sustainability

Article “Predator-In-First”: A Preemptive Biological Control Strategy for Sustainable Management of Pepper Pests in Florida

Vivek Kumar 1,* , Lucky Mehra 2, Cindy L. McKenzie 3 and Lance S. Osborne 1 1 Mid-Florida Research and Education Center, IFAS, University of Florida, 2725 S. Binion Road, Apopka, FL 32703, USA; lsosborn@ufl.edu 2 Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA; [email protected] 3 Horticultural Research Laboratory, Subtropical Research Unit, USDA-ARS, U.S., 2001 South Rock Road, Fort Pierce, FL 34945, USA; [email protected] * Correspondence: [email protected]

 Received: 30 June 2020; Accepted: 18 September 2020; Published: 22 September 2020 

Abstract: The early establishment of a biocontrol agent in the production system, whether in the greenhouse, nursery, or field, is essential for the success of the biological control program, ensuring growers’ profitability. In an effort to develop a sustainable pest management solution for vegetable growers in Florida, we explored the application of a preemptive biological control strategy, “Predator-In-First” (PIF), in regulating multiple pepper pests, Bemisia tabaci Gennadius, occidentalis Pergande, and Polyphagotarsonemus latus Banks under greenhouse and field conditions during different growing seasons. In these studies, two bell pepper cultivars (7039 and 7141) and the phytoseiid mite Amblyseius swirskii Athias–Henriot were used as a model system. Pepper seedlings (~8 week) of each cultivar were infested with varying rates of A. swirskii (20 or 40 mites/plant or one sachet/10 plant) and allowed to settle on plant hosts for a week before planting in pots or field beds. Results showed a comparative consistent performance of the treatment with the high rate of phytoseiids (40 mites/plant) in regulating B. tabaci and F. occidentalis populations in greenhouse studies, and B. tabaci and P.latus pests under field conditions. During two fall field seasons, higher marketable yields of 12.8% and 20.1% in cultivar 7039, and 24.3% and 39.5% in cultivar 7141 were observed in the treatment with the high rate of phytoseiids compared to the untreated control, indicating yield benefits of the approach. The outcome of the study is encouraging and demonstrates that PIF can be an important tool for organic vegetable growers and a potential alternative to chemical-based conventional pest management strategies. The advantages and limitations of the PIF approach in Florida pepper production are discussed.

Keywords: Amblyseius swirskii; Bemisia tabaci; Frankliniella occidentalis; IPM; horticulture; phytoseiid

1. Introduction Within the United States, the southeastern region has certain climatic advantages such as an extended winter photoperiod, mild temperature, and high light intensities conducive for optimal growth of a wide range of crops throughout the year [1]. In Florida, these parameters offer growers a strong foundation for the development of a multi-billion-dollar horticultural industry, ranked second in the country after California [2]. However, favorable climatic conditions, along with diverse flora present in this coastal state with 2170 km of shoreline and over 20 port-of-entries for plant products, also favor the introduction and establishment of a long list of invasive pests. Based on the Florida Department of Agriculture and Consumer Services (FDACS) records, between 1986 and 2000, >100 species of invasive established in Florida at the rate of ~1 species/month [3]. The continuous influx of invasive

