The Plant Microbiome

One of the most exciting fields of research is uncovering the ancient relationship between microbes and plants. Communities of microbes live around, on and inside plants. Composed of bacteria, fungi, viruses and nematodes, this consortia of microorganisms play several crucial roles in the maintenance of ecosystem function such as the cycling of carbon and nutrients as well as sustaining plant growth.

Similar to how crucial the gut microbiome is for maintaining human health, the nuances of plant-microbe interactions have many direct and indirect benefits for plants. This fascinating bidirectional communication between benevolent microbes and plants is known to:

• boost plant growth and improve root architecture through cycling and delivery of nutrients.
• manipulate the hormonal signaling of plants.
• trigger defense responses and protect against malevolent invading pathogens.
• increase protection against herbivory.
• protect against abiotic stresses such as changes in temperature and rainfall.
• repel or outcompete pathogenic microbial strains.

The earliest evidence of the ancient alliance between plants and microbes comes from 400 million year old fossils capturing the presence of arbuscular (branched finger-like hyphae) fungal structures inside plants.

Their alliance is as ancient as plants themselves with molecular evidence pointing to fungal associations with green algae being fundamental to the evolution of land plants around 700 million years ago [1].

Unfortunately soil degradation, land management practices and changes in climate patterns are threatening the integrity of the plant microbiome.

To protect and stimulate the resurgence of the microbiome, future agricultural practices will need to encompass the microbial environment surrounding the plant and not consider the plant as an organism in isolation.

Uncovering the multifactorial roles of microbial association with plants, the biochemical and hormonal signalling pathways involved and how microbes confer advantages to plant growth is a pandora’s box of possibility for engineering agricultural and environmental security.

What is the microbiome?

The plant microbiome is a holiobiont – an assemblage of the plant and all the microorganisms living around, on or inside it. Together the plant and its encompassing microbes form one ecological unit.

In addition to the taxa present, it consists of the bidirectional communication between microbial metabolites and the substances secreted by plants.

Plant root secretions are called exudates and encompass a wide variety of metabolites which adjust and influence the microbiome in the rhizosphere (on and around the plant roots).

The root system deposits up to 40% of the plants photosynthetically fixed carbon into the rhizosphere [2], making this thin zone around the roots one of the most energy rich habitats on Earth [3].

The composition of the microbiome is determined by extrinsic factors (soil conditions, climate, land management) and intrinsic factors (vertical transfer through seeds, plant characteristics, plant organs, and plant–microbe interactions).

A plant’s microbial community is considered an asset for survival as microbes can protect plants against:

• Biotic threats (pests and pathogens) and 
• Abiotic stresses (extremes in temperature, drought, chemical contaminants).

Some interesting facts we now know about the plant microbiome:

• Signalling or cross-talk between above-ground parts of the plant with the rhizosphere is extensive and influences the microbiome composition.
• Plants are able to cry for help under attack and selectively attract beneficial microbes to come to their aid [4].
  • Berendsen et al., 2018 inoculated Arabidopsis leaves with a downy mildew pathogen and measured increased numbers of Microbacterium, Stenotrophomonas and Xanthomonas sp. in the rhizosphere.
  • Verbon et al., 2017 viewed an increased resistance to the downy mildew pathogen in Arabidopsis leaves after manually adding these bacteria to the soil.
  • Plants grown in the same soil had reduced susceptibility to the downy mildew disease in successive generations.
• Drought shifts the composition of the plant microbiome, especially inside the plant [5].
• Microbes secrete antifungal metabolites which confers resistance against pathogenic fungi.
  • Pseudomonas fluorescens produces the antifungal compound diacetylphloroglucinol (DAPG)
• Plants coordinate the performance of microbes to strategically deal with either pathogen attack, nutrient deficiencies, or abiotic stresses such as heat or drought.

Plant mineral nutrition

In the soil, nutrients such as Nitrogen (N), Phosphorous (P), and Sulfur (S) are tied up in organic molecules and are not readily available for plant absorption [6].

Plants depend on microbes which possess the metabolic machinery to depolymerize and mineralize organic forms of N, P, and S.

