Microbial Inoculum Development for Ameliorating Crop Drought Stress: A Case Study of Variovorax paradoxus 5C-2

Introduction

Drought affects plant hormonal homeostasis and root-to-shoot signaling.
Plants have a close relationship with soil microbes, including plant growth-promoting rhizobacteria (PGPR), that modulate plant hormonal homeostasis.
PGPR often shows promise in greenhouse experiments, but field applications are less predictable.
A model for successful field application of PGPR is reviewed, using Variovorax paradoxus 5C-2 as an example.
The world faces a water crisis due to unsustainable water use and altered hydrological cycles.
Climate change will increase water demands in agricultural regions.
Food production needs to increase by 50% to meet demands, potentially exacerbating freshwater loss.
Changes in agricultural practices are necessary, based on scientific research and collaboration.
Classical plant breeding has limitations in developing drought-tolerant crops.
Drought tolerance is controlled by complex multi-gene systems that are difficult to manipulate.
There are logistical challenges in producing GM crops due to variations in drought-tolerant genes across species.
PGPR can enhance crop fitness under drought conditions by interacting with plant root systems.
This review focuses on using PGPR to improve crop performance under drought stress.
Variovorax paradoxus 5C-2 is presented as a case study to match agricultural scenarios with successful PGPR activity.
Plants respond to low soil water status (below -0.05 MPa) by changing root metabolism.
Plants produce the volatile hormone ethylene in response to abiotic stresses.
Elevated ethylene inhibits growth, photosynthesis, and seed set, decreasing yield.
Ethylene promotes fruit ripening but accelerates leaf senescence and inhibits leaf growth.
Ethylene induces lateral cell expansion in stems and roots and increases root hair density and length while inhibiting primary root growth.
Manipulating ethylene levels in plants is an attractive biotechnological target.

ACC as a Key Inter-Kingdom Signal Molecule

Ethylene biosynthesis in terrestrial plants involves the methionine metabolic pathway (Fig. 1).
The conversion of 1-aminocyclopropane-1-carboxylate (ACC) to ethylene is a rate-limiting step.
ACC can be converted to malonyl-ACC (M-ACC), γ-glutamyl-ACC (G-ACC), and jasmonoyl-ACC (JA-ACC).
Plant and microbial enzymes (ACC deaminase, gene notation acdS) can transform ACC into 2-oxobutanoate and ammonia.
ACC integrates plant and microbial metabolic networks, linking root-to-shoot signaling and drought adaptation.
Microorganisms inhabiting the root surface can influence root-to-shoot signaling processes.
PGPR producing ACC deaminase can modulate root ACC levels and ethylene evolution.
Plants exposed to bacterial ACC deaminase in drying soil develop longer root systems and increased biomass and yield.
Wild barley plants in stressful environments have a higher proportion of ACCd-containing rhizobacteria.
Withholding irrigation from wheat plants increases the abundance of acds-containing rhizobacteria.
Understanding the regulation of PGPR recruitment can enhance plant drought tolerance.
Agricultural practices and climate warming reduce soil biodiversity, affecting ecosystem services.
Richness and diversity in soil communities are important for inducing plant drought fitness.
The rhizosphere is the soil adjacent to roots, influenced biologically, chemically, and physically by the root system.
The rhizosphere is enriched with certain soil microbes compared to bulk soil.
qPCR and Illumina sequencing show that plants preferentially recruit acdS alleles from bulk soil.
Impaired microbial biodiversity may compromise plant selection of acdS alleles.
Understanding rhizosphere microbial dynamics is crucial for PGPR application in drought-stressed crops.

