Soil Microbiome Impacts on Floral Rewards and Implications for Pollinator Nutrition

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 plant growth promoting rhizobacteria and mycorrhizae on floral rewards by measuring flower number, size and phenology; pollen nutritional content and grain size, and nectar sugar concentration in plants grown with and without inoculation with live beneficial microorganisms.

Prediction 1.1: Plants inoculated with either mycorrhize or rhizobacteria will exhibit faster growth rates, bloom earlier, and produce larger inflorescences compared to plants not inoculated with microorganisms. Plants inoculated with both mycorrhizae and rhizobacteria simultaneously will have faster growth rates and bloom earlier than those receiving a single inoculate.

Prediction 1.2: Plants inoculated with either mycorrhize or rhizobacteria will produce pollen with larger average grain size and will differ in amino acid concentration and/or composition compared to pollen from plants that were not inoculated. Innoculation with both mycorrizae and rhizobacteria may impact nutritional composition but will not further increase pollen grain size. Nectar sugar concentration will be higher in inoculated plants compared to controls, but will not differ between inoculated treatments. 

Prediction 1.3: Inoculated plants will receive a higher number of insect visitors than control plants, but insect visitation will not differ between inoculated treatments. Diversity of insect visitors will not differ between control and treatment plants.

2. Examine the effects of glyphosate drift on floral rewards by comparing flower number, size and phenology; pollen nutritional content and grain size; nectar sugar concentration; 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.

Introduction:

The purpose of this project is to investigate factors that impact the nutritional quality of bee forage in the agricultural and garden settings with a goal of providing growers 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.

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 amendment of soil with just a few of the many potentially beneficial groups of rhizosphere microorganisms. 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 draw attention to the system and spur further study of mechanisms and influential microbes, ultimately leading 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, or plan their use to minimize those costs. 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

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  • Dr. Harsh Bais (Educator and Researcher)
  • Dr. Charles Mason (Educator and Researcher)
  • Dr. Jeffrey Buler (Educator and Researcher)

Research

Materials and methods:

Impediments and Changes Made:

In 2020 we at first had difficultly preventing fungal growth in the soil of inoculated plants of Objective 1. As discussed in the 2020 annual report, in 2019 and 2020 we attempted several adjustments to the protocols, and ultimately moving plants from the growth chamber to the greenhouse and watering less and more frequently seemed to solve this problem. However, after moving forward with this strategy we were not happy with the growth and vigor of plants in the greenhouse, particularly the flower size and number compared to field plot plants. We believe this could be related to autoclaving the soil and were not able to rectify the issue by adjusting fertilizer or increasing pot size to 1.5 gallons. Plants in objective 2, which were not grown in autoclaved soil, were healthier but would probably benefit from a larger than 1 gallon pot size.  Therefore, to simplify the design, avoid having to autoclave our growing medium, and increase sample size without increasing necessary space and supplies, we have made changes that we feel improve the overall experimental design. 

For both objectives, we have decided to replace sunflower with the smaller aster, dandelion (Taraxacum officinale), which can be grown in containers of much smaller volume, allowing for a larger sample size. Like sunflower, dandelion produces large quantities of pollen and is visited by a wide range of insects for floral rewards.

In Objective 1, rather than field collected soil microbes we have decided to test the addition of two commercial plant growth promoting biofertilizers.  Not only will this greatly reduce the risk of unwanted fungal growth, but it will also allow us to test the effects of particular strains of rhizobacteria and mycorrhizal fungi. We feel this will be more informative to stakeholders than an unknown collection of microbes from field soil. 
 

In Objective 2, we have decided to test the effects of glyphosate drift at the initiation of bolting rather than glyphosate as a pre-planting soil treatment. While we did not expect a strong direct impact of glyphosate many weeks after application due to its rapid breakdown in soil, we were interested in indirect effects through alteration of soil microbiome or early impacts on vegetative growth. However, in a preliminary field trial (not included in this project), we saw no effect of pre-planting glyphosate on flower phenotype, bloom time, or insect visitation. If glyphosate has impacts on flowering that are relevant to flower visiting insects, exposure close to or during bloom may have the strongest effect. Therefore, we think it more applicable to look at the effects of foliar glyphosate exposure at drift equivalent doses.  All other aspects of this experiment, with the exception of plant species as explained above, remain the same.

Because asters have small, shallow nectaries, they are difficult to sample nectar from.  We are currently testing a few other plant species, as potential study plants for nectar. All three species, lacy phacelia (Phacelia tanacetifolia), partridge pea (Chamaecrista fasciculata), and annual coreopsis (Coreopsis tinctoria), are North American native annuals producing large volumes of nectar. One of the challenges of collecting pollen and nectar is the difficulty in predicting the quantity produced and ease of collection. Therefore, we will use these plants to determine if any of them produce enough nectar that can be easily sampled without damaging the plant. Additionally, we will attempt to collect pollen from these plants with the goal of analyzing both nectar and pollen from a single species. Pollen has previously been collected from C. tinctoria successfully in our lab in another study. 

