Over the past decade grubs of the Asiatic garden beetle, Maladera castanea Arrow, have emerged as an early season subterranean root feeding pest of corn in Indiana, Michigan, Ohio and Virginia. M. castanea was initially introduced to the United States in New Jersey in 1921 and would eventually cause sporadic economic damage to turf and ornamentals. The first report of M. castanea in Ohio occurred in 1994, and by 2012 the grubs were first observed heavily infesting corn in sandy soils in the northwestern counties. There are currently no successful management tactics or rescue treatments available for management of M. castanea in corn, often leaving farmers to replant (at great expense) should infestations become too severe. Entomopathogenic (insect-killing) nematodes are naturally occurring in the environment and could be a sustainable control agent for use against M. castanea in corn. Recent advancements in rearing techniques and easy-to-use formulations have lowered production costs and increased overall usage. The goal of this research is to assess M. castanea susceptibility to locally isolated entomopathogenic nematodes in Ohio field corn. To accomplish this, entomopathogenic nematodes will be isolated from the soil samples collected at farms affected by M. castanea in northern Ohio.
Isolated nematodes will subsequently be mass reared “in vivo” using economical and low input technologies. Local M. castanea populations will be collected from local farms and evaluated for susceptibility to these nematode species in greenhouse and field trials. The results from this research will help develop a sustainable management plant for M. castanea in corn using entomopathogenic nematodes. This proposal includes an outreach component to educate farmers about the safety and persistence of these nematode species, and to provide demonstrations regarding rearing nematodes at home and applying them with modified spray equipment. Contingency plans have been devised for the research portion of this study, while extension events including talks and workshops will be evaluated pre- and post-workshop surveys.
The learning outcomes stemming from this project are that farmers will be able to: 1) understand how entomopathogenic nematodes provide a sustainable, low input, and economical management tool with minimal off-target effects, 2) learn which entomopathogenic nematodes M. castanea is susceptible to, 3) identify nematodes based on cadaver color and texture, and 4) learn how to rear and applying entomopathogenic nematodes at home for personal use.
The action outcomes from this study are that farmers interested in utilizing entomopathogenic nematodes for management of M. castanea in corn will be able to; 1) rear their own entomopathogenic nematodes for application on their personal farms, 2) use local entomopathogenic nematodes for management of M. castanea in corn, and 3) minimize insecticide use for management of M. castanea in corn.
Over the past decade grubs of the Asiatic garden beetle (AGB), Maladera castanea Arrow, have emerged as an early season subterranean root feeding pest of corn in Indiana, Michigan, Ohio and Virginia (DiFonzo 2007, Krupke et al. 2007, Hammond 2013). AGB was initially introduced to the United States in New Jersey in 1921 and would eventually cause sporadic economic damage to turf and ornamentals (Hallock 1929). The first report of AGB in Ohio occurred in 1994, and by 2012 the grubs were first observed heavily infesting corn in sandy soils in the northwestern counties. There are currently no successful management tactics or rescue treatments available for management of AGB in corn, often leaving farmers to replant (at great expense) should infestations become too severe. Entomopathogenic (insect-killing) nematodes are naturally occurring in the environment and could be a sustainable control agent for use against AGB in corn. Recent advancements in rearing techniques (Testa and Shields 2017) and easy-to-use formulations have lowered production costs and increased overall usage. The goal of this research is to assess AGB susceptibility to locally isolated entomopathogenic nematodes in Ohio field corn. To accomplish this, entomopathogenic nematodes were first isolated from the soil samples collected at farms affected by AGB in northern Ohio. Isolated nematodes were subsequently mass reared “in vivo” using economical and low input technologies. Local AGB populations were collected from local farms and evaluated for susceptibility to these nematode species in preliminary greenhouse trials. Field trials evaluating entomopathogenic nematode efficacy against AGB grubs will occur in 2019. The results from this research will help develop a sustainable management plant for AGB in corn using entomopathogenic nematodes.
