Final report for GW24-015
Project Information
Irrigated hay meadows are an integral, but under-performing component of livestock operations in the Rocky Mountain West. Flood irrigation, high elevation, and cool temperatures common to these systems result in the concentration of organic materials near the soil surface, constraining microbial-mediated nitrogen cycling, and impeding forage yield. Efforts to improve hay meadow productivity sustainably while maintaining soil health are critical in these semi-arid ecosystems. The soil microbial community is crucial in maintaining various ecosystem functions and services including nutrient cycling through mineralization, primary production, and litter decomposition. Soil microbial community’s abundancy, diversity, and activity is therefore essential to maintain the functioning of terrestrial ecosystems such as the high elevation meadow systems. Microbial inoculants (biofertilizers) are a promising
alternative to increase forage productivity and quality of hay in these meadow systems while cutting down or eliminating chemical fertilizers but have not been evaluated in these systems. Biofertilizers are active strains of microorganisms which stimulate microbial activity and improve mobility of nutrients in soil, and hence may potentially enhance mineralization in the organic horizon of these meadow systems. Our goal is to develop alternative hay meadow management practices that can improve nutrient bioavailability through enhanced microbial activity to reduce reliance on chemical fertilizers, optimize yield, foster carbon sequestration, and promote soil health. We will assess effects of biofertilizers on the soil microbial community’s abundance, diversity,
and function, soil nitrogen availability, and quantity and quality of hay in high elevation hay meadows in both field and controlled environment and disseminate research findings to stakeholders in Wyoming and Colorado. We will use complementary microbial inoculation combinations focused on plant nutrient management through fixation, solubilization or transformation in the soil. Treatments will include control, combinations of microbial inoculants, and mineral fertilizer. Findings are crucial for the producers to make informed decisions in forage management practices.
Overall objective: Evaluate the effects of microbial inoculants on the soil microbial community, nitrogen pools and mineralization, and forage quantity and quality in high-elevation hay meadows in the field, and in a controlled environment and disseminate research findings to stakeholders in Wyoming and Colorado.
Specific objectives
Research objective
- Evaluate the response of the soil microbial community, nitrogen pools and mineralization, and forage quantity and quality to biofertilization in a field setting.
- Evaluate the response of the soil microbial community, nitrogen pools and mineralization, and forage quantity and quality to biofertilization in a controlled environment.
Education objectives
- Assess the current knowledge on the use of biofertilizer, create a common understanding among stakeholders of the proposed project and invite their participation in the project.
- Establish open communication and continuously engage farmers and ranchers in the project progress.
- Advance/share research findings from this project with producers, research scientists, agricultural educators, and 4-H youth groups in Albany County Wyoming, and Walden Colorado
Cooperators
- - Producer
- (Educator and Researcher)
Research
Research objectives
- Evaluate the response of the soil microbial community, nitrogen pools and mineralization, and forage quantity and quality to biofertilization in a field setting.
- Evaluate the response of the soil microbial community, nitrogen pools and mineralization, and forage quantity and quality to biofertilization in a controlled environment.
Field study design
An experiment was established at the Laramie Research and Extension Center (LREC) in an existing forage stand that is under long-term flood irrigation. The site is located between 1980- and 2440-m elevation in a semiarid climate with most of the 260 to 380 mm precipitation falling in summer months, temperatures are cold, with a short 75- to 90-day growing season that frequently has sporadic freezing temperatures. The study design was a Randomized Complete Block Design (RCBD) in which we established twenty-one sampling plots 7. 5m2 in an existing foxtail forage stand, with seven treatments and three replicates. Treatments included Control, synthetic fertilizer (Urea), AMF (Glomus mosseae and Glomus intraradices), Azospirillum brasilense, and AMF+ Azospirillum brasilense, High rate: Accomplish Max + BlackMaxx 22 + HyprCycl High, and Low rate: Accomplish Max + BlackMaxx 22 + HyprCycl
Treatments were applied by hand at the recommended rate in early June.
Controlled environment
A growth chamber experiment was conducted at the University of Wyoming Greenhouse facility. The study employed a completely randomized block design (CRBD) with six treatments and five replicates per treatment, resulting in 30 pots per irrigation regime. The soil used in this study was collected from the field study site: the long-term flood-irrigated and unfertilized hay meadow at the Laramie Research and Extension Center (LREC) to ensure consistency between field and growth chamber conditions. The soil was unsterilized to preserve native microbial communities, simulating near-natural field conditions. Field soil samples were collected to a depth of 10 cm, homogenized, and air-dried prior to use. Each plastic pot was filled with 500 g of the air-dried, homogenized soil and adjusted to 60% water holding capacity using deionized (DI) water. Foxtail seeds were sown at a seeding rate equivalent to 2 lb pure live seed per acre, following USDA guidelines. Plants were grown at a temperature of 25°C and watered after every two days or as needed to maintain target soil moisture. Thinning was performed one week after germination to retain the five most vigorous seedlings per pot. Treatments mirrored those used in the corresponding field study. In addition to the 30 pots maintained at 60% field capacity, another set of 30 pots was established to simulate field flood irrigation, with water applied for the same duration as in the field trial. This resulted in a total of 60 pots: 30 under consistent soil moisture conditions and 30 subjected to flood irrigation, enabling direct comparison of management effects across irrigation regimes.
