Balancing weeds, nitrogen, and soil health in cover crop mixtures

Progress report for GNE22-298

Project Type: Graduate Student
Funds awarded in 2022: $14,998.00
Projected End Date: 07/31/2024
Grant Recipient: Penn State University
Region: Northeast
State: Pennsylvania
Graduate Student:
Faculty Advisor:
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Project Information


Increasing functional diversity is essential to enhance ecosystem services and reduce agriculture’s environmental footprint while simultaneously meeting a growing demand for food. One way to achieve this is through the integration of cover crop mixtures into farming rotations. Cover crops provide a multitude of ecosystem services to cropping systems, including nitrogen retention and fixation, weed suppression, reduction in pest pressure, and increased profitability of cash crops. Here, we propose to evaluate how different cover crop treatments grown in mixtures and monocultures impact weed seed mortality, nitrogen fixation and uptake between species, and soil labile carbon for the following cash crop. These services are microbially mediated and likely enhanced by increasing species functional diversity. We will also investigate the relationship between species abundance (above and belowground) and ecosystem service functions to better inform grower mixture choice. Results from this study will be communicated to the grower community and agricultural professionals through an extension newsletter article, education modules, research field tours, and a Pennsylvania Sustainable Agriculture Conference presentation.

Project Objectives:

Objective 1: Evaluate how weed seed mortality differs among cover crop mixtures and monocultures.

Hypothesis 1: We expect that weed seed mortality will be greater in cover crop mixtures compared to monocultures. Presumably, the greater functional diversity and abundance of roots in mixtures will increase weed seed mortality by increasing microbial diversity and activity. Additionally, we expect that both legumes and brassicas will increase weed seed mortality relative to grasses; legumes increase N, and in turn microbial activity, and brassicas associate with saprophytic fungi which are primarily responsible for breaking down recalcitrate organic matter (such as weed seeds) in the soil.


Objective 2: Evaluate how biological nitrogen fixation (BNF) of legumes in mixtures compares to legume monocultures, and determine how much N from fixation is shared between species within mixtures.

Hypothesis 2: Relative to cover crop monocultures, legume species will have a greater percent of N derived from BNF when mixed with grass and/or brassica due to the drawdown in soil available N. Other species planted with legumes will also have higher N than those planted in monoculture or with other non-legumes due to acquisitioning BNF–derived N exuded from legume nodules.


Objective 3: Evaluate how labile carbon differs among cover crop mixtures and monocultures.

Hypothesis 3: Relative to cover crop monocultures, mixtures will promote higher labile carbon pools due to the greater diversity of cover crop residue inputs (varying in chemical composition) incorporated into the soil.


Objective 4: Evaluate whether cover crop mixtures can increase the diversity and breadth of the ecosystem services they provide (i.e. can mixtures increase multifunctionality).

Hypothesis 4: Relative to cover crop monocultures, cover crop mixtures will provide a greater suite of ecosystem services. We predict that the provisioning of any one service will be lower in mixtures compared to the best performing monoculture. However, when considering all of our measured services mixtures will exhibit greater performance.


Objective 5: Determine if there is a relationship between cover crop species abundance within mixtures and the provided ecosystem service(s) relative to monoculture treatments.

PI Rice has designed a molecular method to quantify relative root abundance of cover crop species in mixtures from field-collected soil cores. Prior to this method, it was impossible to determine the composition of cover crop species in the root biomass. This method is ready to be applied to the field to examine whether ecosystem services are linked to cover crop species composition and abundance.


The purpose of this project is to examine whether various cover crop monocultures or mixtures promote three microbially-mediated ecosystem services: weed seed mortality, biological nitrogen fixation (BNF), and soil labile carbon (C). This research will also contribute to informing the relationship between cover crop species abundance (above and belowground) and services provided in mixtures compared to monocultures. These services are all tightly linked to microbial activity in the soil, which is elevated in the areas surrounding plant roots1–3, therefore, having information on root biomass by species is integral to characterizing how mixtures provide our three ecosystem benefits.

Cover crop adoption is highest in the northeast, partly driven by incentive programs and strict regulations in the Chesapeake Bay watershed4,5. Soil health and weed management are the primary motivators growers cite for using cover crops6. The proposed research will investigate a minimally studied aspect of weed suppression, weed seed mortality, as well as providing additional information on BNF and labile C (an important metric of soil health) to better inform grower selection of cover crop species monocultures and mixtures. This research contributes to agricultural sustainability by evaluating whether the use of diverse cover crop species in crop rotations will achieve a suite of ecosystem services to growers, which will be essential to meet a growing food demand while reducing the environmental footprint of agricultural systems7–9.

