Effect of biopesticides on different Tetranychus urticae Koch (Acari: Tetranychidae) life stages

Crop Protection 128 (2020) 105015

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Effect of biopesticides on different Tetranychus urticae Koch (Acari: Tetranychidae) life stages Julian R. Golec 2, Brianna Hoge 1, James F. Walgenbach * Department of Entomology and Plant Pathology, NC State University, Mountain Horticultural Crops Research and Extension Center, 455 Research Drive, Mills River, NC, 28759, USA

A R T I C L E I N F O

A B S T R A C T

Keywords: Twospotted spider mite Biorational pesticides Integrated pest management Acaricide resistance management

Tetranychus urticae Koch management in many specialty crops relies on intensive pesticide use and has led to rapid evolution of acaricide-resistant populations. These concerns, along with public concern regarding con­ ventional pesticide safety, has generated interest in the use of biopesticides for managing T. urticae. Biopesticides evaluated included the bacterial products Grandevo DF 2 (Chromobacterium subtsugae strain PRAA4-1) and Venerate EP (Burkholderia spp. strain A396), the botanical-based product GOS Neem 7-way (azadirachtin), and the emulsified mineral oil product TriTek. Bifenazate (Acramite 50WP), a synthetic acaricide, and a watertreated control were included for comparative purposes. Contact and residual effects were evaluated using equivalent labeled field rates against T. urticae egg, larval, nymphal and adult life stages. Exposure route (contact or residual) was significant for eggs and larval and nymphal mortality after 24-h exposure. GOS Neem 7-way, TriTek, and Acramite were ovicidal under contact exposure only. Most biopesticides caused >50% larval mor­ tality, yet only Acramite achieved 100% morality. Biopesticides were less toxic to nymphs relative to larvae, and Acramite and Grandevo DF 2 caused >50% nymphal morality. Only Acramite caused significantly higher adult mortality than the control 24-h post treatment. Except for Venerate, all treatments significantly reduced fecundity of surviving adults, with the greatest reduction by Acramite and Grandevo. With the exception of Acramite and a high rate of Grandevo, all treatments exhibited exponential population growth over five gen­ erations. However, most biopesticides evaluated exhibited significant mortality on various T. urticae life stages, and if incorporated into management schemes may be useful tools in acaricide resistance management programs for T. urticae.

1. Introduction Twospotted spider mite, Tetranychus urticae Koch (Acari: Tetra­ nychidae), is a major pest of >150 fruit, ornamental, vegetable, and field crops worldwide (Jeppson et al., 1975, Migeon and Dorkled, 2019). Active immature life stages (larvae, protonymphs and deutonymphs) and adults feed by inserting their stylet into mesophyll and epidermal � ska, 1985). This feeding activity effectively cells (Tomczyk and Kropczyn reduces chlorophyll content and alters the functionality of epidermal and stomatal guard cells, resulting in decreased rates of photosynthesis and transpiration, and increased water stress (Deangelis et al., 1982; 1983; Sances et al., 1979). Damage from T. urticae feeding can result in decreased plant growth and yield, leaf bronzing, necrosis, and

distortion, and in some cases plant death (Deangelis et al., 1983; � ska, 1985; Attia et al., 2013). Moreover, this can Tomczyk and Kropczyn cause cosmetic injuries that reduce the commercial value and market­ ability of fruit, flowers, or foliage (Johnson and Lyon, 1991; Attia et al., 2013). For instance, cosmetic damage appears as gold flecking on to­ mato (Solanum lycopersicum L.) (Meck et al., 2012) and scars on clem­ entine mandarin fruits (Citrus clementina Hort. ex. Tan.) (Pascaul-Ruiz et al. 2014) rendering such fruit undesirable for fresh market retail. Low tolerance for cosmetic damage by growers and retailers results in affected fruit being downgraded or culled leading to significant eco­ nomic losses for growers (Meck et al., 2012). While biological control of T. urticae using predatory mites has been successful in protected growing operations (e.g. greenhouses), pesticides

* Corresponding author. MHCREC, 455 Research Drive, Mills River, NC, 28759, USA. E-mail address: [email protected] (J.F. Walgenbach). 1 Current address: Texas A&M AgriLife Extension Viticulture & Fruit Lab, 259 Business Court, Fredricksburg, TX 78624, USA. 2 Current Address: Corteva Agriscience, 7521 W California Ave, Fresno, CA 93706. USA https://doi.org/10.1016/j.cropro.2019.105015 Received 29 July 2019; Received in revised form 4 November 2019; Accepted 8 November 2019 Available online 10 November 2019 0261-2194/© 2019 Elsevier Ltd. All rights reserved.

