Progress report for GNE19-219

Project Type: Graduate Student
Funds awarded in 2019: $14,984.00
Projected End Date: 11/30/2022
Grant Recipient: University of Delaware
Region: Northeast
State: Delaware
Graduate Student:
Faculty Advisor:
Dr. Deborah Delaney
University of Delaware
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Project Information

Project Objectives:

This project has two primary objectives. 

1. Identify the effects of experimental removal of the soil microbiome on floral rewards by measuring flower number, size and phenology; pollen nutritional content and grain size; nectar sugar concentration; and plant organic volatile compounds in plants grown in sterile soil versus soil inoculated with a field collected community of bacteria and fungi.

Prediction 1.1: Plants grown in sterilized soil will bloom later and produce inflorescences with reduced number of flowers compared to plants grown in inoculated soil, but inflorescence number will not differ.

Prediction 1.2: Pollen produced by plants grown in sterilized soil will exhibit smaller average grain size and will differ in amino acid concentration and/or composition compared to pollen from plants grown in inoculated soil.

Prediction 1.3: Flowers of plants grown in sterilized soil will produce smaller volumes of nectar, and nectar may differ in sugar concentration compared to plants grown in inoculated soil.

Prediction 1.4: Plant volatiles will differ between plants grown in sterile soil and plants grown in inoculated soil

2. Examine the effects of glyphosate treatment on floral rewards by comparing flower number, size and phenology; pollen nutritional content and grain size; nectar sugar concentration; plant organic volatile compounds; and insect visitation between treated and untreated plants.

Prediction 2.1: Plants treated with glyphosate will exhibit delayed flowering time compared to control plants.

Prediction 2.2: Plants treated with glyphosate will differ in the volume and sugar content of nectar produced compared to control plants.

Prediction 2.3: Plants treated with glyphosate will produce pollen with smaller grain size and pollen crude protein and amino acid profiles will differ between treatment and control plants.

Prediction 2.4: Plant volatile organic chemical composition will differ between treated and untreated plants.

Prediction 2.5: Glyphosate treated plants will attract a lower abundance and diversity of flower visiting insects compared to control plants.

We have chosen sunflower (Helianthus annus) as a study plant because 1) it is grown both as a crop plant and bee forage plant in hedgerows and pollinator gardens, 2) it produces large quantities of pollen and is used as a food source by a wide range of native and managed bee species, 3) it has a composition flower with large inflorescence containing many flowers per plant, and 4) dwarf varieties able to be grown in growth chambers are available.

Introduction:

The purpose of this project is to investigate factors that impact the nutritional quality of bee forage in the agricultural setting with a goal of providing farmers a better understanding of how to sustain populations of both wild and managed pollinators.  Sustainable agriculture is dependent on sufficient pollination services, and therefore providing abundant, nutritious forage for bees and other pollinators is foundational to maximizing crop yields and seed production. While bee pollinated crops themselves provide forage and many farmers provide supplementary forage in and near their fields, little is known about how environmental conditions and management practices alter the production of floral rewards (pollen and nectar) provided by these plants. One component of the agricultural system that has yet to be studied for its impact on floral rewards is the soil microbiome. Soil bacteria and fungi impact plant health and have been shown to affect pollen viability and seed set, but these soil mediated impacts on pollen production have yet to be investigated from the perspective of changes in nutritional quality for pollen feeding insects. We expect that changes impacting germination of pollen grains also affect the composition of protein, sugars, lipids, vitamins and/or minerals available to foraging bees. The role of the soil microbiome in nectar production has never been studied, but given the influence of plant-soil interactions on other aspects of the plant reproductive system we expect to see changes to nectar volume and/or sugar concentration as well. Previous studies suggest that changes to plant physiology as a result of these interactions may also affect bee forage by shifting bloom time or duration and flower morphology, though this has rarely been studied, and never under the lens of impact on pollinators. Given these effects on plant chemistry, we also hypothesize that plant volatile organic compounds (VOC’s) involved in attraction of pollinators to the flower, will also be impacted by changes to the soil microbiome.

Herbicides are one management tool that is known to have consequential effects on soil microbes and plant reproduction. Shifts in microbial communities as well as changes to pollen viability and bloom time have been tied to addition of glyphosate, but again, no studies to date have explored the effects of herbicide on insect visitation, pollen nutrition, or nectar production. By examining the soil-plant-insect interactions we aim to improve pollinator management strategies in sustainable agriculture. Shedding light on the role of the soil microbiome in pollinator health will spur development of new tools to enhance forage in crop fields, such as addition of certain bacteria or fungi to improve pollen and nectar content and yield. Studying the implications of herbicide use for pollinator health will provide farmers with direct knowledge of how their management strategies affect pollination services to their crops. In addition to researching these questions, we aim to equip farmers with the informational toolkit needed to understand sustainable promotion of pollination in agriculture.