With heightened awareness of the need for integrated nutrient, pest, and soil management in farming, cover crops are serving a multitude of functions to growers. They not only diversify cropping rotations, but also reduce erosion, retain nitrogen (N), add N through fixation, suppress weeds, reduce pest pressure, and increase profitability of cash crops7,8. However, cover crop species vary in their provisioning of ecosystem services. For example, grasses are particularly effective at scavenging inorganic nitrogen and suppressing weeds, while legumes provide N via BNF. Combining diverse cover crop species enables a wider array of services to be achieved, otherwise referred to as ‘multifunctionality’8.

In the proposed research I will focus on three essential, but relatively less studied ecosystem services: weed seed mortality, BNF, and labile C. We selected these three services because they have the potential to greatly increase crop productivity and profitability. Thus far, much of the work characterizing cover crop effects on these ecosystem services have evaluated either monocultures or grass-legume bicultures with limited work evaluating how more diverse cover crop mixtures may affect these ecosystem services. Additionally, the three ecosystem services we will evaluate within this study are all microbially mediated, and there is an abundance of work showing that plant diversity increases microbial diversity and functioning1–3,10. Therefore, we hypothesize that more diverse cover crop mixtures will enhance the functioning of these ecosystem services.


Materials and methods:

Site characterization and experimental design (Objectives 1-5): All research objectives will be performed in the existing organic long-term cover crop cocktails experiment (CCC) located at the Russel E. Larson Agricultural Research Center at Rock Springs, PA. The existing CCC experimental design is comprised of a three-year corn, soybean, winter wheat rotation19. The experiment was established in 2011 at Penn State University to determine whether diverse cover crop mixtures, as opposed to single-species cover cropping, can enhance ecosystem functions in a corn-soybean-wheat cash crop rotation that produces organic feed. The selected cover crop mixtures target nutrient supply, nutrient retention, weed suppression, and management ease to investigate whether diverse mixtures provide these functions better than cover crops in monoculture. Currently, CCC is measuring total N, C:N in cover crop aboveground biomass, soil nitrous oxide emissions, weed and pest suppression, and cash crop production and profitability. We will be adding the services of weed seed mortality, BNF, and labile C to the service measurement suite, as well as measuring root biomass.           

We will use a subset of eight existing treatments that only contain cover crops which over-winter or tolerate mild winters (in the case of Austrian winter pea).  In addition to overwintering, these four species were selected based on their functional diversity20. Cover crop treatments include three mixtures and four monocultures (listed below). Each treatment is replicated across four blocks and organized in a random split plot design.

Cover crop treatments:

  1. triticale
  2. crimson clover
  3. Austrian winter pea
  4. canola
  5. two species mixture (triticale and Austrian winter pea)
  6. three species mixture (triticale, crimson clover, and Austrian winter pea)
  7. four species mixture (triticale, crimson clover, Austrian winter pea, and canola)
  8. fallow control

Using a combination of one, two, three, and four species mixtures, we can determine how plants are interacting in mixtures with an increasing number of species and relate that to the ecosystem service measurements. This will contribute new information to growers on what cover crops are most appropriate to plant to maximize one service or derive a mixture of nitrogen fixation, weed seed mortality, and increase of labile C services. The experimental design of this project was created through a collaboration between scientists at Penn State and organic growers on the board of this project to ensure that the design yields meaningful information and is addressing grower questions.   

2022 Update: Thus far we have set up the cover crop treatment plots, weeded all plots, and deployed the weed seed mortality assessment bags. The first year of ecosystem service measurements will be executed in the spring and summer.

Plant tissue sampling and processing (Objectives 2 + 5): Within each treatment plot we will establish two 1 m2 subplots, one for fall and one for spring sampling. Subplots will be hand-weeded once a season (we will weed the 1m2 subplot as well as an additional area of approximately 0.5 m in each direction) to ensure that any differences in ecosystem services are due solely to the cover crops, and not due to variation in the weed community within cover crop stands. Within the 1 m2 subplots, we will collect stand counts in fall, and aboveground biomass at both the fall and spring time points. Aboveground biomass will be sorted by species, dried, and weighed. Soil core samples will be taken from all treatments aside from the fallow control. Sampling will be conducted over two time points: at fall frost, and before cover crop termination in the spring (Figure 1). At fall and spring sampling, two cores are taken in the planting row and two between planting rows for an accurate root composition estimate20. We will separate cores to three depths (0-5, 5-20, 20-40 cm) to understand how roots are dispersed vertically. Collected soil core samples will be washed, dried, and weighed.   

