Development of Above and Below Ground Strategies Using Entomopathogenic Fungi and RNAi Technologies for the Control of Root Crop Pests

2024 Progress Report

Hypothesis I: Benchtop granular formulations of B. bassiana and M. burnneum will significantly attract and infect wireworms and CPB larva and result in reduced pest pressure

The formulation was tested at the University of Vermont Horticultural Research and Education Center and University of Vermont Greenhouse.  The on-farm trails were conducted in four fields in Vermont—Intervale Community Farm in Burlington (North West), Last Resort Farm in Hinesburg (Central West), Bear Root Farm in Williamstown (Central), and Shelburne Farm in Shelburne (West)—to evaluate the efficacy of Beauveria bassiana (BotaniGard) in managing CPB populations (Figure 1). Plots were established with treatment areas ranging from 50 to 200 feet, with an equivalent-sized control plot assigned in each farmer’s field. BotaniGard was applied three times at 5- to 7-day intervals, and data on egg masses, larvae, adults, and defoliation rates were recorded in both the treatment and control plots.

Results varied by site North West site was flooded and therefore there is no result from that particular site.

At the Central Location, while no significant difference was observed in the number of larvae per plant, the BotaniGard treatment significantly reduced defoliation. Infected individuals in the BotaniGard-treated plots likely fell ill and reduced their feeding, contributing to the observed decrease in plant damage (Figure 1).

At the Western Site, plots treated with BotaniGard initially had higher CPB pressure compared to control plots. Following treatment, larval numbers decreased and remained low throughout the season. Defoliation in BotaniGard-treated plots also stabilized for three weeks after application (Figure 1 ).

At the Central West Site, BotaniGard-treated plots showed significant reductions in both larval numbers and defoliation. After the storm, defoliation increased in both treated and control plots. However, BotaniGard-treated plots produced significantly higher potato yields compared to the control, demonstrating the efficacy of the treatment even in challenging conditions (Figure 1, 2).

Figure 1. Study sites for Beauveria bassiana (BotaniGard) bioassay trials in Vermont.

Figure 2. (left) Mean number of CPB larvae, adults, and defoliation rank on the last day at 3 different commercial organic farms. (right) Change in the number of CPB larvae, and defoliation rank over the time at 3 different commercial organic farms.

Figure 3. Potato crop yield comparison between plots treated with fungus compared to untreated control.

Hypothesis II: Farmers will independently produce their granular formulation of fungi using a low-tech protocol 

This was done in the second year.

Hypothesis III: RNAi products will significantly reduce pest pressure associated with CPB infestations in conventionally managed potato crops.

We employed the same setup for the treatments and plant damage recording as our initial bioassay (Figure 4). In our initial bioassay, B. bassiana (GHA) exhibited higher mortality and a shorter LT50 compared to M. brunneum (M52). In the subsequent bioassay, our objective was to optimize dosing. The treatments included a control, RNAi yeast applied at 2.5 kg/ha and 5 kg/ha, and B.  bassiana (GHA) applied at 10^6 conidia/ml and 10^8 conidia/ml. Additionally, we tested combination treatments: RNAi yeast at 2.5 kg/ha with B.  bassiana (GHA) at 10^6 conidia/ml, and RNAi yeast at 5 kg/ha with B.  bassiana (GHA) at 10^8 conidia/ml.

The LT50 results revealed that the most effective treatment was the combination of RNAi yeast and B.  bassiana (GHA), which achieved 50% mortality by approximately Day 7. Moderate effectiveness was observed for RNAi yeast alone, B.  bassiana (GHA) alone, and RNAi yeast combined with a half-dose of B.  bassiana (GHA), all reaching LT50 around Day 8. Slower responses were recorded for the half-dose of B.  bassiana (GHA) at approximately Day 12 and the half-dose of RNAi yeast at approximately Day 10. The combination of RNAi yeast and B.  bassiana (GHA) provided the fastest action with the lowest LT50. Both RNAi yeast and B.  bassiana (GHA) treatments consistently reduced plant damage compared to the control, although the lower concentration of B.  bassiana (GHA Half) did not effectively reduce plant damage. Despite LT50 values ranging from 8 to 12 days, treated groups experienced significantly less plant damage, demonstrating the effectiveness of these treatments in mitigating crop losses (Figure 5-left). Plant damage was significantly lower in all the treatments compared to the untreated control (Figure 5-right).

