Biofertilization of Bermudagrass: A step toward sustainable forage production

Final report for LS19-307

Project Type: Research and Education
Funds awarded in 2019: $221,115.00
Projected End Date: 09/30/2022
Grant Recipient: Auburn University
Region: Southern
State: Alabama
Principal Investigator:
Dr. Leanne Dillard
Auburn University
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Project Information


Sustainable production of livestock from both grazed and conserved forage production is critical to economic and environmental viability of the livestock industry. Nitrogen fertilizer accounts for the majority of variable-input costs associated with forage production, and potentially can negatively impact soil, water, and air resources. Bermudagrass is an extremely drought-tolerant and persistent warm-season perennial grass that is used widely throughout the southeastern U.S. for hay production and grazing. It is also very responsive to N fertilization and can produce more than 10 Mg of biomass per hectare. Plant growth-promoting rhizobacteria are beneficial soil microbes applied to agricultural and horticultural crops as inoculants. Growth promotion from PGPR application can result from N fixation, phosphate solubilization or internal changes in hormone signaling pathways associated with some strains. Preliminary data indicate that PGPR may also hold promise as an alternative to synthetic N fertilizer for biofertilization and forage quality enhancement of bermudagrass for livestock.

The objectives of this study are to evaluate plant growth-promoting rhizobacteria for use in hay and grazed forage systems on six Alabama farms to determine its effectiveness as a N-fertilizer supplement or replacement. Measurements of forage yield, quality, insect damage and spring green-up will be taken throughout the growing season over two years. Two field days and a cumulative, hands-on workshop will be held over a 3-year period to disseminate the finding to bermudagrass producers throughout the state. Findings from the study will also be published using established social media outlets and online websites. Extension and peer-reviewed articles will be submitted during Year 3 of the proposed project. Transfer of knowledge to producers and stakeholder groups will be assessed to evaluate the impact of the Extension/outreach program. New knowledge gained from this project could contribute toward significant economic savings and improved efficiency of forage nutrient management in the future, and suggest potentially fruitful areas of new basic and translational research on beneficial plant X microbe interactions.

Project Objectives:
  • Determine forage yield and quality of ‘Russell’ bermudagrass fertilized with Blend 20, Blend 20 + N-fertilizer, N-fertilizer, or no fertilizer and either hayed or grazed by beef cattle.
  • Assess insect damage in ‘Russell’ bermudagrass fertilized with Blend 20, Blend 20 + N-fertilizer, N-fertilizer, or no fertilizer and either hayed or grazed by beef cattle.
  • Determine the differences in initiation of spring growth in ‘Russell’ bermudagrass fertilized with Blend 20, Blend 20 + N-fertilizer, N-fertilizer, or no fertilizer and either hayed or grazed by beef cattle.
  • Disseminate the knowledge gained to a broader audience of stakeholder by organizing producer-driven regional workshops, development of Extension publications, social media posts, and publishing of peer-reviewed abstracts and manuscripts.


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Materials and methods:

Objective 1:

Three ‘Russell’ bermudagrass hay producers and 3 beef cattle producers grazing ‘Coastal’ bermudagrass have already been selected and agreed to allow the investigators to conduct research on their farms. Participating farms were also selected based on location providing one North, Central, and South Alabama location each for hay production and grazing farms.

Two different bermudagrass varieties are being used because of the likelihood that investigators will be unable to find 6 farms that use the same variety. ‘Russell’ bermudagrass was released by Auburn University and has exhibited more cold tolerance than other bermudagrass varieties. Whereas it is highly productive and nutritious, it is not as grazing-tolerant as other varieties, and thus most producers prefer it for hay production only. ‘Coastal’ bermudagrass was released by the USDA and University of Georgia Coastal Experiment Station. It is currently the most widely grown bermudagrass variety in the Southeast. It is not as high-yielding or nutritious as ‘Russell’, but is more persistent under grazing pressure and is preferred for grazing by Alabama forage producers. The investigators acknowledge that some parameters to be measured (i.e., yield, forage quality, and bermudagrass stem maggot infestation) will be confounded by the use of  different varieties in haying vs. grazing systems; however, the current experimental design represents real-world, on-farm production practices that are currently used by producers.


