Applying ecological treatments to boost yields among restoration target species of seed production areas

Progress report for GNC19-293

Project Type: Graduate Student
Funds awarded in 2019: $14,942.00
Projected End Date: 10/29/2022
Grant Recipients: University of Illinois at Urbana-Champaign; University of Illinois at Urbana-Champaign
Region: North Central
State: Illinois
Graduate Student:
Faculty Advisor:
Dr. Jeffrey Matthews
University of Illinois at Urbana-Champaign
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Project Information


Title: Applying ecological treatments to boost yields among restoration target species of seed production areas

Ecological restoration seeks to repair or assist the recovery of damaged or degraded ecosystems and yields benefits to society. Seed production areas (SPAs), which are plantings of wild plant species that apply agricultural and horticultural practices, are critical to supply these seeds for ecological restorations. SPA managers face challenges when cultivating wild plant species, leading to financial difficulties and unreliable seed supplies. Thus, restoration practitioners are commonly restricted by expensive or unavailable seed. SPA management techniques must be improved to boost seed yields to benefit stakeholders and facilitate ecological restorations. A common method to boost yields is to use chemical fertilizers similar to traditional agricultural techniques. Fertilizer addition has potential negative consequences such as nutrient runoff and promoting weed invasion, however. A promising technique is inoculation with arbuscular mycorrhizal fungi (AMF). AMF are soil fungi which associate with plant roots, providing water, nutrients, and pathogen defense in exchange for plant carbon. Recent studies suggest that soil inoculation with AMF could be an alternative and beneficial management technique that boosts plant growth. Yet, the value of AMF inoculation in commercial settings has not been evaluated.

To study SPA management, I will test nutrient addition and AMF inoculation strategies in experimental SPAs. Using a randomized block design, I will determine how the treatments affect seed yields and managerial input for three restoration species in two study sites across three growing seasons. Learning outcomes of the project include increasing knowledge of SPA management techniques, and generating awareness of the potential of AMF as a sustainable management strategy. Long-term action outcomes include implementation of production techniques which will increase seed yield in SPAs and provide a more reliable source of native plant seed for ecological restorations. I will evaluate the outcomes of this project by a combination of personal interaction with stakeholders at targeted conferences, as well as a follow-up survey of a technical report that I will send directly to producers in the native plant industry. My project will enhance the quality of life for SPA managers in the native plant industry, improve seed supplies imperative for restorations, and make this seed production more sustainable.  

Project Objectives:

My outcomes will target stakeholders in the native plant nursery, particularly native seed producers. This project will create critical knowledge about SPA management techniques for native growers. Such knowledge would improve the abilities of managers to reliably produce their crop. Results could encourage native growers to boost yields using sustainable inoculation techniques rather than fertilizer addition. It will promote a greater awareness about the value of soil manipulation through arbuscular mycorrhizal fungal inoculation. My participating stakeholders will apply both methods to their experimental SPAs. Long-term action outcomes include implementation of production techniques which will increase seed yield in SPAs and provide a more reliable source of native plant seed for ecological restorations


Materials and methods:


Field site descriptions

Three sites were used for the project, representing different stakeholders for SPAs (Fig. 1): Barefoot Nursery & Restoration (Springfield, IL, and hereafter, “Barefoot”), Heritage (Champaign, IL, and hereafter, “Heritage”), and Lake of the Woods Forest Preserve (Mahomet, IL, and hereafter, “LOTW”). These locations represent a private enterprise, a local organization, and a regional conservation organization, respectively.

Figure 1: Locations of the three field sites of experimental SPAs in Illinois.

Barefoot is a private nursery which specializes in native plants, including existing plots for seed production. The site consists of small-scale infrastructure for growing and producing seed mixes. The SPA at this site was established on a 1-acre section of a corn-soy farm field, which was adjacent to the nursery. Soils at the site range from sandy loam to pure sand due to historical deposition from the nearby Sangamon River. Thus, some sections in the SPA appear unusually nutrient-poor, as well as prone to heating and desiccation. The Heritage location is former turf grass managed by the Champaign Park District. The site is marginal property for the Park District, with low pedestrian and recreational traffic. The site is flanked by a prairie restoration to the east, and Copper Slough to the west. The soils are Drummer silty clay loam, and have a low weed seed bank. Growing conditions at Heritage seem the least stressful (e.g., high quality soil, high water retention). LOTW is a nature preserve managed by the Champaign County Forest Preserve District (CCFPD), consisting of a matrix of woodland, prairie, and savanna habitats. The SPA was established on an acre of old field adjacent to the popular Buffalo Trace Bike Trail. Previously, the site was dominated by Bromus inermis (smooth brome), Trifolium pratense (red clover), and ruderal species. Soils at the SPA are Dana silt loam, and appear to be of intermediate growing quality when compared to Barefoot and Heritage.