Although beyond the scope of this project, PI Rice has developed a molecular method to estimate the relative composition of plant species in root biomass of diverse species mixtures. In a separately funded study in the CCCs project, PI Rice is currently quantifying the relative abundance of the cover crop species within root biomass of soil cores extracted from the CCC plots and using those estimates to calculate root abundance in each 1m2 plot. In brief, to do this she is using amplicon sequencing of chloroplast DNA and relating the number of reads identified as a particular species to an abundance estimate21. Monocultures are used to determine the maximum biomass of each species (in the absence of competition) and to create calibration curves relating known quantities of root biomass to sequencing read number. Therefore, we will relate the ecosystem services measured within this study (weed seed mortality, BNF, and labile C) to the relative abundance of the cover crop species in root biomass to evaluate to what extent cover crop composition in mixtures predicts ecosystem services.  


Weed seed mortality (Objective 1): We selected Amaranthus powellii (broadleaf) and Setaria faberi (grass) as target weed species because they are summer annual weeds which are problematic during growth of summer annual cash crops. Using the weed seed burial bag method22, we will install mesh bags in each plot (2 bags/species/plot; 128 total) containing 150 seeds combined with 10 g soil. Soil will be collected in the fall directly from each plot where the bag will be installed to ensure the soil in the weed seed burial bags reflects the soil chemical and microbial composition of the soil within each cover crop treatment. Bags will be buried at 10 cm soon after fall planting and strategically located where all species in the mixture are present. The burial depth was selected so that seeds are in an area of high microbial activity. To minimize the possibility of disturbance from field equipment, bags will be placed close to the planting row. All bag locations will be marked with flags, and as an extra precaution, have an attached stainless-steel washer so that they can be located using a metal detector (Figure 2).  

We will evaluate weed seed mortality at two timepoints: 1) prior to cover crop termination: to evaluate the effect that living cover crops have on weed mortality; and 2) at corn harvest, to evaluate the effect that cover crops have on weed seed mortality after incorporation into the soil. At spring cover crop termination all seed bags will be retrieved, but half of the bags will be processed at that time (timepoint 1 above)), and the second half kept in dark conditions mimicking those in the soil before being redeployed into the same treatment plot following corn planting (Figure 1). The remaining seed bags will be retrieved just prior to corn harvest (timepoint 2). Using sieves (0.5 - 1mm) and hand picking, we will separate seeds from the soil and test a subset of seeds for viability using the seed crush test method23. The crush test method involves imbibing seeds in water and crushing them with a consistent weight, and if the seeds crush, they are considered non-viable.    


N fixation (Objective 2): We will estimate the proportion of N derived from N fixation within legume plants and their neighbors using the 15N natural abundance approach, which is commonly used to estimate N fixation of legumes grown in both monocultures and mixtures.

For all mixtures containing legumes, dried shoot tissue is ground separately by species (52 samples/year) and sent for analysis at UC Davis Stable Isotope Laboratory. We will then use the equation below to calculate the percent of nitrogen derived from the atmosphere (%Ndfa) using the following equation to estimate the total N contribution by BNF24:   


%Ndfa = 100[(δ15Nref  - δ15Nleg) / (δ15Nref  - B)]  


In equation one, δ15Nref  is the 15N relative natural abundance of the reference plant (triticale grown in monoculture and averaged from the four blocks, %Ndfa=0) to estimate standard background δ15N3,25. δ15Nleg is the legume’s 15N relative natural abundance, and B is the δ15N relative natural abundance of the legume when it is grown in a N-free medium (%Ndfa=100). The B was calculated previously for both crimson clover3,24,26 and Austrian winter pea3. Once we have calculated the relative natural abundance (%Ndfa), we will quantify BNF using the following equation:  


BNF (kg ha-1) = biomass (kg ha-1) • plant N concentration (%)/100 • %Ndfa/100  


In summary, we will calculate the biological nitrogen fixation (BNF) in kilograms per hectare by multiplying the plant biomass with the proportion plant N concentration and multiplying that with the previously calculated proportion atmospheric derived N. This final output of BNF will be compared by species between treatments to see if certain mixtures promote higher BNF rates. Ultimately, we will be able to determine how much nitrogen is being contributed by legumes through BNF in the mixture and taken up by the neighboring cover crops.  