Sustainability 2020, 12, 7816; doi:10.3390/su12187816 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 7816 2 of 20 pests in the state, with growing concerns of using certain chemical classes, is an important driver pushing the need for alternate control strategies for both conventional and organic growers. For the success of a sustainable biological control program, the establishment of the selected agents in the production system (greenhouse, nursery, field) is of paramount importance. In the past, there have been multiple strategies proposed for the long-term establishment of beneficials in the production system. Some of these include “Open Rearing” (also known as Banker Plants) systems [4–6], “Pest-In-First” strategies [7–9], “Slow Release” systems [10], and the use of artificial diets attracting natural enemies [11]. Although these strategies are known for their role in the conservation of beneficial agents, in turn, suppressing target pests, each of these has certain limitations impacting their broader acceptability. These may include the screening of non-crop (non-host pests) banker plant varieties; the release of non-pest species in the production system; dispersal of biological agents from sachets; and finding the chemicals selectively supporting the beneficial populations [12]. Considering the limitations of the above biological control strategies and needs of the Florida vegetable industry, we explored the utility of a relatively less known biological control concept, “Predator-In-First” (PIF), proposed by Ramakers [13]. The PIF approach combines aspects of inundative and conservational control strategies and aims to establish biological agents on the hosts during the seedling stage or early post-transplanting period before the arrival of any pests. The underlying theory behind the PIF approach is based on the characteristics of generalist predators’ ability to survive on plant provisioned food (pollen, nectar) and morphological host plant characteristics, such as domatia that provide refugia for breeding and development, that allow establishment in the agroecosystem before the initial infestation of prey/pest populations. This is significant because natural enemies generally do not establish until later in the season when or other pest numbers have built up [14–23]. In our studies, pepper plants were used as a model crop system, and the key component of this method involved the release of specific predatory mites Amblyseius swirskii Athias–Henriot on un-infested seedlings before transplanting for commercial production in the greenhouse or the field. The selection of the plant host and the predator species was influenced by our years of experience in assessing multi-trophic interactions with the ornamental banker plant system and the pest spectrum associated with commercial pepper production in the region, including Bemisia tabaci Gennadius, Frankliniella occidentalis Pergande, and Polyphagotarsonemus latus Banks [12,19,24–26]. In addition to causing direct damage to its hosts, these particular whitefly and thrips species hold the potential to vector plant damaging viruses and propensity to develop resistance against chemical insecticides. Thus, in order to meet the demand for vegetable production and related jobs, it is essential to simplify growers’ concern by delivering new management strategies. In the past, Kutuk and Yigit [27] showed the importance of pre-establishment of A. swirskii on pepper plants using Pinus brutia (Ten.) pollen in reducing F. occidentalis populations under greenhouse conditions. However, apart from a few greenhouse studies [13,27], the PIF approach has never been tested extensively under field conditions with multi-pest infestations. In the first part of the project [26], we focused on screening commercially grown pepper cultivars (hot and sweet) for their ability to sustain A. swirskii populations (with or without pollen), and assessed methods for mass inoculating seedlings with A. swirskii before planting in the greenhouse. Based on these studies, two out of 29 pepper cultivars were selected for further testing. Thus, in the current study, we evaluated the application of the PIF approach in managing multiple pests in bell peppers under commercial grower production standards. The specific objectives of this research were to assess the optimal rate (0, 20, 40 mites per plant) and overall performance of phytoseiid mites against multiple pests using the PIF approach with the top two pepper cultivars under two different environments (greenhouse and field) and growing seasons (fall and spring) in Florida.

2. Materials and Methods A series of research studies were conducted in a protected greenhouse and outdoor field conditions at the United States Horticulture Research Laboratory (USHRL), USDA-ARS, Fort Pierce, FL (27.43 N, Sustainability 2020, 12, 7816 3 of 20

80.40 W). The climatic conditions under the greenhouse were affected by outdoor temperature and relative humidity. Amblyseius swirskii populations used in the study were obtained from Koppert Biological Systems, USA (Howell, MI). Upon arrival, mites were stored at the most for 24 h in a growth chamber maintained at 12 2 C, RH 60 5%, and 14:10 h L:D until the day of release. Two commercial ± ◦ ± bell pepper cultivars—7039 (Sakata Seed America Inc., Salinas, CA, USA) and 7141 (Seminis Vegetable Seeds, Inc., St. Louis, MO, USA)—were selected based on the results obtained in previous screening studies [26]. These pepper cultivars showed promise in supporting the survival of phytoseiid mites in the absence of any prey for a considerable amount of time.