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Crop Protection 128 (2020) 105015

play a critical role in managing T. urticae in field crops (Leeuwen et al., 2010, 2015). However, the short life cycle and high reproductive po­ tential of T. urticae often results in intensive use of pesticides. This management system has led to the rapid evolution of pesticide-resistant populations, and as of 2018 there were 501 documented cases of T. urticae resistance to 95 active pesticide ingredients (Whalon et al., 2017). Often, new chemical compounds with novel modes of action are sought to replace chemistries that are no longer efficacious; yet, increased public concern about environmental and health effects of pesticides has renewed interest in biorational pesticides (biopesticides) as an alternative to conventional synthetic chemistries (Copping and Menn, 2000; Mar�ci�c and Medo, 2014; Reddy, 2016). Furthermore, use of alternative management tactics that can help suppress mite populations on crops and reduce the need for conventional pesticides, which can be an effective resistance-management practice on high-value crops. In this regard, biopesticides represent a potentially environmentally respon­ sible and sustainable tactic that can be useful in conventional and organic pest management schemes (Myers et al., 2006; Attia et al., 2013). Biopesticides are pest control products that are generally considered to be reduced-risk, and may be microbial, botanical, mineral, or syn­ thetic in origin. Many are certified by the Organic Materials Review Institute (OMRI) for use on organically produced crops and are highly compatible with IPM. Several botanical and microbial biopesticides have proven effective against T. urticae adults and are commercialized for use against this pest (Copping and Menn, 2000; Attia et al., 2013). Despite these advances, there is a paucity of information regarding differences between contact and residual toxicity as well as stage-specific mortality of T. urticae from different biopesticides. Un­ derstanding these factors can help to define efficient and appropriate use patterns. This study evaluated the toxicity of contact and residual exposure of several OMRI approved microbial, botanical, and mineral oil biopesticides to T. urticae eggs, larvae, nymphs, and adults. Addi­ tionally, the effect of these products on population growth of T. urticae was assessed.

mineral oil TriTek (Brandt Consolidated, Inc., Springfield, IL.). The synthetic acaricide Acramite® 50WP (bifenazate, Arysta LifeSciences, Cary, NC) was included for comparative purposes. Pesticides rates were based on recommended field rates on labels and were mixed at the equivalent application rates of 467 L water per ha. Pesticide descriptions and rates tested are shown in Table 1. It should be noted that the bife­ nazate rate was equivalent to only about 10% of the recommended field rate; however, this caused 80–95% mortality of adult females in topi­ cally applied bioassays (JFW, unpublished data). All test solutions were prepared using distilled water (pH 5.4) containing 0.01% Latron B1956 (J. R. Simplot Company, Lathrop, CA). GOS Emulsifier (80% d-Limo­ nene, 20% surfactant) at 0.25% was added to the GOS Neem solution. 2.2. Bioassay methods The effect of biopesticide toxicity towards T. urticae life stages and fecundity was assessed via contact and residual exposure bioassays. For contact exposure, motile mite stages were transferred with a camel hair brush onto 2.0 cm diameter bean leaf discs punched out from green beans (Phaseolus vulgaris) grown under greenhouse conditions. Leaf disks were placed on moist cotton in a 5.5 cm diameter petri dishes, and then test materials were topically applied to leaf discs with an artist air brush (Paasche model H-set airbrush, Chicago, IL) in 50 μl aliquots, which was equivalent to 2.67 � 0.17 μl/cm2, operating at 34,474 Pa. For residual contact exposure bioassays, leaf discs were dipped into test solutions and then placed on paper towels for 1 h to dry. Leaf discs were then placed on moist cotton in 5.5 cm diameter petri dishes and mite stages were placed on discs. For both bioassay methods, leaf discs were placed on moist cotton (abaxial surface up) in petri dish bottoms that were covered with tops that had a 2-cm diameter screened hole, and all discs with mites were incubated in a growth chamber at 25 � C, approximately 60% RH, and 16:8 L:D photoperiod. 2.2.1. Adult bioassays For adult bioassays, 10 adult females were placed on each leaf disc and mortality was recorded at 24 and 48 h. The number of eggs laid was also recorded and expressed as number of eggs per female per day by living adults. One replicate consisted of 5 leaf disk subsamples (each with 10 females) and each treatment was replicated 3 times (¼ 150 fe­ males per treatment).