Inoculants
All inoculants were commercially sourced and applied consistently across both the field and growth chamber experiments, either as a soil drench or mixed directly into the soil, following manufacturer recommendations. Application rates were based on the specific guidelines provided for each product. Urea was applied at a standard agronomic rate of 70 lb/acre of 46% nitrogen. To ensure uniform starting moisture conditions, control pots and plots received an equivalent volume of water in place of treatment solutions.
Forage Harvest and Analysis
Field forage was harvested in mid-July 2024, coinciding with the typical regional hay harvest period and approximately 75% black seed maturity. Within each treatment plot, biomass was collected from a one-meter length using a hay mower, positioned approximately 45 cm from the plot edge to capture representative forage. Harvested material from the entire plot section was composited, and subsamples taken per plot for further analysis. Samples were oven-dried at 60°C for 72 hours to determine dry weight. Moisture content was calculated and used to adjust bulk plot samples to a dry weight basis. Ground subsamples were analyzed for dry matter crude protein (CP), acid detergent fiber (ADF), and total digestible nutrients (TDN).
In the growth chamber experiment, forage was grown for 120 days and harvested by cutting plants at the soil surface per pot. Harvested samples underwent the same drying, weighing, and nutrient analysis procedures as the field experiment.
Soil sampling
Field soil samples were collected in late July 2024, by randomly taking three soil cores per plot to a depth of 10 cm. These cores were homogenized into a composite sample per plot for microbial and chemical analyses. Samples were maintained on ice during transport to the laboratory. Baseline sampling at the Laramie Research and Extension Center (LREC) provided prior data on microbial biomass, soil carbon and nitrogen pools, and forage characteristics.
Growth chamber soil sampling was conducted once immediately after forage harvest. Soil from each pot was thoroughly mixed, and roots were removed for assessment of mycorrhizal colonization.
Within 24 hours, soil subsamples from field and controlled study were processed as follows: samples for microbial assays were frozen at –20°C; samples for nitrogen pool analyses were refrigerated at 4°C and analyzed the following day; and samples for protein assays were air-dried.
Laboratory assays
Microbial biomass carbon
Microbial biomass carbon (MBC) was measured to assess the response of soil microbial communities to biofertilizer application. MBC was determined using the chloroform fumigation-extraction method, a widely accepted approach for quantifying microbial biomass in soils. Briefly, soil samples were fumigated with ethanol-free chloroform for 24 hours in a vacuum desiccator, followed by extraction with 0.5 M K₂SO₄. Extractable organic carbon from both fumigated and non-fumigated samples was quantified using a TOC analyzer, and microbial biomass carbon was calculated as the difference between fumigated and non-fumigated extracts, using an appropriate kEC factor. This method provided a reliable quantitative indicator of microbial biomass dynamics in response to treatment effects across both field and growth chamber conditions.
Microbial functional analyses (Enzymes)
Extracellular enzymes play an important role in energy transfer, environmental quality, and crop productivity21. Enzyme activities are therefore proxies for microbial activity, rates of organic matter decay, and the availability of substrates for microbial or plant uptake. The activities of eight hydrolytic enzymes that are involved in C, N, P and S cycling i.e., α-glucosidase (AG), β-glucosidase (BG), cellobiohydrolase (CBH), β-xylosidase (BX), nitrogen N-acetyl glucosaminidase (NAG), leucine aminopeptidase (LAP), phosphatase (PHOS), and arylsulfatase (SUL) were measured fluorometrically using MUB-linked substrates. Briefly, soil was mixed with a 50-mM acetate buffer solution and the pH was adjusted to the mean soil pH samples within 0.5 units as enzymes are sensitive to pH. We also assayed oxidative enzymes colorimetrically using L-dihydroxyphenylalanine (L-DOPA). We followed the protocol by20, 22, and analysis are still ongoing.
Microbial diversity (DNA)
DNA-based assays were used to evaluate the response of soil microbial communities to biofertilization in forage meadow systems. all labwork has been performed with data analysis ongoing. These analyses will enable the comparison of the relative abundances of microbial taxa across treatments and provide insights into the persistence and viability of introduced microbes through biofertilizers, as well as any unintended effects on overall microbial community composition.