2.1. Greenhouse Studies Seeds of two bell pepper cultivars were grown in Premier Pro-Mix General Purpose Growing Medium in three separate transplant trays (SpeedlingTM, Ruskin, FL, USA) (total 6 trays). SpeedlingTM trays were placed inside plexiglass screened cages (61 cm 91 cm 61 cm) for ~8 weeks to prevent insect contamination × × before experimentation. Plants selected for the study were healthy, young, vigorous, and free of arthropod pests. Eight-week-old seedlings of each pepper cultivar (7039 and 7141) were inoculated with three different rates of A. swirskii: (1) 20 A. swirskii/plant, (2) 40 A. swirskii/plant, and (3) untreated control—without A. swirskii, making a total of six treatments. Amblyseius swirskii counts in bran (carrier material) were standardized and released on pepper seedlings following the protocol of Kakkar et al. [28]. The bran volume was quantified to determine the desired number of predatory mites (20 and 40/plant) by repeated drawings from the bottle in which the product was received from the company. The carrier material ensured initial support to the phytoseiid mites needed to settle on the host. They were allowed to establish on pepper seedlings for seven days before transplanting into 3.78 L plastic pots. The experiment was conducted in a randomized complete block design with six replicates, and each plot consisted of three plants (18 plants per treatment). For each treatment, potted seedlings were placed in a large saucer filled with water, and the plants did not touch each other. Plants were irrigated as needed (~3 times a week) and fertilized with 800 mL/pot of Peters Professional® 20-10-20 (325 ppm) (Scotts Co., Marysville, OH, USA) once a week. For the efficacy evaluation of the PIF approach, pepper plants were moved to a greenhouse harboring natural populations of B. tabaci (Middle Eastern Asia Minor 1, MEAM1) and F. occidentalis. Developmental stages of arthropods including B. tabaci (eggs and nymphs), F. occidentalis (larvae), and A. swirskii (eggs and motile) were recorded weekly on five randomly selected leaves from the top of each pepper plant by direct count sampling method using a 30 handheld lens for a period of × eight week. Once plants reached flowering stage, three flowers/plant were collected in 50 mL flat top Falcon tubes (USA Scientific, Ocala, FL, USA) and brought to the USHRL laboratory. Foliage samples were rinsed in 20 mL of 70% ethanol for 30 min to dislodge F. occidentalis larvae and A. swirskii motile. The contents in the tube were separated using a 25-µm grading sieve (USA Standard Testing Sieve, W. S. Tyler, Inc., Mentor, OH, USA). Mites trapped inside the sieve were flushed with 75% ethanol into 6 cm Petri dishes (USA Scientific) with artificial grids, and the number of various life stages was counted under a stereomicroscope at 12.5 (Olympus SZX12). × 2.2. Field Studies Field evaluations of the PIF approach were conducted over a two-year period during three consecutive pepper growing seasons (fall, spring) in Florida. The two main objectives of the field trials were to optimize the rate of phytoseiid mites released per plant and determine the best mode of mite application: (1) direct application—mites released directly on the seedlings as in the greenhouse assay or (2) indirect application—mites released using commercial slow-release sachet (Swirski-Mite LD, Koppert Biological Systems). The slow-release sachets can provide protection and food to predators and improve their survival in unfavorable environmental conditions. Seeds of individual bell pepper cultivars were sown in 128 cell SpeedlingTM transplant trays following a similar protocol mentioned above. The method of plant propagation, maintenance, and mite treatment was similar to the Sustainability 2020, 12, 7816 4 of 20 greenhouse trial except that four different rates of mites were used and two methods of application making a total of eight treatments. Rates were (1) 20 A. swirskii/plant, (2) 40 A. swirskii/plant, (3) 1 mite sachet/10 plants, and (4) untreated control—without A. swirskii.