2. Materials and methods 2.1. Chemicals Biopesticides used in this study were chosen because they represent products that are labeled against a wide range of crops and are regis­ tered for both organic and conventional agriculture. They included two products of bacterial origin, Grandevo DF2 (Marrone Bio Innovations, Davis, CA) that contained Chromobacterium subtsugae strain PRAA4-1 (Martin et al., 2007), and Venerate EP (Marrone Bio Innovations, Davis, CA.) that contained heat-killed Burkholderia spp. strain A396. Additional biopesticides included the botanical-based pesticide GOS Neem 7-Way Georgia Organic Solutions LLC, Blakely, GA), a cold-pressed neem oil, and the pre-emulsified petroleum distillate

2.2.2. Deuto- and protonymph bioassays Because of the similar appearance of deutonymphs and proto­ nymphs, these nymphal stages were not differentiated. One replicate consisted of 5 leaf disk subsamples (each with 10 deutonymphs and/or protonymphs) and each treatment was replicated 3 times (¼ 150 deu­ tonymphs and/or protonymphs per treatment). Mortality was recorded after 24, 48 and 72 h.

Table 1 Products and rates evaluated against T. urticae. Product

Active ingredient

Grandevo DF2

a

Venerate EP

b

GOS Neem 7-Way TriTek Acramite WP

c

a b c

30% Chromobacterium subtsage strain PRAA4-1 94.46% heat-killed Burkholderai spp. strain A396

gm/liter azadirachtin 80% mineral oil 50% bifenazate

Company Marrone Bio Innovations, Davis, CA Marrone Bio Innovations, Davis, CA Georgia Organic Solutions, Blakeley, GA Brandt Consolidated, Springfield, IL Arysta LifeSciences, Cary, NC

Field application was assumed to be made at 467 liters/ha. Grandevo DF2 contained not less than 1,000 cabbage looper killing units per mg of product. Venerate EP contained not less than 1,500 beet armyworm killing units per mg of product. 2

Equivalent field ratea per ha

per liter

3.36 kg 2.24 kg 1.12 kg 18.7 L 9.3 L 1% 1% 0.089 kg

7.2 gm 4.8 gm 2.4 gm 40.0 ml 20.0 ml 1% 1% 0.19 gm

AI per liter 2.16 gm 1.44 gm 0.72 gm 37.8 ml 18.9 ml 0.03 gm 0.8% 0.09 gm

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Crop Protection 128 (2020) 105015

2.2.3. Larval bioassays Due to the small size of T. urticae larvae, it was impractical to transfer larval stage individuals with a camel hair brush. Therefore, to expose larvae, five adult females were placed on leaf discs for a 4–6 h oviposi­ tion period and then removed. For contact exposure, eggs were incu­ bated for 4 d at 25 � C (time interval for eggs to hatch) at which time the number of larvae on each disc were counted, and then treated with test solutions using the airbrush. For residual bioassays, leaf discs were dipped into test solutions, air dried for approximately 1 h, and then five adult females were placed on discs for a 4–6 h oviposition period. A replicate consisted of 5 leaf disk subsamples and each treatment was replicated 3 times. Larval numbers were counted at 4 days when eggs hatched (pre-treatment number), and mortality was recorded at 24, 48, 72 and 96 h after treatment.

thus, individual full factorial ANOVA’s (α ¼ 0.05) were used to examine the main effects of biopesticide, exposure method (contact vs. residual), and their interactive effects on each life stage parameter. When signif­ icant interactive effects of exposure x treatment were detected, inde­ pendent t-tests were performed to determine differences between exposure method and treatments of dead adults, larvae, and proto- and deutonymphs across all time intervals. When significant differences were detected among any of the main effects and their interactions, Tukey’s Honestly Significant Difference procedure (HSD) was used to separate mean values. All percentage data were transformed using arcsine √x to normalize variance, while numerical data were √x transformed to normalize variance. All analyses were conducted using JMP Pro v 14.0 (SAS Institute Inc., 2013). 3. Results

2.2.4. Egg bioassays To establish cohorts of eggs, adult females were placed on leaf discs for 4- to 6-h oviposition periods as described above. For contact activity bioassays, eggs were treated with test solutions via the artist air brush to 24-h old eggs. For residual activity bioassays, ovipositing adults were placed on leaf discs for a 4–6 h oviposition, 24 h after they were dipped into test solutions. One replicate consisted of 5 leaf disk subsamples and each treatment was replicated 3 times. Leaf discs were checked 7 d after exposure and the percentage of eggs hatching was calculated via counting the number of hatched eggs, dividing by the total number of eggs, then multiplying by 100.

