Determining Efficacy of Bacillus thuringiensis galleriae STS-502 (BeetleGone ®) Against the Larvae of Alfalfa Weevil Hypera postica Gyllenhal under Montana Conditions

Principle Investigator: Gadi V.P. Reddy1 Collaborators: Stefan T. Jaronski2 and Tatyana Rand2

Project Personnel: Govinda Shrestha1 and Rob Schlothauer2

1Western Triangle Agricultural Research Center, Montana State University, 9546 Old Shelby Rd., P.O. Box 656, Conrad, MT 59425, USA

2United States Department of Agriculture, Agricultural Research Service, Northern Plains Agricultural Research Laboratory, 1500 N. Central Avenue, Sidney, MT 59270

Aim of the Study

The aims of this study were: 1) to evaluate the efficacy of Bacillus thuringiensis galleriae STS- 502 (BeetleGone ®) against the larvae of alfalfa weevil Hypera postica Gyllenhal under Montana Conditions and 2) to determine the impact of B. thuringiensis on parasitization rate of Bathyplectes spp. against H. postica larvae.

Fig. Life cycle of alfalfa weevil

Materials and Methods

Alfalfa fields

The research reported here was conducted in 2016. Two commercial alfalfa fields with a moderate to heavy history of H. postica infestations were selected in Valier and Conrad locations, in the Golden Triangle area of Montana, United States. Before selecting two fields for experiments, five fields (one and four fields in Valier and Conrad respectively) were checked during first week of June 2016, to determine whether which alfalfa field had moderate or high infestation level of alfalfa weevil for imposing treatments. Two persons were walked inside each alfalfa field, starting from 5 m away from the field edges. Each person collected six stems and a stem was collected at a distance of 5 m away from another collection point. All collected stems were kept in Ziploc bags and brought immediately to lab for the assessment of number of larvae per stem.

A randomized complete block design with four replicates was used, with 6 × 6 m treatment plots separated from other plots by 3 m buffer zones to avoid any overlap of treatment effects. Plots were positioned at least 6 m from field edge Furthermore, treatment plots were demarcated by using 1 m tall marking red painted wooden sticks.

BeetleGone ® Ag Phyllom Bioproducts application

A commercial formulation of BeetleGone® Ag Phyllom bioproduct B. thuringiensis galleriae STS-502 was used for the study. Two concentrations of this product was used at the rates of

907.19 grams (low dose) and 1814. 27 grams (high dose) per 4046.86 m2 in 94635.3 ml. NuFilm 17 (236.588 ml/4046.86 m2) was further added in each dose rate to enhance the activity of product, considered here as a surfactant. A product material was first mixed with water, then surfactant was added, and the final suspension was well agitated before spray application. NuFilm 17 with water was considered as a control treatment. The treatments were applied using a CO2 pressure sprayer, delivering the volume of 102.206 L/ 4046.86 m2 and two-man 3.66 m boom with nozzles spaced 0.46 m apart. Furthermore, each plot was sprayed in two swaths and the sprayer was calibrated for ground speed of 20 m in 6 seconds at 35 psi. The spraying activity was performed between 6- 8 am.

Sampling

Alfalfa weevil larvae population

Hypera postica larvae were sampled from BeetleGone or control treatment plots to determine whether B. thuringiensis application had an impact on alfalfa weevil populations.The times of sampling were 2 day before treatment and 3 or 7 days post application. Ten samples, consisting of 3 stems from each sample were collected from each plot and the sampling was performed along an N-shaped transect beginning 1-5 m into the plot. The stems were grabbed at their base and cut off, placing the stems, inverted, into a labeled 6 L plastic bag. The collected 30 stems were placed into one bag, closed and kept in picnic cooler. The samples were returned to lab and dislodged the larvae from foliage by vigorous shaking in a plastic bucket. The larvae were collected, sorted by age class-‘young’ (L1-L2) and ‘old’ (L3-L4) and counted.

Parasitization rate of Bathyplectes spp.

The parasitism of Bathyplectes spp. against H. postica larvae were determined in experimental plots by rearing of alfalfa weevil larvae that were collected at 7 days post application by stem cut or sweep net method. Sweeping was conducted with a standard sweep net (180o arc), and 20 sweeps were made per each treatment plot. The sweeping was performed along an N-shaped transect beginning 1-5 m into the plot as a similar method described above for alfalfa stem sampling. The collected larvae from each treatment plot were kept in a plastic ziploc® bag with alfalfa foliage and transported immediately to laboratory. In the lab, H. postica larvae from each treatment were treatment were transferred into a large paper bag with a paper towel in the bottom. Fresh alfalfa foliage (usually 1 -2 healthy stems) was placed in each bag, top of the bag was folded multiple times and secured with a large binder clip. Fresh foliage was added every other day as needed and dried out foliage was left in a bag in order to avoid risk of losing insects. All bags were kept at room/lab temperature for 14 days at which time most insects had pupated/emerged.

Fig 1. Alfalfa weevil parasitoid

Data analysis

The data were analyzed in R 2.15.1 (R Development Core Team, 2012). For all data, a test with a normal quantile-quantile plot was performed to confirm normality of the data and equality of variance. Where appropriate, Tukey contrast pairwise multiple comparisons were used to test for significant differences in means (Hothorn et al., 2008). Furthermore, the data were subjected to angular transformation prior to statistical analysis.

Alfalfa weevil population

The percentage reduction of alfalfa weevil population was calculated relative to the initial larval population (assessed 2 days before spraying) as follows:

Alfalfa weevil density reduction (AWDR) (%)

Where AWDRst0 represents the number of alfalfa weevil larvae recorded at each treatment plot before the BeetleGone® application and AWDRst1 is the number of alfalfa weevil larvae recorded at each treatment plot in each sampling time (3 days or 7 days after BeetleGone® or NuFilm 17 treatments).