Microbes release this mineral content through turnover of metabolism and cell lysis, or via protozoic predation.

Through these processes, microbes convert N, P and S into the bioavailable ammonium, nitrate, phosphate and sulfate – a plant’s desired nutrients.

Anatomy of THE plant microbiome

The microbes occupying the plant microbiome can be broadly categorized into 2 classes:

• Epiphytes live on or around plant structures.
• Endophytes occupy the internal tissues of the plant.

The plant microbiome space is subdivided into 3 zones:

• Rhizosphere – the thin region of soil influenced by plant roots.
• Phyllosphere – plant aerial surfaces.
• Endosphere – the internal tissues of the plant (also called endorhizosphere).

Each of these are unique environments with varying microbial compositions.

Rhizosphere

The rhizosphere, or plant-root interface, is the narrow band of soil surrounding the roots. The rhizosphere can be further subdivided into the:

• Endorhizosphere – the parts of the cortex and endodermis where the presence of microbes and cations occupy the apoplast. The apoplastic space occurs between cells.
• Rhizoplane– the zone directly adjacent to the root which includes the root epidermis and mucilage.
• Ectorhizosphere– the extension of soil outwards away from the roots.

In the rhizosphere, microbial community structures are very inconsistent in place and time. This is due to the gradient of exudates released along the root structure.

Inconsistency is also driven by factors such as soil type, soil moisture and geographical location as well as differences in plant species, plant genotype and developmental stage.

Root exudates encompass a wide variety of chemical compounds, predominantly organic acids and sugars, but also also amino acids, fatty acids, vitamins, growth factors, hormones and antimicrobial compounds [7].

Other components of rhizodeposition include a sloughing (shedding) of root cells, a release of mucilage and the plant cell poylmers cellulose and pectin.

In this dark and damp environment, plant roots provide carbon to the rhizosphere microbes for energy as well as a structure onto which microbes can attach.

The ability to degrade cellulose is widespread among microbial residents in high-organic-matter soils and results in nutrient acquisition from this polysaccharide. Decomposition of pectin releases methanol which can be used as a carbon source by other types of microbes.

Rhizosphere composition

Studies of rhizosphere microbiomes have revealed remarkably similar distributions of microbial phyla which makes me wonder if they can be similarly grouped into enterotypes as found in the gut microbiome.

The Proteobacteria dominate rhizosphere samples, especially the α and β classes. Other major groups present include Actinobacteria, Firmicutes, Bacteroidetes, Planctomycetes, Verrucomicrobia and Acidobacteria.

Of particular interest in the rhizosphere are plant-growth-promoting rhizobacteria which act through a variety of mechanisms.

Plant growth promoting rhizosphere microbes have been grouped under the umbrella terms:

• PGPR – Plant growth promoting rhizobacteria.
• PGPF – Plant growth promoting fungi.

A lot of what is known about symbiosis and plant growth promoters in the rhizosphere comes from studying:

• nitrogen-fixing symbioses between Rhizobium and legumes and
• the mutualistic partnership between mycorrhizal fungi – ectomycorhizal (EM) and the arbuscular mycorrhizal (AM) fungi of the model organism Arabidopsis, grasses and trees.

Nitrogen fixing bacteria

Nitrogen-fixing bacteria, including those that are free-living (such as Azotobacter spp.) and symbiotic (such as root-nodulating Rhizobium spp.)

• provide a source of fixed nitrogen for the plant,
• can increase bioavailability of phosphorous-containing minerals.

Arbuscular mycorrHizal Fungi

Arbuscular mycorrhizal (AM) symbiosis is one of the most widespread mutualisms on Earth, occurring between soil fungi and the roots of 71% of all vascular plant species .

It is estimated that there are ~50,000 fungal species forming mycorrhizal symbioses with ~250,000 plant species [8].

In AM host plants it increases their exploratory capacity for water and mineral nutrients, triggers changes in root architecture, growth, development and stress tolerance.

In return the plant supplies the fungus with lipids and sugars, at a price of up to 20% of the carbon fixed by photosynthesis [9].

Measuring MicrobIAL density

The density of microbes living within the plant microbiome is measured in CFU (colony forming units).