Characteristics and Dynamics of the Plant Holobiont

Each plant species and its habitat have a unique microbial consortium, the microbiota.
The microbiota's genetic information is the microbiome, and the combination of host and microbiota is the 'holobiont'.
Co-evolution coordinates metabolic processes, increasing holobiont fitness.
The 'hologenome' is the aggregate of microbiome and host genomes.
Integration of genetic material from diverse organisms forms superorganism genetic systems.
Altering the holobiont composition may affect inter-kingdom signaling, changing host fitness/growth.
High-throughput sequencing shows consistent repetition at the phylum level in rhizospheric microbiota.
Bacteria comprise 90% of the holobiome, with dominant phyla including Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Verrucomicrobia, and Acidobacteria.
Different bacterial species occur even between different plant cultivars in the same soil.
Bacterial population dynamics are influenced by the environment, like soil water availability.
Drought-affected soils are enriched with desiccation-tolerant bacteria.
Spore-forming phyla Actinobacteria and Firmicutes are drought-resistant.
Verrucomicrobia are drought-tolerant but not yet described as having ACC deaminase activity.
Drought-stressed plants select for the occurrence of acdS alleles in their rhizosphere.
Isolating and culturing free-living PGPR containing the ACC deaminase gene is of interest.
When developing microbial inocula for agriculture, it is crucial to understand the factors influencing PGPR activity in agricultural ecosystems to mitigate plant drought stress responses.

Challenges of Using PGPR to Alter Hormonal Dynamics in Planta

Soil inoculation with PGPR to promote crop yields has shown promising results.
PGPR techniques are environmentally friendly, with positive or null impacts on rhizospheric community dynamics.
PGPR application is generally accepted by farmers due to the history of using rhizobacterial inoculants.
Most PGPR products are marketed for bio-control (70%) and bio-fertilizers (25%), with only 5% for enhancing plant stress tolerance.
PGPR inoculation sometimes fails under field conditions due to diminished inoculum fitness in competition with native microbes.
Successful inoculation requires that the soil supplies minimum bacterial physiological requirements to grow and colonize the appropriate niche: the rhizosphere, rhizoplane or plant root cells.
Three major environmental components drive holobiont assemblage in agricultural plants.
- Soils determine physical and chemical characteristics, via structure, porosity, texture, nutrients, and pH.
- Plant genotype governs the quantity and quality of nutrients and chemical signals accessible to the microorganism.
- Agricultural practices/field history (e.g. application of herbicides and soil amendments such as lime, manure and microbial inoculants) will affect microbial populations and community diversity.
An example PGPR bacterium, Variovorax paradoxus, is used to demonstrate how environmental components affect microbial inoculation success.
This species shows valuable traits such as osmotic stress resistance, resistance to some biocidal compounds applied in agriculture and high competitiveness.
The genetic diversity of V. paradoxus was probed by studying the pan-genome, the total collection of genes within the genomes of seven V. paradoxus strains, which contains an average of 6000 genes (Table 1).
The pan-genome of V. paradoxus strains shares 3400 genes in common (the core genome), but also has an average of 2000 genes shared by some strains (the variable or accessory genome), and an average of 65 unique genes for each strain.
The percentage of genes covering functions in metabolism of amino acids, carbohydrates, lipids, xenobiotic biodegradation and motility are shared by both core, variable and unique pan-genome (Fig. 2).
Pan-genomes and core genomes of V. paradoxus strains are functionally linked to metabolic versatility and plant growth promotion traits.
Multi-drug resistance features present in V. paradoxus confer pesticide-antibiotic cross-resistance, implying the organism can adapt to different environmental constraints.
V. paradoxus 5C-2 was first isolated from the roots of Brassica juncea L. Czern plants growing in soils contaminated with cadmium, based on its growth of ACC as a sole nitrogen source.
It is a facultative oligotrophic, Gram negative bacterium that can grow in both nutrient- poor and nutrient-rich media.
To compete, endure and settle in the rhizosphere, V. paradoxus 5C-2 displays quorum sensing (QS).
V. paradoxus 5C-2 may shape the bacterial population around the root as it can also degrade QS signals and utilize AHLs as a carbon source (A.A. Belimov, unpublished data).
The N-Acyl homoserine lactone lactonase (AIIA) activity of V. paradoxus provides “quorum quenching”, allowing the bacteria to degrade and grow on acyl-HSL signals.
V. paradoxus 5C-2 has multiple PGPR features including the synthesis of indole-3-acetic acid (IAA), the expression of ACC deaminase activity and enhancing nutrient uptake and partitioning in planta.
Successfully applying V. paradoxus 5C-2 as a soil inoculant will depend on its adaptive capacity to new, potentially hostile soil conditions, e.g. temperature, pH and water content, and competition with native organisms [48].
Ultimately, the physiological needs of the inoculum must match those it will encounter in the new environment.

