Entomopathogenic Nematodes Combined with Adjuvants Presents a New Potential Biological Control Method for Managing the Wheat Stem Sawfly, Cephus cinctus (Hymenoptera: Cephidae)

Principle Investigator: Gadi V.P. Reddy Project personnel: Scott L. Portman

Western Triangle Agricultural Research Center,

Montana State University,

9546 Old Shelby Rd.,

P.O. Box 656, Conrad, MT 59425

Aim of the Study

We tested the hypothesis, in the laboratory and the field that treating wheat stubble with entomopathogenic nematodes (EPNs) solutions containing adjuvants will result in higher wheat stem sawfly (WSS) mortality compared to EPN treatments mixed with water alone.

Fig. Life cycle of wheat stem sawfly

Materials and Methods

EPN infection assay

To determine if WSS was susceptible to EPN infection, diapausing WSS larvae were exposed to three species of EPNs: Heterorhabditis indica, Steinernema kraussei, and Steinernema feltiae.

Wheat stubble containing overwintering WSS was collected from a harvested Judee winter wheat field in Teton County, Montana (N47° 52.1916’ W112° 35.5956’). Permission to collect wheat stubble samples was granted by local private landowners: James Bjelland (Podera county, MT), Ken Johnson (Podera county, MT) and Dan Schuler (Teton county, MT). The research activities reported here did not involve, pose a risk to, or harm any endangered or protected species. Using a scalpel, wheat stems were sliced open along the long axis and larvae were gently removed with forceps or a dissecting needle. Care was taken not to injure the larvae during removal and all larvae were inspected under a stereomicroscope to ensure they had no prior injuries that could affect their mortality or susceptibility to infection by EPNs. EPNs were obtained from Becker Underwood Inc. (now BASF Corp., Ames IA) and stored at 4°C.

Seventy-five WSS larvae were placed singularly in 55mm plastic Petri dishes (Bioplast Manufacturing L.L.C., Bristol, PA) containing two pieces of moistened 55mm Whatman® filter paper (GE Healthcare Bio-Sciences, Malborough, MA). To test different concentrations of infective juveniles (IJs) against WSS, IJs from each EPN species were added to distilled water at concentrations of 200, 400, 800 and 2000 IJs/ml. Using a pipette, EPNs were applied by placing a 25ul droplet of EPN solution onto the filter paper next to the WWS larva – EPN application rates were 50, 100, 200, and 500 IJ/larva. Five WSS larvae were treated with each EPN solution (3 EPNs × 4 concentrations × 5 larvae). Applications using 25ul of distilled water without nematodes served as negative controls. After treatment, Petri dishes were sealed with Parafilm M® (Bemis Company Inc., Neenah, WI) and moved to a 25°C incubator.

Larval mortality was assessed every day, for three days following EPN applications. Dead larvae were immediately moved to fresh Petri dishes lined with moist filter paper. EPN infected WSS larvae rapidly turn reddish-brown in color; thus, they can be easily distinguished from uninfected larvae. EPN infections were confirmed using the “white trap” method (White 1927). After 7 days, all white traps were evaluated for the presence of IJs under a stereomicroscope. Following mortality assessments, the experiment was repeated (N=2) to confirm the results. Daily percent mortalities were averaged within treatments to obtain mean larval percent mortalities two and three days after EPN exposure.

Adjuvant absorbance assay

To test the ability of different chemical solutions to absorb into the hydrophobic plugs, we made artificial plugs from natural plug material and measured the rate of absorbance for each solution. Artificial plugs were used because there is a large amount of variability in the size of natural plugs (0.2-1.0 mg) and natural plugs are extremely fragile and crumble easily during removal.

Wheat stubble containing WSS larvae were collected from two harvested Judee winter wheat fields in Pondera county MT (N48°10.567’ W111°32.872’; N48°11.397’ W111°25.843’) and one in Teton county MT (N47°52.360’ W111°40.324’). Dirt and debris were removed from each stem and clean stems were kept in 473 ml plastic deli containers; deli containers with stems were stored in an incubator at 8o C. To create the artificial plugs, ~200 natural plugs were removed from the wheat stubble and ground into a powder of uniform consistency. Plug material was slightly moistened with distilled water and the open ends of Wilmad-Lab Glass® capillary tubes, which approximated the size of a wheat stem (2.2 mm ID, 2.5 mm OD; SP Industries Inc., Warminster, PA), were gently pushed into the moistened plug material. Artificial plugs were allowed to dry overnight inside the capillary tubes; plugs were removed from the tubes the following day. Artificial plugs were 4-5 mm in length and weighed an average of 3.1 mg.