Labile Carbon (Objective 3): To measure labile C, soil samples will be taken from 0-15 cm depth using a 2.5 cm diameter soil probe in each cover crop treatment two weeks after cover crops are incorporated into the topsoil (Figure 1). Twelve soil cores will be randomly collected from each cover crop plot, combined, and homogenized. For each plot, we will then mix 20 ml of potassium permanganate (KMnO4) with 2.5 g of soil, agitate for 2 minutes, and allow all solid material to settle on the bottom of the tube27,28. The liquid directly above the solid material will be collected and sample absorbance read with a SpectraMax M5 spectrophotometer at 550 nm17. Labile soil organic matter plays a key role in crop nutrient acquisition and is more likely to show changes following management adjustments on a shorter time scale compared to renewable stable nutrient pools. 


Data analysis  

Objectives 1-3: For each ecosystem service measurement, we will perform an analysis of variance (ANOVA) to compare the cover crop treatments to the response variable (weed seed mortality, BNF, labile C absorbance measurement) with treatment as a fixed effect and block as random effect. Additionally, we will visualize the response for each treatment and perform pairwise comparisons to evaluate treatment differences.   


Objectives 4 + 5: We will take a two-step approach to data analysis. First, we will use partial least squares regression to predict how aboveground and estimated belowground biomass of each species across all mixtures predicts each of our quantified ecosystem services. Through this approach, we can determine the relative importance of aboveground versus belowground composition, enabling me to infer the relative contribution of belowground composition to predicting each ecosystem service. Secondly, we will determine how functional diversity of the selected species impact ecosystem services based on relative Rao’s quadratic entropy7,8,29,30. We will calculate indices on the number of functionally distinct species in a treatment and measure the distribution of traits (fall growth potential, spring growth potential) using FDiversity software8,31 . We will then use linear mixed-effect models (service fixed, block nested, year random) with R statistical software (Vienna, Austria) to test whether increasing functional diversity or species richness influences ecosystem service number based on the cover crop treatment. Ultimately, we are interested in making comparisons between functional diversity aboveground, belowground, and combined to determine to what extent the prediction of multifunctionality can be improved within cover crop mixtures by including belowground diversity and composition.  

NE_SARE Methods Figure 1


Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

The results of this research will be shared by the graduate student Emma Rice with growers, extension agents, and other researchers in the Northeast. Findings will be shared through the following avenues:

Field Crop News Article This article will reach Pennsylvania growers directly and detail our findings on how well each species and species mixtures provided individual services and service suites. I will also highlight the role that roots and microbes play in facilitating the provided services. This newsletter is published bi-weekly by the Penn State Field and Forage Crops Extension team.

Educational Modules – The CCC project has a webpage with educational modules on cover crop treatments and the ecosystem services they provide ( I will incorporate my findings into the existing modules, which are designed to engage undergraduate students, but are free to access and easily modified for other audiences.

Pennsylvania Sustainable Agriculture (PASA) Conference in 2024 – I will present research findings to growers, extension agents and industry professionals who are interested in sustainable agriculture. Attendees will likely have a foundation of knowledge on how cover crops facilitate ecosystem services. I hope that my findings will enhance their current knowledge and encourage consideration of additional services cover crops can provide.

Penn State’s Ag Progress Days - I will also host field day tours of the CCCs project to share the findings of this research during Penn State’s Ag Progress Days. This event is held every August and is Pennsylvania’s largest outdoor agricultural exposition with 45,000+ attendees, an estimated 60% of whom are engaged in agriculture or in professions related to agriculture. This field tour will include a walk-through of the experiment, summary of ecosystem services provided by each cover crop treatment, and an informal discussion to identify audience interest in certain ecosystem services. Due to the timing of Ag Progress Days (early August) cover crops will not be planted in the experimental plots, to account for this we will plant a small representation of each treatment in plots adjacent to crops in June so that participants can see the cover crop mixtures and monocultures. We will also grow cover crops in rhizoboxes (Figure 3) so that participants can see the distinct root functional traits of each species to better understand the important role roots play in facilitating key ecosystem services. I am particularly excited about sharing this activity with field day participants, because hearing how species’ roots differ from one another and seeing it with one’s own eyes has a different impact. I have had a lot of success demonstrating these differences through images but living plants in rhizoboxes will elevate this lesson even more.

Published in a peer reviewed journal – The main audience for this manuscript are researchers and extension agents. The hope is that our work will promote future cover crop research investigating the relationship between plant functional diversity and microbially and root mediated ecosystem services.

NE-SARE Outreach Figure 3

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.