Microbiome engineering as a tool to increase crop production in the 21st century
Microbiome engineering as a tool to increase crop production in the 21st century
Author: Falko Mathes, Daniel Murphy & Peter Keating. (Bioscience Pty Ltd) | Date: 12 Feb 2021
Key messages
- Microbiome engineering is a novel approach that recognises the complexity of the soil and plant-associated microbial communities and seeks to increase crop production by enhancing plant-beneficial microorganisms and functions.
- Two products with different modes of action, (i.e. super-absorbent polymers and Bioprime®) increased the abundance of known, beneficial rhizobacteria and predicted plant-growth promoting functions in pot and field trials.
- Wheat yields increased between 2.4% and 26.6% for polymers and between 3.4% and 10.4% for Bioprime across several field trials and sites, which equated to increased net farm income of up to $129.79 per hectare for polymers and up to $104.58 per hectare for Bioprime.
Aims
To improve wheat yields by engineering the existing indigenous soil and plant-associated microbiomes using novel, commercially available amendments rather than microbial inocula.
Introduction
Since the advent of high-throughput sequencing (Margulies et al 2005), our understanding of the microbial diversity present in soils (Fierer, 2017) and associated with plants (Trivedi et al 2020) has increased significantly. These taxonomically diverse microbiomes contain several plant-beneficial functional traits that, for example, help nutrient acquisition (Hartman and Tringe 2019), pathogen defence (Carrión et al 2019) or drought stress tolerance (Glick 2014; Naylor and Coleman-Derr 2017). However, reliable tools to engineer crop microbiomes and to enhance plant-beneficial taxa and traits were hitherto lacking. Here, we present two different products that reliably alter crop-associated microbiomes and their predicted functions to increase wheat yields. The first product contains super-absorbent polymers consisting of poly-acrylic acid (PAA-SAP). When added to soil, these polymers take up soil water and nutrients, and are rapidly colonised by in situ microorganisms (Mathes et al 2020). The dynamics and selectivity of ingress and colonisation of plant-beneficial bacteria can be further improved by adding organic co-polymers, (e.g. mannan and mannose; PAAL-SAP; Pham et al 2017; Mathes et al 2020). The second product is Bioprime®, a patented ferment of molasses (Keating 2013), that contains carbon compounds known to either stimulate plant growth directly, (e.g. plant hormones 2,3-Butanediol and acetoin; Ryu et al 2003) or indirectly via the plant microbiome, (e.g. microbial signalling molecules and labile carbon). Bioprime can be applied as a seed coating, directly into the seed bed or as foliar and soil spray. These two products use different modes of action (microbial adhesion for polymers and signalling chemistry for Bioprime) to achieve their aim of engineering in situ microbiomes and their plant-beneficial functions. Results from pot and field trials with the two products are presented in this paper.
Methods
Bioprime pot trial
In a pot trial, sandy soil (pHCaCl2 4.8, 3.6mg/kg NH4-N, 1.3mg/kg NO3-N, 0.4mg/kg PO4-P, 0.2% total carbon) was planted with five Bioprime seed-treated (2L/tonne of grain) and five untreated wheat seeds (var. Mace), fertiliser was applied in excess (50kg N/ha and 25kg P/ha), and each treatment was replicated eight times. After four weeks, the trial was harvested, and the fresh shoot biomass was determined. Rhizosphere soil was removed from the roots by washing with a phosphate-buffered saline solution containing Tween 20. Soil was frozen at -20°C until DNA extraction.
Field trials
Three polymer field trials were conducted over two growing seasons (Table 1). Treatments included control plots (no amendment) and plots to which polymers were added (10 to 20kg/ha) in-furrow and approximately 3cm below the seed. Sampling was conducted at four weeks (seedling growth, GS10-19) and 12 weeks (tillering, GS 20-29) after seeding. Seven Bioprime field trials were conducted over five growing seasons (Table 1). Treatments included seed treatment, soil application or foliar spray (at tillering) of Bioprime (Table 1). All treatments were applied in a randomised block design using a small plot air seeder where each plot was 2m × 12m with eight rows of crop. Trials were managed according to best district practice as advised by the contractor including seeding method, fertiliser application and date of sowing. At maturity, plants were harvested and grain yields determined.
DNA extraction, microbial community analysis, functional prediction, and statistical analysis
DNA was extracted from polymers, rhizosphere and bulk soil using the the PowerSoil® DNA Isolation Kit (MoBio, Carlsbad, CA, USA) following the manufacturer’s protocol. The extracted DNA was quantified using a fluorescence approach (Qbit® 2.0 Fluorometer, Life Technologies, Mulgrave, Victoria, Australia) and diluted to a concentration of 1ng/µL before PCR amplification of bacterial 16S rRNA genes followed by either Ion Torrent PGM (LSBFG, Perth, Australia) or Illumina HiSeq sequencing (AGRF, Melbourne, Australia). Sequences were analysed using QIIME (Caporaso et al. 2010). Metagenomic functions were predicted in silico to detect potential plant-growth promoting traits (Langille et al 2013). Statistical analyses of the microbial community structure were performed using the software PRIMER-E (Clarke and Gorley 2015). ANOVAs and post-hoc comparisons were performed with jamovi (The jamovi project, 2020).