Finally, we have had to remove the volatile organic compound sampling from the project. We had planned to use equipment belonging to a faculty member who has since left the University of Delaware, and now no longer has access to the necessary tools. While this would have been an interesting addition to the project, measuring insect visitation will still provide us with some information on plant attractiveness to insect visitors.

Experimental Design:
 

Study Species and Growth Conditions: Dandelion (T. officinale) plants are being grown in a greenhouse maintained at approximately 24°C with a 16:8 light:dark cycle. Several seeds per cell were sown in 3”x3”x5” cell trays in a standard potting mix, fertilized weekly with a 20-20-20 NKP fertilizer and watered as needed. After germination, seedlings were thinned to one plant per cell.

Objective 1 Treatments: Mycorrhizal fungi and Plant Growth Promoting Rhizobacteria (PGPR): We are testing two commercial biofertilizers available through ARBICO Organics (www.arbico-organics.com): one mycorrhizal inoculant and one rhizobacterial inoculant. The mycorrhizal product, ARBICO Organics® Root Build 240, contains four endomycorrhizae (Rhizoglomus intraradices, Funneliformis mosseae, Rhizoglomus aggregatum, and Glomus etunicatum), as well as the ectomycorrhizae, Pisolithus tinctorius.  This product was chosen because it contains only microorganisms and inert carrier material, without artificial fertilizer or other nutrients that could obfuscate the specific impacts of the inoculant. Of the many potential PGPR, we will focus on Bacillus spp., many of which have been shown to impact various floral traits of plants (see literature review).  The commercial product we are testing, Sumagrow, was chosen because, like the mycorrhizal product, it is comprised of only microorganisms and inert carrier, and contains four Bacillus spp. strains (B. amyloliquefaciens, B. subtilis, Bacillus pumilus, and B. lichenformis).

 Plants were assigned to one of four treatments: 1) Mycorrhizal innoculant only, 2) Bacillus spp. inoculant only, 3) both products applied simultaneously, and 4) controls with no PGPR product applied. Both products were applied to soil prior to planting according to manufacturer’s instruction and at the recommended application rate. Each trial consists of 30 replicates per treatment. As in objective 1, the current trial will focus on phenotype, phenology and pollen collection. A second trial of the same design and sample size will be initiated in spring to coincide with insect foragers and measure insect visitation. Plant phenotypic traits will be collected during both trials.

Objective 2 Treatments: Simulation of Glyphosate Drift: Plants were assigned to one of 3 treatments: control with no glyphosate added, glyphosate at 1.0% field dose, and glyphosate at 6% field dose.  Glyphosate will be applied to leaves using a pressurized backpack sprayer when flower buds begin to form, but before flowers open.

Nectar Trials for both objectives: Once a nectar plant has been selected from those identified above, we will repeat all experiments with identical treatments. Sample size for this experiment will depend on the pot size and space needed for the chosen plant species, but will be no less than 10 replicates per treatment. At the start of flowering, nectar plants will be moved outside the greenhouse to measure insect visitation (as described below). During this time, nectar will be sampled using microcapillary tubes 3 times per plant during flowering, at the same time of day for each sample. Sugar concentration will be measured in percent using a handheld refractometer.

Data Collection for both Objectives: Plant growth rate will be measured by recording length of the longest leaf and total leaf number for each plant weekly. Floral traits will include days to pollen dehiscence, flower number (or inflorescence number for composites) recorded weekly after bolting, and inflorescence width in dandelion and tickseed measured as an average of 10 random plants per treatment weekly upon bloom initiation. In the winter trial, 10 flowers or inflorescences per treatment and species will be bagged for 24 hours to collect pollen. Samples will be pooled within treatments to obtain an adequate volume for nutritional analysis. Pollen collection will be repeated throughout blooming until at least 1mL has been collected per treatment. A small subsample from each treatment will be slide mounted to measure pollen grain size (average of 200 grains per treatment), 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. Plant growth and bloom metrics and pollen nutritional content will be analyzed by multivariate analysis of variance (MANOVA)

In the spring trial, plant growth metrics and floral traits will be recorded as before. However, rather than bagging flowers for pollen collection, plants will then be moved outside on the University of Delaware farm at the start of flowering to measure insect visitation. On three days per week during bloom, ten inflorescences per treatment will be visually observed for 10 minutes each, during which time all insects coming in contact with either anthers or foraging for nectar (including robbing) will be counted and identified to morphospecies. Morphospecies categories will include: honey bee (Apis mellifera), bumble bee (genus Bombus), large carpenter bee (genus Xylocopa), sweat bee (family Halictidae), small dark bees, 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 non-metric multidimensional scaling (NMDS). We were able to test this methodology in a separate field experiment over summer 2021.

Research results and discussion:

Preliminary Results

As explained in the methods, we have made some changes to the protocols for both objectives. Therefore, results are likely to change in future trials. However, results from trials we have completed, despite their setbacks, may indicate some general trends and have helped inform our protocol adjustments.