Field Preparation and Treatment Evaluations Studies were conducted at the USHRL experimental farm site where six raised beds (150 m long, 0.91 m wide, 0.15 m high, and 1.5 m spacing between centers) of Oldsmar sand (sandy, siliceous, hyperthermic Alfic Arenic Alaquod) were prepared. An alternative method to methyl bromide fumigation–anaerobic soil disinfestation combined with soil solarization was adopted to control weeds and soil-borne pathogens, as well as plant-parasitic nematodes following the protocol of Butler et al. [29]. Each of the six beds was divided into eight 12 m long treatment plots with double rows of pepper planting. A 6.09 m long buffer zone was maintained between two adjacent treatment plots. All eight treatments (two cultivars and four mite rates) were arranged in a randomized complete block design with six replications. Irrigation, fertilization, weed, and disease management were achieved following standard cultural practices [30]. In order to control caterpillar damage (Spodoptera spp., Helicoverpa spp.), Bacillus thuringiensis-based insecticides Xentari DF® (var. kustaki) at 1.2 L/ha (Valent Biosciences Corporation, Libertyville, IL, USA) and DiPel DF® (var. kurstaki) at 1.1 kg/ha [31] were used. No other chemical insecticides were used in these studies. Sampling was conducted weekly from each plot for a period of eight weeks during two fall studies and 10 weeks in the spring study. The sampling unit consisted of randomly selecting 10 top leaves per plot, collected in labeled plastic Ziploc® bags (17 22 cm). Once flowering was initiated, 10 flowers/plot × were collected every week in 50 mL Falcon conical tubes. Foliage samples were brought to the USHRL laboratory and processed following the protocol mentioned above. Leaf and processed flower samples were observed for various life stages of B. tabaci (eggs and nymphs), F. occidentalis (larvae), P. latus (eggs and motile), and A. swirskii (eggs and motile) using a stereomicroscope at 12.5 (Olympus SZX12). × During the first fall season of field trials, we received >30 cm of rain, beginning just days after transplanting. Upon completion of the first three samplings, an augmentative release of mites was done based on the low retrieval of A. swirskii in the treatment plots due to very heavy storms coupled with strong winds for prolonged periods negatively impacting mites and especially sachets. Normal seasonal rain was experienced in the second and third trials, but the second augmentation was repeated for trial comparisons. In the plots receiving A. swirskii through the direct release method, the same number of A. swirskii were released on the plants at the beginning of the study. Two treatments with an indirect release through sachets were supplemented by 1 banker plant/10 field plants. Banker plants were made by using the same aged group pepper seedlings (kept in arthropod-free environment) transplanted in 3.78 L plastic pots and inoculated with 100 mites/plant. Mites were allowed to establish on the host plant for seven days before adding in the treatment plots. Treatments with 7039 and 7141 peppers received banker plants from respective pepper varieties. The decision to replace mite sachets with banker plants as an indirect release method was influenced by (1) the observation on the impact of wind and rain on mite sachets and (2) efficiency of pepper banker plants as a reservoir and medium of mites release reported in our associated studies [19,20,25,26]. Challenges from additional arthropod pests were encountered during the three growing seasons of commercial bell pepper field production that we evaluated. Apart from two target pests (B. tabaci and F. occidentalis), there was a heavy infestation of P. latus observed during both fall growing seasons. Since A. swirskii is a generalist predator, the fortuitous incidence of P. latus benefited the scope of the trial by adding another pest to the spectrum when evaluating the utility of the PIF approach. During the spring season, an extremely high pest pressure of pepper weevil, Anthonomus eugenii Cano was observed, which in the absence of appropriate chemical remediation measures severely impacted pepper yield (Supplementary Figure S1). Consequently, yield data is presented only for the two fall Sustainability 2020, 12, 7816 5 of 20 pepper growing seasons. Extrapolated yield per treatment (kg/ha) for two fall seasons were calculated by converting yield obtained per treatment by using the formula below:

Extrapolated yield (kg/ha) = [Yield obtained per treatment (kg/acre) 2.4 × (conversion unit for acre to Kg/ha)]/(Total treatment size = ~0.033 acre per treatment)

2.3. Statistical Analysis Data from greenhouse and field efficacy experiments were analyzed independently for each season using a generalized linear mixed model with the SAS® [32] procedure GLIMMIX. In respective experiments, a generalized linear mixed model was fit to the data to determine the effect of treatment, sampling period (time in weeks), and their interaction (treatment * week) on arthropods growth stage counts, separately. The sampling week was considered a repeated measure, and replicate was treated as a random effect in the linear mixed model. The first-order autoregressive correlation structure (type = ar (1)) was applied to account for the correlation in observations, generated by re-sampling the same experiment unit over time. The data were normalized using square root transformation to stabilize heterogenous variance before analysis. When the interaction of treatment and time was found to be significant, mean separations were run for differences in treatments in the same time period. In two out of the three field seasons, the effect of treatments was not observed on the F. occidentalis population recorded in flowers. For comparison, the seasonal mean of F. occidentalis and A. swirskii counts is presented in the supplementary figures. Pepper yield from the two fall field trials was analyzed using a one-way analysis of variance. Differences among treatment means were separated using Fisher’s LSD test (α = 0.05). The data presented are the untransformed means.

3. Results For the sake of simplicity, results presented below include life stages of B. tabaci (eggs + nymphs), and A. swirskii (eggs + motile) observed on the leaves and F. occidentalis (larvae) and A. swirskii (motile) recorded in the flower samples over various sampling weeks.