In March 2020, an initial farm visit will be conducted with each participating farmer. The farmer will be given a survey to determine historical management of the bermudagrass stand (i.e., fertilization practices, herbicide and pesticide treatments, cutting frequency and height, grazing method and duration). A 0.05-hectare site will be chosen by the investigators and the farmer to conduct the research, and ground-level markers will be placed to delineate the area. A visual assessment will be made and recorded by the investigators to determine percent forage stand, weed presence, and any other conditions of the site (e.g., soil type, GPS coordinates, slope, etc.).


January through March of Yr 1 and 2, the biofertilizer will prepared in the Auburn University PGPR lab. Three Bacillus strains, Bacillus pumilus AP 7, B. pumilus AP 18, and B. sphaericus AP 282, will be evaluated as a Blend of equal proportions of each strain, herein referenced as Blend 20. Bacterial strains are stored at -80 °C.  When needed, they will be transferred from cryovials to plates of TSA media and allowed to grow at 28 °C in an incubator until spore formation (usually 3-7 d). Bacterial lawns with spores will be scraped with inoculating loops, transferred to sterile centrifuge tubes (50 ml, VWR, Radnor, PA) containing 40 ml of de-ionized water, and vigorously shaken to distribute bacterial cells. Bacterial populations (number of colony forming units [CFU]) in the suspensions will be determined by plating 50 µl of the serial dilution onto TSA plates, incubating plates for 24­-48 h and then counting the number of bacterial colonies on each plate. These populations in solution will be used to prepare stock solutions of each bacterium with a final concentration of 6.0 x 109 CFU per ml of each strain. When needed, Blend 20 will be prepared by mixing each parts of each bacterial strain in solution.


In April, investigators will return to each farm and lay out the experimental plots. Each plot will be 3 × 3 m with 2 replications at each site in a completely randomized design. Fertilizer treatments will be applied to each plot and all plots will be lightly hand watered to move the treatments into the root zone. Blend 20 will be applied using a backpack sprayer (Solo, Newport News, VA) that delivered 500 ml / m2 of freshly-prepared aqueous bacterial suspension of 6 x 109 CFU per ml. After treating, the plots will be hand watered with 12.7 L of water / m2 to move the bacteria into the root zone.  


Hay-cutting interval will be determined by the producer, and the producer will contact the investigators 72-96 hrs prior to scheduled hay harvest. Investigators will return to the farm, and a forage sample will be clipped from each plot for determination of forage yield. Subsamples will be taken to the Auburn University Ruminant Nutrition lab and analyzed for crude protein (AOAC, 2006), neutral detergent fiber, acid detergent fiber, acid detergent lignin (Van Soest et al., 1991), and micronutrient concentrations (Hue and Evans, 1986). In vitro digestibility will also be determined by the Van Soest (1991) modification of the Tilley and Terry procedure (1963) using the Daisy II® incubator system (Ankom Technology, Macedon, NY).


On grazing farms, animals will be excluded from plots using temporary electric fencing with a solar-powered charger after the initial fertilization application. Forage samples will be clipped from each plot every 30-45 days throughout the summer prior to grazing using a 0.1-m2 quadrat from each plot. Animals will be allowed access to the experimental area for 24 h. After 24 h, animals will be again excluded from the area, and post-grazing samples will be taken. For both pre- and post-grazing samples, forage yield will be determined. Subsamples will be returned to Auburn University Ruminant Nutrition Lab and analyzed for crude protein (AOAC, 2006), neutral detergent fiber, acid detergent fiber, acid detergent lignin (Van Soest et al., 1991), micronutrient concentrations (Hue and Evans, 1986), and in vitro digestibility by the Van Soest (1991) modification of the Tilley and Terry procedure (1963). Regrowth forage will continue to be sampled for as long as satisfactory regrowth occurs. Forage yield and quality data will analyzed using the PROC MIXED procedure of SAS 9.4 (SAS Int., Cary, NC). Multiple harvests from the same farm will be analyzed as repeated measures. Least squares means will be used for means separation with α = 0.05.


