Progress report for GNE22-294
Project Information
As more farms in the US Northeast adopt conservation agriculture practices such as reduced tillage and cover cropping, new challenges to pest management continue to emerge, reinforcing the axiom that “no good deed goes unpunished”. Slugs, particularly the gray garden slug (Deroceras reticulatum) and the marsh slug (Deroceras leave) have emerged as prominent pests of corn and soybean under no-tillage systems in the United States. Chemical control using molluscicide baits is the most common control method. However, these baits are expensive to apply, ineffective in many field conditions, and harmful to humans and wildlife. Biological control represents an important alternative approach to slug management for growers practicing conservation agriculture. Slug-parasitic nematodes can be potent biological control agents, and one species is already being deployed in Europe. However, slug-parasitic nematodes are so far unknown and unutilized in the US Northeast. This is because there are only a few “locally” isolated strains in the United States (from the West Coast) and importation of such species is highly restricted. Therefore, the proposed research aims to: 1) Identify species of slug-parasitic nematodes and their prevalence in no-till corn and soybean production systems in Delaware and Maryland, and 2) Determine the effectiveness (% mortality induced, impact of infection on slug feeding behavior) of these slug-parasitic nematodes against slugs. This represents an important step toward developing an effective and sustainable slug management strategy for Northeastern grain production and the project will provide significant training for the student committed to promoting integrated pest management in the USNortheast.
Objective 1: Identify species of slug-parasitic nematodes and their prevalence in no-till corn and soybean production systems in Delaware and Maryland
Objective 2: Determine the effectiveness of slug-parasitic nematodes against gray garden slugs and marsh slugs:
2a) Determine percent slug mortality caused by different slug parasitic nematode species using different concentrations (lethal doses) under laboratory conditions
2b) Define the amount of time it takes for nematode-infected slugs to stop feeding
The purpose of this project is to determine environmentally and ecologically sound management options for gray garden slugs and marsh slugs using locally isolated slug-parasitic nematodes, reducing dependency on chemical molluscicide baits without sacrificing pest control. Slugs, especially gray garden slug and marsh slug, have emerged as major pests in corn and soybean production systems under conservation practices (Douglas & Tooker, 2012). Both species feed on a wide variety of cultivated and non-cultivated plants in addition to cover crop residues (Gall & Tooker, 2017). In no-till corn and soybean, slug damage is severe in the early growing season, usually when juvenile slugs feed on seeds and emerged seedlings (Dively & Patton, 2022). Severe damage has been reported when planting date aligns with slug hatch, which exposes seeds, seedlings, or young plants to a rapidly growing slug population (Douglas & Tooker, 2012).
Slug damage is associated with leaf shredding in corn and destroyed cotyledons or seedling death in soybean (Brichler, 2020). High levels of injury cause yield reduction and in some cases require replanting of entire fields (Douglas & Tooker, 2012). Damage is most prominent in young plants because older plants can outgrow slug injury (Abendroth, et al., 2009; Douglas & Tooker, 2012). The control of slugs through the use of chemical molluscicides applied in granular baits is difficult because the baits are easily thwarted by rain, have a short field life-span, and need to be applied in close synchrony with pest activity (Henderson & Triebskornz, 2002; Castle, et al., 2017). Thus, there remains a need for effective and environmentally sound slug management strategies.
Slug-parasitic nematodes represent a powerful potential biological control agent because they inhabit the soil, actively locate cryptic hosts in their environment, and harbor symbiotic bacteria with potent molluscicidal properties (Tan & Grewal, 2001). Even though the Western US (California, Oregon) has reported isolation of local strains of a slug-parasitic nematode species ( (Mc Donnell, De Ley, & Paine, 2018; Mc Donnell, Colton, Howe, & Denver, 2020), the Northeast has yet to isolate and record presence of any species or strains. In Europe, one slug-parasitic nematode species is already commercialized, but importation of nematodes is greatly restricted in the US (Mc Donnell, De Ley, & Paine, 2018; Mc Donnell, Colton, Howe, & Denver, 2020). Furthermore, slug-parasitic nematodes can be highly species-specific, with most control failures following from the use of unsuitable nematode strains or unfavorable environmental conditions (Rae, Robertson, & Wilson, 2009). Slug-parasitic nematodes thus represent uniquely adapted biological control agents whose efficacy must be examined and optimized within the environmental and geographical context where management is needed. This project will identify nematode species present in the US Northeast and their efficacy against the two economically important slugs in this region, and represents an important step toward alleviating the apparent trade-off between conservation agriculture and effective slug management.
