The goal of this project is to determine the benefits and sustainability of neonicotinoid seed treatment (NST) use in mid-Atlantic grain production. NSTs are widely used in field crops to provide protection from early season soil and foliar pests; in 2011, 34-44% of soybean and 89-100% of corn was planted using NSTs. However, research suggests an overuse of NSTs. An EPA report from 2014 concluded that NSTs do not provide economic benefits in US soybean production. EPA survey data indicated that in soybean, roughly 65% of growers use NSTs without targeting specific pests, which goes against the principles of IPM. NSTs are often used as a form of insurance, because growers cannot anticipate levels of many pests at the time of planting. Additionally, in most crops, the active ingredients from NSTs remain active in plant tissue for four to six weeks after planting, so they only provide protection against early season soil and seedling pests. NSTs offer benefits in systems with regular early season pest problems, such as mid-South soybean. However, in areas without consistent pest pressure, NSTs provide inconsistent or no yield improvement in soybean, field corn, sunflower, wheat and oilseed rape production. In many cases, growers have to use NSTs even if they do not benefit from them, because of the difficulty of acquiring untreated seed. The mid-Atlantic region is one area where grain crops do not experience frequent early season pest problems. One of the primary goals of this project is determining whether the use of NSTs in Maryland grain crops reduces pest populations and improves yield, allowing growers to make informed decisions about the best way to protect their crops and optimize yield.
In addition to providing inconsistent benefits, NSTs can also have various non-target impacts. Neonicotinoids have been found to negatively affect beneficial arthropods including natural enemies like predators and parasitoids. At high doses, neonicotinoids are acutely toxic, and at lower doses, they may have sublethal impacts on behaviour and physiology that can reduce biological control. This can cause secondary pest outbreaks. While many studies have focused on how neonicotinoids and NSTs affect specific taxa, their effect on the overall arthropod community is less well studied. By using several different methods to sample arthropods at several stages, I will evaluate the impact on NSTs on both pest and beneficial arthropods, and how that impact changes over time. Soil dwelling arthropods are especially at risk from NSTs because the majority of active ingredients remain in the soil, where they may persist for long periods, and could accumulate due to repeated use. Thus, I will focus on both the foliar and soil arthropod communities.
Arthropods are not the only organisms at risk from neonicotinoid residues in soil; other important soil organisms are also negatively impacted by neonicotinoids. Neonicotinoids are highly toxic to earthworms, which perform several important functions including decomposing organic matter, increasing soil porosity and aeration, and facilitating water and nutrient cycling. Although studying the impacts of NSTs on earthworms is difficult, we can measure soil parameters that could be altered by changes in the abundance, behavior and physiology of earthworms and other soil invertebrates, such as pH, aggregate stability, active carbon and available nitrogen. Many of these parameters also depend on soil microbes; the soil microbial community is integral to organic matter breakdown and nutrient cycling, and neonicotinoids have been found to alter their abundance, diversity and activity. Most studies on neonicotinoids and soil microbes have been conducted in the lab; I will use quantitative PCR and Illumina sequencing of the 16S ribosomal RNA gene from field-collected soil to determine how the use of NSTs alters abundance and community structure of soil prokaryotes, and whether specific functional groups such as N2-fixing bacteria are altered. By evaluating the impacts of NSTs on soil microbes and important soil parameters, my goal is to determine whether repeated use of NSTs could lead to a potential decline in soil health and quality.
The overall goals of this project are to further our understanding of the non-target impacts of NSTs, and to determine whether their use is beneficial in mid-Atlantic grain production. Given that NSTs do not provide consistent yield benefits, and can have many non-target impacts, their widespread use may not be warranted in this region. If NSTs do impact the ecosystem functions performed by beneficial arthropods, soil microbes and other organisms, they could damage agroecosystem health, and cause a decline in agricultural productivity over time. Due to the immense variability of factors such as soil, climate, crop varieties and biodiversity, costs and benefits of NSTs need to be determined separately for different areas and systems. Much of the current research on neonicotinoids is from Europe, where NST use has been severely restricted since 2013 due to concerns about pollinator health. The findings of those studies may not be applicable when making decisions about NST usage in this country. Through this research project, I aim to determine whether the use of NSTs is warranted in mid-Atlantic grain production, and to evaluate their impacts on this agroecosystem. By furthering our understanding of how NSTs can affect non-target organisms, including those that perform essential ecosystem functions, I will facilitate their effective and sustainable use.