Figure 4. (left) Greenhouse bioassay setup and (right) plant damage assessment reference.

Figure 5. (left) Bioassay results: Mortality probability by different treatments compared to untreated control . (right ) Mean plant damage in different treatments compared to untreated control .

Field trials were conducted in 10-foot by 10-foot plots with six replicates per treatment at the University of Vermont Horticultural Research and Education Center (Figure 6). Treatments were applied three times at 5-day intervals. Monitoring involved counting eggs, larvae, and adults on 10 stems per plot and assessing plant damage before and after each application. The treatments mirrored those tested in the bioassay: control, RNAi yeast at 2.5 kg/ha and 5 kg/ha, B.  bassiana (GHA) at 10^6 conidia/ml and 10^8 conidia/ml, and their combinations. At the Horticulture Research Center, the trial demonstrated a clear and consistent reduction in CPB populations. However, following a storm on July 10th, potato plants began to decline due to a fungal disease, resulting in minimal tuber production despite the reduced pest pressure (Figure 7). 

Figure 6. Plot setup for the bioassay at the University of Vermont Horticultural Research and Education Center.

Figure 7. Result of two different dosages of each treatment on CPB larvae and adults population at the research field trail.

Our results were presented at several professional and farmer-focused meetings, including:

• Nouri-Aiin, M. (2024, December 17). On-farm innovations for Colorado potato beetle management beyond Entrust. New England Vegetable & Fruit Conference, Manchester, NH, USA.
• Nouri-Aiin, M., Lewins, S., & Izzo, V. (2024, November 13). Eco-friendly pest management strategies for late-season root crops: Integrating entomopathogenic fungi and RNA interference in Northeastern diversified farms. Entomological Society of America Annual Meeting, Phoenix, AZ, USA.
• Eller, K., Izzo, V., & Nouri-Aiin, M. (2024, November 11). Wireworm populations in Vermont. Entomological Society of America Annual Meeting, Phoenix, AZ, USA.
• Vermont Entomology Participatory Action and Research Meeting with Farmers: This meeting engaged 19 farmers, though 9 attended, with 2 unable to join due to emergencies. We presented our findings, discussed experiments, and received valuable feedback to refine our protocols.

Due to the flood and excessive humidity leading to fungal disease in potatoes, some of the results from this year were inconclusive. As a result, an extension request was submitted and approved, allowing for further investigation and validation of our findings in subsequent trials.

Figure 8. Potatoes plant affected by white wall sclerotia (Sclerotium sp.)

2023 Progress Report

 Hypothesis I: Benchtop granular formulations of B. bassiana and M. burnneum will significantly attract and infect wireworms and CPB larva and result in reduced pest pressure

Throughout April, May, and June, an extensive array of traps baited with sprouted wheat enclosed in nylon stockings were deployed at the Intervale Community Center, Bear Root Farm, and Burnt Rock Farm to assess wireworm activity. Subsequently, all captured wireworms underwent morphological identification and were relocated to an insect cage within a greenhouse setting. In January 2024, a bioassay is planned to be carried out on the wireworm population in the colony, utilizing both Beauveria bassiana and Metarhizium burnneum.

Prior to and during the experiments conducted in April, May, and June, wireworm traps were deployed at the Intervale Community Center, Bear Root Farm, and Burnt Rock Farm. The results from April and May at the Intervale Community Center and Bear Root Farm are displayed in Figure 1. Conversely, no significant wireworm pressure was observed at Burnt Rock Farm. Furthermore, by June, the wireworm population had diminished to such an extent that a significant number were no longer detected in the traps, and as a result, the data for this month is not presented. Morphological identification of wireworms was performed for each field separately to determine species, as depicted in Figure 2A and B. The composition of the wireworm population varied between the fields, with Bear Root Farm exhibiting a greater variety of species. Notably, Limonius infuscatus was the most abundant species at the Intervale (Figure 2A), while Athous sp. predominated at Bear Root Farm (Figure 2B). It is worth noting that identifying the specific species of Athous requires DNA barcoding.