Results
Polymer microbiome analysis and effects on wheat yield
Bacteria readily ingressed into polymers (Figure 1A), attached to their micro- and nano-structures (Figure 1B), and interacted with intersecting plant roots (Figure 1C). Both the wheat rhizosphere and the polymers recruited certain rhizobacterial taxa from the soil microbiome, with some of them being further enriched in the polymers (e.g. Oxalobacteraceae [Betaproteobacteria], Streptomycetaceae [Actinobacteria], and Sphingobacteriaceae [Bacteroidetes]; Figure 2A). A number of plant-growth promoting traits including soil P acquisition (3-phytase, P < 0.01; Figure 2B) and plant stress response suppression (ACC deaminase or acdS gene, P < 0.05 after 4 weeks; Figure 2C) were significantly higher in polymers than in the rhizosphere. Yield responses were largely positive (Figure 2D), except for Badgingarra where soil water repellence (MED 2.4) lowered water infiltration rates that could not be overcome by small quantities of polymers added to the root zone. (For more details, please refer to Mathes et al., 2020).
Effect of Bioprime on wheat rhizosphere microbiome, plant growth and yields
Over a four-week pot trial, Bioprime seed treatment significantly increased wheat shoot biomass by 27% (P = 0.004; Figure 3A) and elevated abundances of rhizobacterial taxa belonging to a range of phyla (Figure 3B). This was associated with increases in a number of predicted plant-growth promoting functions (Figure 3 C) such as secondary metabolite production (including signalling molecules), disease suppression, (i.e. siderophore production) and nutrient acquisition, (i.e. 4-phytase). In field trials, Bioprime increased yields between 3.4% and 10.4% across different modes of application, trial locations and growing seasons (Figure 3D, Table 1).
Return on investment
Product and import costs of the PAA polymers are $8,000 per tonne. Even at the low application rate of 10kg/ha, this product adds a substantial input cost to growers ($80/ha). A positive return on investment is only achieved when grain yields increase by at least 250kg/ha, (e.g. 10% yield increase of 2.5t/ha crop or 20% yield increase of a 1.25t/ha crop). At the maximum yield benefit of 26.6% at Dandaragan (Figure 2D), the net ROI equates to $129.79 per hectare. The product is likely to perform its function for more than one season increasing the ROI but is eventually biodegraded (see below).
Bioprime is manufactured in WA and priced very competitively. Relatively low application rates (Table 1) allow a positive ROI even at modest yield improvements, especially when seeds are treated with Bioprime. Thus, the trial at Badgingarra (2017; Table 1; Figure 3D) had the highest net ROI of $103.14 per hectare.
Ecological safety of the products
The two products presented here have been designed to minimise adverse effects on the environment. The polymers do not contain the toxic (poly-) acrylamide but the deamination product polyacrylate. The latter is commonly used in nappies, sanitary and medical products, and does not irritate skin. Like all chemicals, they should be handled with care and the appropriate PPE should be worn (e.g. gloves). Polymers persist in soils but are slowly degraded into short-chain carboxylic acids by bacteria and fungi that produce extracellular enzymes such peroxidases (Stahl et al., 2000) and /or monooxygenases (Nyyssölä and Ahlgren, 2019). Short-chain carboxylic acids are labile and commonplace in soils and within cells, and readily metabolised (Hassanpour and Aristilde, 2021). Bioprime is a natural ferment of molasses containing both complex carbon molecules as well as short-chain carboxylic acids which are biodegradable.
Conclusion and outlook
The 21st century has been touted as the ‘The Century of Biology’. With continuous advances in DNA sequencing and bioinformatic analyses, we will improve our understanding and consequently be able to precisely engineer the complex interactions between vastly diverse microbiomes, crop plants, and soil. The research presented in this paper shows that the two products, which can be purchased by Australian growers in 2021, can reliably and predictably alter crop-associated microbial communities and increase grain yields while utilising two very different modes of action (microbial adhesion for polymers and signalling chemistry for Bioprime). Future work will include more field trials to assess the robustness of results across soil types, and employ functional metagenomics to further elucidate which microbial groups and activities are responsible for increasing crop yields.
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Contact details
Dr Falko Mathes
Bioscience Pty Ltd
488 Nicholson Road, Forrestdale, WA 6112
Phone: 08 9397 2446
Email: falko.mathes@biosciencewa.com
GRDC Project Code: POL00001,