SPA preparation and experimental design

Experimental SPAs were created using a randomized block design, with a 2×2 factorial treatment combination, with AMF inoculation and fertilizer addition as treatments (Fig. 2). Each block was 32 ft x 43 ft and consisted of 4 25-ft rows. To prevent treatment spillover, buffers between each row were 6 ft; buffers between rows within blocks were 7 ft (Fig. 2a). Buffers were seeded with turfgrass at Barefoot and LOTW due to the lack of suitable vegetation at these sites.

Figure 2: The dimensions and experimental design of the SPAs at the three sites. A) A visual representation of the dimensions of each block and its components. B) An example of the experimental design, showing randomized treatments within two example blocks. Each block has randomized positioning of the study species within each row.

Figure 3: SPA establishment at Barefoot in April 2020. Spray paint on the landscape fabric marked study species identity (i.e., randomization within rows).

SPAs were established in early April 2020 by using 4’ Dewitt Sunbelt weed barrier fabric to smother existing vegetation (Figs. 3-4).

Figure 4: SPAs following establishment at LOTW in May 2020. Note the presence of Trifolium pratense and weedy mustard species, as well as the initial fence posts for deer fencing.

Study species descriptions, cultivation, and field transfer

I chose four study species: Asclepias tuberosa (butterfly weed), Echinacea pallida (pale purple coneflower), Eryngium yuccifolium (rattlesnake master), and Parthenium integrifolium (wild quinine). These species were selected because 1) they are herbaceous flowering target species in restorations and are valuable for use in USDA programs such as the Conservation Reserve Program and CP-42 pollinator plantings (National Conservation Practice Standards 2015), 2) they were also previously shown to have positive growth responses to indigenous AMF treatments in experimental settings (Koziol et al. 2017).

I purchased seed of the four study species in January 2020, from Prairie Moon Nursery (Winona, MN). Prairie Moon Nursery sells sourced seed (i.e., local ecotype) from a network of producers throughout the Midwest. All seed was cold moist stratified in double-sterilized sand for 60 days.

I applied the nurse plant method for inoculation. This method pre-infects seedlings or plugs prior to field establishment, which can boost inoculum survival and have infected plants act as nuclei to nearby plants (Koziol et al. 2017). In late March, 2020, seeds were sown into flats containing a mixture of 50-50 sand and general-purpose soil mix (1:1:1 soil/peat/perlite), which was double sterilized using steaming. There were one to two rest days in between each sterilization event. After germination and the appearance of cotyledons, seedlings were transferred to 72-cell growing trays. Thereafter, for all the study species, half of seedlings were put into -AMF growing trays (60-40 double-sterilized sand/potting soil mixture), while the other half were transferred to +AMF growing trays. +AMF growing trays consisted of 45-45 double sterilized sand/potting soil mixture, and 10% by volume of MycoBloom AMF. 10% of AMF mixture by volume is considered a liberal volume by MycoBloom instructions. I used the 50-50 sand/potting soil mixture as the base in growing trays in order to increase nutrient stress to promote +AMF seedling inoculation. Because AMF infection occurs over 2 weeks, seedlings were grown for 4-6 weeks before field transfer to promote root infection. Seedlings were grown in temperature-controlled conditions (24°C) at the University of Illinois Agricultural and Consumer Sciences Plant Care Facility (40° 6′ 7″ N, 88° 13′ 26″ W).

Most plugs were planted in-field between late April through June in Heritage and LOTW. Plugs were planted in late June and early July at Barefoot to allow the grass seed in the buffers to stabilize the soil. At each site, a particular species was always planted on a single day to minimize priority effects. Dead seedlings were replaced within 2 weeks of the initial establishment to have more complete rows, while avoiding substantial time elapsed to lead to priority effects. In each row, 6 plugs of each species were planted per row, totaling 24 plants per row across the four study species. Plugs were planted by staggering at a 1.4-ft distance (Fig. 2a); plants were staggered to avoid overcutting the center of the fabric. The fabric was cut by a 15-cm “X” in the plastic to transfer the plugs.    

Field management

Throughout the growing season, buffers within the blocks were mown approximately once every 10 days. Rows were weeded as needed, generally monthly for Heritage and Barefoot, and biweekly for LOTW. In August, 2020, I began fertilization treatments across all +nutrient rows in the three sites. The fertilizer used was Peters Excel 15-5-15 (NPK) fertilizer; a high N:P ratio was sought out because P fertilization is less important with respect to seed production (Ken Fromm, personal communication). Fertilization treatments consisted of, and will consist of for the duration of the experiment, adding 350 mL at 125 ppm (N) concentration to each individual plant within the +fertilizer rows.

Data collection in fall, 2020

Figure 5: Data collection at the Heritage site of plant survival, size, and flowering status in November 2020. Measured in the foreground is E. pallida; P. integrifolium is behind it.