3.1. Evaluation of the PIF Approach under Greenhouse Conditions

3.1.1. Fall Greenhouse Study During the fall greenhouse study, both the main effects (treatment and time) had a significant effect on B. tabaci and A. swirskii populations recorded on the pepper leaves (Supplementary Table S1). The effect of treatments on the abundance of pests and the predator varied over time, explaining the treatment * week effects. The B. tabaci population was low at the beginning and increased rapidly after weeks 4–5. A significant suppression in the B. tabaci population was observed in all phytoseiid treatments compared to two untreated plots for all sampling dates between weeks 2 and 8. (Figure1a). No significant differences in the activity of two phytoseiid rates (20 vs. 40) were observed except in weeks 5 and 7. The overall B. tabaci suppression (over 8 weeks) in the phytoseiid treatments of two pepper cultivars compared to untreated plots of the respective cultivars were 75.9% and 89.4% in treatments 7039_20 and 7039_40 (compared to 7039_0), and 70.5% and 80.3% in 7141_20, and 7141_40 (compared to 7141_0). The population of A. swirskii followed a similar trend to B. tabaci, and it was low during the initial week and peaked towards weeks 6–7 (Figure1b). A significantly higher number of A. swirskii was reported in all phytoseiid treatments than in the two untreated plots on all the sampling dates between weeks 2 and 8. No significant differences in the abundance of A. swirskii in two phytoseiid treatments (20 vs. 40) were observed except in weeks 5 and 8. Sustainability 2020, 12, x FOR PEER REVIEW 6 of 22 Sustainability 2020, 12, 7816 6 of 20

Figure 1. Mean number ( SE) of (a) Bemisia tabaci and (b) Amblyseius swirskii life stages observed Figure 1. Mean number (±SE)± of (a) Bemisia tabaci and (b) Amblyseius swirskii life stages observed per per plot on pepper leaves in various sampling weeks during fall greenhouse study. The first part of plot on pepper leaves in various sampling weeks during fall greenhouse study. The first part of the the treatment name represents pepper cultivar, and the second part shows the number of A. swirskii treatment name represents pepper cultivar, and the second part shows the number of A. swirskii received per seedling before transplanting in the pots. Treatment bars within a sampling period with received per seedling before transplanting in the pots. Treatment bars within a sampling period with different letters are significantly different (Fisher’s LSD test, p < 0.05). different letters are significantly different (Fisher’s LSD test, p < 0.05). During the fall trial, flower samples were collected from weeks 2–8. There was a significant effect of treatmentDuring the rate fall and trial, time flower on F. samples occidentalis werelarvae collect anded A.from swirskii weeksmotile 2–8. There population was a significant recorded ineffect the flowerof treatment samples, rate whereas and time the on eff F.ect occidentalis of treatment larvae * week and interaction A. swirskiiwas motile observed population only onrecordedF. occidentalis in the (Supplementaryflower samples, whereas Table S1). the Except effect forof treatment 7141_40, none* week of interaction the phytoseiid was observed treatments only showed on F. occidentalis significant suppression(Supplementary in F. occidentalisTable S1). Exceptcounts for compared 7141_40, to none two untreated of the phytoseiid plots on twotreatments sampling showed dates (Figuresignificant2a). However,suppression overall, in F. occidentalis a grower acceptablecounts compared reduction to two rate untreated of >70% (7039_20plots on two= 74.5%, sampling 7039_40 dates= (Figure81.4%, 7141_202a). However,= 87.0, overall, and 7141_40 a grower= 95.7) acceptable in the reductio thrips larvaln rate abundance of >70% (7039_20 was observed = 74.5%, in 7039_40 the phytoseiid = 81.4%, treatments7141_20 = 87.0, compared and 7141_40 to respective = 95.7) untreated in the thrips plots. larval A significantly abundance higher was observed number ofin A.the swirskii phytoseiidwas reportedtreatments in compared the treatment to respective 7039_40 untreate than twod plots. untreated A significantly plots on all higher the sampling number of dates A. swirskii except was on weeksreported 5 andin the 7, treatment whereas in7039_40 7141_40 than such two di untreatedfference was plots observed on all the in sampling weeks 5, dates 6, and except 8 (Figure on weeks2b). No5 and significant 7, whereas diff erencesin 7141_40 in thesuch abundance difference of wasA. swirskiiobservedin twoin weeks phytoseiid 5, 6, and treatments 8 (Figure (20 2b). vs. No 40) weresignificant observed. differences Overall, in significantly the abundance higher of A.A. swirskii swirskii incounts two phytoseiid were observed treatments on treatments (20 vs. 40) 7039_40, were 7141_40,observed. and Overall, 7039_20 significantly than 7141_20 higher (p < 0.05). A. swirskii counts were observed on treatments 7039_40, 7141_40, and 7039_20 than 7141_20 (p < 0.05). SustainabilitySustainability2020 ,202012, 7816, 12, x FOR PEER REVIEW 77 of of 22 20