DNA was extracted from soil subsamples using a Qiagen DNeasy PowerSoil Pro kits following manufacturer protocol. The 16S rRNA gene (for bacteria) and ITS region (for fungi) were amplified from extracted DNA to characterize bacterial and fungal communities, respectively. Sequence data will be processed and analyzed using the phyloseq package in R, with alpha-diversity (within-sample diversity) and beta-diversity (between-sample dissimilarity) metrics computed to assess treatment effects.
Analysis of variance (ANOVA) will be used to test for differences in alpha-diversity, while PERMANOVA will be applied to evaluate beta-diversity and overall shifts in microbial community composition across treatments. To identify differentially abundant taxa (amplicon sequence variants, ASVs), we will apply multiple statistical approaches, including ANCOM-BC2, as recommended for compositional microbiome data analysis.
Mycorrhizal colonization
At the conclusion of the greenhouse experiment, root samples were carefully harvested and gently washed with distilled water to remove adhering soil particles. Roots were then stained with 5% ink-vinegar stain following standard procedures. Mycorrhizal colonization levels were assessed using established protocols from the Van Diepen Lab. This analysis is currently ongoing and will provide valuable insights into the extent of arbuscular mycorrhizal fungi (AMF) colonization under the various treatment conditions.
Soil protein
Soil protein, measured as autoclaved-citrate extractable (ACE), represents the largest pool of organic nitrogen in soil and is a key indicator of the N reservoir that can be depolymerized and subsequently mineralized. In this study, the ACE assay was performed as a functional and sensitive indicator of this organic N pool following the procedures described by Hurriso, 2018 and as routinely applied in the Van Diepen Lab with modifications.
Briefly, 2.0 g of soil was placed into Erlenmeyer flasks and extracted with 24 mL of 0.02 mol L⁻¹ sodium citrate (pH 7.0). The soil–citrate suspensions were autoclaved at 121°C and 15 psi for 30 minutes. After cooling, samples were centrifuged at 3,100 × g for 15 minutes, and 10 µL of the supernatant was transferred into each well of a 96-well plate containing 200 µL of sodium bicinchoninate (BCA) reagent. Two hundred microliters of undiluted Bradford reagent (Bio-Rad G-250 dye) was then added to each well, and plates were incubated at 60°C for one hour. Absorbance was measured at 590 nm using a microplate reader. Protein concentrations were determined by comparing absorbance values to a standard curve generated from bovine serum albumin (0–500 µg mL⁻¹) prepared in 0.02 mol L⁻¹ sodium citrate (pH 7.0) and diluted in phosphate-buffered saline (PBS).
Ammonium and nitrate
Ammonium (NH₄⁺) and nitrate (NO₃⁻) are the primary forms of plant-available nitrogen and play a vital role in plant growth and development. To evaluate the effects of microbial inoculants on nitrogen mineralization, concentrations of these inorganic N forms were measured in soil extracts.
Fresh soil samples were extracted with 2 M KCl, and the extracts were analyzed colorimetrically using a Lachat flow injection autoanalyzer following established protocols in the Van Diepen Lab. The Lachat system utilizes the salicylate-hypochlorite method for NH₄⁺ and the cadmium reduction method for NO₃⁻, providing accurate and sensitive quantification of soil nitrogen availability across treatments.
Data analysis
Data were analyzed using R statistical software. Treatment effects on soil and plant variables were evaluated using analysis of variance (ANOVA) when assumptions of normality and homogeneity of variance were met. When these assumptions were violated, the Kruskal–Wallis test, a non-parametric alternative, was applied. When significant differences were detected, Tukey’s Honest Significant Difference (HSD) test was used for post hoc pairwise comparisons. Statistical significance was determined at α < 0.05
Objective 1. Evaluate the response of the soil microbial community, nitrogen pools and mineralization, and forage quantity and quality to biofertilization in a field setting.
Soil Microbial Biomass Carbon (MBC)
We evaluated the effects of biofertilizer and synthetic nitrogen (N) treatments on soil microbial biomass carbon (MBC) in a flooded field system. Although MBC did not differ significantly among treatments (p > 0.05; Fig. 1A), clear trends were evident. The arbuscular mycorrhizal fungi (AMF) treatment yielded the highest MBC, suggesting that AMF may enhance microbial growth by stimulating root exudation and modifying the rhizosphere environment. Similarly, the HyperCycle high-rate treatment resulted in elevated MBC values, potentially reflecting improved nutrient availability or relief from microbial carbon limitation. Conversely, the HyperCycle low-rate treatment showed the lowest MBC, indicating that insufficient nutrient inputs constrained microbial growth. Urea-treated plots exhibited intermediate MBC, consistent with previous reports that synthetic N fertilizers boost plant productivity but do not proportionally increase microbial biomass due to limited organic carbon inputs. Azospirillum alone yielded relatively low MBC; however, co-application with AMF produced a modest increase, though not exceeding AMF alone. Interestingly, the unfertilized control maintained intermediate MBC, highlighting the resilience of native microbial communities under long-term flooding.