At each site, I determined each individual plant’s survival, flowering status, and size (Fig. 5). Size was assessed by measuring a plant’s height and its estimated leaf area. For each plant, leaves were subjectively counted as “small,” “medium,” or “large” based on the species. For each species, I estimated the area of each size class by averaging twenty samples of each species-leaf size combination. Leaf size class counts were then multiplied by the calculated average, and then summed for each plant. Seed was hand-harvested by row from the first crop in fall 2020. Seeds were weighed in January 2021 (Fig. 6). For plant size estimators and seed yield, I averaged the values across species and within rows, yielding per capita averages.  

Figure 6: An IGNITE intern in the lab processing and weighing wild quinine seeds from the 2020 field season.




National Conservation Practice Standards. 2015. Conservation cover (Ac.): Code 327. (

Koziol L., Schultz P.A., Bever J.D., et al. 2017. User Manual: A practical guide to inoculation with arbuscular mycorrhizal fungi in ecological restoration. SERDP Project RC-2330.

Research results and discussion:

Preliminary findings

Here, I present some preliminary analyses of the data from the 2020 field season. For simplicity, I report on survivorship among the study sites. I also present an analysis of the plant size and seed yield for Heritage only, due to this site’s better reliability of data due to superior plug establishment (survival per species per row was always >4). The species E. pallida and P. integrifolium were analyzed since these species had the best establishment and growth during the season.

Figure 7: Survivorship of planted plugs in the 2020 field season across the three sites.

Survivorship among the plugs was generally >75% across all species and sites (Fig. 7). Plug establishment was most successful (i.e., greatest survival and flowering) at Heritage, followed by Barefoot. LOTW had the lowest survival across all species due to deer and rodent herbivory (see Challenges of 2020 field season). Establishment was the highest for E. pallida (93% survival across all sites). A. tuberosa, P. integrifolium, and E. yuccifolium had survival percentages of 80, 81, and 83%, respectively.

Figure 8: (Log+1)-transformed per capita seed production at Heritage in 2020. The black line represents the median; the box represents the interquartile range; the whiskers represent the data distribution; dots represent outliers.

To measure seed production at Heritage, I used per capita seed yields, averaged per individual per row, to account for differential survival within rows. Yields were log+1 transformed to assist with visualization and analysis. E. pallida produced marginal seed yields, while P. integrifolium had the highest yields and appeared fully mature (Fig. 8). For both study species, E. pallida and P. integrifolium yields were the lowest in control rows.

I used an ANOVA for randomized complete block design to analyze per capita seed production at Heritage, using block, the two flowering species (E. pallida and P. integrifolium), and treatment as explanatory variables. Species was highly significant and explained most of the variation (F=195.18; p<0.001; Table 1). There was no evidence to suggest that per capita seed yield had significantly different means among the groups, nor was there evidence of a block effect (p>0.1; Table 1).

Model term df Sum squares F p






Block number















Plant size, as measured through per capita leaf area and height, followed a similar result to per capita seed yield. Leaf area was lowest among control plots for both E. pallida and P. integrifolium (Fig. 9a); height was lowest in control plots for E. pallida, whereas +Nutrient and control plots had generally lower heights than the other treatments (Fig. 9b). However, ANOVAs on the leaf (Table 2) and height (Table 3) data suggested no significant difference (p>0.1) with respect to treatment. However, there was a significant effect of the species in both models.

Figure 9: Per capita plant size of E. pallida and P. integrifolium at Heritage reflected by A) log-transformed leaf area or B) log-transformed height. The black line represents the median; the box represents the interquartile range; the whiskers represent the data distribution; dots represent outliers.

Model term df Sum squares F p






Block number















For the first year, the estimated value of wild quinine seed at Heritage was approximately $750.00, whereas at LOTW P. integrifolium seed was valued at approximately $175.00. The financial value of P. integrifolium seed at Barefoot was marginal, amounting to <$20.00 due to low flowering at this site. E. pallida seed was valued at approximately $50.00 at Heritage and $45.00 at LOTW.

Table 3: A summary of the ANOVA examining average total leaf area for E. pallida and P. integrifolium.

Model term df Sum squares F p
Species 1




Block number 1




Treatment 3




Residuals 38




Participation Summary
3 Farmers participating in research

Educational & Outreach Activities

1 On-farm demonstrations
1 Tours
1 Workshop field days
1 Boy Scout field day (see description)

Participation Summary

Education/outreach description:

The physical locations of the study sites were conducive to informal outreach. Public outreach and accessibility were one of the primary reasons to expand the project to the LOTW location, where both experimental and volunteer SPAs are adjacent to a bike trail popular among Champaign County residents. Over the season, I spoke to ~30 patrons about the project between the Heritage and LOTW sites. The CCFPD has erected an interpretive sign at the site, which describes SPAs and the project with respect to prairie restorations at LOTW.