Figure 2. Mean number ( SE) of (a) Frankliniella occidentalis larvae and (b) Amblyseius swirskii motile Figure 2. Mean number± (±SE) of (a) Frankliniella occidentalis larvae and (b) Amblyseius swirskii motile observed per plot in pepper flowers on various sampling weeks during fall greenhouse study. The first observed per plot in pepper flowers on various sampling weeks during fall greenhouse study. The part of the treatment name represents pepper cultivar, and the second part shows the number of first part of the treatment name represents pepper cultivar, and the second part shows the number of A. swirskii received per seedling before transplanting in the pots. Treatment bars within a sampling A. swirskii received per seedling before transplanting in the pots. Treatment bars within a sampling period with different letters are significantly different (Fisher’s LSD test, p < 0.05). period with different letters are significantly different (Fisher’s LSD test, p < 0.05). 3.1.2. Spring Greenhouse Study 3.1.2. Spring Greenhouse Study In the spring season, similar to the fall trial, there was a significant effect of treatment, time, In the spring season, similar to the fall trial, there was a significant effect of treatment, time, and and their interaction (treatment * time) on B. tabaci and A. swirskii populations (Supplementary Table S1) their interaction (treatment * time) on B. tabaci and A. swirskii populations (Supplementary Table S1) recorded on the host leaves. A significant suppression in the B. tabaci population was observed in all recorded on the host leaves. A significant suppression in the B. tabaci population was observed in all phytoseiid treatments compared to two untreated plots on the majority of sampling dates (weeks 3–8) phytoseiid treatments compared to two untreated plots on the majority of sampling dates (weeks 3– (Figure3a). No significant di fferences in the activity of two phytoseiid rates (20 vs. 40) were observed in any sampling period. The overall B. tabaci suppression (over 8 weeks) in the phytoseiid treatments Sustainability 2020, 12, x FOR PEER REVIEW 8 of 22 Sustainability 2020, 12, 7816 8 of 20 8) (Figure 3a). No significant differences in the activity of two phytoseiid rates (20 vs. 40) were observed in any sampling period. The overall B. tabaci suppression (over 8 weeks) in the phytoseiid of two pepper cultivars compared to untreated plots of the respective cultivars were 79.6% and 76.2% treatments of two pepper cultivars compared to untreated plots of the respective cultivars were 79.6% in treatments 7039_20 and 7039_40, and 70.6% and 73.8% in 7141_20 and 7141_40. During the spring and 76.2% in treatments 7039_20 and 7039_40, and 70.6% and 73.8% in 7141_20 and 7141_40. During A. swirskii season,the spring a higher season, density a higher of density ofwas A. reportedswirskii was on reported host leaves on host compared leaves compared to the fall to greenhouse the fall study.greenhouse The population study. The of A.population swirskii was of A. low swirskii at the was beginning low at the of beginning the study of andthe study increased and increased rapidly and peakedrapidly during and weekspeaked 5–6during (Figure weeks3b). 5–6 A (Figure significantly 3b). A significantly higher number higher of numberA. swirskii of A.was swirskii reported was in all phytoseiidreported in treatments all phytoseiid than treatments two untreated than two plots untreated on all theplots sampling on all the dates sampling between dates weeks between 4 and 8. Aweeks significantly 4 and 8. higherA significantlyA. swirskii higherabundance A. swirskii was abundance recorded was on recorded 7039_40 on than 7039_40 the restthan ofthe the rest treated of plotsthe on treated weeks plots 5 and on 6. weeks No significant 5 and 6. No di significantfferences indifferences the abundance in the abundance of A. swirskii of A.in swirskii two phytoseiid in two treatmentsphytoseiid (20 treatments vs. 40) were (20 observed. vs. 40) were observed.

Figure 3. Mean number ( SE) of (a) Bemisia tabaci and (b) Amblyseius swirskii life stages observed per Figure 3. Mean number± (±SE) of (a) Bemisia tabaci and (b) Amblyseius swirskii life stages observed per plot on pepper leaves in various sampling weeks during spring greenhouse study. The first part of plot on pepper leaves in various sampling weeks during spring greenhouse study. The first part of the treatment name represents pepper cultivar, and the second part shows the number of A. swirskii the treatment name represents pepper cultivar, and the second part shows the number of A. swirskii receivedreceived per per seedling seedling before before transplanting transplanting in in the the pots.pots. Treatment Treatment bars bars within within a sampling a sampling period period with with differentdifferent letters letters are are significantly significantl diy ffdifferenterent (Fisher’s (Fisher’s LSDLSD test,test, p < 0.05).0.05).