Soil Temperature

Soil temperature is a crucial factor affecting rhizosphere dynamics.
Microbial activity rate doubles for every 10 °C rise in temperature until enzyme structure and cellular material are disrupted.
Temperature changes physical properties like surface adhesivity, affecting microbial motility.
Soil temperature depends on irradiation energy, soil reflectivity (albedo), rainfall, and topology, and it increases during drought events.
Soil temperature decreases with depth, which may influence the inoculation technique used in arid environments.
V. paradoxus 5C-2 is a mesophile microorganism, with optimal growth at 28 °C (Fig. 3A).
Maximum motility occurred at 30 °C, with no measurable motility at temperatures ≤ 15 °C or ≥ 37 °C (Fig. 3A).
Knowing the optimal temperature will help determine the correct ecosystem climate zone and time of the year to encourage its proliferation.
It will proliferate at spring-summer minimum soil temperatures averaging 18 °C, avoiding agricultural practices triggering temperatures above 28 °C.
Surface soil temperature can exceed 28 °C in crops grown in black plastic mulch, where V. paradoxus 5C-2 inoculation may be ineffective.
Drought events combined with high air temperatures trigger surface soil temperatures higher than 40 °C in lower latitudes.
Thermophile PGPRs, instead of mesophile, may be better able to establish following inoculation, depending on the method of inoculation.

Soil pH

Soil pH affects the availability of nutrients and metals, affecting microbial composition and biomass.
Bacterial species exhibit various tolerances to pH, with most growing in a range between pH 6 and 9, but generally growing best at neutral conditions.
pH effects on bacterial motility and fitness will mediate the rhizosphere bacterial community.
Alkaline pH (> 8.5) suppresses bacterial motility, while catabolic reactions that consume acids are favored at acidic pH (< 6.5).
Alkaline pH represses chemotaxis systems, perhaps due to their energetic cost.
Minimal growth of V. paradoxus 5C-2 occurred at pH 4 and 9, while growth and motility were maximal between pH 5 and 7 (Fig. 3B).
The optimal pH embraces the range recommended by agronomists (pH6-7) for good crop nutrition.
Highly leached tropical soils are often acidic, which mobilizes aluminium in the soil solution which can be toxic for plant growth and inhibits root elongation.
Since ethylene is involved in root growth inhibition under aluminum toxicity, the possibility that acdS-containing rhizobacteria such as V. paradoxus 5C-2 can overcome Al-mediated root growth inhibition merits further research.

