Plant defense elicitors: plant fitness versus wheat stem sawfly

Insects

Wheat stubble containing diapausing WSS larvae were collected from winter wheat fields in Pondera and Chouteau counties, Montana, from December 2016 to February 2017. Infested WSS stems (8–10 cm long) were stored in 473 ml round plastic deli containers and maintained in a climate cabinet (5 ± 1 °C) for up to 3 months in the dark to facilitate the completion of obligatory larval diapause (Holmes, 1977). Afterward, WSS-infested stems were transferred to new deli containers that had been half filled with garden soil, with the stem inserted upright into the soil. A total of 50–60 stems were placed in each container, kept inside insect cages (12 × 10 × 10 cm) and held at 19–21 °C, 50–60% RH and a 16:8 h L:D photoperiod at the Western Triangle Agricultural Research Center (WTARC) of Montana State University, USA.

Containers were lightly moistened twice a week with tap water to minimize desiccation of the WSS larvae. Cages were checked daily, and newly emerged WSS adults were transferred into new empty cages with pieces of cotton moistened with a 10% of honey water solution until they were used for experiments. Approximately 100 WSS adults were held in each cage, with a 60:40 female:male ratio. To minimize the effect of host deprivation time, laboratory bioassays were performed with WSS females within 48 h of emergence. In this rearing system, adults emerged within 3–4 weeks from the WSS infested stems.

Plants

All experiments (field and laboratory) used the winter wheat variety “Yellowstone” (Bruckner et al., 2007), because this is one of the most common wheat varieties grown in Montana. This variety is high yielding, has excellent baking and noodle qualities, moderate resistance to plant diseases (dwarf smut and stripe rust), and is highly susceptible to WSS damage (USDA NASS, 2017).

The winter wheat seedling nursery plot was established at WTARC before the laboratory bioassay experiments because wheat plants require exposure to cool temperatures for stem elongation and reproduction. The consideration for establishing a nursery plot was mainly because of low winter wheat seedling survival rate when a refrigerator was used to complete the vernalization process (G. Shrestha, personal observation, 2016). The plot was seeded at a rate of 194 live seeds per m² in September 2016. Nutrient and irrigation management followed standard local growing practices. When the plants completed the vernalization process in spring 2017, the seedlings (two unfolded leaves) were transplanted into tapered square pots (13 × 13 × 13.5 cm) at a density of three plants per pot. Plants were maintained in a greenhouse at 18–20 °C, 50–60% RH and natural light conditions until used for experiments. Each pot contained 1.62 kg of prepared soil mixture, which was an equal proportion of sand, vermiculite, and peat moss, and N, P and K level of 17.13, 11.49, and 8.10 g/100 kg of soil mixture, respectively. Plants were watered four to five times weekly, and fertigated with Peters General Purpose Fertilizer (JR Peters, Allentown, PA, USA) at 100 ppm in aqueous solution at fortnight intervals. Nutrient application started when plants reached the third leaf stage (Zadoks, Chang & Konzak, 1974). Plants used for the laboratory experiments were at developmental stage “Zadoks 33” at which two to three nodes are visible, since WSS female adults prefer this stage for oviposition (Holmes & Peterson, 1960).

Chemical source and rate

Actigard 50WG^® was obtained from Syngenta (Fargo, ND, USA) as a water-dispersible granular formulation containing 50% active ingredient. cis-jasmone was obtained from Sigma-Aldrich (St. Louis, MO, USA) as a soluble liquid formulation with 85% purity. Azadirachtin^® (extracts from neem), as a 1.2% emulsifiable concentration, was obtained from Gowan Company (Yuma, AZ, USA). For both laboratory and field experiments, the chemicals were applied at concentrations of 0.75 ml/l (Actigard), 0.5 ml/l (cis-jasmone) and 2.88 ml/l (Azadirachtin), the concentrations recommended by manufacturers for agricultural use.

Laboratory experiments: sawfly adult settling preference

Bioassay experiments were performed to determine the settling preference of WSS female adults between wheat plants treated with chemicals (as above) and an untreated control. For the application of chemicals, potted plants established in the greenhouse were transported to a spraying room. Spray application was made to individual plants using a 750 ml hand-held sprayer, with a spray volume of 20 ml per plant. After chemical application, plants were allowed to dry for 1 h, and then placed in collapsible cages. Control plants were treated with tap water using the same device.