3.1. Adult mortality and fecundity Biopesticide treatment and time were both significant factors in assessing mortality in adult bioassays, but exposure method and inter­ active effects were not significant (Table 2). Overall mean � sem per­ centage mortality increased significantly from 18.5 � 2.4% to 33.1 � 2.8% (n ¼ 2,700 adults) between 24 and 48 h post exposure (t ¼ 4.10; df ¼ 106; P < 0.0001). Acramite resulted in the highest adult morality compared to all other treatments, although all three rates of Grandevo and GOS Neem 7-way had significantly higher mortality than the control at 48 h (Fig. 1). Adult fecundity after 48 h was significantly affected by biopesticide, but not exposure method (Table 2). Mean � sem fecundity in the control after 48 h was 6.73 � 1.1 eggs/female/day, which was significantly higher than surviving adults in the Acramite treatment (0.96 � 0.4) and

2.3. Population growth estimates The cumulative effects of the various treatments on population growth or decline of a generation of T. urticae was estimated by calcu­ lating the number of f1 adults (Af1) generated after exposing an initial cohort of 100 females and their progeny to an application of test ma­ terials according to the equation:

Table 2 Category, factors, degrees of freedom (df), F-values (F), and P-values (P) from Analysis of Variance results of experiments comparing the effect of biopesticides on twospotted spider mite life stage mortality.

Af1 ¼ (Af0*ASC)*(F*5)*ESR*LSR*NSR where, Af0 ¼ size of initial cohort of adults (100 in this example), ASC ¼ adult survival rate from contact exposure of the biopesticide, F ¼ fecundity of surviving adults (assume oviposition for 5 days), ERS ¼ egg survival rate following residual contact of biopesticide, LSR ¼ larvae survival rate following residual contact of biopesticide, and NSR ¼ nymphal survival rate following residual contact of biopesticide. This scenario assumed that the initial cohort of adults (Af0) was the only life stage exposed to contact effects, and all other life stages experience residual effects. It is also assumed that adults surviving exposure to the biopesticide survived for 7 days and their fecundity remained constant during this time. Finally, population growth over five generations was also estimated, with the assumption that the initial cohort of adults and successive generation of adults were exposed to a single application of biopesticide.

Category

Factor

df

F

P

Adult mortality

Time Biopesticide Exposure Time x biopesticide Time x exposure Biopesticide x exposure Time x Biopesticide x exposure Biopesticide Exposure Biopesticide x exposure Biopesticide Exposure Biopesticide x exposure Time Biopesticide Exposure Time x biopesticide Time x exposure Biopesticide x exposure Time x biopesticide x exposure Time Biopesticide Exposure Time x biopesticide

1, 72 8, 72 1, 72 8, 72 1, 72 8, 72 8, 72

35.46 16.16 0.001 1.289 0.045 1.160 0.336

<0.0001 <0.0001 0.975 0.263 0.853 0.335 0.949

8, 36 1, 36 8,36 8, 36 1, 36 8, 36 2, 72 8, 36 1, 36 16, 72 2, 72 8, 36 16, 72

28.60 1.22 1.83 33.10 1.933 2.373 233.5 29.03 0.723 4.733 3.1447 4.734 1.318

<0.0001 0.276 0.104 <0.0001 0.173 0.036 <0.0001 <0.0001 0.4007 <0.0001 0.0491 0.0005 0.2107

3, 108 8, 36 1, 36 24, 108 3, 108 8, 36 24, 108 8, 36 1, 36 8, 36

138.3 78.23 0.121 4.016

<0.0001 <0.0001 0.730 <0.0001

7.619 2.875 4.0726

0.0001 0.0139 <0.0001

5.571 48.15 2.619

0.0001 <0.001 0.0227

Adult fecundity Eggs per cohort Deuto/protonymph mortality

2.4. Statistical analysis All bioassays were set up as factorial experiments in a completely randomized design. Leaf disks subsamples for each replicate of each treatment and life stage was averaged before analyses. Data on percent adult morality were subjected to factorial analysis of variance (ANOVA, α ¼ 0.05) to examine the main effects of biopesticide, exposure method (contact vs. residual), time (24 and 48 h), and their interactive effects. Because data on percentage mortality of larvae and proto- and deuto­ nymphs were collected over 3 (24, 48 and 72 h) and 4 (24, 48, 72, and 96 h) time points, respectively, these data were subjected to full factorial repeated measures ANOVA (α ¼ 0.05) to detect differences across time. Data on adult fecundity, total eggs per cohort, and percentage of eggs hatching to larvae were collected at a single time point after treatments;

Larval mortality

7 d egg mortality

3

Time x exposure Biopesticide x exposure Time x biopesticide x exposure Biopesticide Exposure Biopesticide x exposure

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Crop Protection 128 (2020) 105015

Fig. 1. Mean (�SEM) percentage mortality of twospotted spider mite adults resulting from contact and residual biopesticide exposure. Bars with different lower- and uppercase letters indicate significantly different means via Tukey’s HSD (P ¼ 0.05) within contact and residual exposure methods, respectively. For all treat­ ments, n ¼ 300.