The primary goal of this study is to examine the effects of repeated use of neonicotinoid seed treatments (NSTs) in a three-year crop rotation of full season soybean, winter planted wheat, double cropped soybean and corn. Specifically, we are studying the impacts of thiamethoxam and imidacloprid treated seed on:
- Arthropod pests and beneficial arthropods – data from visual counts has been analysed for all four crops. Data from litter, pitfall traps and sticky cards is being processed and will be analysed in 2018.
- Crop growth parameters and yield – neonicotinoid treated seeds had some early season effects on stand density in full season soybean and corn, but there were no consistent impacts. Neonicotinoid seed treatments did not significantly impact yield in full season soybean, double cropped soybean, winter wheat or corn.
- Soil health, including physical soil parameters and microbial abundance, which will be measured through quantitative PCR and Illumina sequencing – soil parameters such as compaction, aggregate stability and percent carbon, oxygen and nitrogen were measured at the start of the study in 2015, and soil has been collected for end of study measurements. Soil samples have been collected and stored for quantitative PCR and Illumina sequencing, which will be conducted in 2018.
- We are also testing whether commercially available Solvita soil respiration test kits can detect differences in soil respiration due to NSTs, and comparing the sensitivity with results from qPCR results for microbial abundance. When soil was collected for qPCR, Solvita tests were carried out on soil from the same samples immediately after collection.
Funding from this grant, which began in July 2016 will be used specifically for objectives 3 and 4. In 2018, qPCR and Illumina sequencing will be conducted on DNA extracted from stored soil samples from wheat, soybean and corn samples.
SARE funding was secured during the second year of a three-year study. In order to provide a complete description of the research, methods and results from all three years will be included.
The experiment was replicated at two sites, the Wye Research and Education Center (WREC, Wye) in Queenstown, MD, and at the Central Maryland Research and Education Center (BREC, Beltsville) in Beltsville, MD. Four treatments were planted at each site: untreated seeds, fungicide only, fungicide + Gaucho 600 Flowable (imidacloprid), and fungicide + Cruiser ® 5FS (thiamethoxam) treated seeds for each crop. Because commercial NSTs also include fungicides, a fungicide only treatment was included in addition to the untreated control to separate the impacts of the insecticides from those of the fungicides. Soybean and corn seeds were treated at low and medium rates respectively, which are most commonly used in Maryland. Because NSTs are not commonly used in wheat in Maryland, we treated wheat seeds with a medium rate. At both sites, 16 plots were planted in a Latin square design, with each plot measuring 30ft by 50ft.
Before planting, baseline soil measurements were taken for each plot. Measurements included soil compaction, which was measured using a penetrometer, and analysis of soil samples for wet aggregate stability, texture, percent soluble salts, soil pH and carbon, hydrogen, and nitrogen content. These measurements were repeated in fall 2017 at the end of this three-year study, to measure and compare the cumulative effects of the crop rotation for each of our seed treatments. Soil compaction was not measured because soil moisture significantly impacts penetration force, and soil moisture is low in the fall.
2015 Soybean Rotation: Commercially treated soybeans (Variety 93Y8F, Dupont Pioneer) were used at both sites. At Beltsville, soybeans were planted at a 17.8 cm (7-inch) row spacing with a drill calibrated to 155,000 seeds per acre on May 14th into a field that was previously used to grow soybeans. Soybean first emerged around May 26th and were harvested on October 22nd. At Queenstown, soybeans were planted at a 19.1 cm (7.5-inch) row spacing with a drill calibrated to 150,000 seeds per acre into a field that was previously corn on May 26th. First emergence occurred around June 6th, and soybeans were harvested on October 22nd.