Objective 1 Results: Effects of Inoculation with Soil Microbes: Given that beneficial bacteria and fungi in soil can act as plant growth promoters, we predicted that inoculation with a field collected microbial community would increase the growth rate and cause earlier bloom time in sunflower. However, based on a one-way multivariate analysis of variance (MANOVA), there was no difference between inoculated treatment plants and control plants grown in pasteurized medium in plant height, leaf number, or bloom time (Fig.1; F(1,28)=1.21, p<.05). However, the difference in diameter of pollen grains (average diameter of 200 grains per treatment) between the two treatments was significant in a one-way analysis of variance (ANOVA), with inoculated plants producing larger pollen grains (Fig. 2; F(1,98)=338.3,p<.05). While these pollen samples were not sent for nutritional analysis, the alteration of grain size indicates an effect on pollen grain development that may also involve a difference in protein or other nutrient contents of pollen.

Figure 1: Inoculation with field collected soil microbes had no effect on bloom time, plant height, or leaf number in sunflower.
Figure 2: Plants grown in inoculated soils produced larger pollen grains on average than plants grown in sterile soil.

Objective 2 Results: Effects of Glyphosate:

As an herbicide, glyphosate is lethal to many plants at adequate doses, but has also been known to stimulate growth at low doses, and to have varying impacts on the soil microbiome. Therefore, it may be difficult to predict how glyphosate might impact floral rewards. Results from our completed greenhouse trial with sunflower showed no difference between treatments when comparing phenotypic variables together using a MANOVA (F(4,52)=1.3432,p>0.05). However, a correlation between treatment and plant height that may be important was identified using ANOVA (Fig. 3; F(2,26)=6.106, p<0.05), with plants treated at the recommended field rate being tallest on average. These field dose plants also developed smaller pollen grains compared to both control and high dose plants (Fig. 4; F(2,147)=16.68,p<0.05). We hypothesize that the accelerated vegetative growth in low dose glyphosate application may divert resources away from pollen development. Bloom time (F(2,26)=2.1580,p>0.05) and leaf number (F(2,26)=0.423,p>0.05) ANOVAS showed no significant difference between treatments.

Figure 3: While a MANOVA showed no effect of glyphosate treatment on variables measured, there may be a biologically significant correlation between glyphosate dose and plant height. Treated plants were taller than untreated plants, although plants treated with a higher dose (150% field dose) were shorter than plants treated at the recommended field dose (F(2,26)=6.106, p<0.05).
Figure 4: Field dose plants produced smaller pollen grains than untreated controls or plants treated with a high dose of glyphosate (F(2,147)=16.68,p<0.05).

Timeline for Project Completion

Winter and Spring 2022:

  • Continue greenhouse trial 1 for Objective 1 (currently underway)
  • Test other plant species for nectar collection and to establish growing conditions (currently underway)
  • Greenhouse trial 1 for Objective 2 with dandelion
  • Hiring of 1 undergraduate assistant to help with summer data collection. The technicians will work 30-40 hours per week for 8 weeks in June and July. (paid with another funding source)
  • Start trial 2 for each objective in early April to coincide with summer bloom time

Summer 2022

  •  complete trial 2 for objectives 1 and 2 (with insect visitation)
  • Begin analyzing data from trial 1 for both objectives
  • End of summer: send out pollen samples for nutritional analysis

Fall 2022

  • Data analysis and preparation of final report
  • Graduate student presents results at annual Entomological Society of America conference

Winter 2022-23

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

Education & Outreach Activities and Participation Summary

2 Curricula, factsheets or educational tools
2 Webinars / talks / presentations
1 Workshop field days

Participation Summary:

125 Farmers
3 Number of agricultural educator or service providers reached through education and outreach activities
Education/outreach description:

Workshop Summary

A day-long workshop was held in person on August 7, 2021 for 20 participants recruited through the University of Delaware Master Gardener Program. While the majority of participants were Master Gardeners, we had a few open seats which were offered to and filled by members of a local native plant gardening group on Facebook. The workshop consisted of two presentations (attached) with interspersed hands on activities. Each participant received a folder containing a business card sized bee identification quick reference guide and a flyer summarizing the workshop content on gardening for pollinators (attached). Additionally, the folder included several relevant factsheets and plant lists from the Xerces Society (www.xerces.org) publication library. Participants were first exposed to the diversity and ecology of flower visiting animals with a particular focus on bees. They were given a box of numbered specimens which they had the opportunity to observed under the microscope as different taxonomic groups and physical characteristics were discussed.

After lunch, workshop participants were given a tutorial on using the app iNaturalist to observe and identify plants and insects. One hour was then spent practicing using the app and observing some of the bees discussed in the morning presentation. Our afternoon presentation focused on resource needs of flower visiting insects, variation in floral rewards, and simple steps for creating quality pollinator habitat.

Online presentations

After receiving positive feedback from workshop participants, the extension office asked us to give the presentations in the form of an online training for first year Master Gardeners in September 2021.  Two trainings, each approximately two hours, were conducted over zoom with a combined audience of 105 trainees and 3 University of Delaware extension professionals. These attendees did not receive the print resources, but were given digital version of all workshop materials. Additionally, these resources were shared with a handful of members of the native plant gardening Facebook group, who requested them upon learning that the workshop was full. The resources were later made publicly available on the graduate student's professional website.

Academic Presentations

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 2023 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.

 

Information Products

    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.