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.
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.
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. Soil innoculants were prepared using 75g samples of soil collected from the top 10cm of a sunflower field on the University of Delaware farm. Samples were stored at 4°C until preparation of inoculant. Soil samples were homogenized by sifting through a 1.18mm mesh sieve to remove stones and break up large soil particles, and then suspended in 1L sterile phosphate buffered saline (PBS). This solution was then vortexed and sonicated for 20 seconds to release microorganisms from soil particles. The inoculant was then centrifuged at 4000rpm for 15 minutes to pellet microorganisms and the supernatant was poured off. The pellet was then resuspended in PBS, and centrifugation, removal of supernatant and resuspension were repeated. The remaining inoculant was stored at 4°C.
A standard potting soil was sterilized by autoclaving twice for 20 minutes at 121°C, with a 24 hour cooling period between. Because in our first few attempts we discovered that the inoculant contained a high content of fungal spores that rapidly established in pots, we modified our protocol as described in the initial proposal by filtering to remove fungal spores. Innoculant for treatment plants was vacuum filtered using 8um filter paper to remove fungal spores but retain soil bacteria. For control plants, the inoculum was then vacuum filtered through 2um filter paper to remove all microorganisms but preserve any chemical properties of the inoculum given to treatment plants.
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. Standard 1 gallon greenhouse pots were filled with sterile potting soil and bottom saturated with dilute inoculant (100ml of either control or treatment inoculant in 900ml sterile water). 10ml of undiluted inoculant was added to the top of each pot. Three seeds were planted per pot and thinned to one plant per pot after germination. Plants are being grown at 24°C with 16hr photoperiod and watered as needed with sterile water. Due to the spatial constrains of the growth chamber, each trial consists of 18 total plants, 9 per treatment. Three trials total will be conducted for a total of 54 plants.
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.
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 inoculant. The third trial, which is currently underway, used the modified protocol described above which seems to have eliminated this problem. Once this trial is complete, we will repeat with two additional trials.
Our first glyphosate trial is currently being established in the greenhouse, and will be followed by two additional trials this spring and summer.
We have also applied for funding (Garden Club of America Pollinator Fellowship, $4000) to test the effects of glyphosate use on colony health in the eastern bumble bee (Bombus impatiens) in an experiment planned for summer 2020. If funded, this experiment will provide direct evidence that the effects (if any) of glyphosate on floral rewards have real implications for bee health.
Education & Outreach Activities and Participation Summary
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 to be held during summer 2020. 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. Additionally, an undergraduate student will be applying for funding through the University of Delaware Extension Scholars program to assist in planning, production of educational materials, and presentation/coordination of the workshop. Development of the workshop will begin in February of this year, with the workshop itself held in June 2020.
The graduate student will be presenting a poster at the Delmarva Soil Summit (February 26-27, 2020) outlining the study objectives, methods, and preliminary results. In fall 2020 she will give an oral presentation at the Entomological Society of America national meeting. Final results will also be presented at the Entomological Society of America national meeting in 2021. Upon completion of the project, we anticipate producing at least two publications to peer-reviewed scientific journals.
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.
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.