Soil samples were taken before planting, one week after soybean emergence (VC-V2), six weeks after planting (V5) and twelve weeks after planting (R3). Solvita tests (Woodbridge laboratories) were used to measure soil respiration immediately after soil collection, and a subsample of the remaining soil was placed in a deep freeze (-80oC) for future analysis of the soil prokaryote community using quantitative PCR and next generation Illumina sequencing of the 16S ribosomal RNA gene. In addition to soil sampling, above ground and epigeal arthropod abundance was measured periodically throughout the season, as well as plant growth, plant germination, and final yield at soybean harvest.
2015/2016 Winter Wheat: Wheat was planted on October 26 at Beltsville and October 27 at Queenstown, with 7-inch (17.8cm) and 7.5-inch (19.1 cm) row spacings, respectively. The drill was set to a seeding rate of 1.75 million seeds/acre at both sites. The same seed treatments were planted in the same plots used for the soybean rotation, using winter wheat seeds (variety MBX14K297, Mercer) that we treated in a cement mixer. Wheat harvest occurred on June 28 at Queenstown and June 30 at Beltsville; yield, moisture and test weight were recorded.
At Feekes stages 4, 5-6 and 9-10, soil cores were collected from each plot and used to conduct Solvita respiration tests; a portion of each soil sample was stored at -80°C for later microbial analysis. Visual counts, sticky cards, pitfall traps and litter extractions were used to measure arthropod abundance. Stand density, Normalised Difference Vegetation Index (NDVI), and tiller counts were used to evaluate wheat growth and vigor.
2016 Double-Cropped Soybean: Commercially treated soybean (variety P39T67R, Pioneer) was planted on July 7 at Queenstown with a 7.5-inch row spacing and a seeding rate of 123,000 seeds per acre and July 8 at Beltsville with a 7-inch row spacing and a seeding rate of 200,000 seeds per acre. Soybean was harvested at both sites on November 2, and yield, moisture and test weight were recorded. Soil was collected for Solvita respiration tests and stored for qPCR and Illumina sequencing at the VC-V2, R1 and R3 stages. Stand count, growth stage, and plant height were measured in each plot. Visual inspection, sweep nets, pitfall traps, litter samples, and sticky cards were used to sample epigeal and foliar arthropods.
2017 Corn: Commercially treated field corn was planted on May 4 at Beltsville and May 8 at Queenstown, using TA506-22DPRIb seeds. Corn was planted with a 30-inch row spacing at both sites, with a planting rate of 30,000 seeds per acre at Beltsville and 33,000 seeds per acre at Queenstown. Corn was harvested on September 27 at Queenstown and on October 5 at Beltsville, at which time yield, moisture content and test weight were recorded. Soil for Solvita and qPCR was collected pre-planting, and at the V3-V4, V10-V12 and R3-R4 stages. After corn was harvested, soil was collected for testing soil quality parameters. Stand density and plant height were measured early in the growing season. Visual inspection, pitfall traps, litter samples, and sticky cards were used to sample epigeal and foliar arthropods at various points over the course of the season.
Microbial Activity Analysis
Solvita Test: The Solvita test kit measures basal respiration by measuring the rate of CO2 emission from the soil, providing a snapshot of soil microbial respiration. Soil samples were collected by taking 30 soil cores from each plot, with cores taken both from within and between rows, using a soil probe of 23mm diameter and a depth of approximately 12 cm. The soil was mixed thoroughly in a bucket and brought back to the lab in cloth soil bags. In lab, roots, invertebrates and other debris were removed from the soil and 100g of soil was weighed out into the plastic jars provided by the company on the same day the soil was collected. Gel probes were placed in each jar following the instructions provided and the samples were placed in a growth chamber at a constant temperature of 22°C. These probes work on the principle of the Beer-Lambert Law and change color in proportion to the concentration of CO2. After 24 hours, the probes were removed, and CO2 emission was measured using the Solvita Digital Color Reader, a portable digital spectrometer that reads the results from the probes.