Additionally, I managed a volunteer day at the Lake of the Woods site with a dozen Boy Scouts on August 22, 2020. Scouts were socially distanced and wearing masks. Scouts assisted with planting plugs in an additional, volunteer seed production section, weeding, and fixing fencing damaged by deer. Prior to work, I gave a short lecture talking to the Scouts about seed production areas, their utility to ecological restoration, information about the study species, and the importance of plant biodiversity. I was also working with stakeholders at all three sites throughout the season; stakeholders assisted me by providing labor, advice, and materials. For example, the owner of Barefoot suggested the layout for planting plugs and fabric cutting (Fig. 4a). I was working closely with an intern from the University of Illinois IGNITE Undergraduate Research Program, who received valuable research and field experience from the project.

Project Outcomes

1 Farmers changed or adopted a practice
1 Grant received that built upon this project
1 New working collaboration
Project outcomes:

Seed production areas (SPAs) are important to secure seed supplies for ecological restoration. Despite this importance, management practices to boost SPA yields are not well understood. The objectives of the GNC19-293 project are to study the agronomic production of native prairie wildflowers using arbuscular mycorrhizal fungi (AMF) inoculation and fertilizer application. I established experimental SPAs across three sites in a complete randomized block design, applying the treatments factorially to four prairie wildflower species. The project generally proceeded as planned despite the pandemic, and expanded in scope (namely, SPA establishment on an additional site, and an additional study species). I concluded my first year of data collection in the establishment year. Plant survival varied across sites and species, with deer and rodent herbivory damage being a larger challenge than anticipated at one of the sites. While per capita seed production and plant size was generally greater in treatment rows compared to control rows, there was no significant difference among the treatments. Approximately 1,000 USD of seed was produced in 2020. I hosted a volunteer field day with the Boy Scouts of America in August; this year I will be organizing additional field days and composing a report to send directly to stakeholders.

The 2021 field season and beyond

I plan several academic and outreach projects for the duration of the project. Because deer herbivory has been more intense than expected, I will be fixing deer fencing at LOTW and Barefoot in early spring, including replacing it with sturdier material. The growing season will continue normally, including general maintenance, monthly fertilization of the +nutrient rows, and eventual data collection (e.g., seed mass, plant size, and survival) in summer through fall. This year, I plan to take root samples to assess AMF infection among treatments; lab space for this component of the project was precluded by Covid-19.

I have plans for two major outreach efforts this year. First, I will coordinate with the Boy Scouts of America again to make one or multiple field days at the LOTW SPAs, which will include general maintenance and seed harvesting activities. Second, I will be drafting a short and interpretive report about AMF and their potential value to native plant nurseries, both directly (e.g., cultivating AMF strains to sell to restoration ecologists), and indirectly (e.g., potentially improved plant growth/survival). I have a list of over 65 native plant nurseries I developed for a separate project; this list will be used as a mailing list for the written product. Furthermore, I am interested in hosting a field day at the Barefoot Nursery site, where local farmers have been curious about the project. Similarly, I have been in contact with the University of Illinois Extension to conduct a field day and demonstration with Master Naturalists and Master Gardeners at Heritage. However, these tentative field days will be contingent upon the end of the Covid-19 pandemic.

Knowledge Gained:

Surprises and challenges of 2020 field season

Several challenges have complicated the research of the 2020 pilot season. The Covid-19 pandemic led to the cancellation of a scientific internship planned at the Barefoot site, in partnership with the University of Illinois at Springfield. The Covid-19 pandemic caused minor delays to beginning the project, though the research was designated as essential due to its time sensitive manner and proceeded.

However, the most substantial challenge of the field season was seedling mortality being higher than initially expected, mostly due to the intensity of herbivory and vandalism from deer. At LOTW, deer herbivory was frequent, and multiple deer fence kits were ruined over the course of the season, amounting to over $1,000 in damages. In addition, the former old field status of the LOTW site encouraged seedling mortality by rodents. Herbivory caused multiple rounds of seedlings to be killed, including an entire row of plantings mere days after field transfer. Therefore, establishment at the LOTW site was poor, particularly for butterfly weed and wild quinine. Seedling mortality was also high at Barefoot, though desiccation and poor soil quality was the apparent and primary reason. Deer herbivory and vandalism was also occasional at Barefoot.

Figure 10: Robust establishment and flowering of the Heritage site. Pictured is the general view of the Heritage site (left), and a typical row in September 2020, showing E. pallida in the foreground, and P. integrifolium in the background (right).

However, my stakeholders and I were pleasantly surprised that established plants produced seed in their first growing season (Fig. 10). The seed has been donated to the Champaign Parks District and CCFPD to be used in local ecological restorations. This season, I was not expecting to acquire any flowering or yield data.


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.