Objective 1. Screen local soils for presence of naturally occurring entomopathogenic nematodes
Experimental Design. nematodes, I worked closely with Ohio State University Extension Educators Eric Richer (Fulton and Lucas Counties), John Schoenhals (Williams Co.), Garth Ruff (Henry Co.) and Mike Gastier (Erie Co.) to meet, discuss plans with, and obtain permission from 9 local farmers across 5 Ohio Counties to sample for nematodes and AGB on their land (5 from Fulton County, 2 from Henry and Williams and 1 from Lucas and Erie). Several of these farmers are fervent supporters of on-field extension research conducted previously by Ohio State University research staff. In addition, land at the Ohio State University’s Ohio Agricultural Research and Development Center in Wayne County, Ohio was also sampled for nematodes. Although we did not reach our goal of 40 fields due to time constraints, efforts will be made next year to continue sampling in the same fields over time to assess population dynamics of entomopathogenic nematode species with respect to insect pest populations and other environmental characteristics of each field and across fields. Any observed patterns could inform us as to which nematode species might be better adapted to different fields based on abiotic factors alone. From each field, bait stations or soil cores (3 cm diameter x 20 cm long) were placed or taken within 20 plots pre-established plots that were arranged in a 5×4 grid within a 4-5-acre area; these plots were already being used for an AGB spatio-temporal sampling network and will further compliment the research being conducted in this study.
To sample for entomopathogenic nematodes, two methods were assessed to isolate entompathogenic nematodes either in the field or in the lab. First, bait stations were set out for one week in June to attract and isolate entomopathogenic nematodes in the field. Each bait station consisted of a wire mesh tube-shaped enclosure (2.54 cm diameter x 7.64 cm long) filled with 10 waxworms and were buried under soil near the surface and left for one week. After one-week bait stations were collected and brought back to the lab. Discolored, dead waxworms (e.g. infected by nematodes or other microbials) were then placed on a White trap (see Objective 2 for description) and surrounded by distilled water to promote emergence of nematodes from the waxworm cadavers into water. White traps were maintained at room temperature (~23OC) in the dark (under cardboard on a counter) for as long as it took for nematodes to emerge (~3-7 days). Emerged nematodes could be identified by the yellowish and cloudy appearance of the water. Following emergence, water from each dish was evaluated under the microscope for the presence of nematodes. Unfortunately, a combination of raccoons, ants, and parasitic fly species consumed the waxworms from a significant number of bait stations and several locations so soil cores, which are described in more detail in the following paragraph) were and will continue to be used for the remainder of this work.
During the week of September 23, 2018, 60 soil cores (2.54 cm diameter x 20 cm long) were randomly sampled from each field (3 per plot) with each core being split among two containers based on soil depth as different nematode species are known to persist at different depths; the top 5 cm and bottom 15 cm of each soil core were placed into labeled 12-oz and 32-oz deli containers, respectively. Containers were then brought to the lab and 5 or 10 greater wax moth, Galleria mellonella (L.), larvae were added to the small and large deli containers, respectively, as they are highly susceptible to entomopathogenic nematode infections (Testa and Shields 2017). Containers were ventilated by poking holes through the lid. Each container was flipped upside down to encourage contact between nematodes and waxworms. After 72 hours, G. mellonella mortality rates and cadaver discoloration were assessed for each core subsample. Nematodes were then isolated from the hosts as aforementioned for the bait stations. Additional field characteristics including soil type and composition, annual rainfall and temperature, previous and current crops, and proportion of land covered by weeds were also assessed for future analyses evaluating spatio-temporal dynamics of entomopathogenic nematodes in field cropping systems of northwest Ohio.
