Abiotic stresses including water deficits, salinity, low phosphate and acidity can negatively impact soybean growth and number of flowers per plant, pods per plant, seeds per pod, and weight of pods (1). Moreover, the soybean genes induced by these stresses indicate activation of reactive oxygen species (ROS) pathways in response to these stresses (1). ROS pathways are similarly activated in other crops (2). ROS are oxidative products, including radicals, that are highly reactive and cause cellular damage. Whereas ROS in non-stressed plants serve as signal molecules, high levels can be toxic to cells. Recently, microbial inoculants on diverse crops like rice, citrus and potato were found to induce antioxidant activities that confer stress protection (3-5), but this has not yet been explored in soybean. We propose to take multiple approaches to explore the potential for microbes to serve as protectants for plants under ROS-generating stressful conditions.
(1) Peláez-Vico et al. (2024) Plant J 117:1728-1745; (2) Zafar et al. (2023) Front Plant Sci 14:1265700; (3) Singh et al. (2020) Sci Advances 10:4818; (4) Mamun et al. (2024) Horticulturae 10:102; (5) Want et al. (2023) Front in Plant Sci 14:1247342

Task A) Evaluate microbes and microbial consortia for improving soybean stress tolerance. Individual microbes can enhance the stress tolerance of a range of crops. We have recovered microbes and microbial communities from the endosphere of roots of soybeans grown under water-deficits. We proposed that microbes and microbial communities selected by stressed soybeans have a strong probability of promoting plant health when used as inoculants. Here, we propose to use our collection of microbes, both singly and in combination, and our collection of recovered microbiomes to evaluate the potential for microbes to enhance the resilience of soybeans to abiotic stresses. We will compare inoculated and uninoculated plants for their growth under a range of water deficits, salinity, and low phosphate conditions, using growth parameters that we have validated as indicators of abiotic stress. These results will test the benefits of inoculants as stress protectants for soybeans. These results will also test whether soybeans select for microbial communities that are beneficial under stressful environmental conditions, with this new knowledge providing microbiome targets for soybean breeding aimed at stress tolerance.

Task B) Explore the influence of ROS on interactions within the root microbiome. ROS may influence microbial interactions within microbiomes in various ways, and knowledge of these interactions would be useful to strategies to amplify target beneficial microbes. We hypothesize that stress-induced systemic ROS in soybean enrich for specific microbes by killing those that are not ROS tolerant. Alternatively, ROS in soybean may enrich for specific microbes by activating the production of antibiotics or predation functions that kill non-target microbes. We propose to use our collection of microbial isolates from roots of water-limited soybeans in lab assays to evaluate ROS tolerance and the impact of ROS on antibiotic production and predatory activities. We will use this information to help optimize the inoculants identified in Task A, such as by evaluating the impact of enrichment via ROS exposure to increase their survival in stressed plants. Understanding the processes by which ROS alter root microbiomes is critical to using these processes to direct or reshape soybean microbiomes, as we propose to do with Bradyrhizobium inoculants, below.

Task C) Use ROS-based strategies to improve the efficacy of Bradyrhizobium japonicum inoculants under stressful environmental conditions. Biological nitrogen fixation due to the Bradyrhizobium-soybean symbiosis is highly sensitive to water deficits and high salinity. We hypothesize that stress-associated ROS signaling in soybean contributes to the sensitivity of the symbiosis to these stresses, and that this sensitivity can be attenuated by various ROS-based strategies. We propose to test this, first, by enhancing the tolerance of B. japonicum to ROS by passaging cultures through increasing ROS levels in culture. We predict that pre-adaptation of the symbiont to ROS will enable earlier nodulation and nitrogen fixation, and the resulting fixed nitrogen will help promote continued plant growth under at least low levels of stress. Second, we will screen for microbial isolates that enhance the oxidative stress tolerance of B. japonicum in culture, and will co-evolve B. japonicum in mixed cultures under ROS stress, with cross protection likely resulting from the secretion of antioxidants and enzymes. We predict that inoculants with B. japonicum in mixtures with antioxidant-producing bacteria or in cultures co-evolved to be ROS tolerant will enable earlier nodulation and nitrogen fixation. These results have the potential to boost nitrogen fixation under water deficits, as well as in the presence of other ROS-generating stresses including salinity, low phosphate and acidity.