Soil Water Availability

Soil water availability is one of the most influential factors that shape a microbial community.
Drought destabilizes the bacterial community network, changing the trophic network dynamics.
The community tends to reorganize their populations towards drought tolerant bacteria, like Verrucomicrobia and Alphaproteobacteria.
Soil drying decreases water potential, decreasing bacterial internal water potential.
Bacterial accumulation of solutes, or osmolytes, is one survival strategy to maintain a favorable water potential gradient, but leaves the bacteria riskily dependent on nutrient availability.
Drought-induced morbidity can result from damage to the outer cell membrane, which may explain the higher resistance of Gram-positive bacteria to desiccation.
Since V. paradoxus 5C-2 lives naturally in the rhizosphere, it can be exposed to a wide range of water potentials, which may be as low as −10 MPa.
To simulate the effects of soil drying on bacteria, glycerol was added to TSB medium to impose a range of osmotic potentials (-0.14 to −12 MPa).
There was no significant effect on V. paradoxus 5C-2 survival but the bacteria were not motile (Fig. 3C).
Motility steadily decreased from -0.17 MPa to -0.24 MPa, and bacteria were immobile below -0.54 MPa.
After cells were exposed to different osmotic potentials in liquid media for 12 h, motility tests were conducted to study whether V. paradoxus 5C-2 survives at low osmotic potential but loses its ability to resume motility (Fig. 3D).
Exposure to even the lowest osmotic potential tested (−12 MPa) did not diminish subsequent V. paradoxus 5C-2 motility.
Compared to some other bacteria, V. paradoxus 5C-2 seems more resistant to osmotic shocks.
In Rhizobium sp., the growth is minimal at −5 MPa while this osmotic stress had no effect in V. paradoxus 5C-2.
Lower osmotic tolerance of R. leguminosarum is correlated with its production of low molecular weight external polysaccharides (EPS).
While decreased rhizobial motility and growth at low soil water potentials may decrease nodulation frequency, co-inoculation with V. paradoxus 5C-2 partially alleviated negative effects of drought on nodulation.
Taken together, these data suggest that V. paradoxus 5C-2 survives drought conditions, and recovers its motility when a favorable water regime is re-established, which may assist rhizosphere colonisation following inoculation of plants grown in drying soil.

Plant Influences on the Microbial Community

Rhizosphere microbial activity can influence plant nutrient status while, vice versa, plants can also influence the physiology of microbial communities with their root exudates.
Root exudates vary from one species to another and are affected by plant age and nutrient status.
Plants may invest more than the 20 % of the total carbon fixed in photosynthesis in rhizosphere carbon efflux.
This can benefit the plant if root exudate composition ‘selects’ beneficial microorganisms that colonize the roots.
Soil porosity will affect the amount and distance which root exudates can travel, in turn affecting the chemo-attraction of the rhizobacteria.
Since bacterial response time, or detection limit for root exudates, may alter bacterial fitness, bacteria utilize chemotaxis to assess a cocktail of exudates.
Root exudation varies along the root surface, determined by different root cell characteristics, thus affecting rhizospheric chemical composition from tip to base.
Moving away from the root tip defines different zones such as root tip, with the root cap and the meristem, the elongation zone without any cell division, the maturation zone with differentiated xylem vessels and root hairs and finally the mature zone with dead root hairs (Fig. 4C).
For maize, root surface pH changed along the root axis.
Root tip pH was 7.6 and decreased linearly within the first 4 mm distance by 0.75 units and then increased linearly by 0.50 pH units.
Pseudomonas species preferentially colonise the root tip and the elongation zone of wheat.
Rhizobium leguminosarum and the fungal pathogen Nectria haematococca are attracted to the maturation zone of pea roots while Bacillus sp. preferentially colonize the Arabidopsis root elongation zone.
Confocal microscopy is often used to determine the location of fluorescently labelled bacteria on the roots of plants grown in artificial media in vitro.
The numbers and distribution pattern of V. paradoxus 5C-2 along the roots of both pea (Pisum sativum cv. Progress No. 9) and corn (Zea mays cv. ZB677) was measured, since inoculation increased growth of both species.
Colonization of pea roots was minimal near the tip and maximal near the base of the root where root hairs were present (Fig. 4A, C).
Colonization of corn roots showed no specific spatial distribution and was 10-fold lower than in peas (Fig. 4B).
Differences in the relative concentrations of organic components such as amino acids and sugars exuded from corn and pea roots were correlated with bacterial attraction to the roots of each species.