Four wheat plants were placed inside each cage. Two plants were those that had been treated with one of the test chemicals, while two were controls treated with water. A total of 10 female adults (48 h old) were released in the center of each cage and allowed to settle overnight. However, insects were released 5 days after the Actigard treatment application to allow plants to induce resistance against insect pests (Benhamou & Bélanger, 1998). The following day, the position of each WSS adult (on chemically treated plants, water treated plants, or elsewhere) inside the test cage was recorded, once in the morning (9:00–10:00 am) and once in the afternoon (15:00–16:00 pm).

A similar procedure was followed to assess the settling behavior of WSS female adults under a no-choice condition, in which a single plant treated with one of the chemicals or with water (control) was placed in a cage and seven insects were released. The caged plants were held at 19–21 °C, 50–60% RH, and a 16:8 h L:D photoperiod. Each treatment was replicated four times (each insect cage = one replicate) per experiment, and the experiment was conducted twice (N = 8).

Locations of winter wheat fields used in field trials

The field experiment was conducted at three locations: Knees (N 48°00′08.5 W 111°21′51.8), Conrad (N 48°18′29.0 W 111°55′23.1), and Choteau (N 47°59′36.0 W 112°06′49.9), in the Golden Triangle, Montana, USA. All experimental locations were known to have had moderate to high WSS infestations for many years. The experimental plots were seeded between the first and second week of September 2016 at a rate of 194 live seeds per m². The seeds were planted in four rows, with 30 cm between rows. Glyphosate (Roundup Powermax^®) was applied at the rate of 2.5 l/ha (the active ingredient of 540 g/l of acid glyphosate) before seeding to control weed growth. Fertilizers N, P, and K at 224.2, 0, and 22.4 kg/ha were broadcasted while planting, and an additional application of 12.3, 25.2, and 0 kg/ha of these three nutrients were applied through the seed plot drill.

At each field location, treatments were arranged in a randomized complete block design with four replicates per treatment. Plots for treatments were 3.6 × 1.2 m separated by 0.60 m buffer zones to avoid cross contamination of treatments.

Monitoring of wheat stem sawfly adults

WSS has a single generation per year. Adults have mean life expectancy of only 5–8 days (Criddle, 1922), but adult emergence can occur over an extended period of time, generally about 3 weeks. WSS female adults usually begin to deposit eggs inside developing wheat stems when stem elongation starts during the spring season (Knodel, Shanower & Beauzay, 2016; Fulbright et al., 2017). Multiple eggs can be laid within a stem, but only a single larva survives up to maturity. Eggs hatch about 5–7 days after being laid. Larval development includes three to four stages and will last for about 1 month. As wheat plants mature, the larva moves toward the base of the stem to prepare for a period of dormancy called diapause (Fulbright et al., 2017). Further information regarding WSS life cycle can be obtained from Knodel, Shanower & Beauzay (2016) and Fulbright et al. (2017).

Estimating the ideal application time for synthetic plant defense elicitors and a botanical insecticide (Azadirachtin^®) can be one of the critical factors for WSS management. Currently, no degree-day model has been established for determining adult emergence (Knodel et al., 2009). Therefore, two methods were used for monitoring the emergence of adults: (1) dissection of WSS-infested stubble to determine the stage of immature development (Nansen et al., 2005) and (2) sweep net sampling in the winter wheat fields to detect adults (Knodel et al., 2009). Experimental plots and their adjacent winter wheat fields were scouted weekly from the last week of April until mid-June, 2017.

