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Soil microbes for drought-resilient crops

The microbes in and around an oilseed plant’s roots could be a resource for helping the plant deal with drought stress. That’s the intriguing concept behind a project led by microbiologist Tim Dumonceaux, a research scientist with Agriculture and Agri-Food Canada (AAFC) in Saskatoon.

“Plants use a variety of methods to condition the soil environment around their roots to try to get microbial populations that will benefit the plant,” says Dumonceaux, outlining some of the science underlying this concept. “One really important way is by excreting various compounds, called root exudates, from their roots into the soil.”

He explains that these root exudates influence the composition of the microbial community around the roots by attracting and encouraging microbes that thrive under these soil conditions. In turn, these microbes can influence the plant, for better or worse. For instance, some of the microbial species might bring nutrients to the plant, some might cause disease in the plant, some might help the plant survive weather stresses like drought.

So, a plant’s root-associated microbial community is intimately linked to the plant’s growth, survival and productivity. In a sense, the plant and its microbiome form a single, interdependent unit.

Dumonceaux suspects that the microbial communities associated with the roots of drought-tolerant oilseed plants might be an important part of the ability of those plants to withstand drought. His project is seeking to shed light on the role these communities play in an oilseed plant’s response to drought and on how to enhance the microbial contribution to improved drought tolerance.

Funded by the Canola Agronomic Research Program (CARP), his three-year project is investigating the below-ground microbial communities of drought-tolerant and drought-sensitive lines of canola and other brassica oilseed crops.

Project activities include analyzing the species composition of the root-associated microbial communities of these oilseed lines when grown under different moisture conditions; experimenting with some of the individual microbial strains in lab and greenhouse trials; and analyzing the root exudates of the different lines and looking at how the different exudates relate to the plant’s drought tolerance and the microbiome.

Interestingly, just as root-associated microbes and their plant host are interdependent, Dumonceaux’s microbial project is tied in with a related project about drought tolerance in oilseed crops from a plant perspective.

That related project is led by two of his AAFC-Saskatoon colleagues, research scientists Christina Eynck and Isobel Parkin. Their project is one of several projects that together make up a major five-year effort on drought adaptations in Canadian agricultural ecosystems, which is funded by the Genomics Research and Development Initiative (GRDI), a federal interdepartmental program.

Eynck is a breeder of brassica oilseed crops and Parkin is a molecular genomics researcher focusing on brassica species. Their GRDI project is investigating the molecular biology and phenotypic aspects of drought stress in canola (Brassica napus), Ethiopian mustard (Brassica carinata) and camelina (Camelina sativa).

“The CARP project is working hand-in-glove with this GRDI project and relies on it for samples and [brassica] expertise,” notes Dumonceaux. Both projects started in 2022.

Analyzing microbial DNA
One component of Dumonceaux’s project involves using DNA sequencing to identify and quantify the below-ground microbes associated with drought-tolerant and drought-sensitive oilseed lines. His research group collects and analyzes samples from the plant roots, the rhizosphere, which is the soil immediately next to the roots, and the bulk soil, which is the soil a little farther away from the roots.

They extract the total DNA from each sample and then look for certain DNA sequences, known as taxonomic markers, which help identify which types of microbes are present. However, some taxonomic markers are better than others at differentiating between closely related things, like different species in the same genus or different subspecies in the same species. “So, as part of our year-one work, we looked at different taxonomic markers and different preparation methods to achieve the most accurate microbial community profile we can,” says Dumonceaux.

Consequently, his group is using two common markers, one for differentiating bacteria and the other for differentiating fungi, as a first pass to get a feel for the microbial community – at least down to the genus level. Then, if they need higher-resolution species information for certain samples, they’ll use a different marker, called chaperonin-60, which helps in getting down to the species or even the subspecies or strain level in some cases.

Drought response and microbial communities
In 2022, Eynck and Parkin’s GRDI project team started the task of screening many different canola, Ethiopian mustard and camelina lines for response to water-limited conditions in greenhouse experiments. Through this phenotyping work and previous studies, the team has already identified a number of drought-tolerant and drought-sensitive lines. In 2023, the GRDI team planted those lines in a field trial at AAFC-Saskatoon’s research farm. To compare the effects of water-sufficient and water-stressed conditions, half of the trial was irrigated and the other half was dryland.

In these plots, Dumonceaux’s group targeted four oilseed lines: a drought-tolerant canola, a drought-sensitive canola, a drought-tolerant Ethiopian mustard and a drought-sensitive Ethiopian mustard. They sampled the root, rhizosphere and bulk soil of the four lines in both the irrigated and dryland plots.

At present, his group is analyzing the DNA from hundreds of samples collected from the 2023 plots.

Once they have the microbial DNA data and the GRDI oilseed phenotypic data for the lines, Dumonceaux and his group will use standard and new statistical tools to tease out which microbes or groups of microbes are associated with and possibly contributing to improved drought tolerance in the oilseed plants.

“For example, an exciting recent statistical tool provides a way to look at which microbes tend to appear together in different circumstances,” he says. “Essentially, it allows you to identify taxa that occur together either in a positive or a negative relationship. And you can characterize how these microbes relate to one another in the whole network of microbial taxa. This tool has a lot of potential for identifying strains that are very interesting to us.”

Root exudate analysis
Root exudates are organic acids made by plants. “Plants fix atmospheric carbon dioxide using the energy from sunlight and synthesize small organic acids. They transport those organic acids down to their roots and exude them into the soil immediately around the roots, to encourage the growth of the microorganisms that benefit from those organic acids,” Dumonceaux explains.