Nine commercial adjuvants (Adigor®, Advantage®, Alypso®, Penterra®, R-11®, Silwet L-77®, Sun Ag Oil®, Sunspray 11N®, and Syl-Tac®) were mixed according to the manufacturers’ recommendations; Barricade (Barricade International Inc, Hobe Sound, FL ), Tween 80®, Triton X-100®, and Urea (Thermo Fisher Scientific, Waltham, MA) were mixed at concentrations of 1.0%, 1.0%, 1.0% and 5.0% respectively (Table 1). Because Sun Ag Oil and Sunspray 11N contain mostly mineral oil, which does not readily dissolve in water, 0.05% Triton X-100 was added to both as an emulsifier. 5.0 ml of each solution was poured into 55 mm glass petri dishes– distilled water served as the control. Artificial plugs were released singularly into each solution and a stop watch recorded the time (seconds) required for the plugs to become completely saturated – recording did not continue past 300 sec. The assay was performed three times (N=3) for each solution (Table 2) and absorbance times were averaged to obtain mean saturation times.

Laboratory assay of EPNs with carrier solutions

To determine if EPN solutions containing different chemical additives would allow EPNs to pass through the plug formed by the WSS and come into contact with the insect, we applied carrier solutions containing EPNs to the tops of wheat stubs. Although H. indica was previously found to cause high mortality in WSS larvae (Table 3), H. indica was not used for further testing because this species prefers warm moist environments and is generally only found in tropical or subtropical climates. H. indica was replaced with S. riobrave because this species survives in dryer climates – such as the semi-arid climate of the northern Great Plains. Pilot trials tested six species of EPNs (H. bacteriophora, S. carpocapsae, S. feltiae, Steinernema glaseri, S. kraussei, and Steinernema riobrave). However, only H. bacteriophora, S. feltiae, and S. riobrave produced significant mortality (>30%), thus, subsequent trials only included these three species. All species of EPNs used in this experiment were commercially available and included both cruisers and ambushers. Commercial availability of an EPN was an important selection criterion because we wanted to test only species that growers could readily obtain in large numbers.

Distilled water and thirteen different chemical carrier solutions were prepared according to Table 1 and stored at 4o C. H. bacteriophora, S. feltiae, and S. riobrave were obtained from a commercial supplier (Sierra Biological, Pioneer CA) and stored at 4o C. EPNs were allowed to equilibrate to room temperature (22o C) before being added to 4 ml of each carrier solution.

Solution volumes were adjusted to achieve concentrations of approximately 2000 IJ/ml.

Soil was collected from an onsite field plot, rocks and other debris were removed manually, and distilled water was added to bring the soil moisture level to ~30%. The soil was sterilized at 125o C for 45 mins in an autoclave. Previously collected wheat stubble, which housed diapausing WSS, was removed from cold storage (8o C) and 15-20 individual stems were inserted into 473ml deli cups containing approximately 150 ml of the moist autoclaved soil. Using disposable pipettes, solutions containing EPNs were mixed thoroughly and applied to the wheat stems by placing a single droplet (~20 ul) on top of the stem’s plug. To determine if WSS were previously infected by naturally occurring EPNs, subsets of stems were treated with distilled water containing no EPNs (negative control). The order of treatment applications was randomized and treated stems were incubated at 25o C in a growth chamber (14:10 L/D, 50% RH) for 7 days.

Following incubation, stems were sliced open with a scalpel along the long axis and larvae or pupae were carefully removed with forceps or a dissecting needle. Both larvae and pupae were found because the insects were slowly developing during the four months in cold storage.