Field trial: application of chemicals

The field experiment included three treatment types: (1) initial application of chemicals when WSS eggs expected to be present inside stems (treatment # 1), (2) delayed initial application of chemicals when WSS young larvae are expected to be present inside stems (treatment # 2) and (3) initial and delayed initial applications of chemicals (i.e., two times) when WSS eggs and larvae are expected to be present, respectively, inside stems (treatment # 3) (Fig. 1). The goal of the treatment categories was to determine the most vulnerable insect stages and the response of wheat plants to the spray timing. All chemicals were applied on the same date (treatment 1: May 29, 2017; and treatment 2 & 3: June 6, 2017) at the Knees and Choteau field trial locations. However, for the Conrad location, spraying started 10 days later because of the late emergence of WSS adults. Treatments were applied using a SOLO backpack sprayer (SOLO, Newport News, VA, USA) calibrated to deliver about 150 l of spray solution/ha based on nozzle flow and walking speed. Plants treated with water served as untreated control plots. At all field trial locations, chemicals were applied at the wheat stage with four to six nodes.

Collection of wheat stems

Wheat stems were sampled in all plots to measure treatment effects during the growing season. Samples were collected 3 days before treatment application, and 10, 20, 30, and 50 days after initial treatment application. Three random samples were taken from the two central rows of each treatment plot, with five stems/sample. Wheat stems were cut from the base of plants with a scissors, placed into a single zip-lock plastic bag, and transported from the field site in coolers. During the final sampling, clumps of stems were removed randomly at each of three sampling points in the two middle rows of each plot with the help of a shovel to collect whole, mature plants. This technique was used mainly because diapausing WSS larvae prefer to remain at the base of the wheat stem.

Samples were taken to the laboratory, where stems were dissected lengthwise with a fine bladed scalpel to measure (1) WSS infestation levels, including the presence of WSS immatures, or WSS frass inside dissected wheat stems at each sampling time, (2) WSS diapausing larval body weights and their mortality (number of dead larvae inside stems) at the final sampling time, and (3) WSS larval parasitism percentage, as measured by the presence of parasitoid cocoons vs unparasitized WSS stages inside wheat stems.

Sampling of adult WSS and parasitoids

To determine whether synthetic plant defense elicitors or Azadirachtin repelled WSS adults and what further impacts these compounds might have on WSS parasitoids, a sweep net was used to sample adult insects, both WSS and the two Bracon parasitoid species. Sweeping was done with a standard sweep net (180° arc), collecting 15 sweeps from each treatment plot. Netting was done 10, 20, and 30 days after initial treatment application. Collected insects were stored in a freezer until they were examined and counted in the laboratory.

Stem lodging level at harvest

Wheat stem lodging was determined by visually classifying locations in pots on a rating scale from 1 to 10, with 1 indicating that all plants in a plot were vertical and 10 indicating that all plants in a plot were horizontal during crop harvest time.

Yield and quality

A Hege 140 plot combine (Hege Maschinen GmbH, Lichtenstein, Germany) was used to harvest the wheat from treatment plots. Care was taken to avoid the borders and any overlap of treatment effects on wheat yield and quality. Each plot length was measured, and the wheat grain threshed from each plot. Wheat grains were cleaned with a seed processor (Almaco, Avenue Nevada, IA, USA) and weighed on a scale to determine yield. Test weight was measured on a Seedburo test weight scale (Seedburo Equipment Co, Des Plaines, IL, USA). The protein percentages of seeds were determined with NIR grain analyzer, IM 9500 (Perten Instruments, Springfield, IL, USA).

Data analysis

The data were analyzed using R 2.15.1 (R Development Core Team, 2011). For all data, a normal quantile–quantile plot was performed to validate normality of data and equality of variance. Wherever appropriate, Tukey’s contrast pairwise multiple comparison was run using the function “multcomp” to compare significant differences in means (Hothorn, Bretz & Westfall, 2008).

Adult settling behavior

Data was pooled over the two experimental runs as there was no significant difference (>0.05) between two experimental runs. Two-way analysis of variance (ANOVA) was used to determine the effects of treatment and time on WSS adults settling behavior when adults were exposed separately to each chemical and control inside insect cages. Paired t-test was used to assess the effect of treatment on WSS adults settling behavior at morning or evening observation, when adults were exposed simultaneously to each chemical treated and untreated plants inside cages.