“Plants produce a whole range of these root exudates. So, the plant conditions the soil through its metabolic activity.”

The technical capacity to analyze these exudates is increasing these days. A few years ago, Dumonceaux was one of the principal investigators in a CARP study completed in 2021 that looked at root-associated microbes in various canola rotations. At that time, his group had access to analytical capacity for quantifying five different root exudates. However, thanks to some new equipment and newer methods at the University of Saskatchewan’s Soil Science department, his group can now quantify 13 root exudates.

In 2022, Dumonceaux’s group refined the protocols for using the new equipment to quantify quite small amounts of exudates. Then they tested their protocols with some drought-tolerant and -sensitive lines grown under water-sufficient conditions in a greenhouse experiment. This test confirmed they could quantify all 13 exudates. Having better exudate data should help in developing a clearer picture about things like the exudate differences between the tolerant and sensitive lines, how a plant’s exudate profile is affected by water-sufficient and water-limited conditions and how the different exudates relate to microbial community patterns.

Interestingly, this test also found statistically significant differences in the profiles of the root exudates produced by the tolerant and sensitive lines.

“This is really important,” says Dumonceaux. “Since the plants were not under drought stress, these differences suggest there is a different genetic potential for producing these root exudates between the different lines. That lends credence to the idea that the drought-tolerant and drought-sensitive lines differentially condition the soil environment to each get a distinct microbial community.”

Dumonceaux’s group is currently analyzing the root exudates of the drought-tolerant and -sensitive lines in the 2023 samples.

Evaluating individual strains
To get another angle on the plant-microbe relationships, Dumonceaux and his group are planning to isolate, identify and culture some of the microbial strains associating with the drought-tolerant oilseed lines. They’ll use these cultures in greenhouse trials to study the mechanisms by which these microbes may contribute to a plant’s ability to withstand moisture-limited conditions.

He says, “Having the actual microorganisms in our hands, whenever possible, will allow us to experiment with the microbes. For example, we can apply the root exudates and see which genes are induced [in the microbes], how that affects the drought-tolerant phenotype and that sort of thing.”

The GRDI project
Eynck and Parkin’s project includes several components related to their different areas of expertise.

For instance, one component involves developing and using sophisticated phenotyping tools to screen diverse collections of canola, Ethiopian mustard and camelina germplasm to identify drought-tolerant and drought-sensitive lines. The project’s ongoing greenhouse screening work includes collecting a range of data – such as transpiration rate, biomass accumulation and water-use efficiency – for the different oilseed lines, grown under various soil moisture conditions.

Based on the screening results, the team is selecting a few of the most drought-tolerant and the most drought-sensitive lines of each oilseed species. They are assessing these extreme lines more intensively, for example, looking at differences in photosynthesis and shoot and root system architecture, under controlled conditions simulating Prairie drought events.

“These assessments use state-of-the-art phenotyping tools, including 2-D and 3-D root imaging systems, multi-spectral shoot imaging sensors and deep-learning computer vision,” notes Dumonceaux. “They have already noticed some differences in the root architecture of the tolerant and sensitive lines.”

In 2024, the GRDI team will also be continuing the field trials with sensitive and tolerant lines under irrigated and dryland conditions at Saskatoon. As well, starting this fall, they will be growing the lines in Arizona in collaboration with United States Department of Agriculture researchers.

“By growing these lines in Arizona, we can collect field data during our winter season, and we can take advantage of the dry conditions there. Although we have dryland plots in the Saskatoon trials, we might get lots of rain in some years. We’re pretty sure the Arizona plots will not get much water unless we add it.”

For the field trials, the GRDI team is developing digital imagery methods, such as drone flyovers, to rapidly quantify detailed phenotypic characteristics of the different lines. “Such methods could really upscale the ability to screen all the different lines for the phenotype of interest,” says Dumonceaux.

One of the other project components is investigating the genetic mechanisms and biomarkers related to adaptation to water-limited conditions in the oilseed lines. This work includes various analyses such as examining which genes are expressed and which metabolites are produced by the different lines when exposed to drought stress.

Dumonceaux adds, “This will be combined with the microbial work to give a holistic sense of the drought-tolerant lines’ responses to water-stressed conditions.”

Future possibilities
Ultimately, combining and analyzing the data from the GRDI and the CARP projects could point the way to innovative strategies for improving the ability of canola, camelina and Ethiopian mustard crops to survive and recover from very dry conditions.

Dumonceaux sees various possible ways the findings from his CARP project might contribute to increased drought tolerance in the three oilseed species. He notes that these possibilities have parallels to the probiotic and prebiotic approaches used in adjusting the gut microbiome of humans and agricultural animals to improve their health.

“Probiotic approaches are where you add a microorganism to obtain benefits. Prebiotic approaches are where you add something to the feed to influence the composition of the microbial communities in the gut,” he explains.

Dumonceaux’s research collaborators on the CARP project include Jennifer Town with AAFC-Saskatoon, Bobbi Helgason at the University of Saskatchewan and Sean Hemmingsen of the National Research Council Canada at Saskatoon. Collaborators with Eynck and Parkin on the GRDI project include Raju Soolanayakanahally, Sally Vail and Hema Duddu at AAFC-Saskatoon, and Steve Shirtliffe at the University of Saskatchewan.