For each location, isolated nematode species were pooled by cadaver color and reared separately following Testa and Shields’ (2017) protocol. Emerging nematodes were isolated from G. mellonella cadavers using White traps which consisted of a small plaster of Paris disk (~5 cm) placed inside a larger petri dish which is then topped with a filter paper and ten infected cadavers per bait station and soil core sample. (NOTE: For the remainder of this work only nematodes isolated from soil cores will be utilized.) Distilled water was added to each petri dish to surround the cadavers and facilitate natural emergence of nematodes away from the waxworm host. The emerged nematodes, cadavers, and traps were rinsed with approximately 25 ml distilled water into a beaker. Standard serial dilution was used to attain a concentration of approximately 15,000-30,000 nematodes per 25 ml. The 25 ml of nematodes was added to plastic containers (~450 ml) filled ¾ full of sawdust and 250 G. mellonella larvae, closed with a vented lid, and monitored daily for nematode emergence. Each container was then rinsed with distilled water through two stacked mesh sieves (Nos. 40, 20) for 2-3 minutes. The flow through was collected and standardized to 15,000-30,000 nematodes per 25 ml. Autoclaved soil was inoculated with 25 ml of nematode stock and stored in the dark at room temperature for long-term reservoirs (~6-12 months).
Entomopathogenic nematode species were also obtained from Elson Shields at Cornell University and added to the stocks of our nematode species to increase genetic diversity. All nematode species were augmented in isolated areas on the OARDC campus to maintain long-term natural populations that could be used at any time.
V2 organic corn plants were grown in the greenhouse at a temperature of 20oC ± 3. Individual corn seeds were sown into 32 oz deli containers; this size container is nearly identical to the volume of soil we use to sample for AGB in the field. Soil was either Millgrove loam or Haskins fine sandy loam, both collected from the same field in Henry County, OH to identify whether soil type influenced nematode infection rates. AGB 2nd and 3rd instar grubs were collected from an infested field in Henry County, OH on October 8 and stored in a refrigerator in soil at 10oC prior to experimentation. Prior to efficacy tests, AGB grubs were placed at room temperature for 24 hours, after which 2 of either 2nd or 3rd instar grubs were added to each replicate and allowed to acclimate for 24 hours. On October 11, 2018, one of the five treatments (water control, Hb, Hm, Sc, and Hb + Hm) was then applied to the appropriate containers at a conservative rate of approximately 1000 IJs per 25 ml water for a total of 12 plant replicates per treatment. This rate was chosen as it is often used to evaluated entomopathogenic nematode infection potential against insects in the lab. After 7 days, grubs were evaluated for entomopathogenic nematode induced mortality, and plant shoots and roots were separated, dried, and weighed. Grub mortality, root, shoot, and total plant weights were compared for entomopathogenic nematode treatments in separate one-way ANOVAs with Lsmeans ≤ 0.05 and Tukey-Kramer post-hoc tests.
Objective 1. Screen local soils for presence of naturally occurring entomopathogenic nematodes
Based on the resulting waxworm cadaver colors and the presence of nematodes under the microscope we successfully isolated 3 entomopathogenic nematode species from all of the field sampled. These include Hb, Hm, and Sg. In addition, the entomopathogenic fungi Metarhizium anisopliae was also isolated. Efforts are currently being made to confirm the identification of these species using the following protocol that was developed by Camila Hofman et al. (in press) to sequence the mitochondrial cytochrome oxidase subunit I (COI) gene:
Nematode Identification: DNA barcoding. Single nematodes for each cadaver color will be placed in 1.5 ml microcentrifuge tubes, then onto glass coverslips in 18 ml of sterile water. DNA extractions will consist of the maceration of individual nematodes using a micropipette tip (Powers et al. 2014). Mashed nematodes in water will then be stored at -20°C in 0.25 ml PCR reaction tubes until PCR. The primer set: COI-F1KF (29bp, 5’- CCTACTATGATTGGTGGTTTTGGTAATTG-3’) and COI-R2KF (23bp, 5’- GTAGCAGCAGTAAAATAAGCACG-3’) (Kanzaki and Futai 2002) will be used to amplify the mitochondrial cytochrome oxidase subunit I (COI) gene. Excluding the primers, amplification products should yield 658-bp for resulting sequences. PCR amplification reactions will consist of 6.4 ml of ddH2O, 1.8 ml of each 20 mM primer, 15 ml of 2XJumpStart RED