Period 1: Oct 1, 2024 – March 31, 2025
Aims Milestones
All • Hire and onboard a postdoctoral researcher to perform the work
1 • Select microbes and design microbial consortia to screen as protectants for soybean under stress
• Select soybean germplasm to use in the screening assays
2 • Identify the optimal technical approaches for applying and measuring ROS stresses in microbial cultures
3 • Generate a collection of commercial soybean inoculants (from companies) and soybean rhizobia (from research labs)

Period 2: April 1, 2025 – Sept 30, 2025
Aims Milestones
1 • Optimize assays for soybean growth under water deficits, salinity and low phosphate conditions
• Optimize measurements for evaluating soybean growth performance
• Evaluate the impact of at least 50 individual microbes on plants grown under at least one stressful condition
2 • Evaluate the ROS tolerance, impact of ROS on antibiotic production, and impact of ROS on predatory activities of the collection of isolates selected in Aim 1
3 • Perform the passaging of cultures of soybean rhizobia through increasing ROS levels in culture to enhance their ROS tolerance

Period 3: Oct 1, 2025 – March 31, 2026
Aims Milestones
1 • Evaluate the impact of at least 20 microbial consortia on plants grown under at least one stressful condition
• Complete studies to assess the extent of benefits that these inoculants can provide as stress protectants for soybeans
2 • Implement information on the ROS impacts on microbes to modify and optimize microbial consortia used in Aim 1 and to provide insights into the results from Aim 1
3 • Screen for microbial isolates that enhance the oxidative stress tolerance of soybean rhizobia when grown in mixed cultures and/or under conditions in which the microbes are co-
evolved under increasing ROS concentrations
• Identify the mechanisms contributing to potential cross protection in culture, including the secretion of antioxidants and antioxidant enzymes

Period 4: April 1, 2026 – Sept 30, 2026
Aims Milestones
1 -2 • Summarize the findings on ROS-based microbial strategies to enhance soybean health under stress in a manuscript and submit for publication
3 • Evaluate whether pre-adaptation of the symbiont or microbial consortia to ROS enables earlier nodulation, better nitrogen fixation, and/or improved growth of soybean grown under
water deficits, salinity, low phosphate or high acidity conditions
• Summarize the findings on ROS-based strategies to improve soybean inoculant performance under stressful conditions in a manuscript and submit for publication

Updated April 29, 2025:
A postdoctoral researcher was hired to work on all aims of the project but was only able to start late in this quarter of the project. A database was created of root isolates with curated sequences indicating their phylogeny, indicating the inclusion of over 200 isolates representing over 75 genera, and also metadata on their origins, including the drought versus non-drought conditions of the plant of origin. Efforts to select soybean germplasm (Aim 1), technical approaches for measuring ROS in microbial cultures (Aim 2) and commercial soybean inoculants (Aim 3) are underway.

Updated March 31, 2026:
Project Title: Using soybean microbes to enhance the growth and resilience of soybeans

Objective 1. Evaluate microbial isolates and microbial consortia for improving soybean stress tolerance

In this reporting period we developed and deployed a drought screening protocol for microbes. The protocol involved subjecting a drought-sensitive soybean genotype to a two-phase drought treatment consisting of (i) reduction of soil moisture to 40% field capacity (FC) and (ii) complete withholding of irrigation once plants reached the unifoliate stage. Microbial strains were screened that had been isolated from soybean rhizosphere communities under drought and represented microbial taxa that were known to be enriched in soybean roots under drought. After introducing microbes as seed inoculants, the impact of the microbes was evaluated based on plant shoot height, shoot weight, and two measures of photosynthetic performance, quantum yield (photosystem II efficiency) and electron transport rate, all of which were significantly lower for drought-stressed than non-stressed plants in the assay.