all three rates of Grandevo (Fig. 2). Numerically, increasing rates of Grandevo resulted in decreasing rates of fecundity, but these differences were not significant. Both rates of Venerate were the only treatments to not significantly reduce fecundity below the control. Total number of eggs per surviving cohort was significantly affected by treatment and the interaction of treatment x exposure (Table 2). Mean � sem residual exposure to the lowest rate of Grandevo (158.3 � 29.5) resulted in significantly (t ¼ 2.31; df ¼ 4; P ¼ 0.047) fewer eggs per cohort compared to contact exposure (270.0 � 24.9) (Fig. 3), and accounted for the significant treatment x exposure inter­ action. All three rates of both contact and residual exposure to Grandevo and contact exposure to GOS Neem 7-way significantly reduced mean eggs per cohort below that of the control. Acramite resulted in almost 90% fewer eggs (147 � 24.0) compared to the control (1309 � 54.8), and the high rate of Grandevo had approximately 78% fewer eggs (285 � 58.4) than the control (Fig. 3).

mortality (Table 2). With the exception of the high rate of Venerate, mortality increased significantly over the 24 to 72-h exposure period for the other treatments (Table 3). Across all treatments, mean � sem mortality significantly increased from 14.9 � 2.4% to 32.7 � 2.9% (n ¼ 2,700 deutonymphs þ protonymphs) from 24 to 72 h post exposure (df ¼ 2, 159; F ¼ 14.09; P < 0.0001). Acramite was most toxic to deuto- and protonymphs, while all rates of Grandevo DF2 and GOS Neem 7-way also had significantly higher mortality than the control at 72 h (Table 4). The significant treatment x contact interaction was the result of Acramite having higher mortality via contact (92.0 � 2.0%) versus residual (57.3 � 6.4%) exposure (t ¼ 5.08; df ¼ 16; P < 0.001). 3.3. Larval mortality Treatment, time, and the interactions of time x treatment, time x exposure, exposure x time, treatment x exposure, and treatment x exposure x time all had a significant effect on larval mortality (Table 1). Acramite was the only treatment that resulted in >95% larval mortality at 24 or 48 h post exposure (Table 5). Across all treatments, mean � sem larval mortality increased significantly from 27.9 � 4.0% to 50.8 � 3.6% from 24 to 96 h post exposure (df ¼ 3, 212; F ¼ 6.15; P ¼ 0.0005),

3.2. Nymphal mortality Treatment, time, and the interaction of time x treatment, time x exposure, and treatment x exposure had significant effects on nymphal

Fig. 2. Mean (�SEM) fecundity of twospotted spider mite adults surviving exposure to different biopesticide residues. Bars with different letters indicate significantly different means by Tukey’s HSD (P ¼ 0.05). For all treatments, n ¼ 300. 4

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Crop Protection 128 (2020) 105015

Fig. 3. Mean (�SEM) number of eggs per cohort of twospotted spider mite adults surviving contact and residual exposure to biopesticides. Bars with different lowerand uppercase letters indicate significantly different means via Tukey’s HSD (P ¼ 0.05) within contact and residual exposure methods, respectively. * indicates significant difference between exposure methods via t-test (P ¼ 0.05). For all treatments, n ¼ 150 adults per replicate (50 per replicate). Table 3 Category, treatment, factor, F-values (F), and P-values (P) from repeated mea­ sures Analysis of Variance results of experiments comparing twospotted spider mite over time within each biopesticide treatment.

Table 4 Mean (�SEM) percentage mortality of twospotted spider mite proto- and deu­ tonymphs (combined) at 24, 48, and 72-hrs post biopesticide exposure (contact and residual combined) resulting from various biopesticides. Timea 24 h

48 h

72 h

2.4 g

8.3 � 2.0 Ab

4.8 g

10.7 � 1.2 Ab 15.3 � 2.0 Ab 7.3 � 1.2 Ab

23.0 � 6.1 Bbc 26.7 � 3.8 Bb

38.0 � 5.4 Cbcd 41.3 � 4.2 Cbc

30.3 � 3.5 Bb

43.7 � 4.4 Cb

11.3 � 1.8 ABbc 14.3 � 1.9 Abc 18.0 � 5.2 Bbc 12.7 � 2.7 ABbc 66.3 � 8.5 Ba

17.0 � 1.7 Bef

7.3 � 1.3 Bc

10.7 � 1.8 Bf

Treatment

Factor

F

P

Larval mortality

Grandevo (2.4 g/L) Grandevo (4.8 g/L) Grandevo (7.2 mL/ L) Venerate (20 mL/ L) Venerate (40 mL/ L) GOS Neem 7-way TriTek (1%) Acramite WP (0.2 g/L) Control Grandevo (2.4 g/L)