Taq ReadyMix (Sigma-Aldrich, Inc. St. Louis, MO) and 5 ml DNA template from the macerated nematode, for a total reaction volume of 30 ml. PCR cycling protocol will consist of a hot-start and an initial denaturation of 5 minutes at 94°C followed by 45 cycles of 15 seconds at 94°C (denaturation), 15 seconds at 55°C (annealing), 60 seconds at 72°C (extension) and a final extension step of 5 minutes at 72°C. All PCR will be conducted in a thermal cycler at the Ohio State University Department of Entomology. To confirm successful amplifications, 3 ml of PCR products will be loaded into 1% agarose gels stained with GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA) in 1× Tris-acetate-EDTA (TAE) buffer. Gels will be placed into electrophoresis with 0.5X Tris-Borate-EDTA (TBE) running buffer for 35 minutes at 155V. DNA templates will be sequenced via bidirection Sanger sequencing at Functional Biosciences (Madison, WI), then edited and aligned on Geneious (Biomatters Inc., Newark, NJ). Resulting sequences will be submitted to NCBI GenBank following publication.
The resulting sequences will provide positive controls for us to confirm additional isolated nematode species. We will continue to sample each field for soil cores as performed this fall and evaluate the spatio-temporal dynamics of entomopathogenic nematode species with respect to environmental variables including climate, precipitation, soil type and composition, and biotic factors including insect species populations. Sampling will occur once in the Spring and once in the Fall of 2019 and of 2020. I am currently learning how to use GIS to accompany these analyses. We will also use PCOrd2 to perform a Principal Component Analysis to see whether certain combinations of environmental variables contribute to species distribution, while robust multiple regression will be used to identify whether certain variables are better predictors for species presence and distribution. A manuscript will be prepared from these results and submitted to PLOS ONE following completing of this work.
Objective 2. Rear isolated entomopathogenic nematode species using established “in-vivo” techniques
Each species was successfully reared following the protocol established by Testa and Shields (2017). Each species is currently being reared/preserved in several ways: populations were established in vegetative areas around the OARDC in Wooster, OH. Autoclaved soil was inoculated with each species and stored in vented containers in the dark at room temperature. Nematode colonies are being maintained in waxworms in the lab. These species will be mass reared using waxworm cups prior to field trials in the spring.
Objective 3. Evaluate M. castanea susceptibility to these nematodes in corn
Using a conservative rate of ~11,824 nematodes per ft2, all four nematode treatments successfully infected and killed AGB, albeit at different rates. (It is important to note that the number of grubs used in this study was a limitation as we are unable to rear the species in a controlled setting and we must drive 2-3 hours to collect grubs from the field. In addition, grubs are approximately the size of sand grains in the summer months, so we had to wait until mid-Fall to collect grubs that were a reasonable size to work with as our bait stations failed due to other animal species, which occurred around the time grubs were turning into adults). Hb and Hb + Hm treatments caused the highest mortality around 40% and were significantly different from the control which received no nematodes. Sg and Hm alone killed approximately 10% and 20% of grubs, respectively, but did not vary from the water control. Despite mortality rates below 50%, the rate used was only a fraction of what would be applied in a production setting, suggesting that actual field mortality rates may be much higher. Additional factors that may have impacted nematode infection rates (e.g. soil type and grub instar) were compared in separate two-way ANOVAs using proc reg in SAS 9.4; grub infection rates did not vary for sandy and loamy soils or among 2nd and 3rd instar grubs (P>0.05). These results were largely consistent with those evaluating commercially available nematodes against AGB in turf grass systems. Unlike these studies, our study isolated nematodes from the local environment as these species will be better adapted to local environmental extremes such as drought and temperature extremes and should not require annual augmentation into the field; previous studies in upstate New York showed that locally isolated entomopathogenic nematodes can reduce pest populations in field cropping systems and persist for over 7 years after initial application.