Individuals that appeared infected with EPNs were dissected under a stereomicroscope to confirm the presence of EPNs; individuals that appeared healthy were placed in small 59 ml portion cups and monitored for seven days for latent signs of infection. WSS percent mortalities were calculated from groups of 15-20 stems contained in each deli cup. The assay was subsequently repeated two more times on different dates (N=3). Mortality was assessed for a total of 1173 larvae and 288 pupae (15-20 stems × 14 carrier solutions × 3 EPNs × 3 repetitions). Percent mortalities from each repetition were averaged within treatments (carrier solutions × EPNs) to obtain mean percent mortality values.

Field trials of EPNs with carrier solutions

The previous experiment demonstrated that Penterra, Silwet L-77, Sunspray 11N, and Syl-Tac performed better at allowing EPNs to enter stems compared to all other adjuvants, thus, these four carrier solutions, as well as, Barricade and distilled water were selected for field tests.

Although water and Barricade were not top performers in the laboratory assay, they were included in our field tests because EPNs are typically mixed with water for spray applications, and Barricade has been used successfully to increase the efficiency of EPNs against above- ground insects. All three species of EPNs were tested with the six different carrier solutions at three field locations (3 × 6 × 3 Randomized Complete Block design) – untreated stems served as negative controls to determine if any WSS were infected with indigenous EPNs. In early May 2016, field plots were established in three previously harvested (fall 2015) Judee winter wheat fields; two locations (Bjelland Farm and Johnson Farm) in Pondera county MT (N48°10.567’ W111°32.872’; N48°11.397’ W111°25.843’) and one location (Schuler Farm) in Teton county MT (N47°52.360’ W111° 40.324’). Permission to conduct field trials was granted by local private landowners as mentioned above. Soil type at each location consisted of silty clay loam. Field plots were 1 m2 and contained 3-4 rows of wheat stubble. The corners of the plots were marked with orange painted wooden stakes. To minimize variation in WSS densities [21], plots were arranged linearly approximately equal distances from the edges of the fields. Individual plots were spaced ~8.0 m apart to avoid effects from overspray or migration of EPNs and plot order was randomized at each location.

Carrier solutions were prepared fresh and EPNs added at a concentration of 1000 IJs/ml –the lower EPN concentration more closely simulated real-life application conditions. After adding EPNs, treatment solutions were kept at 8o C prior to transporting to the field sites in order to conserve the EPN’s energy reserves and minimize their temperature related stress response. In the field, 100 ml of the treatment solutions were added to 3.79 L pressurized hand sprayers (H.D. Hudson Manufacturing Company Chicago, IL) – this volume also more closely simulated real-life application conditions of. All sprayers were pressurized with 25 pumps of the handle (>100 psi) which provided enough pressure to apply the more viscous 1.0 % Barricade but still below 200 psi which can cause mortality to EPNs. To standardize the spray rate and spray pattern, a single spray nozzle was interchanged between sprayers for all treatments. The nozzle was adjusted to provide an even cone-shaped spray pattern ~15 cm wide at a height of 15-20 cm.

Between each treatment, the nozzle was rinsed for 3 sec each with soapy water, then tap water, which thoroughly removed any remaining solution from the previous treatment. Treatment solutions were applied evenly to each plot by holding the tip of the nozzle ~15-20 cm above the soil level and moving the nozzle back and forth in a sweeping motion until the liquid was exhausted. To minimize UV exposure and high daytime temperatures, treatment solutions were applied just before sunset. Average air temperatures during treatment applications were 17.2o C, 15.2o C, and 17.2o C at the Bjelland, Johnson, and Shuler Farms, respectively. Average daily air temperatures and daily RH for the five day treatment periods were 10.6o C; 79% RH, 10.0o C; 78% RH, and 12.2o C; 81% RH at the Bjelland, Johnson, and Shuler Farms, respectively.