Among the 12 microbial isolates tested, a single Streptomyces isolate showed promise at mitigating drought stress based on modest increases in shoot weight and both measures of photosynthetic performance compared with uninoculated, drought-stressed control plants. However, this strain did not provide consistent improvement in plant performance in subsequent repeated experiments. In addition to individual isolates, 12 mixtures of microbes, termed microbial consortia, were constructed, with 11 consortia constructed based on their relatedness at the genus and family levels and one consortium with >30 diverse microbes. None of these consortia provided significant improvement in plant performance under drought. Given the labor-intensive nature of the assay and the lack of promise based on these results, we shifted subsequent efforts to other aspects of the project.

Objective 2. Explore the influence of reactive oxygen species (ROS) on interactions within the root microbiome

We hypothesize that stress-induced systemic ROS in soybean enrich for specific microbes by killing those that are not ROS tolerant. To evaluate this hypothesis, we characterized collections of microbes isolated from drought-stressed and non-stressed soybean roots for their tolerance to ROS using hydrogen peroxide as a model ROS compound. Based on measurements of the minimum inhibitory concentration (MIC) of hydrogen peroxide for each strain, we classified 39 strains as ROS tolerant and 55 strains as ROS sensitive. The majority of the ROS tolerant strains were in the class of bacteria that was most strongly enriched in drought-stressed soybean roots, namely the Actinomycetes (Fig 1 in attached report). These results support the possibility that bacterial ROS tolerance enhances persistence in drought-affected rhizosphere environments, and highlights the potential for antioxidant strategies to improve the growth, persistence, and/or performance of microbial inoculants, such as Bradyrhizobium spp., in roots with accumulated ROS.

Objective 3. Use of ROS-based strategies to improve the efficacy of Bradyrhizobium inoculants under stressful environmental conditions.

Nodulation of soybean is known to be negatively impacted by drought stress. We quantified this impact by evaluating the growth and nodulation properties of soybean plants with and without inoculation with Bradyrhizobium diazoefficiens USDA110. The plants were subjected to two levels of drought, modest (40% FC) and severe (20% FC), and also to well-watered conditions (80% FC). Although inoculation significantly increased the shoot weight of the plants under each of the conditions as compared to the uninoculated plants, the number of nodules was reduced and the nodule mass was dramatically reduced under the drought conditions as compared with well-watered inoculated plants (Fig 2, see attached report). The negative impact of water deficits on nodule development highlights the opportunity to improve nodulation under these conditions, and this could be done by exploiting the ability other microbes to improve inoculant performance.

We evaluated the potential for antioxidants to improve the growth of Bradyrhizobium strains. We first tested six Bradyrhizobium strains for their sensitivity to oxidative stress and found that they were all highly sensitive (MIC values for hydrogen peroxide of 3 to 6 mM). We then evaluated the impact of exogenous antioxidants on Bradyrhizobium growth at various levels of stress, finding that the antioxidants glutathione and Trolox provided some protection, whereas the antioxidant ascorbic acid did not.

Microbes can confer protection to other microbes against oxidative stress via a variety of mechanisms beyond antioxidant production. We are currently developing a screen to evaluate the direct impact of microbes on the growth of Bradyrhizobium using an engineered Bradyrhizobium diazoefficiens (previously B. japonicum) strain that exhibits fluorescence based on expression of a green fluorescent protein (Ledermann et al. 2015). We are optimizing this screen to identify microbes that can enhance either the growth of B. diazoefficiens in the absence of stress, or its growth and/or stress tolerance in the presence of oxidative or salinity stress. The assay is rapid, as B. diazoefficiens growth is monitored based on fluorescence, overcoming the necessity of differentiating B. diazoefficiens from the candidate helper strains when co-cultured in microplates. By screening ROS-tolerant and/or potential antioxidant-producing Actinomycete strains from drought-stressed soybean roots, along with a broad collection of other soybean root isolates, our goal is to identify microbial helper strains that can enhance the resilience of Bradyrhizobium inoculants on roots under soil water deficits and potentially other stressful conditions.

References
Ledermann, R., I. Bartsch, M.N. Remus-Emsermann, J.A. Vorholt and H.-M. Fischer. 2015. Stable fluorescent and enzymatic tagging of Bradyrhizobium diazoefficiens to analyze host-plant infection and colonization. Mol Plant-Microbe Interact 28:959-967.

View uploaded report

Soybean microbiomes are an under-utilized resource for advancing soybean health, and more knowledge of how best to exploit this resource is critical to the future of the industry.