WSS fitness: larval body weight and mortality

The overall data on larval body weight was fitted to a mixed model with chemical type and application time as fixed factors, the location as random effect (1|Unit) and the larval body weight per plot as response variable using the function “lmer.” The model was subsequently reduced if applicable, with stepwise elimination of factors having no effect. Kenward–Roger test was carried out to compare the models with the function “KRmodcomp” (Halekoh & Højsgaard, 2012). In addition, data of WSS larval body weight for each location was fitted to a generalized linear model with the function “glm” and family “gaussian.” Two categorical variables of data, chemical type and application timing were converted into factors. The mean larval body per treatment was the response variable. The model was then modified with stepwise removal of factors having no effect and the F-test was performed to compare the models. A similar procedure was followed for analysis of WSS larval mortality data.

Winter wheat plant fitness: lodging, infestation, yield and quality

Stem lodging data were analyzed with a similar method as described for WSS fitness. One-way ANOVA was carried out to determine effect of treatment on: (1) WSS infested stem damage percentage at each sampling time including pre-treatment data and (2) grain yield and quality.

Wheat stem sawfly adults, and parasitoid adults and their parasitism level

The numbers of WSS and parasitoid adults, and parasitism (Bracon spp. pupae inside stems) percentage data were non-normally distributed even after log transformation, and a non-parametric one-way ANOVA (Kruskal–Wallis test) was used to examine application timing of each treatment on (1) numbers of WSS and parasitoid adults at each sampling time and (2) parasitism percentage at the final sampling time. Mann–Whitney U-tests were used as post hoc multiple comparisons between treatment means.

Wheat stem sawfly adult settling preferences

Wheat stem sawfly adults were able to settle (preparatory to oviposition) on winter wheat seedlings when plants treated with each chemical and untreated control presented to adults, either separately or together inside insect cages. In no-choice experiments, average adult settling percentages were higher on plants treated with untreated control (53%) followed by cis-jasmone (50%), Actigard^® (49%) and Azadirachtin^® (42%) (Fig. 2). However, there was no significant difference between in adult settling percentage between the treatments (F = 1.20; df = 3, 56; P = 0.32). The observation time had further no effect on adult settling percentage (F = 1.49; df = 1, 56; P = 0.22) (Fig. 2). There were no significant interaction effects between treatments and observation time (F = 0.57; df = 3, 56; P = 0.63).

In contrast, when WSS adults were exposed simultaneously to each chemical treated and untreated plants inside cages, they significantly preferred untreated control over Azadirachtin-treated plants both in the morning (t = −5.79; df = 7; P = 0.001) and evening (t = −3.00; df = 7; P = 0.02) observation periods (Fig. 3). Adults also showed significantly higher preference to the untreated control than the Actigard-treated plants in the evening (t = −3.66; df = 7; P = 0.01), but without effect in morning (t = −1.47; df = 7; P = 0.18) observation period (Fig. 3). Adults showed no significant preferences between untreated control and cis-jasmone treated plants in either the morning (t = −0.11; df = 7; P = 0.90) or evening (t = 0.28; df = 7; P = 0.78) observation periods (Fig. 3).

Infestation level

Pre-treatment data is presented in Table 1. At the Knees location, WSS infested damage stem percentage levels were generally low in chemical-treated plots compared to untreated control plots, irrespective of sampling time (Table 1). However, there were no significant differences in WSS infested damage percentage levels between treated and untreated control plots for most of the sampling times (10, 20, and 50 days after initial treatment applications) (Table 1). Except for third sampling time (i.e., 30 days after initial treatment application), Actigard^® two applications (WSS egg and larval stages) had a significantly lower stem damage percentage levels (mean ± SE; 35.00% ± 9.18) than the untreated control plots (67.00% ± 7.20 to 77.00% ± 1.92) (Table 1).

Similarly, at the Conrad and Choteau locations, treatments had no significant effects on WSS infested damage percentage levels at any sampling time (Table 1). Except, at the final sampling time (i.e., 50 days after initial treatment application) at Choteau location, stem damage levels were found to be significantly lower on wheat plots treated with two times applications (WSS egg and larval stage) of Actigard^® (mean ± SE; 80% ± 1.50) compared with untreated control plots (92.00% ± 4.52 to 96.00% ± 1.65) (Table 1).