V. paradoxus 5C-2 Dispersal in a Soil Mesocosm

As with other bacteria, V. paradoxus can form biofilms on the plant root.
Bacterial dispersal occurs when they detach from the biofilm, switching to a planktonic lifestyle allowing new plant roots to be colonised.
The dispersal process is complex and can be regulated by nutrient status and QS signals.
It involves several steps such as the up-regulation of flagellar and chemotactic proteins and down-regulation of EPS.
Bacterial dispersal was tested in a mesocosm with field soil to determine bacterial movement in the rhizosphere and bulk soil.
When peas were grown in the mesocosm (Fig. 5), V. paradoxus 5C-2 moved gradually away from the initial zone of inoculation, such that after 30 days, it was recovered from pea roots over 50 cm distant from the initial site of inoculation (Fig. 5A).
In the absence of plants, it moved only 30 % of this distance and no change in movement was detected from 12 days after inoculation (Fig. 5B).
Since V. paradoxus 5C-2 has a high dispersal capacity by moving between pea roots, field experiments were designed with substantial buffer strips between inoculated and uninoculated plots to prevent contamination.
In other systems, runoff from sloping soil may risk cross-contamination of un- inoculated plots and should be considered in field trial design.

Field Application of a PGPR Inoculum

A field experiment was designed to understand the physiological requirements of V. paradoxus 5C-2 and determine the effects of V. paradoxus 5C-2 on pea (Pisum sativum) growth and development under soil drying conditions.
It was performed in a field situated at 53°50′44.42″N and 2°46′23.55″W (Lee Farm, Bilsborrow, Lancashire, UK).
The mineral content of this soil at this site was predominantly sandy (93 %), while silt and clay were 2.8 % and 4.2 % respectively.
The organic matter content of the soil was 7.7 % with a pH of 5.8 (water extraction).
The field site (13 m x 7 m) was covered by a semi-transparent plastic roof (“poly-tunnel”) to exclude rainfall and the irrigation system installed.
Soil moisture was monitored by an irrigation controller and data logger (Model GP1, Delta-T Devices, Burwell, UK) connected to soil moisture sensors (Delta-T Devices, Burwell, UK, SM-200 sensors).
Half of the 24 plots were randomly assigned for V. paradoxus 5C-2 inoculation.
During this field experiment, soil temperature at 5 cm depth varied between 15 °C (week 1, 01/09/2009) and 19 °C (week 5, 06/10/2009), while soil pH and water availability matched those optimal for V. paradoxus 5C-2.
Pea (Pisum sativum ‘Progress No. 9’) plants were germinated in seedling trays filled with an organic substrate (Levington’s M3, Levington, UK) in a greenhouse at the Lancaster Environment Centre.
The inoculation was done on the day of transplanting by submerging the peat blocks containing one seedling per block) in a bacterial solution 1 L, 10^{10} CFU. ml^{-1}.
At the field site, seedlings were transplanted on 01/09/2009 and well watered initially, then water was withheld (reaching soil water potential of −0.06 MPa at 20 cm depth) from half the plants 3 days after transplanting.
Shoot fresh weight was determined 21 and 47 days after transplanting.
Roots were also sampled at these times to determine V. paradoxus 5C-2 colonizing the rhizosphere, using selective agar growth medium containing the antibiotics rifampicin, kanamycin and streptomycin to which this strain is resistant.
Colonies that resembled authentic V. paradoxus 5C-2 colonies were counted, and samples were taken to spread again on selective medium containing ACC as a sole nitrogen source for confirmation that bacteria contained acdS.
While uninoculated plants were not colonized (since the plot arrangement reflected the results of Fig. 5), V. paradoxus 5C-2 root colonization decreased 20-fold by the end of the experiment (Fig. 6C).
Well-watered plants inoculated with V. paradoxus 5C-2 had 50 % more nodules than uninoculated controls (Fig. 6B).
Drying soil decreased nodule number by 30 %, but the addition of V. paradoxus 5C-2 restored nodule number to a level that exceeded control plants grown in well-watered conditions.
V. paradoxus 5C-2 inoculation increased shoot biomass by 23 % in well-watered soil (Fig. 6A).
Dryer soil significantly decreased shoot biomass of uninoculated controls by 22 %, but applying V. paradoxus 5C-2 increased biomass above that of the well-watered controls.
Thus in the field experiment V. paradoxus 5C-2 colonization in the roots was not adversely affected by drought, while drought decreased the number of nodules by 40 % (Fig. 6B).
These results were qualitatively similar to those obtained in greenhouse experiments, even though soil type (and thus likely indigenous bacterial community) was very different.