The scientific literature examining the role of soil microbes and effects of herbicides on crops has identified measurable effects on plant growth rates and seed set, yet previous studies have failed to address pollination ecology. This study is novel in that it aims to identify how these two factors influence the quality of forage for pollinating insects. The economic value of pollination services to U.S. crops by wild and managed bees has been estimated at $16 billion annually (Ritten et al. 2018).  In sustainable agriculture, sufficient pollination of crops leading to high yields and seed set hinges on maintenance of diverse, abundant populations of wild bees and/or healthy managed bee colonies. For efficient and cost-effective management of resources for these bees in the agricultural landscape, we must first understand how the quality of floral rewards vary in relation to conditions in which the plants are grown.

Addressing this gap requires two approaches 1) studying the ecological interactions between soils, plants, and insects feeding on floral rewards on a basic level, and 2) exploring specific management practices and how they impact these interactions. Guided by the first approach, we ask a fundamental question which has yet to be explored: Is the soil microbiome important in the production of floral rewards, and do its impacts on pollen and nectar production have implications for pollinator health? As the first study to investigate this topic, we aim to provide foundational knowledge by looking at the soil microbiome as a whole to draw attention to the system and spur further study of mechanisms and influential microbes. Bacteria and fungi found to enhance vegetative plant growth have been used as soil inoculants to increase crop productivity, therefore we expect this work to lead to the development of new management tools aimed at increasing the value of flowering plants as bee forage.

The second approach takes a more targeted look at a widely used herbicide, with the goal of providing information that can be directly applied by farmers, beekeepers, and hobby gardeners. While the effects of herbicides and pesticides have been studied for their lethal and sublethal effects on insects, the study of their indirect impacts by altering floral rewards is new. Growers who have invested resources in either managed bee pollination or forage for wild bees must be able to weigh the potential costs to these investments when selecting weed management strategies. If significant impacts on floral rewards are observed in this study, we hope to further investigate if these effects can be lessened by adjusting treatment time and dosage in the future to develop best management practices for growers.

In addition to providing novel information on soil-plant-insect interactions, we draw attention to the need for a better understanding of factors affecting floral reward quality.  The full suite of factors impacting vegetative growth are potentially important for pollen and nectar production, and we expect our investigation of the soil microbiome and herbicide treatment to open the door to further explorations of other conditions impacting sustainable agriculture.

Cooperators

Click linked name(s) to expand
  • Dr. Harsh Bais (Educator and Researcher)
  • Dr. Charles Mason (Educator and Researcher)
  • Dr. Jeffrey Buler (Educator and Researcher)

Research

Materials and methods:

The central focus of this project and my dissertation as a whole is exploring the impacts of belowground conditions on the production of floral rewards and their nutritional value to pollinating and flower visiting insects. Specifically, we are testing whether removal of the natural soil microbiome or application of the herbicide glyphosate impact plant growth, bloom time, flower number, nectar concentration, pollen grain size, pollen nutritional content, insect visitation, and the chemical profile of plant volatile chemicals released during flowering that may aid in attraction of pollinators.

Impacts of the Soil Microbiome

To address these questions we designed a series of greenhouse experiments. To test the role of the soil microbiome in floral reward production, we compare dwarf sunflower (Helianthus annus Big Smile, Johnny’s Seeds) grown in sterile soil to plants grown in soil inoculated bacteria extracted from sunflower field soils. Sunflower was chosen as a study subject because it is a high pollen production plant visited by a wide range of pollen and nectar feeding insects and is grown both as an agricultural crop and in pollinator gardens. We chose a dwarf variety that could be grown in growth chambers.

Because autoclaving soil can alter soil chemistry, we grew all plants in sterilized potting soil either with or without an inoculant containing field collected soil bacteria. We initially extracted innoculants from field soil, which resulted in fungal growth in pots. To solve this problem we are instead using field soil directly to innoculate sterilized soil. Soil innoculants were prepared using 75g samples of soil collected from the top 10cm of a sunflower field on the University of Delaware farm. Soil samples were homogenized by sifting through a 1.18mm mesh sieve to remove stones and break up large soil particles and stored at 4°C until planting. A standard potting soil was sterilized by autoclaving twice for 20 minutes at 121°C, with a 24 hour cooling period between. This sterilized substrate was then placed in 30 1 gallon nursery pots. A 75g field soil sample was mixed with potting substrate to innoculate with soil microorganisms. Innoculants for control plants were autoclaved prior to mixing with the substrate as described above.