CFU is estimated by making varying dilutions of the sample (usually factors of 10), growing the microbe dilutions on a petri dish medium (with a number of replicates for each dilution), and then using a program such as OpenCFU to count the number of colonies per photographed plate.

In the rhizosphere:

• 10¹¹ cfu of prokaryotic cells comprising more than 30,000 species.
• 10^7 cfu of fungi per gram of fresh root.

Rhizoplane:

• 10^7 cfu per gram of fresh material were calculated.

Phyllosphere

The phyllosphere, comprising the aerial surfaces of the plant, is a more dynamic environment than the rhizosphere. It is subject to extremes in temperature, moisture, radiation, precipitation and wind over the course of night and day. These abiotic factors also indirectly affect the phyllosphere microbiome through changes in plant metabolism.

Compared to the rhizosphere, the phyllosphere is nutrient poor and accommodates less microbes. Leaf structures are colonized by up to 10^7 microbes per cm2.

At high microbial taxonomic levels, phyllosphere microbiomes of different plants can seem similar, but at the species and strain levels differences in composition are dramatic, reflecting the selection pressures of living in the phyllosphere [10]. 

Soil microbiome activity affects the Phyllosphere

Interestingly, experiments have shown that cross-talk or communication occurs between the aerial and root parts of a plant where leaf metabolite profiles of Arabidopsis were altered by the addition of soil microbes to the roots.

In the 2013 paper Potential impact of soil microbiomes on the leaf metabolome and on herbivore feeding behavior scientists investigates how different soil microbiomes affect plant growth and the leaf metabolome (collection of all plant and microbial metabolites).

They collected soils from 11 locations and 4 overstory (aerial layer of plant foliage) conditions from Arabidopsis, pine, corn and potato. They replicated the types of soils (i.e. 4 potato soils from different locations) and analyzed their respective nutrient compositions for pH, total carbon, total nitrogen, nitrate, phosphate, potassium, zinc, iron, copper, manganese, and boron.

They grew Arabidopsis plants to 14 day old seedlings from sterilized seeds. To these seedlings soil slurries were added (50g of each previously collected soil type with additional 250ml Hoagland solution). Controls had sterilized soil which removed the majority of microbes present.

2 weeks after, 4 week old plant shoots and roots were harvested for biomass analyses. After processing, the compounds present were separated and identified using GC-MS and were grouped into the categories – amino acids, phenolics, sugars, sugar alcohols and unknown.

Significant differences in leaf metabolites were observed between experimental pots and controls.

They found the difference in root and shoot biomass was more significant in the managed corn and potato soils compared to controls than the unmanaged pine and Arabidopsis soils (P=0.012) and that the soil mixture nutrient composition didn’t vary dramatically between collected soils and microbe sterile control soils.

Pyrosequencing analysis found soil microbiomes clustered by plant overstory type, consistent with previous studies showing variations in microbiome bacterial communities depending on soil type, plant/crop type and geographical location.

By adding insect larvae to the leaves of the Arabidopsis plants and then measuring their weight after 1 day of feeding, they found reduced insect herbivory in plants pots with the collected soil compared to the control. This was especially evident in all 4 potato soils and both corn soils suggesting a beneficial and significant effect of their microbial community in preventing their leaves being eaten by enhancing resistance to herbivory.

Larval weight was positively correlated with leaf content of b-alanine (r = 0.804), cytosine (r = 0.731) and L-phenylalanine (r = 0.719).

These findings suggested a beneficial role for the endogenous microbes present in the managed crop soils in both protecting against insect herbivory and stimulating plant growth, as well as proving the microbes in the soil influence the leaf metabolome.

Phyllosphere composition

Bacterial and fungal communities in the phyllospheres of various plants have been profiled using PCR amplification of rRNA genes.

Microbial richness seems to be greater in warmer, more humid climates than in temperate ones. 

Similar to the rhizosphere, Proteobacteria (the α and γ classes) are consistently the dominant bacterial phylum, with Bacteroidetes and Actinobacteria also commonly found.