The overall data were fitted to a linear mixed model with sampling time interval, BeetleGone® treatment dose and alfalfa weevil populations per replicate as fixed effects (categorical variables converted to factors), the variation in alfalfa weevil populations (1|Unit) as random effect and the mean alfalfa weevil populations per treatment as response variable using the function “lmer”. The mean alfalfa weevil population per treatment was calculated using the “Summaryby” work package (doBy). The model was then simplified with stepwise removal of factors having no effect. The Kenward-Roger test was run using the function “KRmodcomp” to compare the models (Halekoh and Højsgaard, 2012).

For the subset data, One-Way Analysis of Variance (ANOVA) was carried out to determine the effect on alfalfa weevil population across treatment levels at each sampling time.

Parasitism level

One way-ANOVA was performed to evaluate whether the spray of BeetleGone® has an effect on parasitism level of Bathyplectes spp. on alfalfa weevil population. The parasitism percentage was calculated as (Numbers of parasitoids pupae formed / Total number of alfalfa weevil larvae reared from collected treatment plots) × 100.

Figure 2. Mean numbers (± SE) of alfalfa weevil larvae recorded 2 days before treatments

Results

Alfalfa weevil population

The mean number (± SE) of alfalfa weevil larvae per 30 alfalfa stems, as quantified 2 days before the BeetleGone® or NuFilm 17 spray on alfalfa plants at two locations; Valier and Conrad ranged from 11.25 -12.75 and 10.75-13.00, respectively across treatment plots (Figure 2). Overall, this field experiment clearly demonstrated significant main effects on alfalfa weevil population for both treatment levels (Valier: F = 13.19; df = 2, 18; P < P < 0.0001; Conrad: F = 15.20; df = 2, 18;P < 0.0001) and sampling times (Valier: F = 6.09; df = 1, 18; P < 0.05; Conrad: F = 5.66; df = 1, 18; P < 0.05). However, no significant interaction effects between the treatments and sampling times was found (Valier: F = 0.1; df = 2, 18; P > 0.05; Conrad: F = 0. 06; df = 2, 18; P > 0.05).

Table 1. Cumulative percentage reduction (mean ± SE) of alfalfa weevil larval population on alfalfa plants after BeetleGone® or Nu-Film (Control) application.

Different letters within a row indicate significant differences between treatments (Tukey test, p<, p< 0.05).

Across the treatment levels, the significant difference on alfalfa weevil population was found at 3 days (Valier: F = 5.32; df = 2, 9; P <0.05; Conrad: F = 6.78; df = 2, 9; P <0.05) or 7 days (Valier:F = 8.93; df = 2, 9; P <0.01; Conrad: F = 9.98; df = 2, 9; P <0.01) after the BeetleGone® spray. The percentage reduction of alfalfa weevil larval populations over the BeetleGone® treatments including the control (NuFilm 17), was found dose-dependent with the highest reduction percentage recorded with highest concentration at both sampling times (Table 1). In Valier location, mean levels of alfalfa weevil larval population in alfalfa plots treated with high dose of BeetleGone® ranged from 33-55 %, 11 to 27 % for low dose of BeetleGone® and 2 to 9 % for NuFilm 17 (3 days or 7 days after treatments, respectively; Table 1). Similarly, in Conrad location, average levels of alfalfa weevil larval population in alfalfa plots treated with high dose of BeetleGone® varied from 38-59 %, 25 to 40 % for low dose of BeetleGone® and 6 to 15 % for NuFilm 17 (3 days or 7 days after treatments, correspondingly; Table 1).

Parasitism level

Alfalfa weevil larval parasitoids Bathyplectes spp. have been found in alfalfa fields at both research locations. Irrespective of sampling methods or locations, the results showed that there were a tendency of higher parasitism of Bathyplectes spp. on alfalfa weevil population when alfalfa plots were treated with NuFilm 17 as compared with BeetleGone® treated alfalfa plots (Table 2). However, only a significantly lower percentage of Bathyplectes spp. parasitism was found when alfalfa larvae were collected from alfalfa fields treated with high dose of BeetleGone® through stem cut at Valier location (F = 5.35; df = 2,9; P = 0.02). In other cases, no significant differences were found on parasitism levels between alfalfa fields treated with NuFilm 17, lower or higher doses of BeetleGone® at both Conrad (stem cut: F = 3.02; df = 2, 9; P = 0.09 and sweep netting: F = 0.87; df = 2, 9; P = 0.45) and Valier (sweep net: F = 2.20; df = 2, 9; P = 0.17) locations. The mean parasitism level at Valier and Conrad research locations varies from 5- 26 % and 17-36 % respectively (Table 2).

Table 2. Parasitism percentage (mean ± SE) of Bathyplectes spp. on alfalfa weevil population 7 days after BeetleGone® or NuFilm 17(Control) applications

Different letters within a row indicate significant differences between treatments (Tukey test, p< 0.05).

Acknowledgements

We would like to thank Connie Miller for assistance with field work. This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Multistate Project S-1052, and the Working Group on Improving Microbial Control of Arthropod Pests Covering Research in Montana under Accession # 232056.

References

Halekoh, U., Højsgaard, S., 2012. pbkrtest: Parametric bootstrap and Kenward Roger based methods for mixed model comparison, 2012. R package version 0.3-4.

Hothorn, T., Bretz, F., Westfall, P., 2008. Simultaneous inference in general parametric models.

Biometrical Journal 50, 346-363.

R Development Core Team., 2012. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.