 Prior to planting, sunflower seeds were surface sterilized by washing with 70% ethanol for 3 minutes, 5% bleach for 3 minutes, followed by 3 rinses in sterile water. Three seeds were planted per pot and thinned to one plant per pot after germination. Plants are being grown at ambient greenhouse temperature (approximately 75°C) with a photoperiod of 16 hours, watered as needed with tap water, and fertilized weekly using a 20-20-20 fertilizer. We are currently in our first trial since adjusting the protocol to eliminate fungal growth, and the problem seems to be resolved. We plan to conduct 3 trials (18 plants each) for this experiment including a total of 54 plants (26 treatment, 26 control)

Plant growth stage, height and leaf number are being recorded weekly prior to blooming. Time of flowering and pollen dehiscence, and inflorescence size (measured as width of the composite flower head, number of ray flowers and number of disc flowers) will be recorded as flower buds emerge. One the third day of pollen dehiscence flowers will be bagged for 24 hours to collect pollen. A small sample will be slide mounted to measure pollen grain size, and the remainder of collected pollen will be sent to the University of Missouri-Columbia Agricultural Experiment Station Chemical Laboratory (AESCL) for analysis of crude protein, amino acid profile, fatty acid profile, and sugar profile. For these analyses, all pollen will be pooled for control and for treatment plants to ensure adequate volume for analyses and obtain a generalized nutritional profile for plants grown under the two different experimental conditions. Plant growth and bloom metrics and pollen nutritional content will be analyzed by multivariate analysis of variance (MANOVA)

        Immediately following pollen collection, volatile organic compounds (VOC’s) will be sampled from all plants. One inflorescence per plant will be bagged for three hours to concentrate released VOC’s. The airspace within the bag will then be pulled through a molecular filter to collect VOC’s. Samples will then be dissolved into Dichloromethane solvent so they can be analyzed in a gas chromatographer fitted with mass spectrometer.  Two internal standards will be used for calibration: n-Octane and nonyl acetate. Software paired with the mass spectrometer provides the chemical composition and relative concentrations of chemicals within the samples. Differences between samples will be analyzed using non-metric multidimentional scaling (NMDS).

After VOC’s are sampled, nectar will be sampled from one flower per plant using microcapillary tubes and sugar concentration will be measured using a handheld refractometer. Sugar concentration will be included in the MANOVA analysis above. For plant trials conducted in spring and summer, plants will then be moved outside on the University of Delaware farm to measure insect visitation. Insect visitation will be measured by visually observing and recording insects coming in contact with the disc or ray flowers of the sunflower inflorescence (insects landing elsewhere on the plant or hovering and not collecting pollen or nectar will not be recorded). Groups of 4 plants will be monitored at a time for ten minutes each, and visitation will be recorded for each individual plant. Insect visitors to each flower during that time will be recorded and identified into the following groupings: bumble bee (genus Bombus), large carpenter bee (genus Xylocopa), sweat bee (family Halictidae), mason bee (genus Osmia), mining bee (genera Andrena and Colletes), butterflies (order Lepidoptera), beetles (order Coleoptera), hover flies (family Syrphidae), and other flies (order Diptera). Insect abundance and diversity will then be calculated for treatment and control plants and compared using analysis of variance (ANOVA), and differences in community structure of visitors to the two treatment groups will be assessed using NMDS.

Glyphosate is the one of the most commonly used herbicides by both farmers and gardeners. In the context of sunflower, it is used as a pre-planting weed control, and it is also used in the establishment of pollinator gardens and supplemental pollinator forage strips. To explore the influence of pre-planting glyphosate use on floral rewards in sunflower, we are growing sunflower in the greenhouse under three glyphosate treatments: no glyphosate, glyphosate applied at the recommended field dosage, and a high glyphosate concentration at 150% the recommended field dose. Each of three trials will include 20 plants per treatment grown in a commercial potting mix. To mimic real world conditions, we will use glyphosate in the form of the commercial product RoundUp. Roundup diluted in sterile water will be applied once to the soil 1 week prior to planting. Control plants will receive only sterile water and no herbicide. As in the experiment above, three seeds per pot will be planted and later thinned to one per pot. Plants will be grown at ambient greenhouse temperature (approximately 75°C) with a photoperiod of 16 hours, watered as needed with tap water, and fertilized weekly using a 20-20-20 fertilizer. Plant growth and blooming metrics, pollen and nectar sampling, VOC sampling, and insect visitation will be measured and analyzed as described in the previous experiment.