Time

53.98 44.76 12.72

<0.0001 0.0002 0.005

7.483

0.019

25.79

0.0008

10.64 12.15 3.748

0.008 0.006 0.079

20.30 117.7

0.002 0.0003

GOS Neem 7way TriTek

1%

59.04 42.78

0.0011 0.002

Acramite WP

0.2 g

10.27

0.027

Control

-

4.520

0.0941

112.7 19.75 25.2

0.0003 0.009 0.0054

23.53

0.0061

Deuto- and protonymph mortality

Grandevo (4.8 g/L) Grandevo (7.2 mL/ L) Venerate (20 mL/ L) Venerate (40 mL/ L) GOS Neem 7-way TriTek (1%) Acramite WP (0.2 g/L) Control

Time

Treatment

Amount/ Liter

Category

Grandevo

7.2 g Venerate

20 mL 40 mL

1%

11.0 � 4.4 Ab 12.7 � 4.5 Ab 7.0 � 2.3 Ab 57.7 � 9.3 Aa 4.3 � 0.9 Ab

19.3 � 2.3 Adef 29.0 � 3.9 Cbcde 20.7 � 2.8 Bcdef 74.7 � 8.3 Ba

Means within rows followed by the same capital letter and means within col­ umns followed by the same lowercase letter are not significantly different via Tukey’s HSD (P ¼ 0.05). a For all treatments n ¼ 300.

3.4. Egg mortality Treatment, exposure method, and their interaction significantly affected egg hatch at 7 d post exposure (Table 2). Overall, percentage egg hatch was significantly (t ¼ 4.97, df ¼ 52, P < 0.001) higher via re­ sidual (mean � sem ¼ 93.0 � 1.4%) compared to contact exposure (68.0 � 5.6%). In fact, under residual contact activity, none of the treatments differed significantly from the control (Fig. 4). Under contact activity, only 17.2 � 5.5% of Acramite-treated eggs hatched, which was significantly lower than the control (70.1 � 5.5%). While only about 44% of eggs hatched when exposed via contact activity to GOS Neem and TriTek, they did not differ significantly from the control. The sig­ nificant treatment x exposure interaction was the result of significantly lower egg hatch via contact versus residual exposure for Acramite (t ¼ 5.56; df ¼ 4; P ¼ 0.012) and TriTek (t ¼ 2.92; df ¼ 4; P ¼ 0.043), while there was no difference between contact and residual exposure for

respectively. With the exception of Acramite, which resulted in high mortality after 24 h, mortality significantly increased over the 96 h period for all treatments (Table 3), and at 96 h all treatments except the low rate of Venerate was significantly higher than the control. The significant treatment x exposure interaction was the result of significantly higher larval mortality following contact versus residual exposure for the highest and lowest rates of Grandevo (mean � sem for contact ¼ 43.3 � 3.2% vs. residual ¼ 33.5 � 2.0%; t ¼ 2.56; df ¼ 22; P ¼ 0.018) and Venerate (contact ¼ 26.1 � 5.1% vs. resid­ ual ¼ 10.1 � 2.3%; t ¼ 3.00; df ¼ 22; P ¼ 0.007), respectively. Conversely, significantly higher larval mortality resulted from residual (60.3 � 5.1%) versus contact exposure (39.5 � 5.1%) to GOS Neem 7way (t ¼ 2.86; df ¼ 22; P ¼ 0.009). 5

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Table 5 Mean (�SEM) percentage mortality of twospotted spider mite larvae at 24, 48, 72, and 96-hrs post biopesticide exposure (contact and residual combined) resulting from various biopesticides. Treatment

Amount/Liter

n

Grandevo

2.4 g 4.8 g 7.2 g 20 mL 40 mL 1% 1% 0.2 g -

198 203 18 218 256 111 148 159 236

Venerate GOS Neem 7-way TriTek Acramite WP Control

Time 24 h

48 h

72 h

96 h

10.0 � 1.5 Ade 17.6 � 3.3 Acde 32.8 � 3.4 Abc 9.3 � 2.2 Ade 8.2 � 1.6 Ade 43.5 � 10.6 Ab 30.0 � 9.1 Abcd 95.5 � 1.5 Aa 4.2 � 1.3 Ae

22.0 � 4.3 Bbc 29.1 � 4.9 Bbc 33.1 � 3.5 Ab 11.2 � 3.1 Acd 18.7 � 5.0 ABbcd 37.6 � 8.0 Ab 33.6 � 8.0 Ab 99.3 � 0.4 Aa 4.1 � 1.3 Ad

30.8 � 4.5 Cbc 41.0 � 4.8 Cbc 38.6 � 2.5 ABbc 21.4 � 7.0 ABcd 37.0 � 8.5 BCbc 51.4 � 4.6 ABb 42.1 � 5.5 ABbc 99.6 � 0.4 Aa 5.9 � 1.4 Ad