Despite the variability, overall MBC stability across treatments reflects fundamental constraints on microbial activity in flooded soils. This aligns with findings by Das et al., 2025, who demonstrated that oxygen diffusion limitation in saturated soils restricts aerobic microbial growth. AMF's relative advantage may stem from its ability to transport oxygen along hyphae, creating localized oxic microsites that enhance microbial activity and phosphorus availability under anaerobic conditions. The positive trend observed with the HyperCycle high-rate treatment suggests that balanced carbon-to-nitrogen ratios can alleviate microbial carbon limitation without causing ammonia toxicity often associated with mineral fertilizers.
Soil ACE Protein Results
In the Field Flooded System, soil ACE protein concentrations varied across treatments (Figure 1B), though statistical analyses indicated no significant differences (NS) among treatments. The AMF and Azospirillum + AMF treatments exhibited relatively higher ACE protein levels, suggesting a trend where bioinoculation may stimulate soil enzymatic activity by enhancing root exudation and improving rhizosphere microhabitats (Smith & Read, 2008; Bashan et al., 2004). The Azospirillum treatment also demonstrated moderately elevated ACE protein compared to the Urea treatment, consistent with observations that Azospirillum promotes microbial activity and enzyme production (Bashan et al., 2004). Conversely, the low rate of HyperCycle fertilizer yielded the lowest ACE protein levels, indicating that reduced nutrient inputs may be insufficient to sustain robust enzyme activity under flooded conditions.
Importantly, despite these trends, ACE protein concentrations remained statistically stable (NS), ranging narrowly across treatments (approximately 20–40 g kg⁻¹). This stability reflects the resilience of soil protein to short-term agricultural interventions and supports the understanding that soil protein largely serves as a long-term nitrogen reservoir governed by legacy organic matter dynamics rather than recent management inputs. The absence of significant treatment effects aligns with findings by Leung et al. (2020), who reported strong correlations between soil protein and historical organic matter inputs across diverse agroecosystems. Thus, while soil ACE protein is a valuable indicator of sustained soil health, its sensitivity may be limited in detecting acute shifts in nitrogen management, particularly in systems characterized by active organic matter turnover.
Field Flooded System Soil Protein
Available Nitrogen (AN)
Significant treatment effects emerged for available nitrogen (AN; p = 0.041; Fig. 1C). The HyperCycle high-rate treatment produced the highest median AN (~110 mg/kg), significantly exceeding Azospirillum + AMF (p < 0.05). These results suggest HyperCycle's high-rate formulation may enhance nitrogen mineralization or reduce nitrogen losses under saturated field conditions through slow-release mechanisms or favorable microbial interactions. Urea treatments showed moderate AN level (~40 mg/kg), statistically indistinct from most treatments except the high-rate HyperCycle, likely due to rapid nitrogen transformation or leaching under flooding conditions (Zhang et al., 2015). Conversely, Azospirillum + AMF plots exhibited the lowest AN (~25 mg/kg), potentially due to rapid plant or microbial uptake. Intermediate AN value from AMF-only and HyperCycle low-rate treatments, comparable to the control, indicate biologically active or low-input amendments can stabilize nitrogen availability effectively.
Field Flooded System Available N
Forage Yield and Quality
Forage yields did not differ significantly among treatments, yet notable agronomic trends emerged (Fig. 2A). Urea-treated plots achieved the highest forage yield, closely followed by Azospirillum treatments, highlighting the potential of biofertilizers to closely match synthetic nitrogen-driven productivity. These findings align with previous research demonstrating that biofertilizers like Azospirillum enhance yield through nitrogen fixation, hormone synthesis, and improved nutrient uptake (Bashan et al., 2004). Furthermore, plant growth-promoting rhizobacteria (PGPR) have consistently shown promise in maintaining productivity with reduced synthetic nitrogen inputs (Adesemoye et al., 2009). Collectively, our results reinforce the viability of Azospirillum and related bioinoculants as sustainable alternatives that support both productivity and long-term soil health in flooded forage systems (Lehmann et al., 2020).
Forage quality parameters, crude protein (CP), acid detergent fiber (ADF), and total digestible nutrients (TDN), also exhibited non-significant differences across treatments (p > 0.05 for all; Fig. 2B–D), though agronomic meaningful trends emerged. Urea treatment achieved the highest median CP (~7.5%), closely followed by Azospirillum + AMF and AMF-only treatments, indicating biofertilizers partially compensate synthetic nitrogen inputs by facilitating nitrogen cycling. The Azospirillum-only and control treatments produced lower CP (~6.0%), reflecting more limited nitrogen assimilation. High ADF values across treatments (~39–42%) with urea plots exhibiting the highest fiber content potentially indicate increased structural biomass growth under elevated nitrogen availability. Control plots demonstrated slightly lower ADF, suggesting less fibrous growth, albeit non-significant. TDN patterns closely paralleled ADF trends, with Azospirillum + AMF and control treatments displaying slightly higher TDN (~53%), while urea and AMF-only treatments had marginally lower values, suggesting possible trade-offs between nutrient content and forage digestibility (Barnes et al., 2003).