Research results and discussion:

Data collection is currently still underway, and results are not yet available. Three trials have been established for the microbiome experiments, although (as described above) the first two failed due to fungal growth from the innoculant. The third trial was halted midway through when the University closed due to Covid-19 and access to labs was restricted. The pandemic has considerably delayed the project, essentially shifting our goals by 1 year. We are currently back in the lab and new trials for the microbial and herbicide experiments are now underway.

 A field study using additional funding outside of this grant has also been added to support the glyphosate greenhouse trials, and will be conducted May-August 2021.

Due to the pandemic delays, we plan to apply for an extension for this grant. The modified plan assuming this extension is granted is outlined below.

Winter and Spring 2021:

  • Trial 1 for objective 1 (currently underway)
  • Trial 1 for objective 2 (currently underway)
  • Hiring of 2 undergraduate assistants to help with data collection. The technicians will work 30-40 hours per week for 8 weeks in June and July. (applications due Feb 18)
  • Plan Pollinator Health Workshop and recruit participants

Summer 2021

  • Trials 2&3 for objectives 1 and 2 (with insect visitation)
  • Field trial (added using additional funding and not described above)
  • Hold Pollinator Health Workshop (July or August)

Fall 2021

  • Data analysis and processing of pollen samples
  • Analysis of workshop questionnaires
  • Graduate student presents current results at annual Entomological Society of America conference
  • Report on progress and current results to SARE by January 15

Spring 2022-Winter 2023

  • Final data analysis and dissertation writing
  • manuscript preparation
  • Final report to SARE by January 15, 2023
Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary

Education/outreach description:

The primary outreach product of this study will be a two-day workshop focused on pollinator nutrition and health aimed at farmers and master gardeners. University of Delaware Cooperative Extension will be assisting with recruiting 20 participants. The workshop will be led by the graduate student and faculty advisor, with assistance from extension personnel and the two undergraduate assistants. The workshop was originally planned for summer 2020, but was delayed due to the pandemic. We expect to be able to hold the workshop in person in July or August 2021, but are prepared to develop a virtual or hybrid design if absolutely necessary. Development of the workshop will begin in March 2021, with the workshop itself held in July or August depending on availability of personnel from cooperative extension.

The graduate student presented a poster at the Delmarva Soil Summit (February 26-27, 2020) outlining the study objectives, methods, and preliminary results. In fall 2022 she will give an oral presentation at the Entomological Society of America national meeting. Upon completion of the project, we anticipate producing at least two publications to peer-reviewed scientific journals.

Project Outcomes

1 Grant applied for that built upon this project
Project outcomes:

Should our experiments reveal significant effects of the soil microbiome and/or glyphosate on floral reward production, we expect these findings to spark a new area of study surrounding both how current practices impact the quantity and quality of floral rewards, and also potential strategies (for example, supplementation with specific microorganisms) for increasing efficiency and production/quality of food for potential pollinators provided by both crops and pollinator gardens. Even in the event that our exploratory study finds no effects on floral rewards, insect attraction and visitation, and bee health, there are many additional questions (effects of inoculation with specific bacterial strains, effects of other agricultural chemicals, effects on other plant species) that will remain to be explored. By opening the door to this area of research in sustainable agriculture, we hope to continue studying the connections between belowground-aboveground interactions that may impact pollinator health and to encourage other researchers to study the potential of these systems to improve pollination services and pollinator conservation.

Knowledge Gained:

As this project is still in the stage of data collection, we can only speculate how the results we obtain, as well as our interactions with growers, gardeners, extension, and the academic community will alter our views of sustainable agriculture as it pertains to pollination health.

My academic career is focused on developing a broad ecological background and conducting research focused on interactions between insects and their environments. After completion of my degree, my goal is to become a tenure track professor of Biology or Ecology with a heavy focus on teaching and extension. As a graduate student, I have found that I am most stimulated to learn and explore via research when doing so through a lens of educating others. I am passionate about mentoring undergraduates as they develop their skills as scientists, and plan to focus my future research on providing undergraduates with the opportunity to think about their study organisms in the context of its role within the ecosystem. As a faculty member in a broad field such as Ecology, I will be able to explore my wider interests in the field by bringing my entomological background to collaborations with researchers studying other taxa to offer a more integrated view of study systems. I am also committed to public education and plan to make outreach and education of laypeople, farmers, gardeners, and beekeepers a central focus of my career.

 

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.