Figure 1. Comparison of wireworm populations collected at the Intervale Community Center and Bear Root Farm during April and May, revealing a greater abundance of wireworms captured in April at both locations.

A)                                                                                   B)

Figure 2. Distribution of wireworm species captured in traps at A) Intervale Community Center and B) Bear Root Farm, expressed as a proportion of the total captured specimens.

A field trial was carried out across four different farms, each with five treatments: B. bassiana, M. burnneum, B. bassiana with rolled oats, M. burnneum with rolled oats, and a control group. Each treatment was replicated five times at each farm. Experimental plots, each 10 feet long, were set up in a randomized complete block design, with four treatments and five replicate blocks. Approximately 20 potato plants were grown in each plot, totaling 100 potato plants per treatment. To minimize plot interactions, buffers were placed between plots within the same row. Each treatment was applied twice with a three-week interval. The initial applications for all treatments were carried out in the third week of June by incorporating them into the furrows in each plot. The second round of applications for all treatments took place three weeks later, using the same method as the first round.

In October 2023, the potatoes were harvested and assessed. Twenty moderately sized potato tubers were extracted from the center of each plot, cleaned, and then examined for damage incidence and severity on each tuber, including the number of galleries.

Field Trial Site Details and Treatment Application Schedule

• Intervale Community Center: The first treatment application was conducted on June 20th, 2023. Our field, along with others at the Intervale Community Center, was flooded prior to the second application, leading to the discontinuation of the experiment.
• Burnt Rock (Hinesburg) and UVM Horticultural Research Center: Due to low levels of wireworm pressure at Burnt Rock Farm and UVM Horticultural Research and Education Center (despite a notable number of adult click beetles at the UVM Horticultural Research Center), no data analysis is reported for these farms. We are considering adding two new sites in the upcoming season at farms with a history of higher wireworm pressure.
• Bear Root Farm: The first application was performed on June 21st, 2023, and the second on July 14th, 2023. Potatoes were harvested and assessed for wireworm damage on October 3rd, 2023.

Our data exhibited a non-normal distribution pattern, as indicated by the Shapiro-Wilk normality test (p-value < 0.05). Therefore, a non-binomial generalized model was utilized to accommodate outliers and the non-normal distribution for analyzing potential differences among treatments. Table 1 presents the results of the non-binomial regression analysis for the treatments at Bear Root Farm. The number of galleries in the B. bassiana treatment was found to be significantly different from the control, while the other treatments did not exhibit any significant differences from the control (Table 1). Following B. bassiana, the Metarhizium + rolled oat treatment demonstrated notably low wireworm damage compared to the other treatments. Figures 4A and 4B depict the severity and incidence percentage of wireworm damage under different treatments. It is evident that B. bassiana and Metarhizium + rolled oat significantly reduced wireworm damage.

Table 1. Non-binomial regression analysis

A)                                                                              B) 

Figure 3. Wireworm damage assessment on 20 potatoes per plot (n=500)grown at Bear Root Farm in 2023 depicting A) average severity, representing the mean number of wireworm galleries per treatment, and B) average incidence, indicating the percentage of potatoes exhibiting wireworm damage.

Hypothesis II: Farmers will independently produce their granular formulation of fungi using a low-tech protocol 

At the  NOFA-VT winter conference 2023, we hosted a workshop titled "D.I.Y Beneficial Fungi: Grow Your Own Fungal Insecticide at Home". The abstract for the workshop was as following:

"The Vermont Entomology and Participatory Action Research Team (VEPART) and Soil Ecology lab at the University of Vermont offered a workshop designed to empower growers to cultivate their own beneficial fungi cultures for controlling insect pests in agricultural settings. The workshop covered topics such as the use of beneficial fungi for pest control, basic cultivation techniques using readily available equipment (most of which can be found in your kitchen), and effective application methods. Participants had the opportunity for hands-on experience in growing beneficial fungi. We also evaluated the practicality of the methods taught for different types and scales of production systems and reflected on our successful field applications of the beneficial fungi."

We provided a handout to participants including a detailed explanation and visual representation of each step involved in growing entomopathogenic fungi. The workshop attracted 28 participants, with 19 of them completing our survey. Feedback from the participants indicated a high level of satisfaction with the information presented during the workshop, and they expressed a strong interest in implementing the method in their own practices (Figure 4).