DNA extraction, quantitative PCR, and Illumina sequencing: Subsamples of the soil collected from the field are being stored at -80°C for future qPCR. Total soil DNA being is extracted using the DNeasy PoweLyzer PowerSoil Kit (Quiagen, Hilden, Germany) according to the manufacturer’s instructions, with the modification that an MP Fast Prep 24 instrument is used to lyse cells (MP Biomedicals, Santa Ana, CA). DNA is being quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA) and diluted to 2.5 ng μl−1. The gene copy number will be determined for the16S ribosomal RNA (rRNA) gene (a prokaryotic barcoding region) through qPCR, allowing us to quantify the overall abundance of soil prokaryotes. qPCR will be performed on a StepOne Plus Real Time Thermocycler (Life Technologies, Carlsbad, CA) using KICQSTART Sybr Green Ready Mix with ROX (Sigma-Aldrich, St. Louis, MO). For each reaction, three replicates will be conducted using 2μl of soil DNA template per reaction. The microbial community will be classified using 16S rRNA Illumina sequencing. Sequencing will be performed on an Illumina MiniSeq (Illumina, Inc., California, USA). Standards, primers, and protocols for qPCR and Illumina sequencing are being provided by Dr. Stephanie Yarwood.
Differences in Solvita test readings were evaluated through analysis of variance, using the Fit Model platform of JMP 13.1.0 (SAS Institute Inc., Cary, NC). Data from both sites were combined, and treatment was a fixed factor, while site, row and column were treated as random blocking factors, with row and column nested within site. Because Solvita readings were taken multiple times per crop, date and treatment-date interaction were also included in the model as fixed effects. Soil quality parameter data will also be analyzed using analysis of variance; the data from before and after the study will be analyzed separately, and time will not be included as a factor, due to the temporal variability in soil quality. Differences in 16S rRNA gene copy number will also be compared using analysis of variance, with treatment, date, and treatment-date interaction as fixed effects.
The assumption of normality will be tested using a Shapiro-Wilk test, and data will be transformed when required. The assumption of homoscedasticity will be tested using Levene’s test, and weighted least squares methods (Weighting factor: (residual variance)-1 of the fixed effect that deviated most from homoscedasticity) will be used when needed. In cases where effect differences are statistically significant (P<0.05), treatment effects will be compared using means comparisons with Tukey’s adjustment.
Sequences generated through Illumina sequencing will be filtered, clustered into Operational Taxonomic Units and assigned to taxa using the QIIME (V1.9.0, http://qiime.org/index.html) software package (Quantitative Insights Into Microbial Ecology). QIIME will also be used to calculate alpha and beta diversity and compare microbial community structure between treatments.
Analysis of variance was also used to test for differences in stand count, yield, plant height, and insect abundance, using the Fit Model platform of JMP 13.1.0 (SAS Institute Inc., Cary, NC). Data from both sites was combined for all analyses. Assumptions were tested and corrected for as described previously, and significantly different treatment effects were compared using means comparisons with Tukey’s adjustment.
Future work: DNA is currently being extracted from stored soil samples; qPCR and Illumina sequencing will be conducted on extracted DNA in 2018. Soil measurements taken before planting in 2015 were repeated using soil that was collected after corn was harvested in 2017, and will be compared in 2018. Arthropods collected from litter samples, pitfall traps, sticky cards and sweep nets in wheat and soybean are being processed in the lab; identification and analysis will be completed in 2018.
Solvita Soil Respiration Test: Soil respiration as measured by the Solvita test kit did not differ significantly between treatments in full season soybean (Treatment F(3,99)=1.5054, P=0.2179), winter wheat (Treatment F(3,71)=0.5662, P=0.6391), double cropped soybean (Treatment F(3,71)=0.4396, P=0.7254) or corn (Treatment F(3,97.9)=0.9328, P=0.4279) (Fig. 1-4).
