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

Field Season 2023:

 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

Lab trail:

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.

Field Trial:

• Wireworm traps

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.

A)

B)

• Fungal treatments

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

1. 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.

2 & 3. 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.

4. 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 (refer to Figure 3).

Results:

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.

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B)

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" (Figure 5). 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 (Figure 6). 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 7).

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 Field Season Report

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. 

Hypothesis 1a: grains inoculated with entomopathogenic fungi (I.e. benchtop culturing) will reduce wireworm pressure when applied as a soil application

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.

Figure 1. Treatments application

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 (Figure 1).

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 2). Mean damage incidence and mean damage severity were designated as dependent variables, and differences among treatments were determined via a generalized linear model.

Figure 2. 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 3, Figure 4). 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.

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

Figure 4. Mean incidence of wire worm damage on 20 sweet potatoes per plot (n=100) grown at the Intervale Community Farm, VT in 2022.