Five days after treatment, five clumps of wheat stubble were randomly collected from each plot and placed in clean zip-lock bags during transport back to the laboratory. Rainy conditions (0.85 cm / day, May 20-22) during collecting caused the wheat clumps to be soggy, thus wheat clumps were allowed to dry for ~24 hrs before separating. Stems containing diapausing larvae or pupae were removed from the wheat clump, cleaned of dirt and debris, and placed in 473 ml plastic deli containers. Stems were stored at 8o C until they could be assayed for the presence of EPNs (<5 days). Twenty stems (various lengths) from each plot were randomly selected and carefully sliced open with a scalpel to expose the larvae (248 total) or pupae (827 total). All larvae and pupae were assayed for mortality. Dead larvae or pupae were dissected under a stereomicroscope to look the presence of EPNs; individuals that appeared healthy were placed in small 59 ml portion cups and observed for 7 days for latent signs of infection. WSS percent mortality was calculated for each treatment plot, at each location, and percent mortalities were averaged across locations (N=3) to obtain mean percent mortality values for all treatments (carrier solutions × EPNs).

Data analysis

Many factors can cause mortality in WSS populations (e.g. environment conditions, parasitoids, fungi, pathogens, etc.). Therefore, treatment percent mortalities from both laboratory and field tests were adjusted using the Schneider-Orelli formula to correct for percent mortalities found in control samples. Initial two-way analysis of variance (ANOVA) showed no significant percent mortality differences in larvae vs. pupae (P=0.12), thus larval and pupal mortalities were pooled among treatments (EPNs × solutions).

For the laboratory experiment, treatment (EPNs × solutions) percent mortalities from each repetition were treated as independent samples (N=3). Two-way ANOVA was used compare differences in WSS percent mortalities among treatments. The ANOVA model (R2=0.47, P<0.0001) for the laboratory experiment included “EPN species” and “carrier solution” as predictor variables. The “EPN × solution” interaction term was not significant (P=0.552) and was removed from the model. Post-hoc multiple comparisons (Dunnett’s test, α=0.05) were used to determine differences in WSS mortality when stems were treated with EPNs mixed with chemical carrier solutions vs. EPNs mixed with distilled H2O (control). Tukey’s HonestSignificant Difference (α=0.05) was used to test for WSS mortality differences among the three EPNs.

For the field experiment, treatment (EPNs × solutions) percent mortalities from each location were treated as independent samples (N=3). Two-way ANOVA was used to compare differences in WSS percent mortalities among treatments. The ANOVA model (R2=0.59, P<0.0001) included “farm”, “EPN species”, and “EPN × farm” interaction term as predictor variables – “carrier solution” was not significant. Post-hoc multiple comparisons (Tukey’s HSD, α=0.05) were used to test for differences in WSS percent mortality for all three predictor variables. All analyses were carried out in JMP v. 12 (SAS Institute, Cary, NC).

Results

EPN infection assay

This test confirmed that three species of EPNs have the ability to infect and kill WSS larvae. H. indica proved to be the most virulent species because WSS mortality was 100% after day 2 for all concentrations of IJs (Table 3). High concentrations of S. feltiae (200, 500 IJ/larva) also produced 100% mortality by day 3. The highest mortality achieved by S. kraussei was 60%, making it the least virulent of the EPNs tested. EPN related differences in WSS mortality suggest that WSS is more susceptible to infection and death from H. indica and S. feltiae, compared to S. kraussei.

Adjuvant absorbance assay

Water alone does not readily absorb into plugs formed by the WSS, therefore, we tested a variety of commercially available adjuvants including: surfactants, wetting agents, oils, and a humectant (Barricade) for their ability to increase absorption. Artificial plugs released into distilled water required more than 5 min to become completely saturated. Plugs would float on the surface of the water for a considerable amount of time (~2-3 min) before the water would begin to absorb – affirming the hydrophobic nature of the plug material. The amount of time required for the plugs to be completely saturated in the different solutions was variable (Table 2); however, saturation occurred most rapidly in R-11 (4.2 ± 0.03 sec). Plugs were also saturated quickly in Syl-Tac and Adigor (6.5 ± 0.85 and 12.4 ± 3.58 sec, respectively). This result indicates that chemical additives would allow EPN suspensions to absorb into the plug >50× more rapidly than EPN suspensions made with water alone.