Multiple Plant Species Colonization

Considering the cost of developing microbial inoculants, an important question is whether they can successfully colonize, and promote growth of, multiple plant species.
Multiple studies have measured V. paradoxus 5C-2 colonisation of the root system in a range of substrates, with levels of root colonisation similar in pea and other species, and between plants that were grown in well-watered versus drying soil, using the technique described above.
Thus V. paradoxus 5C-2 is competitive in the rhizosphere of a range of species, and promoted growth of plants grown in vitro, pot trials and field trials .
In further field experiments, V. paradoxus 5C-2 promoted potato, Solanum tuberosum, shoot growth and tuber yield.
It can show specificity in its interaction and growth promotion according to the host genotype.
Of 4 tomato (Solanum lycopersicum) recombinant inbred lines tested for their growth and physiological responses to drought, only one was more drought resistant following V. paradoxus 5C-2 inoculation.

V. paradoxus Strains

While there is abundant evidence that this specific bacterial strain is a promising inoculant to boost growth of plants grown in drying soils, it is of interest to know if other V. paradoxus strains could have the same effect.
V. paradoxus is a widespread bacterium across different soils and environments and generally promotes plant growth under stress conditions such as drought , salinity, heavy metals and phytopathogens.
Isolation of local strains, their enrichment and inoculation is theoretically possible. However, each strain has different properties and thus effectiveness.
For example, 10 different strains of V. paradoxus were isolated, in which ACC deaminase activity in vitro varied by an order of magnitude .
Interestingly, in vitro root growth promotion also differed by circa 25 %.
Thus not all the strains share the same effectiveness, and in the case of V. paradoxus, there will be differences in plant host specificity, that affect the capacity to integrate into the holobiome.

Hyper-osmotic Stress

Bacterial mechanisms to cope with hyperosmotic stress comprise complex genetic regulation involving a large set of genes arranged in operons.
The V. paradoxus core genome possesses genes that configure the bacteria for coping with hyper-osmotic stress (Table 2), with two main mechanisms identified.
First, osmotic adjustment allows the accumulation of physiologically compatible organic osmolytes utilising betaine-aldehyde dehydrogenase, and choline/betaine ABC transporters.
Secondly, selective influx of inorganic ions such as potassium and chlorine occurs via trans-membrane channels.
Conserved genetic material across multiple strains suggests that V. paradoxus is well-adapted to proliferate in drying soil, it is less clear whether all strains can promote growth of plants in drying soil.

Final Remarks and Future Outlook

One concern of introducing a new organism to the field is that it may alter microbial community structure: displacing some organisms and/or favouring the activity of plant pathogens.
However, this risk is likely minimal since bacterial colonization of the root systems decreased with time following inoculation (Fig. 6C).
Soil inoculation with PGPR sometimes facilitates their integration within soil trophic networks.
Microbial stress tolerance traits are of pivotal importance for PGPR success under abiotic stress conditions and such information can help design field applications to mitigate drought-induced crop losses.
PGPRs often possess more than one beneficial characteristic and complete genomic information can identify multiple potential growth- promoting mechanisms of each isolate .
Putative PGPRs can be identified by their specific genes rather than conventional bioassays.
Information on the species dwelling in the plant holobiont (metagenomics) can help understand rhizosphere microbial community dynamics of a specific crop.
With metatranscriptomics, it is possible to foresee the active bacteria or target genes and determine synergistic behaviours of the inoculum with native microbial population.
Integrating the working model with improved understanding of the microbial ecology of droughted agroecosystems may increase the reliability of microbial inocula, thereby enhancing grower acceptance.