Previous studies on the impact on neonicotinoids on soil microbes have found that neonicotinoids have a greater impact on community composition than on overall microbial abundance or richness. Thus, even though the Solvita results did not show a treatment difference, there could be differences in the composition of the soil microbial community. Additionally, the Solvita test measures overall soil respiration, which encompasses various types of organisms. By conducting qPCR of the 16S rRNA gene, we will quantify differences in prokaryotic abundance, which may be different than the overall CO2 measured by the Solvita test. Illumina sequencing of the 16S rRNA gene will allow us to evaluate the impact of NSTs on prokaryotic community composition and relative abundance of different groups. It will provide insight into how NSTs may impact important ecosystem functions performed by soil prokaryotes.
Our results from Obj. 1 and 2 (not funded by this grant), so far suggest that the use of neonicotinoid seed treatments may not always be beneficial in Maryland soybean, wheat and corn. Seed treatments did provide some pest protection early in the growing season in full season soybean and wheat, but not in double cropped soybean or corn. In full season soybean, thiamethoxam had a greater impact than imidacloprid on both pest and beneficial insects. Pest pressure was low throughout the study, and did not reach treatment threshold for any pest in all four crops. Subsequently, seed treatments did not lead to an increase in yield in any of the crops. Our results are in keeping with an EPA report that found that the use of neonicotinoid seed treatments in soybean does not provide economic benefits in most cases, and the northeast was identified as a region where this is especially true. We have also found that seed treatments do have a negative impact on some beneficial arthropods; we will have a greater understanding of the impacts on NSTs on beneficial arthropods after analysing data from sweep net samples, sticky cards, pitfall traps and litter extraction. By sharing our findings with growers, we will help them make management decisions about how to best use neonicotinoids in a way that is both sustainable and economically beneficial.
Education & Outreach Activities and Participation Summary
Results from this study will be used to make recommendations to Maryland grain producers about using neonicotinoid treated seeds in a sustainable and beneficial manner. This project is partially funded by grants from the Maryland Soybean Board and the Maryland Grain Producers Board, and reports on the progress and results of the project are also submitted to these stakeholder organizations.
In 2015, information from this study was presented to ~285 farmers, agricultural educators, and service providers at various extension events. In 2016, results from this study were presented to ~580 farmers and extension professionals at several extension events, including the Mid Atlantic Crop School, Northern Maryland Field Day, Maryland Commodity Classic, Eddie Mercer Field Day, and Small Grain Twilight Tour. Surveys of Mid Atlantic Crop School attendees (45 responders) found that 80% will use the information presented about this project, 35% planned to change how they use seed treatments, and 87% would share the information with others. In 2017, findings from this study were shared with ~156 farmers and others at extension events. All 1021 participants are listed as farmers above because we were unable to divide the audience at extension presentations and field days into growers and educators, although the majority were growers. Extension articles on this work were published in the research edition of the University of Maryland Extension Agronomy News in 2015, 2016, and 2017, and reports were provided to the Maryland Grain Producer’s Utilization Board and Maryland Soybean Board.
Talks about this study were presented to a scientific audience as part of the Student Competitions at the 2016 International Congress of Entomology, and at the 2017 Eastern Branch meeting of the Entomological Society of America. This research was also presented at the Northeastern IPM Center’s 2017 IPM Online Conference, which can be accessed online. Findings from this study will continue to be communicated to both growers and researchers at extension events and scientific conferences in coming years.
The results from this project will guide mid-Atlantic grain producers in making pest management decisions regarding the use of neonicotinoid seed treatments (NSTs). Our results so far indicate that in the absence of early season pest pressure, the use of NSTs in mid-Atlantic grain production may be unwarranted. While it is difficult to acquire untreated corn seeds, NSTs are not yet widely used in soybean and wheat in Maryland. Our findings will allow growers to make informed decisions about whether or not to use NSTs in soybean and wheat. When surveyed about extension presentations on our findings, 76% of growers reported that the information was beneficial to them, 48% said that they would implement our recommendations or change their practices, and 50% said that they would share the information with others. Thus, our results are contributing to better agricultural practices in Maryland grain production, economically benefitting our stakeholders by improving their ability to manage insect pests.