Wheat stem sawfly and parasitoid population level

In general, WSS adult populations were found to be higher at the Choteau location followed by the Knees and Conrad locations (Table 2). The highest number of adults were observed at 10 and 20 days after initial treatment. Regardless of location, treatments did not have a significant impact on WSS adult populations, at any sampling time (Table 2).

Higher Bracon spp. adult population levels were observed at the Knees location followed by Choteau and Conrad locations (Table 2). There were no significant differences in parasitoid adult population levels between treatments at each sampling time at the Knees, Choteau, and Conrad locations (Table 2). In addition, the study did not deduce any significant difference on parasitism levels between treatments (Table 2). The overall average parasitism at the Knees, Choteau, and Conrad locations varied from 7.77% to 25.05%, 0.00–3.49%, and 7.67–21.94%, respectively (Table 2).

Diapausing larval body weight

Overall, diapausing WSS larvae body weights were observed higher at the Conrad location, followed by Choteau and Knees locations (Fig. 4). Mean levels of body weight in chemical-treated or untreated control plots ranged from 12.66 to 19.00 mg for Conrad, 11.88–16.90 mg for Choteau and 9.67–13.46 mg for Knees locations (Fig. 4). Overall, chemical type (F = 9.13; df = 3, 135; P < 0.0001) and timing of treatment application (F = 3.78; df = 2, 135; P = 0.02) had significant effects on diapausing larval body weight, based on the linear mixed model analyses. No significant interaction (F = 1.91; df = 6, 129; P = 0.80) between the chemical type and timing of treatment application was detected and consequently, the parameter estimates from the additive mixed model (chemical + application timing) were presented (Table 3).

This study demonstrated main significant effect on larval body weight for both chemical type and timing of treatment application at two of the study locations: Knees (chemical type: F = 6.38; df = 3, 44; P = 0.001; timing of treatment application: F = 3.35; df = 11, 36; P = 0.002) and Conrad (chemical type: F = 4.43; df = 3, 44; P = 0.008; timing of treatment application: F = 4.89; df = 11, 36; P = 0.0001) (Fig. 4). There was a significantly lower larval body weights on Actigard-treated plots with one application (WSS egg stage) and two times applications (WSS egg and larval stages) compared to untreated control plots at the Knees location (Fig. 4). At the Conrad location, lower body weight was significantly observed with Actigard^® two applications made at WSS egg and larval stages compared to most of the untreated control plots (Fig. 4). In contrast, there was no significant main effect on larval body weight for chemical type (F = 2.22; df = 3, 44; P = 0.09) and timing of treatment application (F = 1.85; df = 11, 36; P = 0.08) at the Choteau location. There were significant interactions between chemical type and timing of treatment application at the Conrad location (F = 4.10; df = 8, 44; P = 0.001), but not at the Knees (F = 1.84; df = 8, 44; P = 0.10) or Choteau (F = 1.62; df = 8, 44; P = 0.15). Regardless of treatment application timing, cis-jasmone and Azadirachtin^® treatments had no effect on larval body weight at the Knees and Choteau locations (Fig. 4).

Diapausing larval mortality

Overall, linear mixed model analyses indicated that chemical type (F = 6.31; df = 9, 130; P = 0.0001) and application timing (F = 3.57; df = 8, 130; P = 0.0001) had significant effects on diapausing larval mortality. Significant interaction (F = 4.63; df = 6, 130; P < 0.0001) was found between the chemical type and application timing and the parameter estimates from interaction model (chemical type × application timing) are presented (Table 3).

This study depicted significant main effect on diapausing larval mortality for both chemical type (Knees: F = 9.32; df = 3, 44; P = 0.0001 and Choteau: F = 3.40; df = 3, 44; P = 0.02) and application timing (Knees: F = 4.78; df = 11, 36; P = 0.0001 and Choteau: F = 4.79; df = 11, 36; P = 0.0001) (Fig. 5). There were significant chemical-application timing interactions at both Knees (F = 2.27; df = 8, 44; P = 0.04) and Choteau (F = 4.50; df = 8, 44; P = 0.001) locations.