Objective 2:

During each sampling event, each plot will be assessed for insect (i.e., fall armyworm and bermudagrass stem maggot) infestation with the cooperation of the landowner prior to grazing or mowing. Fall armyworm larvae and stem maggot flies will be sampled by sweeping two 0.1-m2 areas with a sweep net in the morning (Flanders and Treadaway, 2018). Percent of insect damaged grass in two 0.1-m2 samples will also be recorded for each plot. Producers will be provided a sweep net and 75% alcohol containers and will be asked to sweep plots at 2-week intervals between sampling dates. This will provide more accurate data, as well as be an opportunity for producers to learn how to samples for insect pests. Insect data will be compared between treatments and to damage thresholds (e.g., 2-3 fall armyworm larvae per 0.1 m2) when available. The landowner will observe and assist with sampling and a sweep net will be kept at each site during the study for their use. Numbers of fall armyworm larvae or stem maggot flies per sample will be summarized for all samples as well as total for each year. Percentage of damaged grass will be transformed as proportions (0-1) with an arcsine transformation before analysis. Percentages of damaged grass and seasonal abundance of both insect pests will be compared across treatments using ANOVA for repeated measures (JMP, Cary, NC). The seasonal total of each pest species found in each treatment will also be compared using ANOVA. Means will be compared using single degree of freedom orthogonal contrasts similar to Gunter et al. (2018).


Objective 3:

In March and April of 2021 and 2020, investigators will measure rate of spring green-up in each plot. Each plot will be scanned with a hand-held normalized difference vegetative index (NDVI, FieldScout CM 1000, Spectrum Technologies, Inc., Aurora, IL) to determine live green material vs. dormant plant material. Also, plots will be visually rated by three evaluators (landowners and two researchers) for percent green-up every 2 wk. Measurements of NDVI will be analyzed using repeated measures multivariate analysis of variance (MANOVA), with orthogonal contrasts used to compare means (P < 0.05, JMP Version 13. SAS Institute Inc., Cary, NC).

Research results and discussion:

During the COVID pandemic, Auburn University was committed to the continuation of all research programs, specifically federally funded research such as this project. As a result, we were able to begin the research project as proposed in the grant. Scientific manuscripts from this project are currently being submitted, but the results of this project were the focus of 1 M.S. student thesis in 2021 entitled, ‘Influence of Plant-Growth Promoting Rhizobacteria on Arthropod Populations, Forage Biomass, and Soil Health in Bermudagrass Pastures.’ Below is the abstract from this thesis:

Bermudagrass (Cynodon dactylon (L.) Pers.) is a warm-season, perennial pasture grass commonly used in forage production systems in the southeastern United States. Drought tolerance and pronounced yield response to high rates of nitrogen fertilizer applications make bermudagrass the preferred species for hay production and grazing pastures. With growing concerns of negative impacts resulting from high levels of nitrogen fertilizers applications, it is important to explore biological alternatives for fertilizers in the forage industry. Plant growth-promoting rhizobacteria (PGPR) represent a potential sustainable alternative to chemical fertilizers that could significantly reduce the amount of nitrogen in forage production. The overall purpose of this project was to evaluate the potential of PGPR as a biostimulant to reduce chemical inputs in bermudagrass hay fields and grazing pastures. PGPR with and without nitrogen fertilizers were compared to full rates of nitrogen fertilizers to evaluate differences in forage biomass, arthropods populations, and soil health between treatments. A 2-year, large-scale field evaluation in bermudagrass pastures comparing effects of PGPR and nitrogen fertilizers was conducted. Three grazed locations and three hay locations throughout the state of Alabama were established with three replications per location and six treatments per replication. The six treatments include a blend of PGPR (Blend 20), a single strain PGPR (DH44), Blend 20 plus a ½ rate of nitrogen fertilizer, DH44 plus a ½ rate of nitrogen fertilizer, a full rate of nitrogen fertilizer, and a control plot. These treatments allowed comparisons of PGPR to a full rate of nitrogen, and an evaluation of the effect of PGPR when applied with a half rate of nitrogen fertilizer. Two applications of treatments and four harvests were completed each year. At harvests, arthropod populations, forage heights, and forage dry weight data were collected. Treatments only significantly affected biomass weights on the locations managed as hay. Forage weight from plots treated with the full rate of nitrogen fertilizer and both PGPR plus ½ rate treatments was significantly greater than control plots. These results indicate that PGPR plus a ½ of nitrogen fertilizer can yield similar results as a full rate of nitrogen fertilizer in fields utilized for hay production and grazing systems. A 1-year, large-scale field evaluation in bermudagrass pastures comparing effects of PGPR and nitrogen fertilizers was conducted. Two sites at the E.V. Smith Research Center (Macon County, Alabama) were selected and three replicates of six treatments were established on each site. The treatments were Blend 20, DH44, Blend 20 plus a ½ rate of nitrogen fertilizer, DH44 plus a ½ rate of nitrogen fertilizer, a full rate of nitrogen fertilizer, and an untreated control. These treatments allowed comparisons of PGPR to a full rate of nitrogen, and an evaluation of the effect of PGPR when applied with a half rate of nitrogen fertilizer. Two applications of each treatment were applied plots were evaluated four times. At harvests, arthropod populations, forage heights, and forage dry weight data were collected. In September 2020, soil cores and samples were also collected from each plot to analyze and compare populations of the soil mesofauna and soil respiration as indicators of soil health. There were no significant treatment differences for forage biomass or arthropod populations at either site. Soil respirations and populations of mesofauna were numerically greater in plots treated with PGPR but were not statistically significant in oribatid populations.

Participation Summary
6 Farmers participating in research


Educational approach:

Three workshops will be organized throughout Alabama. Each will be located in a different geographical region (i.e., North, Central, and South) and will be held on cooperating producer farms. At each field day, pre- and post-testing methods will be used to evaluate program impact. Two Alabama Cooperative Extension (ACES) publications will be written on proper fertilization management of bermudagrass. Routine social media posts, eNewsletters, and updates on regarding the project and sustainable fertilization practices will be published. Data from this experiment will be presented at the biennial Alabama Forage Conference (Fall 2019 and 2021), and abstracts will be presented at regional and national scientific meetings (e.g., American Forage and Grassland Council, Crop Science Society of America, and American Society of Animal Science), and 1 peer-reviewed scientific manuscript will be submitted.

Educational & Outreach Activities

40 Consultations
4 Curricula, factsheets or educational tools
1 Journal articles
7 On-farm demonstrations
1 Online trainings
4 Published press articles, newsletters
8 Webinars / talks / presentations
2 Workshop field days

Participation Summary:

504 Farmers participated
105 Ag professionals participated
Education/outreach description:

To date, 3 popular press articles have been written and distributed. This included in-state publications (Alabama Cattleman's Magazine and The Season) and 1 national publication (Progressive Forage Grower) with a total readership of over 55,000. Eleven scientific abstracts have been submitted to regional and national conferences.

One peer-reviewed journal article was written and published in 2020, Griffin, M.E., R.B. Muntifering, M.K. Mullenix, D.W. Held, and S.L. Dillard. 2020. Evaluation of plant growth-promoting rhizobacteria on stockpiled bermudagrass. Crop, Forage, and Turfgrass Management 6:e20028. Three more peer-reviewed articles are currently in preparation for submission in Summer 2022. 

