bacteria (Fig. 2B, C), as well as other microbes,¹⁷ influence nutrient flux between the soil and the root through microbial activity.

Figure 3.

Agricultural field of strawberries (Fragaria ananassa) in California, 2015 (A). Photograph of representative field-grown strawberry plants that were either treated with a cocktail of microbes (bacterial biofertilizer “Vesta”) or raised in standard soil as control. Note that, after manufacturer's recommended treatment, the “Vesta-strawberries” are larger than the untreated plants (B).

Further studies are needed to fully understand the biochemical relationships between the plant and attached microbial partners. The agricultural biologicals industry is growing at fast pace to explore novel plant-microbe activities with the goal of developing environmentally sustainable Biopesticides, Biostimulants and Bacterial Biofertilizers.¹⁷

Conclusions and outlook

In summary, we conclude that the principle of plant gnotobiology is of great value as a novel research agenda for the study of host-microbe-associations, reaching from the analysis of symbiotic relationships to pathogenic interactions.^15,16 Moreover, it represents an emerging branch of botany with an enormous potential for the improvement of crop yield and quality.¹⁷ In 1836, J.-B. Boussingault established the first Agricultural Experiment Station in France, and similar institutions were subsequently set up in Germany and other European countries.^2,10 One hundred and 80 y later, the legacy of the founder of plant gnotobiology is still alive and well (Figs. 2, 3).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the Alexander von Humboldt-Foundation, Bonn/Germany (AvH-Stanford 2013/14 to U. K, Institute of Biology, University of Kassel, Germany). The strawberry field-study was supported by the manufacturer of the soil amendment “Vesta” to pursue an independent scientific investigation conducted by I-Cultiver, Inc. (Livermore/Albany, California, USA).