In conclusion, these insights highlight that biofertilizer applications, particularly combined inoculations, may enhance soil microbial functionality, organic nitrogen pools, and sustainable forage productivity, thereby presenting compelling alternatives to synthetic nitrogen inputs in flood-irrigated field systems
Field-Flooded-System-Forage-Yield-Quality
Objective 2:Evaluate the response of the soil microbial community, nitrogen pools and mineralization, and forage quantity and quality to biofertilization in a controlled environment.
We evaluated the response of the soil microbial community, nitrogen pools and mineralization, and forage quantity and quality to biofertilization under controlled environmental conditions, contrasting flooded and moisture-limited dryland systems. The primary objective was to determine the effectiveness of synthetic and biological treatments in enhancing soil health indicators and forage productivity under varying moisture regimes.
Soil Protein
Soil protein concentrations (~9–12 g kg⁻¹) were stable across all treatments in both Controlled Flooded and Controlled Dryland Systems, showing no significant differences (NS; Figure 3). These results indicate that short-term biofertilizer and synthetic nitrogen inputs minimally influenced this long-term indicator of soil nitrogen (N) status. Such stability likely reflects microbial communities' adaptive biochemical processes; whereby proteinaceous compounds are conserved through physical encapsulation within aggregates and chemically bonded to stable soil organic matter fractions. Although not statistically significant, Azospirillum treatments consistently showed a slight elevation in soil protein concentrations in both Controlled Flooded and Dryland systems, suggesting a possible trend toward enhanced microbial activity or root-associated microbial biomass accumulation. This subtle trend supports the potential of Azospirillum inoculants in modestly enhancing soil nitrogen dynamics under different moisture regimes.
Controlled System Soil Protein
Available Nitrogen
Available N exhibited significant variation across treatments and moisture regimes (Figure 4 ). In the Controlled Flooded System, Urea-treated soils had substantially greater available N (~30 mg kg⁻¹), significantly exceeding those in biofertilizer treated soils (Azospirillum and AMF, ~5–10 mg kg⁻¹; p = 0.003). These results underscore the immediate efficacy of synthetic fertilizers under saturated conditions, which facilitate rapid N availability for plant uptake (Geisseler and Scow, 2014). Despite the lower overall available N, HyperCycle high-rate treatments showed slight numerical increases compared to other biological treatments, suggesting modest benefits from this inoculant even under anoxic conditions. In contrast, the limited performance of biological inoculants under flooded conditions likely arises from oxygen scarcity constraining microbial mineralization and N fixation processes, as described by Bashan and Holguin (1997).
In the Controlled Dryland System, treatments significantly influenced available N (p = 0.010), with Urea treatments achieving the highest available N, followed closely by AMF and HyperCycle high-rate treatments, demonstrating comparable effectiveness in enhancing N availability (~125–175 mg kg⁻¹). Azospirillum also showed intermediate effectiveness, reinforcing its potential as a beneficial biofertilizer under moisture-limited conditions. These findings highlight AMF and HyperCycle’s capacity to mobilize and retain N effectively, emphasizing their ecological advantage in promoting sustained N availability through stable nutrient cycling pathways under drought stress (Smith and Read, 2008; Leung et al., 2020). Such resilience underscores biological N sources' adaptive potential, distinguishing them from synthetic fertilizers' typically short-lived nutrient pulse (Erisman et al., 2018).
Controlled System Available Nitrogen
Forage Yield
In the Controlled Flooded System, forage yield responses to fertilization and biofertilizer treatments did not reach statistical significance (NS, p> 0.05), yet discernible trends emerged. The Urea treatment demonstrated the highest median yield, consistent with the field study and established literature on the efficacy of synthetic nitrogen in flood-irrigated forage production (Geisseler & Scow, 2014). Notably, the Azospirillum treatment also exhibited competitive yields, suggesting that nitrogen-fixing bacteria can enhance productivity even in saturated soils, likely through improved rhizosphere nitrogen availability (Bashan et al., 2004). The AMF-only treatment showed moderate performance, a surprising finding given that anaerobic conditions typically suppress mycorrhizal activity (Smith & Read, 2008), possibly indicating some flood-tolerant AMF functionality. In contrast, the Control and HyperCycle high-rate treatments yielded the least, underscoring the limitations of unamended systems and certain microbial consortia under flooding.