At the Knees location, across the timing of treatment application, significantly higher diapausing larval mortality occurred when Actigard^® was applied two times (WSS egg and larval stages) compared to untreated control wheat plots (Fig. 5). Delayed one-time application (WSS larval stage) of Actigard^® had significantly higher larval mortality than untreated control plots, but its initial application (WSS egg stage) had no effect on mortality (Fig. 5). Similarly, at the Choteau location, wheat plots treated twice (WSS egg and larval stages) with Actigard^® had significantly higher larval mortality when compared to untreated control plots. In addition, delayed one-time application of cis-jasmone and Azadirachtin^® had significantly higher larval mortality compared to untreated control plots sprayed with water at the WSS egg stage (Fig. 5).

However, in Conrad location, chemical type had the only significant main effect on larval mortality (F = 3.18; df = 11, 44; P = 0.03), whereas timing of treatment application was without effect on larval mortality (F = 1.96; df = 11, 36; P = 0.06) (Fig. 5). There were no significant interactions between chemical type and timing of treatment application (F = 1.41; df = 8, 44; P = 0.22).

Wheat stem lodging at harvest

Based on overall data fitted to the linear mixed model, chemical type (F = 23.15; df = 3, 136; P = 0.0001) had significant impact on wheat stem lodging, but without any effect of timing of treatment application (F = 2.37; df = 2, 136; P = 0.09). No significant interaction (F = 1.91; df = 6, 130; P < 0.08) was found between the chemical type and timing of treatment application and the parameter estimates from reduced mixed model (chemical type) are presented (Table 3).

This study demonstrated main significant effect on wheat stem lodging for both chemical type and timing of treatment application at two of the study locations: Knees (chemical type: F = 8.12; df = 3, 44; P = 0.0001; timing of treatment application F = 3.02; df = 11, 36; P = 0.001) and Choteau (chemical type: F = 10.66; df = 3, 44; P = 0.0001; timing of treatment application F = 4.20; df = 11, 36; P = 0.0001) (Fig. 6). At both locations, significantly lower mean stem lodging (±SE) occurred when Actigard^® was applied twice (WSS egg and larval stages) (Knees: 3.00 ± 0.40; Choteau: 5.00 ± 0.41) while the remaining treatments showed no significant differences from the untreated controls (Knees: 7.00–7.50; Choteau: 8.75–9.00) (Fig. 6). There were no interaction effects between treatment and timing of treatment application (Knees: F = 1.06; df = 8, 44; P = 0.41 and Choteau: F = 1.45; df = 8, 44; P = 0.20).

At the Conrad location, treatment was the only main factor that significantly affected the wheat stem lodging rate (F = 6.09; df = 3, 44; P = 0.01), but without significant effect for timing of treatment application (F = 1.78; df = 11, 36; P = 0.09) (Fig. 6) and interaction (F = 0.41; df = 8, 44; P = 0.90).

Yield

Irrespective of treatment and location, wheat plots treated with cis-jasmone had higher yields than plots treated with Actigard^® and Azadirachtin^® (Table 4). However, there were no significant differences on yield levels between treatments at any location (Knees: F = 0.635; df = 11, 36; P = 0.78; Choteau: F = 0.50; df = 11, 36; P = 0.89; and Conrad: F = 0.28; df = 11, 36; P = 0.98). Average winter wheat grain yield levels ranged from 3,660–4,077, 3,613–3,972, and 4,349–5,241 kg/ha at the Knees, Choteau and Conrad locations, respectively (Table 4).

Quality

Treatments had no significant impact on kernel weight at any location: Knees (F = 1.67; df = 11, 36; P = 0.12), Choteau (F = 1.09; df = 11, 36; P = 0.40) and Conrad (F = 1.53; df = 11, 36; P = 0.16) (Table 4). Similarly, there were no significant differences in protein percentage between treatment at any study location: Knees (F = 0.70; df = 11, 36; P = 0.73), Choteau (F = 1.49; df = 11, 36; P = 0.17) and Conrad (F = 0.73; df = 11, 36; P = 0.69). The test weight across all treatment ranged from 788–819 kg/m³ for Conrad, 790–795 kg/m³ for Knees, and 755–789 kg/m³ for Choteau locations (Table 4). Protein levels across all treatment varied from 13–15% for Choteau, 13–14% for Conrad and 11–12% for Knees locations (Table 4).