Cooperators
Research
Materials and Methods
Objective 1: Identify species of slug-parasitic nematodes and their prevalence in no-till corn and soybean production systems in Delaware and Maryland
Origin of slugs and nematodes
Marsh and gray garden slugs will be collected from 10 different corn and soybean fields in Delaware and Maryland, during the spring of 2022, 2023, and 2024. Slugs will be collected using shingle traps and carried to the laboratory using plastic containers with perforated lids and lined with moist paper towels. Shingle traps will be placed along two transects for a total of 8 traps per field, sampled weekly from March-July for a two-year period (Figure 1). In the laboratory, slugs will be fed carrots and leafy vegetables, replaced on a daily basis, and the containers containing slugs will be cleaned weekly and slug mortality recorded.
Figure 1. Slug-parasitic nematode isolation and identification workflow.
Isolation, Morphological and Molecular identification
Slug-parasitic nematode isolation will involve dissecting living slugs suspected of nematode infection and recovering internal nematodes from them. Thereafter, a freeze-killed slug will be inoculated with the nematodes and transferred to modified white traps to encourage nematode reproduction (Pieterse, Tiedt, Malan, & Ross, 2017). A White trap is a nematode trapping technique that uses nematodes’ attraction to water to isolate the infective stages of the nematodes (Figure 1) (Orozco, Lee, & Stock, 2014). The modified White trap by Kaya and Stock (1997) consists of a petri dish, on which the host cadaver rests, placed in another larger petri dish containing water. As infective juveniles emerge, they move to the surrounding water trap where they are harvested, and the process can be expanded to commercial production levels (Shapiro-llan & Hall, 2012). Infective juveniles will be harvested within the first week of emergence, after which storage will be through the use of culturing flasks held at 14°C in the laboratory and re-cultured on fresh freeze-killed slugs every 4 weeks. Slug parasitic nematodes needed for molecular analysis will be stored in 100% ethanol, while for morphological analysis, the slug-parasitic nematodes will be killed and fixed with hot TAF (2% triethanolamine, 8% for malin in distilled water) (Pieterse, Tiedt, Malan, & Ross, 2017). Morphological identification of slug parasitic nematodes will be through mounted microscope slides with a paraffin wax ring and the slides containing nematodes will be viewed and the nematodes measured using a compound microscope with a fitted camera and live measurement capacity (Leica DM200, Leica Microsystems). For molecular identification, nematodes will be identified by sequencing cytochrome C oxidase 1 (COI) (Figure 1). First, DNA will be isolated from nematodes using a DNeasy Blood and Tissue Kit (Qiagen). Second, a portion of the COI gene will be PCR-amplified using combination of universal primers following (Mahmoud, et al., 2019). Third, COI amplicons will be submitted for Sanger sequencing at the University of Delaware DNA Sequencing & Genotyping Center. Lastly, resulting sequences will be compared to a global database of nematode COI sequences using NCBI BLAST.
Objective 2: Determine the effectiveness of slug-parasitic nematodes against gray garden slugs and marsh slugs
2a) Determine percent slug mortality caused by different slug parasitic nematode concentrations (lethal doses) under laboratory conditions
Percent slug (gray garden slug and marsh slug) mortality will be assessed using laboratory nematode feeding bioassays. Slugs will be collected from corn and soybean fields in Delaware and starved 48 hours before the experiment. Only visibly healthy/active slugs will be used for the bioassays. Slug-parasitic nematodes will be tested for their ability to kill slug hosts at different concentrations (0, 50, 100, 200 and 250 infective juveniles per slug) following the concentrations used by (Carnaghi, et al., 2017). However, if more than one species of slug parasitic nematode is obtained, only two concentrations of infective juveniles will be used to ensure feasibility. Four replicates with 10 slugs each (total number of slugs = 40) will be tested. Bioassays will be stored at 25 °C and mortality observed daily for three weeks (Pieterse, Tiedt, Malan, & Ross, 2017; Mc Donnell, De Ley, & Paine, 2018). To confirm the cause of mortality, slugs will be examined under microscope to check for the presence of emerging nematodes.