39.7 � 5.8 Ccd 50.6 � 4.6 Cbcd 49.1 � 4.2 Bbcd 30.6 � 8.2 Bde 50.6 � 9.0 Cbcd 67.2 � 4.4 Bb 59.6 � 3.9 Bbc 100 � 0.0 Aa 9.9 � 1.7 Be

Means within rows followed by the same capital letter and means within columns followed by the same lowercase letter are not significantly different via Tukey’s HSD (P ¼ 0.05). Fig. 4. Mean (�SEM) percentage of twospotted spi­ der mite eggs hatching 7 days after contact and re­ sidual exposure to biopesticide residues. Bars with different lower- and uppercase letters indicate significantly different means via Tukey’s HSD (P ¼ 0.05) within contact and residual exposure methods, respectively. * indicates significant difference be­ tween exposure methods via t-test (P ¼ 0.05). For contact exposure n ¼ 139, 146, 143, 157, 141, 144, 133, 143, 140 for Grandevo 2.4 g, 4.8 g, 7.2 g, Venerate 20 ml, 40 ml, GOS Neem, TriTek, Acramite and the control, respectively. For residual exposure n ¼ 73, 81, 77, 111, 136, 63, 71, 59, 117 for Grand­ evo 2.4 g, 4.8 g, 7.2 g, Venerate 20 ml, 40 ml, GOS Neem, TriTek, Acramite and the control, respectively.

the other treatments.

Acramite, biopesticides generally exhibited lower levels of toxicity, with Venerate exhibiting the weakest overall response. Grandevo was moderately toxic to all life stages except eggs, with mortality rates to adults, larvae and nymphs not differing significantly among rates. One of the strongest effects of Grandevo was reduced fecundity of females at all rates. Neem and TriTek were more active against larvae than nymphs or adults, as this was the only life stage in which mortality significantly differed from the control. In addition, GOS Neem, TriTek, and Acramite all exhibited ovicidal activity when eggs were exposed to contact rather than dried residuals. To our knowledge, there are few studies on the effect of the bacterialderived Grandevo DF2 and Venerate EP products to various life stages of T. urticae. The results of this study suggest that reduced fecundity of mites, regardless of exposure route, was an important mechanism of activity for Grandevo. Neither product exhibited particularly high levels of toxicity to nymphs or adults, and only about 50% mortality of larvae occurred 4 d after exposure. Ray and Hoy (2014) similarly noted that Grandevo caused comparatively low adult T. urticae mortality rates 3 d after exposure. Notably, Grandevo effectively reduced adult fecundity and total eggs per cohort by 72.5% compared to the control, which was similar to decreases in these two traits resulting from exposure to Acramite (86.9%). The importance of this fecundity reduction of Grandevo in limiting population growth of T. urticae is well illustrated in Fig. 5A. In contrast, Venerate, which consists of heat-killed Burkholderia spp., exhibited no significant effect on reproductive traits. It should be noted, however, that both Grandevo and Venerate are considered slow acting, potentially taking up to 7 d to show observable effects (Marrone

3.5. Tetranychus urticae population growth The estimated cumulative effect of biopesticides on a generation of T. urticae following exposure of a cohort of 100 females is shown in Fig. 5A. Acramite was the only treatment causing population extinction, which resulted from 100% mortality during the larval stage. None of the biopesticides led to population decline, although the high rate of Grandevo limited population growth to 1.4X the initial cohort of 100 adults generated 137 f1 adults. The generational population increase in the control was 31.5X, compared to 17, 10.8, 3.3, 2.5, 3.5 and 5.8X for Venerate at 20 and 40 ml, Grandevo at 2.4 and 4.8 gm, GOS Neem 7Way, and TriTek, respectively. The estimated impact of exposing five successive generations of T. urticae to biopesticides is shown in Fig. 5B. Under this scenario, population size of Grandevo treated adults increased by a factor of 4.9 over 5 generations, while all remaining treatments exhibited exponential growth over five generations, increasing by factors ranging from a low of 90 in the 4.2 g/liter rate of Grandevo to 3.1 � 107 in the control. 4. Discussion All tested biopesticides exhibited activity against one or more life stages of T. urticae, yet exposure to Acramite, the only non-biopesticide included, consistently resulted in the highest mortality, despite the low rate tested. In contrast to the rather acute response of T. urticae to 6