In addition to highlighting inconsistent yield improvement, findings from this study will also further our understanding of non-target impacts of NSTs, further allowing growers to choose sustainable pest management strategies. We are evaluating the impact on beneficial arthropods which play an important role in biological control and preventing pest outbreaks, and on soil health, which is crucial to long-term agricultural productivity. By studying a three-year rotation of four crops, we will determine whether the repeated use of NSTs has a cumulative impact over time. On completion of this study, we will be able to inform growers about the benefits and drawbacks of NSTs. On a wider scale, studies like this contribute to a body of literature showing the inconsistent benefits of NSTs, which could eventually result in a wider availability of untreated corn seed. If untreated seed becomes more available, and NSTs do not improve productivity, this work could reduce pesticide inputs into the environment, improving the health of the Chesapeake Bay and other environmentally-sensitive areas. The economic and environmental benefits of this project for Maryland and mid-Atlantic grain production will contribute to a more secure and environmentally sustainable food supply for society.
This project has greatly advanced my understanding of sustainable agriculture and the challenges involved in improving agricultural practices. I have come to realize the importance of taking an integrated approach when considering an agriculture system – focusing on individual aspects such as soil health, or controlling a single type of pest is not a sustainable approach, and we should look for solutions that address more than one problem. One the other hand, I have also realized the difficulty inherent to this process, as practices that improve one aspect of agriculture are often detrimental to others, such as the difficulty of managing weeds and insect pests in reduced or no-tillage systems, or the unintended impacts of insecticides on the soil microbial community. I have learnt while my research is primarily focused on insects, I cannot succeed without understanding other aspects of agriculture and thinking about sustainable agriculture as a wh
I have also gained many valuable skills over the course of this study that I will continue to use throughout my scientific career. I have gained experience in planning and conducting field work, managing technicians and improvising when things do not go as planned. I have learnt how to analyse various types of data, write grants and reports, and present my findings to both the scientific community and growers. I am learning how to implement molecular techniques including DNA extraction, quantitative PCR and Illumina sequencing, which have a wide range of applications, and am also becoming adept at arthropod identification and community analysis. The skills I have gained during this project have been invaluable in developing future research projects.
I am continuing my research with a lab study focusing on cereal aphids in winter wheat, which will be conducted in 2018. Although NSTs usually provide benefits in areas with high pest pressure, even small numbers of cereal aphids can spread major diseases of small grains like barley yellow dwarf virus. In my field study, I found that NSTs only controlled cereal aphids in winter wheat in the fall, but in other studies, they have maintained effectiveness well into the spring. One reason for this variation could be differences in the effect of the winter dormancy period of wheat, such as regional variation in winter temperatures. Another reason for variation in aphid control by NSTs in wheat could be the impact of the treatment on aphid natural enemies. Parasitoids, which play an important role in controlling R. padi and other cereal aphids, can be exposed to the active ingredient from NSTs in several ways. Most studies have focused on how neonicotinoids impact adult parasitoids, and their effect on immature parasitoids remains poorly understood. Additionally, many studies focus on survival and abundance, often overlooking sublethal effects that could also impact natural enemies’ ability to control pest populations. To better understand the efficacy of NSTs in winter wheat, I will conduct a laboratory study evaluating how long NSTs are effective against the Bird cherry-oat aphid, Rhopalosiphum padi. I will also evaluate how NSTs impact Aphidius colemani, a parasitoid of R. padi, when adults lay eggs in aphids that have fed on treated wheat, and how those impacts change over the course of the growing season. This study will evaluate NSTs as a tool for protecting winter wheat from cereal aphids and will provide broader insight into this understudied route of exposure for parasitoids. I have applied for funding for this project through the Maryland Grain Producers Utilization Board and am waiting for their determination.