In the Controlled Dryland System, yields were universally lower due to water constraints, yet trends mirrored the hierarchy observed in flooded conditions. Urea again produced the highest median yield, reaffirming the role of nitrogen as a primary yield-limiting factor in arid agroecosystems. The AMF treatment performed moderately well, aligning with its documented role in enhancing drought resilience through improved water and nutrient uptake (Augé, 2001). However, Azospirillum underperformed, potentially due to moisture-limited microbial activity or competition with indigenous soil microbiota. The HyperCycle high-rate treatment showed intermediate results, suggesting variable efficacy under moisture stress.
While statistical non-significance precludes definitive agronomic recommendations, these trends highlight the context-dependent utility of biofertilizers. In flooded systems, Azospirillum may complement synthetic nitrogen, whereas in dryland systems, AMF could mitigate water limitations. Further research should explore strain-specific adaptations and integrated nutrient management to optimize these biotechnologies for sustainable forage production.
Forage Quality
Forage quality in the Controlled Flooded System exhibited significant treatment-dependent variations (Figure 6). Urea application markedly increased crude protein (CP) content (~25% dry matter) compared to biofertilizer treatments (Azospirillum, Azospirillum + AMF, AMF) and the Control (~5–15%; p < 0.001), highlighting the rapid nitrogen availability provided by synthetic fertilization (Geisseler & Scow, 2014). Total digestible nutrients (TDN) showed a non-significant but notable trend toward higher values under Urea (~65–66%; p = 0.047), suggesting improved energy availability for livestock. Conversely, acid detergent fiber (ADF) was significantly lower in Urea-treated forage (~32%; p = 0.046), indicating enhanced digestibility compared to biologically amended treatments (~35–38%). These results are consistent with previous findings demonstrating synthetic nitrogen’s role in optimizing forage nutritive value through efficient nutrient assimilation (Geisseler & Scow, 2014).
In contrast, forage quality in the Dryland System did not differ significantly across treatments for CP, ADF, or TDN (NS), likely due to moisture limitations constraining nutrient uptake and microbial activity. Although Urea maintained a slight edge in CP, the lack of statistical significance suggests that biofertilizers (e.g., AMF, Azospirillum) may provide comparable forage quality under water-limited conditions.
Controlled System Forage Quality
Discussion
Our investigation into the effects of microbial inoculants on soil microbial communities, nitrogen pools, and forage productivity in high-elevation flood-irrigated hay meadows revealed important insights into the potential role of biofertilizers in sustainable agroecosystems. Despite the absence of statistically significant differences in microbial biomass carbon (MBC) and soil protein across treatments, observed trends suggest that arbuscular mycorrhizal fungi (AMF) and Azospirillum brasilense inoculation can enhance microbial activity and nitrogen cycling under both flooded and dryland conditions. These findings align with prior work demonstrating AMF’s ability to improve rhizosphere function and nutrient availability under oxygen-limited and phosphorus-constrained conditions as well as the role of Azospirillum in nitrogen fixation and hormone-mediated plant growth promotion (Bashan et al., 2004).
The higher available nitrogen (AN) observed under synthetic urea treatments in flooded systems reflects the rapid mineralization and bioavailability typical of inorganic fertilizers (Geisseler and Scow, 2014). However, enhanced available nitrogen stability (soil ACE protein) under AMF and microbial consortia in dryland regimes underscores the ecological advantage of biofertilizers in promoting sustained nutrient cycling and mitigating nitrogen losses under moisture stress (Smith and Read, 2008). This context dependency highlights the necessity for tailored fertilization strategies that consider site-specific hydrological regimes.
Forage yield and quality parameters, including crude protein (CP), acid detergent fiber (ADF), and total digestible nutrients (TDN), exhibited non-significant but agronomic relevant trends, with biofertilizers achieving yields comparable to urea. These results support the viability of biofertilizers as alternatives to synthetic nitrogen that can maintain productivity while enhancing soil microbial health. The observed forage quality variations, notably higher CP and lower ADF in urea treatments, likely reflect the immediacy of mineral nitrogen availability, whereas biofertilizer treatments may contribute to longer-term soil fertility and improved forage nutritive value through enhanced microbial function.
Research Outcomes
This study demonstrates the promising role of microbial inoculants, particularly AMF and Azospirillum brasilense, in supporting soil microbial biomass, nitrogen availability, and forage productivity in high-elevation hay meadows subject to flood irrigation and dryland conditions. Although short-term applications did not elicit statistically significant differences in all measured parameters, biological trends indicate meaningful ecological benefits of biofertilizers that justify further investigation. Incorporating microbial inoculants into forage management may reduce dependence on synthetic fertilizers, promote sustainable nutrient cycling, and improve resilience in semi-arid mountain agroecosystems. Future research should focus on optimizing inoculant compositions, dosage, and timing, integrating microbial biofertilizers with conventional management, and evaluating long-term effects across diverse environmental gradients. Our results contribute to the growing body of evidence advocating biodiversity as part of integrated nutrient management frameworks in vulnerable high-elevation ecosystems.