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cumulative impact of stage-specific effects on population growth (For­ bes and Calow, 1999; Stark and Banks, 2003). When T. urticae popula­ tion growth was simulated over five generations, with each generation exposed to single applications of respective biopesticides, population growth was positive for all biopesticide treatments. With the exception of the high rate of Grandevo, population growth was exponential for all other biopesticide treatments, with the 4.8 g/liter rate of Grandevo limiting growth the least. While biotic and abiotic factors will further suppress population growth beyond that predicted in our simplified model, the level of suppression provided by most of the biopesticides evaluated in this study are unlikely to keep T. urticae below damaging levels in high value specialty crops with low tolerances for damage (Park and Lee, 2005; Meck et al., 2013; Pascual-Ruiz et al., 2014). However, the level of suppression may be sufficient to reduce the frequency of chemical acaricide use, or these biopesticides may be useful tools for improving the predatory-prey ratios of either naturally occurring or augmentative releases of predatory mites. For instance, Mar�ci�c et al. (2012) compared the efficacy of biopesticides and conventional syn­ thetic acaricides on two mite species, T. urticae and Panonychus ulmi Koch under greenhouse and field conditions. The authors found that the biopesticides Naturalis (Beauveria Bassiana strain ATCC 74040) and Kingbo (an oxymatrine-based acaricide derived from Sophora flavescens) were nearly equally as effective in reducing population density of T. urticae on greenhouse grown tomatoes and cucumber, and of P. umli density on field grown apples as the conventional synthetic acaricides acrinathrin and spirodiclofen. Also, in whole plant experiments on greenhouse tomatoes, Pavela (2009) reported 100% mortality of a cohort of T. urticae 12-days after spraying a 1% solution of neem. While results are promising, the effect of these biopesticides on key natural enemies should be assessed to determine their compatibility for bio­ logical should in different cropping systems (Cote et al., 2002; Cloyd et al., 2006; Liburd et al., 2007; Ray and Hoy, 2014; Ditillo et al., 2016; Schmidt-Jeffris and Beers, 2017). Both neem (Spollen and Isman, 1996) and mineral oil (Xue et al., 2009) have been shown to be compatible with biological control of T. urticae with the predatory mite Phytoseiulus persimilis Athias-Henriot, but little is known of the effects of Grandevo on predatory mites. Synthetic acaricides will continue to play an important role in managing T. urticae in high value specialty crops. Yet, control failures to numerous active ingredients (Whalon et al., 2017) including newer chemistries such as spirodiclofen, pyridaben, cyenopyrafen, and chlor­ fenapyr (Xu et al., 2018; Wang et al., 2016; Gong et al., 2014; Leeuwen et al., 2010), substantiate the need for alternative approaches that minimize synthetic acaricide applications and thereby delay the onset of resistance in T. urticae populations. Several of the biopesticides evalu­ ated in these studies exhibited significant mortality on various life stages of T. urticae, and if incorporated into management programs may be integral in acaricide resistant management programs against T. urticae. Future research should investigate the use of these biopesticides as rotational products or even as alternatives to conventional synthetic acaricides, as well as their compatibility with key biological control agents, to assess their impact on T. urticae at the population level in protected and field-grown crops.

Fig. 5. Effect of biopesticide exposure to an initial cohort of 100 female two­ spotted spider mites based on life stage survivorship (A), and population growth over five generations with each generation exposed to a single application of respective biopesticides (B). Model assumes contact residue exposure to adults (affecting adult mortality and fecundity rates), and residual exposure to all other life stages.

Bio Innovations, 2018a, b). Hence, our results may have underestimated effects of these products. Several studies have investigated T. urticae life stage mortality resulting from treatment with mineral oil-based products, naturally derived neem oil, and Acramite against T. urticae. Chiasson et al. (2004) reported that direct application of a 0.7% solution of neem oil (Neem Rose Defense EC 50%; San Antonio TX) caused about 22% mortality to adult T. urticae 48 h after application, while egg hatch was reduced by >98% 5 d after application compared to the control (water-sprayed). While adult mortality levels were similar to our results, we observed egg hatch reductions of only 56.3% with a 1% formulation of GOS Neem 7-way when directly applied to eggs. In addition, direct exposure to various mineral oil derived products (i.e., Sunspray Ultrafine, Agro­ aceite, Volck Miscible, and Taxaco D-C-Tron Plus) tested at concentra­ tions ranging from 0.5 to 3.0% resulted in nearly 100% mortality of eggs, nymphs and adults 7 d after application (Chueca et al., 2010). These mortality rates were much higher than what we observed with TriTek, an emulsified formulation of mineral oil. These differences may be associated with volume of water and/or application method used to expose eggs to residues. However, the high toxicity of Acramite that we observed to all life stages is well documented (Kim and Seo, 2001; Kim and Yoo, 2002). While all of the biopesticides evaluated in this study adversely affected one or more life stages of T. urticae, effects were not nearly as acute as those of the synthetic acaricide Acramite. However, assessing results at the population level can aid in better understanding the

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research was supported, in part, by Marrone Bio Innovations and the North Carolina Agricultural Research Service.

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