Proteogenomic analyses of various phyllosphere microbiomes have revealed species that assimilate plant-derived ammonium, amino acids and simple carbohydrates (pectin), implicating these compounds as the primary nitrogen and carbon sources in the phyllosphere. 

The presence of microbial rhodopsins revealed by metagenomic analysis suggests that the bacteria in the phyllosphere may be phototrophic – capable of capturing light for energy – and are not entirely dependent on the host plant for sustenance [11].

ENDOSPHERE

Endophytic bacteria reside inside plant tissues for at least a portion of their lives.

Remember I previously wrote about endosymbiosis and how bacteria entered eukaryotic cells and eventually took up residence in there, adapting to their host’s cellular environment and evolving some form of symbiosis?

Well I just learned that about the existence of modern endophytic bacteria inside plants! These bacteria represent an intersection of coevolution with plants that are performing archaic symbiotic functions.

Many endophytic bacteria belong to the previously discussed group of Plant Growth Promoting Rhizobacteria (PGPR) since they can stimulate plant growth either directly or indirectly by suppressing plant pathogens.

Directly they can promote plant growth by providing plants with nutrients/substrates (e.g., phosphorous, nitrogen and iron) and producing various plant hormones [12]. Indirect benefits come from their blocking or antagonism towards pathogenic bacteria.

I have many questions. What is their role inside the plant cells? Are they parasitic or beneficial to the host cells? Can they travel from the soil into plant cells and back to the soil? What are the mechanisms allowing them entry into plant cells? How do they remain undetected by the host immune response?

I gleamed some answers from this excellent review. Let’s dive in.

Endophytic bacteria are non-pathogenic and occur at much lower densities than their root and leaf dwelling partners, usually ~10^3 cfu. High concentrations of endophytes over 10^8 cfu per gram can trigger the host’s defense response.

Endophytic bacteria can be categorized into 3 groups based on whether they require plant tissue to live and reproduce:

• obligate – cannot survive in soils, must colonize plant cells.
• facultative – widely exist in soil, colonize and infect plant cells under certain conditions.
• passive – lack the inherent ability to colonize or infect cells, enter cells through cracks and wounds on the plant.

They are thought to be a subset of the rhizospheric microbiome, possessing special characteristics which grant them access inside the root.

In this way, plant roots act as ‘gatekeepers’, screening soil bacteria from the rhizosphere and the rhizoplane, with only an exclusive number being granted access inside.

Colonization patterns

Some obligate bacteria may have evolved the capacity to enter root tissue, while other passive bacteria can gain access inside through natural cracks/breaks in the roots and root tips.

More endophytes are found in roots than aerial parts of the plant but their presence in xylem vessels suggesting they travel upwards from the roots to the leaves through transpiration.

Once inside the roots, endophytes colonize the apoplast and dead or dying cells.

Confirmation that certain bacteria are true endophytes must come from microscopy as there are many nooks and crannies on the surface of a plant non-endophytes can protect themselves in even after surface sterilization.

Endophytic bacteria are dominated by the classes:

• Proteobacteria (∼50% in relative abundance),
• Actinobacteria (∼10%),
• Firmicutes (∼10%) and
• Bacteroidetes (∼10%).

ENDOPHYTE characteristics

Distinctly different from the root clinging microbes, among the unique traits conferring them adaptability to the plant’s interior are:

• Motility.
• Chemotaxis.
• Production of cell-wall degrading enzymes.
• Lipopolysaccharide formation.
• ROS scavenging enzymes.

Endophytes may alter their gene expression when colonizing plants. Genes encoding proteins related to bacterial motility, chemotaxis and adhesion that were induced in Burkholderia kururiensis M130 in the presence of rice plant extracts (Coutinho et al., 2015). 

Motility

The bacterial flagellum is a potent microbe-associated molecular pattern (MAMP). When fragments of a flagellum’s molecular signature are detected by host cells it induces a front-line response by the innate host immune system.

The flagellum may be key for endophytic colonization due to its ability to:

• enable movement.
• adhere and anchor to surfaces.

We know this from flagella-deficient mutant studies where Azospirillum brasilense‘s ability to colonize wheat roots were hindered due to decreased adsorption (adhesion) to wheat roots.