During the spring trial, there was a significant effect of treatment rate and time on F. occidentalis larvae and A. swirskii motile population recorded in the flower samples, whereas the effect of treatment * week interaction was not observed (Supplementary Table S1). No significant differences in F. occidentalis counts Sustainability 2020, 12, x FOR PEER REVIEW 9 of 22

During the spring trial, there was a significant effect of treatment rate and time on F. occidentalis Sustainabilitylarvae2020 and, 12 A., 7816 swirskii motile population recorded in the flower samples, whereas the effect of9 of 20 treatment * week interaction was not observed (Supplementary Table S1). No significant differences in F. occidentalis counts were observed in the phytoseiid treatments compared to two untreated plots were observedon any of inthe the sampling phytoseiid dates treatments (Figure 4a). compared However, to overall, two untreated a significant plots suppression on any of the in sampling the thrips dates (Figurelarval4a). population However, overall,was observed a significant in the treatments suppression 7039_40 in the and thrips 7141_40 larval compared population to two was untreated observed in the treatmentsplots. The 7039_40seasonal andthrips 7141_40 suppres comparedsion in the tophytoseiid two untreated treatments plots. of Thetwo pepper seasonal cultivars thrips suppressioncompared in the phytoseiidto untreated treatments plots were of 35.4% two and pepper 65.9% cultivars in treatments compared 7039_20 to and untreated 7039_40, plots and 40.2% were 35.4%and 71.6% and in 65.9% 7141_20 and 7141_40. From the onset of flowering, a low-moderate count of A. swirskii was observed in treatments 7039_20 and 7039_40, and 40.2% and 71.6% in 7141_20 and 7141_40. From the onset of in the flower samples which peaked on week 6 aligning with the thrips population. Significantly flowering,higher a numbers low-moderate of A. swirskii count were of A. reported swirskii inwas the observedtreatments in7039_40 the flower and 7141_40 samples compared which peakedto the on weektwo 6 aligning untreated with plots the on thrips weeks population. 6 and 7 (Figure Significantly 4b). However, higher no numbers significant of differencesA. swirskii werein A. swirskii reported in the treatmentscounts were 7039_40 observed and on 7141_40 phytoseiid compared treatments to thewith two two untreated different rates plots (20 on vs. weeks 40) of 6 application and 7 (Figure (p 4b). However,>0.05). no significant differences in A. swirskii counts were observed on phytoseiid treatments with two different rates (20 vs. 40) of application (p > 0.05).

Sustainability 2020, 12, x FOR PEER REVIEW 10 of 22

Figure 4. Mean number ( SE) of (a) Frankliniella occidentalis larvae and (b) Amblyseius swirskii motile Figure 4. Mean number± (±SE) of (a) Frankliniella occidentalis larvae and (b) Amblyseius swirskii motile observed per plot in pepper flowers on various sampling weeks during spring greenhouse study. observed per plot in pepper flowers on various sampling weeks during spring greenhouse study. The The firstfirst part part ofof the treatment name name represents represents pepper pepper cultivar, cultivar, and andthe second the second part shows part shows the number the number of of A. swirskiiA. swirskiireceived received per per seedling seedling beforebefore transplanting in in the the pots. pots. Treatment Treatment bars bars within within a sampling a sampling periodperiod with with different different letters letters are are significantly significantly di differentfferent(Fisher’s (Fisher’s LSD LSD test, test, p p< <0.05).0.05).

3.2. Evaluation of the PIF Approach Under Field Condition

3.2.1. Fall Field Study 1 In the first fall field season, both the main effects (treatment and time) and their interaction (treatment * time) had a significant effect on the pest B. tabaci, P. latus, and predator A. swirskii populations recorded on the pepper leaves (Supplementary Table S1). Weekly samplings showed overlapping generations of three arthropods on the host plants throughout the study period (Figure 5). The B. tabaci population was low at the beginning of the study and increased rapidly after week 3 and peaked during week 5, gradually decreased at week 7, and then maintained low-moderate levels towards the end. During weekly surveys, significantly lower counts of B. tabaci were observed on treatments 7039_40 and 7141_40 compared to two untreated plots on weeks 6–8 and two other lower phytoseiid direct application treatments (7039_20, and 7141_20) showed impact towards the end of the study on weeks 7–8. Sachet treatments (7039_sachet, 7141_sachet) were efficacious only in week 7 (Figure 5a). The overall B. tabaci suppression (over 8 weeks) in the phytoseiid treatments of two pepper cultivars compared to untreated plots of the respective cultivars were > 55% in the direct application method (7039_20 = 55.3%, 7039_40 = 59.2%, 7141_20 = 60.4%, and 7141_40 = 68.3%), and 42.1% (7039_sachet) and 45.5% (7141_sachet) for the sachet treatments. Sustainability 2020, 12, 7816 10 of 20