Figure 4. Survey results assessing knowledge acquisition during the workshop and gauging interest in implementing this practice by the participants.

Hypothesis III: RNAi products will significantly reduce pest pressure associated with CPB infestations in conventionally managed potato crops. 

At the UVM Horticultural Research Center, a field consisting of 60 plots, each measuring 10 ft with 15 ft buckwheat buffer in between to prevent spraying drift, was prepared. Throughout the week, the number of CPB was diligently recorded to assess population levels ahead of RNAi and EPF applications. However, the presence of a high abundance of predatory insects has prevented the realization of significant CPB pressure required for the application of our treatments.

To tackle this challenge, we've launched a greenhouse bioassay at the UVM Greenhouse, focusing on CPB larvae and employing the same treatments. This ongoing experiment is aimed at generating results that will be presented next year. Currently, we are testing six treatments, including a control, B. bassiana, M. burnneum , RNAi, B. bassiana + RNAi, and M. burnneum  + RNAi, with each treatment consisting of 9 replications. Due to the controlled environment of the assay, we intend to explore various approaches, such as targeting different larval stages and pupae within the soil. Our objective is to determine the most effective method for applying fungi in preparation for the upcoming field season.

2022 Progress Report

Hypothesis I: grains inoculated with entomopathogenic fungi (I.e. benchtop culturing) will reduce wireworm pressure when applied as a soil application

Lab trials were postponed to the winter of 2022-2023 as we were unable to capture a sufficient number of field caught wireworm larvae. After several attempts using suggested bait/trap protocols, we decided to postpone the lab experiments for the winter season and substitute mealworms (an easily reared and similar taxa) for the bioassays.

RNAi trials will also begin in the 2023 season, as the two current commercial sources of the product were unable to supply us with the sufficient product to satisfy our experimental needs. Furthermore, we wanted to calibrate the dosage for the below ground applications (i.e. entomopathogenic fungi trials) before setting up a full above/below ground trial. 

For the 2022 field season we conducted our first field trial at the Intervale Community Farm located in Burlington, VT. This trial looked to test the efficacy of entomopathogenic fungi as a a control for wireworm larvae in sweet potato plantings.

Sweet potato slips were planted in 40’’ wide beds with 4 rows per bed and 1 foot spacing within rows. Experimental plots (10 bedft long) were established in a randomized complete block design with  four treatments and five replicate blocks. Each plot contained ~20 sweet potato plants for a total of 100 sweet potato plants per treatment. We also included buffers between plots within the same row to reduce interactions among plots.

The treatments included an untreated control (no grain), sterilized millet grains (grain control), mycotized millet grains (2g/plant) and the organic grower chemical control Seduce (44 lbs/acre). Seduce is a spinosad based chemical bait used by some organic root crop growers. The first applications of all treatment were made on June 16 by adding them through the opening of the plastic around each plant incorporated into the soil. Second applications of all treatments, August 1^st, and last application on September 14^th in the same manner as above.

Sweet potatoes were harvested and scored in October 2022. At harvest, 20 moderately sized sweet potato tubers were dug from the center of each plot, washed, and then evaluated for damage incidence and severity on each tuber (number of galleries) (Figure 1). Mean damage incidence and mean damage severity were designated as dependent variables, and differences among treatments were determined via a generalized linear model. 

Figure 1. Wireworm damage on a sweet potato

There was no significant difference between the mean incidence of wireworm damage on sweet potatoes with any of the treatments applied (Table 1, Figure 2, Figure 3). However, the severity of wireworm damage (number of galleries) on sweet potatoes was significantly lower in mycotized grains than grain control and seduce. In addition, the number of galleries was lower in mycotized grain treatment damage severity was not significantly different of those in no grain control (Table 1). This could be the result of high variation in the number of galleries.

Table 1. Mean incidence and severity (± SE) of wireworm damage on sweet potatoes at the Intervale Center VT, summer 2022

Figure 2. Mean severity of wire worm damage on 20 sweet potatoes per plot (n=100) grown at the Intervale Community Farm, VT in 2022

Figure 3. Mean incidence of wire worm damage on 20 sweet potatoes per plot (n=100) grown at the Intervale Community Farm, VT in 2022.