Laboratory assay of EPNs with carrier solutions

This assay demonstrated that certain chemical additives improved the ability of EPNs to penetrate the plug and infect the residing WSS larvae or pupae. On average, WSS mortality was significantly higher (F=9.49, df=12, P<0.0001) when EPNs were mixed with Penterra (P=0.015), Silwet L-77 (P=0.043), Sunspray 11N (P=0.002), or Syl-Tac (P=0.008), compared to EPNs mixed with distilled water (Fig 1) – two of these solutions (Silwet L-77, and Syl-Tac) contained silicone-based polymers. There were also EPN related differences in WSS mortality (F=6.69, df=2, P=0.002). On average S. riobrave and S. feltiae inflicted 50.5% and 47.1% mortality, respectively – significantly higher (P=0.002, P=0.019) than 35.0% mortality from H. bacteriophora. This result indicates that S. riobrave and S. feltiae are better at penetrating the plug and infecting diapausing WSS than H. bacteriophora.

Field trials of EPNs with carrier solutions

In the field, solutions containing S. feltiae and 0.1% Penterra increased WSS mortality up to 29% in harvested winter wheat stubble. On average, solutions containing S. feltiae increased WSS mortality (5.1%) more than H. bacteriophora or S. riobrave (F=6.87, df=2, P=0.003; Fig 2), and S. feltiae combined with Penterra, resulted in the highest average mortality (9.78%; Table 4). However, S. feltiae’s effectiveness varied extensively across the three locations (Table 5); hence, location also had a significant effect on WSS mortality (F=14.71, df=2, P<0.0001). WSS percent mortality was higher at the Schuler farm compared to the other locations (P<0.0001). Multiple comparisons of the EPN × farm interaction showed that S. feltiae was more effective at the Schuler farm (15.5%) compared to all other EPN-location combinations (F=9.95, df=4, P<0.0001); no significant location-related mortality differences were found for H. bacteriophora or S. riobrave. These results indicate that spraying winter wheat stubble with solutions containing S. feltiae mixed with 0.1% Penterra may result in a significant decrease in the number of developing WSS larvae and pupae.

Acknowledgements

This work was supported by Montana Wheat and Barley Committee. This material is also based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Multistate Project W3185, # The Working Group Biological Control of Pest Management Systems of Plants under Accession # 231844.

References

White GF. A method for obtaining infective nematode larvae from cultures. Science. 1927; 66:302-303.

Table 1. Adjuvant: product name, manufacturer, main chemical ingredients, and formulation.

Table 2. Number of seconds required for three artificial plugs (avg. length: 4-5 mm; avg. mass:

3.1 mg) to become completely saturated when placed in 5.0 ml of carrier solution. Recordings were stopped after 300 seconds had elapsed.

Table 3. Average (mean ± SE) percent mortality (N=5) of wheat stem sawfly larvae (Cephus cinctus) treated with three species of EPNs (Heterorhabditis indica, Steinernema feltiae, and Steinernema kraussei), 2 days and 3 days after exposure.

Table 4. Average (mean ± SE), minimum, and maximum percent field mortality (N=3) of wheat stem sawfly (Cephus cinctus) from wheat stubble treated with three species of EPNs (Heterorhabditis bacteriophora, Steinernema feltiae, and Steinernema riobrave) combined with different carrier solutions.

Table 5 Average (mean ± SE), minimum, and maximum percent field mortality (N=3) of wheat stem sawfly (Cephus cinctus) from wheat stubble treated with three species of EPNs (Heterorhabditis bacteriophora, Steinernema feltiae, and Steinernema riobrave) at three different locations.

Fig. 1. Mortality of wheat stem sawfly (Cephus cinctus) from wheat stubble treated with three species of EPNs (Heterorhabditis bacteriophora, Steinernema feltiae, and Steinernema riobrave) combined with different carrier solutions. Percent mortalities were pooled across EPN species and bars represent average percent mortality (mean ± SEM) for each treatment solution (N=9).

Asterisks indicate significant differences in percent mortality (Dunnett’s test, α=0.05) compared to controls (H2O).

Fig. 2. Mortality of wheat stem sawfly (Cephus cinctus) from field wheat stubble treated with three species of EPNs (Heterorhabditis bacteriophora, Steinernema feltiae, and Steinernema riobrave). Percent mortalities were pooled across EPN species and bars represent average percent mortality (mean ± SEM) for each species (N=18). Different letters indicate significant differences in percent mortality (Tukey’s HSD, α=0.05).