Plant growth characteristics (root, shoot and total plant weights) were similar for plants of all treatments. Interestingly, plant weights were numerically the smallest for Hb, one of the treatments that caused the highest AGB mortality. When looking at the roots and shoots of plants from this treatment, it was apparent that the grubs fed on the main roots closest to the kernel at a higher rate for this treatment than the others. This suggests there is likely a very low economic threshold level for AGB grubs in corn, thus necessitating the need for novel management tools like entomopathogenic nematodes, especially since insecticide seed treatments appear to be ineffective against this pest. These results were presented at the Entomological Society of America’s 2018 Annual Meeting in Vancouver, BC on November 12, 2018. I was recently invited by Paul Georgen from Pioneer Seed in Indiana to present these results to local farmers in north central Indiana on January 24, 2019. In addition, I will hold a workshop in 2019 to educate farmers in NW Ohio about entomopathogenic nematodes, how to mass produce them, and how to apply them using their own equipment. A formal, easy to use handout will also accompany the workshop session.
Although initially planned for the 2018 field season, the field evaluations will begin in 2019 with cooperating farmers and continue beyond the grant end date. A grant extension was obtained due to a brief teaching appointment during Fall 2017 when this research was proposed to begin. The persistence and efficacy of entomopathogenic nematodes against AGB grubs (and other soil dwelling pests) will be evaluated on 4 fields previously sampled for nematodes. A randomized complete block design with four replicates per treatment will be used for this study. Each plot will be 8 rows wide by 30 feet with rows spaced 30 inches apart. Organic corn will be planted beginning of May and seeded for 28,000 plants per acre. The three most effective nematode treatments from the greenhouse trial and Btg will be evaluated at their most effective rates. Weeds and other pests will be managed per commercial recommendations. Plant stand counts will be recorded from flagged 17.5 feet of two interior rows for each plot until AGB pupates. Grubs will be counted from 5 random soil samples per plot using the cup cutter method. Nematodes will be sampled from 5 AGB traps per plot. These traps will consist of wire-mesh cage containing at least 10 AGB grubs that are inserted into the ground; any nematodes in the surrounding area should infest the grubs. Adult populations will be monitored weekly throughout the summer. A pitfall trap will be placed in the center of each plot to record adult emergence, while milk jug traps with antifreeze that are used for Western bean cutworm will be placed in the center of each side of the field to monitor dispersal. Yield (tonnes/ha) will be recorded from the middle four rows of each plot. For the field evaluations I will hire an undergraduate worker through this grant to provide them training in graduate-level research and to enhance their professional development. These results will be compiled into a manuscript to be submitted to the Journal of Economic Entomology following the completion of all work.
Educational & Outreach Activities
The outreach components which are discussed in the Results and Discussion portion of the progress report will be completed in 2019 and 2020, following the completion of this grant. Prior to conducting the on-field nematode sampling I met and consulted with 9 individual farmers in 5 Ohio counties about my plans for research and answered any questions they had. I am finalizing reports on our findings thus far (in addition to our AGB sampling in these fields).
I have presented my findings thus far at the Entomological Society of America’s annual meeting in Vancouver, BC in 2018. I was awarded 2nd place in the President’s Prize competition for this talk. I will be presenting the findings of my greenhouse study at a meeting for local farmers in north central Indiana on January 24 as I was invited by Paul Georgen who works for Pioneer Seed to speak. i will also be participating in the Fulton County field day in Northwest Ohio and plan to set up a workshop to teach interested farmers in the benefits and utility of entomopathogenic nematodes and how they can isolated, rear, and augment their own nematodes into their fields for biological control. At these events, I will have handouts prepared to answer basic questions about entomopathogenic nematodes in field crops. I also plan to write articles for Ohio State extension (C.O.R.N. newsletter, and eFields on-farm trial reports) about our findings which will be made available to the public.