Genes encoding Type IV pili (TFP), the crucial virulence factor formed by pilin subunits, exist in the genome of endophytic B. phytofirmans PsJN bacteria.

Mutant analysis of Azoarcus sp. lacking TFP were not able to colonize rice roots, findings congruent to adherence being a crucial step for bacterial invasion of plants.

Cell wall degrading enzymes

Cell wall degrading enzymes:

• Break cell walls.
• Translocate compounds to the apoplast.
• Genes encoding plant polymer-degrading:
  • cellulases,
  • xylanases,
  • cellobiohydrolases,
  • endoglucanase,
  • cellulose-binding proteins

were detected in high copy numbers in the metagenome of rice root endophytic bacterial communities.

Oxalotrophy, the capacity of utilizing oxalate as a carbon source, is required for the successful colonization of B. phytofirmans PsJN on lupin and maize plants.

Oxalotrophy may be a unique trait of beneficial bacteria as plant pathogenic or human pathogenic species of the Burkholderia genus are not able to use oxalate.

Lipopolysaccharide formation

An ancient arms race between plants and bacteria has lead to plants developing a highly sophisticated surveillance system to detect signature molecular patterns such as flagellin, chitin, lipopolysaccharides and other glycans or endotoxins occurring endogenously on bacterial surfaces.

This surveillance detection system consists of finely-tuned receptors on plant cell surfaces primed to recognize a wide array of MAMPs (microbe-associated molecular patterns). Structurally homologous to the Toll-like receptors expressed on human immune cells which recognize PAMPS (Pathogen-associated molecular patterns) and DAMPS (Danger-associated molecular patterns), these highly conserved PRR (Pattern recognition receptor) defense systems among species represent the age-old battle between pathogens and eukaryotes.

Endophytic bacteria are thought to produce their own MAMPs which do not significantly trigger plant immune responses.

They also lack the T3SS and T4SS injector systems (the common machinery for injecting effector proteins inside host cells via the pilus). Bacteria with T3SS and T4SS systems stimulate effector-triggered immunity (ETI) upon releasing effector proteins into the host cell.

Since endophytic bacteria can be considered as potential invaders it is important for them to evade triggering a systemic response or to neutralize the immune after-effects.

ROS-Scavenging

When a bacteria enters a plant this cues the plant’s defense response resulting in reactive oxygen species (ROS) generation. A high number and diversity of genes encoding ROS-scavenging enzymes such as superoxide dismutase (SOD) and glutathione reductase (GR) are represented in the metagenome of the endophytic bacterial communities in rice roots (Sessitsch et al., 2012). ROS-scavenging and sequestering is likely a trait of endophytes to buffer the immune response.

Plant-hormone regulated bacteria

A plant’s finely-tuned hormonal system, namely the salicylic acid and jasmonic acid pathways form the backbone of the plant immune signalling network [12].

Emerging evidence reveals that beneficial root-inhabiting microbes hijack the hormone-regulated immune signaling network to establish a prolonged mutualistic association.

Agricultural and commercial benefits

I hope you enjoyed this quick forage into the amazing world of plant microbiomes. The human microbiome project is a brand new field encompassing around 10 years of research. In that short time much has been done to illuminate the importance and signification of gut bacteria for human health.

Similar to how knowledge about the human microbiome has revolutionized medicine I sense there will be a similar shift in agriculture and sustainable practices. Sentiment among farmers is changing. Many are moving towards permaculture and organic farming practices upon understanding the importance of maintaining a strong plant microbial alliance.

The future path will be to understand the microbiome in situ to discover gene expression and metabolic networks in relation to the microbial environment in time. This is called the metaphenome (product of the metagenome and the environment).

Global soil sampling and genomics analyses to add to the growing databases of plant microbes will be essential.

Manipulation of plant microbiome could help to: 

• Overcome antimicrobial resistance in crops.
• Reduce plant disease.
• Restore ecosystem function.
• Increase agricultural production.
• Reduce need for chemical inputs or pesticdes.
• Reduce greenhouse gases by increasing the cycling of carbon.
• Increase sustainability of agriculture practices.
• Discover new antibiotic compounds or useful microbial metabolites.