2b) Define the amount of time it takes for nematode-infected slugs to stop feeding time after exposure
The amount of time required for nematodes to cause slugs to stop feeding will be determined by a greenhouse experiment at the UD Entomology greenhouse kept at 20°C and 85% relative humidity. The experimental will consist of a completely randomized design using plastic trays (54×38×10 cm) planted with soybean or corn and infested with five slugs per tray. Treatments (replicated eight times each) will consist of: 1) soil inoculation with nematodes at plant emergence, 2) soil inoculation with nematodes one week after plant emergence, and 3) a no-nematodes control (El-Danasoury & Iglesias-Piñeiro, 2017). Plant damage will be assessed every two days over a period of three weeks. Slug damage will be quantified using an image processing tool available as a smartphone application (Leaf byte https://apps.apple.com/us/app/leafbyte/id1362985339) to estimate percentage leaf area damaged. The pictures will be taken on a live plant, placing a sheet of white paper in the background to facilitate image processing.
A total of 1,030 slugs consisting of leopard, three-banded, marsh, and gray garden slugs were collected between March 2022 and July 2022 from 15 different sites with no-till corn and soybean production systems in Delaware and Maryland. Most slugs were Marsh slugs (858), followed by gray garden slugs (121), leopard slugs (27), and three-banded slugs (24). Overall, only 30 of collected slugs were parasitized by nematodes (2.91%). The nematodes isolated were from the leopard slugs (0.097%) and (2.82%) marsh slugs. The three-banded slugs and gray garden slugs did not contain or exhibit parasitic nematodes. The isolated nematodes are currently maintained in liquid cultures at the laboratory and DNA has been extracted for sequencing and species identification.
Education & Outreach Activities and Participation Summary
Participation Summary:
I attended and presented a poster at the CANR symposium, a program hosted by the University of Delaware agriculture and natural sciences college. The poster focused on using parasitic nematodes and ground beetles to control slugs. I also attended and presented a poster at the CANR symposium, a program hosted by the University of Delaware agriculture and natural sciences college. The poster focused on using parasitic nematodes and ground beetles to control slugs.
Project Outcomes
Our research project will contribute to future sustainability by identifying local parasitic nematode species that have the potential to provide control of a key early season pest without reliance on costly and environmentally toxic molluscicides. These parasitic nematodes are naturally found in the soil and can actively locate and kill their slug hosts in the field without causing environmental contamination or imbalance. Overall, having a deeper understanding of the mollusk parasitic nematode species present in commercial agricutlural fields and their pathogenicity potential is a cardinal step towards developing a sustainable mollusk management program and mollusk parasitic nematode-based control technologies
Our project's main goal has been working towards effective management of slugs within corn and soybean production systems cultivated under conservation agriculture principles (cover cropping and no-till), by increasing knowledge about the impacts of slug-parasitic nematodes on farms. We have been working on our first objective, which aims to isolate parasitic slug nematodes from the fields in the Northeastern region of the United States. Our preliminary results indicate two different nematode species isolated, and efforts to confirm the species' identity are underway. The future direction for this project is to confirm species identity and conduct laboratory and greenhouse trials to determine their pathogenicity toward slugs, while continuing to collect more nematode strains for further testing. We also plan to communicate our findings through journal publications, conference presentations, workshops, and field days. I am passionate about promoting sustainable agriculture in rural communities. Thus, the knowledge from this project will help me understand alternative pest management approaches and promote them among small-scale farmers. Professionally, this project will enhance my skills in DNA extraction and sequencing, mass rearing of slug parasitic nematodes using in-vitro techniques, and increasing my visibility and experience through publications. The latter will prepare me for my career, whether I pursue a career in academia or industry.