References
Adesemoye, A.O., Torbert, H.A., & Kloepper, J.W., 2009. Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. Microbial Ecology 58(4), 921–929. https://doi.org/10.1007/s00248-009-9531-y
Augé, R. M. (2001). Water relations, drought and vesicular‑arbuscular mycorrhizal symbiosis. Mycorrhiza, 11(1), 3–42.
https://doi.org/10.1007/s005720100097
Barnes, R. F., Nelson, C. J., Collins, M., & Moore, K. J. (Eds.). (2003). Forages: An Introduction to Grassland Agriculture (6th ed.). Ames, IA: Iowa State Press.
Bashan, Y., & Holguin, G. (2004). Azospirillum-plant relationships: environmental and physiological advances (1990–2003). Canadian Journal of Microbiology, 50, 521–577.
https://doi.org/10.1139/w04-035
Bashan, Y., & Holguín, G. (1997). Azospirillum–plant relationships: Environmental and physiological advances (1990–1996). Canadian Journal of Microbiology, 43(2), 103–121.
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Das, A. K., Lee, D.-S., Woo, Y.-J., Sultana, S., Mahmud, A., & Yun, B.-W. (2025). The impact of flooding on soil microbial communities and their functions: A review. Stresses, 5(2), 30.
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Erisman, J. W., Sutton, M. A., Galloway, J., Klimont, Z., & Winiwarter, W. (2008). How a century of ammonia synthesis changed the world. Nature Geoscience, 1, 636–639.
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Geisseler, D., & Scow, K. M. (2014). Long-term effects of mineral fertilizers on soil microorganisms – A review. Soil Biology and Biochemistry, 75, 54–63.
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Smith, S. E., & Read, D. J. (2008). Mycorrhizal Symbiosis (3rd ed.). Academic Press, London.
Walkiewicz, K., et al. (2020). Balanced forage nutrition through AMF–Azospirillum biofertilizer combinations in flooded agroecosystems. Journal of Plant Nutrition and Soil Science, 183, 500–510.
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Education and Outreach
Participation Summary:
Objective 1: Assess the current knowledge on the use of biofertilizer, create a common understanding among stakeholders of the proposed project and invite their participation in the project.
A structured questionnaire and extension meeting
To assess knowledge, perceptions, and practical challenges related to biofertilizer adoption in the target region, a structured questionnaire was administered to key stakeholders, including farmers, gardeners, hay producers, agricultural extension agents, and soil management specialists. The survey was distributed during a local extension meeting in March 2025, facilitating direct engagement with individuals actively involved in agricultural production. Responses revealed a range of familiarity with biofertilizer technologies; while many participants expressed strong interest, hands-on experience was limited. Recurring concerns focused on cost-effectiveness, product accessibility, and compatibility with existing conventional fertilization practices. Stakeholders also emphasized the need for clear, science-based information on efficacy, application methods, and long-term benefits to support informed decision-making.
Following data collection, a stakeholder workshop was convened to present preliminary findings, clarify project objectives, and address knowledge gaps. These interactive sessions provided a platform for open dialogue, allowing participants to share field experiences, voice concerns, and identify practical barriers to adoption. Misconceptions such as perceived risks of yield reduction or incompatibility with chemical fertilizers were addressed through evidence-based discussions. The workshops further enabled collaborative input on project design, ensuring that proposed interventions align with the real-world needs and constraints of the farming community.
These outreach efforts successfully fostered a shared understanding among diverse stakeholder groups and secured commitments for continued participation in project activities.
Questionnaire copy
Biofertilizer Questionnaire _RO_LVD
Objective 2. Establish open communication and continuously engage farmers and ranchers in the project progress
Farm/ study site tour by stakeholders
As part of our commitment to fostering open communication and sustained collaboration with agricultural stakeholders, a field tour of the project study site at the Laramie Research and Extension Center was held in July 2024. The event brought together farmers, extension agents, and other key stakeholders to observe experimental plots firsthand and engage in discussions regarding ongoing biofertilizer trials in flood-irrigated hay meadows.
During the tour, the project team provided comprehensive explanations of the research objectives, methodologies, and preliminary findings. Emphasis was placed on the potential impacts of biofertilizers on soil health, forage productivity, and the long-term sustainability of flood-irrigated forage systems. This interactive format encouraged stakeholders to ask questions, share their on-farm experiences, and offer practical feedback, thereby creating a transparent and participatory environment.
The field tour served to strengthen trust and collaboration between researchers and producers, fostering a sense of shared ownership in the project’s outcomes. This hands-on outreach activity demonstrated the importance of direct engagement in bridging the gap between scientific research and practical implementation.