As the State Forage Extension Specialist, Dr. Dillard has fielded approximately 40 communications related to bermudagrass fertility management and biofertilizer use in forage systems. This number has increased dramatically in the last 6 months due to rising fertilizer prices. This has created an increased interest in biostimulant/biofertilizer products. Furthermore, a peer-reviewed Extension Publication was completed in 2020 entitled 'Bermudagrass in Alabama' and can be found on at Additionally, two non-peer-reviewed publications were published on the website. Due to COVID-19, no workshops or field days were conducted from March 2020 through March 2021. Two field days were help on September 11, 2021 in South Alabama and  and ) and Fall 2021 (Central Alabama) for disseminating first year results of the project and proper N fertility in bermudagrass systems. In September, 2021 the Forage Basics online course was released. This course covers bermudagrass management, as well and fertility. As of May 1, 2022, 301 participants have registered for the course.

In April 2022, Dr. Dillard hosted a multi-state In-service training for 80 Animal Science and Forage Extension processionals from 8 states. Proper forage N fertility  and the use of biostimulants and biofertilizers was highlighted during this training. Dr. Dillard disseminated the results of this project at the Southern Pasture and Forage Crop Improvement Conference as a keynote speaker in July 2022. This conference draws research and Extension professionals from across the Southeast. 

Learning Outcomes

580 Farmers reported changes in knowledge, attitudes, skills and/or awareness as a result of their participation

Project Outcomes

1 Grant received that built upon this project
4 New working collaborations
Project outcomes:

Over the course of the project, over 580 farmers and ranchers in the Southeast have increased knowledge on bermudagrass N fertility and/or plant-growth promoting rhizobacteria. We reached over 120 Extension agents and research scientists through agent trainings and presentation of scientific abstracts on plant growth promoting rhizobacteria. As a result of this project, federal funding was allocated in partnership with the USDA-ARS Soil Dynamics Lab in Auburn, AL to further research the use of PGPR in forage systems. This led to the recent hiring of a USDA-ARS microbiologist that will further investigate the use of PGPR in forage systems. Furthermore, a collaboration with the University of Tennessee was initiated during this project and we currently are conducting research on the use of PGPR in tall fescue and bermudagrass systems in cooler climates (i.e., North Tennessee). In collaboration with the University of Tennessee and the USDA-ARS we submitted a USDA-NIFA AFRI Foundational Integrated Research and Education grant in 2021. This grant was not funded but revised and resubmitted in 2022. We are currently awaiting the result of the grant review panel. We have also started a collaboration with a 3rd party firm to produce PGPR on a larger scale to allow for increased research and field-scale research. While, on-farm adoption of PGPR is still not available due to lack of commercial availability, knowledge of proper N fertility and ‘scam’ products increased for producers at all outreach activities hosted. This project was very timely because as N fertilizer prices have increased in the last two years, more non-verified products have entered the marketplace. Our Extension team has therefore been able to take the lead as a source of science-based knowledge on these products and assist producers and Extension agents with discerning between various products on the market.

            Many of our proposed outreach programs were affected by the COVID pandemic, we likely reached more farmers and ranchers from our novel programing than we would have from the proposed plan.  Below is a list of Extension activities that included bermudagrass fertility and/or PGPR discussions. Notably is the ‘Forage Basics’ online course. This course was created during COVID 19 as a free, online tool to assist beginning farmers with forage management. There is an entire module dedicated to bermudagrass fertility and timing of fertilization. Over 330 individuals viewed this module during the first year after the release of the course. 



Further work needs to be completed on the efficacy of plant growth promoting rhizobacteria on perennial and annual grass systems. Grasslands represent the largest land use in the U.S. and PGPR could provide an economic and environmentally sustainable method of maintaining forage production, while reducing inputs and reducing reliance on fossil fuels and synthetic fertilizers. 

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Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.