Objective 3: Advance/share research findings from this project with producers, research scientists, agricultural educators, and 4-H youth groups in Albany County Wyoming, and Walden Colorado
Outreach Activities: 4-H Youth Engagement on Soil Microbes and Soil Health
On May 21, 2025, as part of our outreach activity, we conducted an interactive outreach session with local 4-H youth to enhance their understanding of soil microbes and their critical role in sustainable agriculture. The session engaged young learners through hands-on activities, visual demonstrations, and discussions, fostering a deeper appreciation for soil health and its impact on forage production.
Participants explored the diverse world of soil microorganisms, including bacteria, fungi, algae, protozoa, and viruses, and learned how these microbes drive nutrient cycling and ecosystem balance. A highlight of the session was the opportunity to view cultured biofertilizer microorganisms, such as Azospirillum bacteria and fungal cells, under microscopes. This direct observation helped youth connect theoretical knowledge to real-world applications in agriculture.
To further their understanding of soil ecosystems, the youth participated in lessons on soil texture and color, discovering how these physical properties influence water retention, aeration, and microbial habitats. The session also featured the construction of Winogradsky columns, which allowed participants to observe microbial stratification in a self-contained ecosystem. Through this activity, they identified aerobic microbes and algae at the surface, sulfur bacteria in middle layers, and anaerobic sulfate-reducing bacteria at the bottom. These observations sparked discussions about microbial habitats, oxygen gradients, and their relevance in agricultural systems like flood-irrigated forage fields and rice paddies.
To reinforce the connection between soil health and plant productivity, youth examined photographs comparing healthy forage grasses with nutrient-deficient plants. This visual comparison underscored the importance of microbial activity and proper soil management in achieving optimal forage quality.
The outreach successfully achieved its goals of increasing youth awareness of soil microbial ecology and its agricultural significance. By combining interactive learning with real-world examples, the session inspired curiosity and empowered participants to recognize the value of sustainable land management practices. These efforts contribute to our broader mission of cultivating informed future stakeholders who will advocate for and implement sustainable agriculture solutions.
Outreach Presentation to Plant Sciences Department, University of Wyoming
In support of our outreach and education objectives, a presentation was delivered to graduate and undergraduate students, faculty, and staff in the Plant Sciences Department at the University of Wyoming. The seminar focused on ongoing research investigating the effects of biofertilizers on soil microbial communities, nitrogen cycling, and forage productivity in high-elevation flood-irrigated hay meadows.
The presentation highlighted key findings from field and controlled environment trials, emphasizing the potential of microbial inoculants as sustainable alternatives or complements to synthetic nitrogen fertilizers. Discussions addressed soil health implications, forage quality outcomes, and practical considerations for adoption in regional agricultural systems.
Attendees engaged actively during the session, asking questions and providing valuable feedback that will inform future outreach and research efforts. This engagement fostered increased awareness and interest in sustainable soil management practices among emerging agricultural professionals and researchers.
Poster Presentation at the Western COA Annual Summer Meeting
As part of the project’s final outreach efforts, a poster presentation was delivered to Western COA Deans, AES Directors, Extension Directors, Academic Deans, and CARET representatives during the annual summer meeting LREC in the Hansen Arena on June 24th 2025. The poster summarized the completed research findings on the effects of biofertilizers on soil microbial communities, nitrogen dynamics, and forage productivity in high-elevation flood-irrigated hay meadows.
The presentation showcased key results demonstrating the potential of microbial inoculants to enhance soil health and maintain forage yields, highlighting their role as viable alternatives to synthetic nitrogen fertilizers. Attendees expressed strong interest in the outcomes and their implications for regional agricultural sustainability.
This event provided an important opportunity to disseminate final project results, engage with influential agricultural leaders, and reinforce collaborative networks to support the continued adoption and application of biofertilizer technologies.
- In-Person Meetings and Workshops
We facilitated face-to-face meetings and interactive workshops to build trust and encourage open dialogue with stakeholders. These sessions included hands-on demonstrations and field tours, allowing participants to observe treatment plots firsthand. This approach promoted active engagement and enabled discussions tailored to stakeholder interests and needs.
- Surveys and Questionnaires
A structured survey was administered to assess stakeholders’ knowledge, perceptions, and challenges related to biofertilizer use. The feedback collected helped inform our communication strategy by identifying knowledge gaps and concerns, ensuring that messages are relevant and targeted effectively.
- Digital Communication
Outreach events were promoted through weekly and monthly email newsletters and social media updates on platforms such as Facebook. These digital channels proved effective in reaching a broad audience and maintaining ongoing engagement.
- Demonstration Plots
On-site demonstration plots showcased the practical benefits of biofertilizer use, providing tangible evidence of effectiveness. These pilot projects facilitated peer-to-peer learning and helped build stakeholder confidence through direct observation of results.
- Extension Services Collaboration
We partnered with local extension agents who are trusted advisors within agricultural communities. Their involvement enhanced outreach effectiveness by tailoring messaging to local contexts and providing ongoing technical support to stakeholders.