3.2. Evaluation of the PIF Approach Under Field Condition

3.2.1. Fall Field Study 1 In the first fall field season, both the main effects (treatment and time) and their interaction (treatment * time) had a significant effect on the pest B. tabaci, P. latus, and predator A. swirskii populations recorded on the pepper leaves (Supplementary Table S1). Weekly samplings showed overlapping generations of three arthropods on the host plants throughout the study period (Figure5). The B. tabaci population was low at the beginning of the study and increased rapidly after week 3 and peaked during week 5, gradually decreased at week 7, and then maintained low-moderate levels towards the end. During weekly surveys, significantly lower counts of B. tabaci were observed on treatments 7039_40 and 7141_40 compared to two untreated plots on weeks 6–8 and two other lower phytoseiid direct application treatments (7039_20, and 7141_20) showed impact towards the end of the study on weeks 7–8. Sachet treatments (7039_sachet, 7141_sachet) were efficacious only in week 7 (Figure5a). The overall B. tabaci suppression (over 8 weeks) in the phytoseiid treatments of two pepper cultivars compared to untreated plots of the respectiveSustainability cultivars 2020, were 12, x FOR> 55% PEER in REVIEW the direct application method (7039_20 = 55.3%, 7039_40 = 59.2%,11 of 7141_20 22 = 60.4%, and 7141_40 = 68.3%), and 42.1% (7039_sachet) and 45.5% (7141_sachet) for the sachet treatments.

Figure 5. Cont. SustainabilitySustainability2020, 202012, 7816, 12, x FOR PEER REVIEW 12 of 2211 of 20

Figure 5. Mean number ( SE) of (a) Bemisia tabaci,(b) Polyphagotarsonemus latus, and (c) Amblyseius Figure 5. Mean number± (±SE) of (a) Bemisia tabaci, (b) Polyphagotarsonemus latus, and (c) Amblyseius swirskiiswirskiilife stages life stages recorded recorded per plotper plot onpepper on pepper leaves leav ines variousin various sampling sampling weeks weeks of of the the first first field field study (fall season).study (fall The season). first part The of first the part treatment of the treatmen name representst name represents pepper cultivar,pepper cultivar, and the and second the second part shows the numberpart shows of A. the swirskii numberreceived of A. swirskii per seedling received beforeper seedling transplanting before transplanting in the field. in An the augmentative field. An releaseaugmentative of A. swirskii releasewas of done A. swirskii after week was done 3. Treatment after week bars 3. Treatment withina bars sampling within a period sampling with period different letterswith are different significantly letters di arefferent signific (Fisher’santly different LSD test, (Fisher’sp < 0.05). LSD test, p < 0.05).

The P.The latus P. latuspopulation population was was not not recorded recorded duringduring initial sampling, sampling, and and its itsoutbreak outbreak occurred occurred duringduring the middlethe middle of theof the study study period period (weeks (weeks 4–5)4–5) (Figure(Figure 55b).b). All All treatments treatments that that received received direct direct phytoseiidphytoseiid application application (7039_20, (7039_20, 7039_40, 7039_40, 7141_20, 7141_20, andand 7141_40) 7141_40) were were efficacious efficacious on the on themajority majority of of sampling dates (weeks 5–8) maintaining a low-moderate pest population throughout the study sampling dates (weeks 5–8) maintaining a low-moderate pest population throughout the study period. period. However, sachet treatments were not consistent in regulating the P. latus population. The However, sachet treatments were not consistent in regulating the P. latus population. The overall overall P. latus suppression (over 8 weeks) in the phytoseiid treatments of two pepper cultivars P. latuscomparedsuppression to untreated (over 8plots weeks) were in> 85% the phytoseiidin the direct application treatments method of two (7039_20 pepper = cultivars 91.6%, 7039_40 compared to untreated= 92.0%, plots7141_20 were = 87.1%,> 85% and in 7141_40 the direct = 94.8%). application In two methodsachet treatments, (7039_20 P.= latus91.6%, suppression 7039_40 =was92.0%, 7141_20comparatively= 87.1%, and low, 7141_40 40.8% =(7039_sachet)94.8%). Intwo and sachet74.8% (7141_sachet). treatments, P.latus suppression was comparatively low, 40.8%During (7039_sachet) initial sampling and 74.8% weeks, (7141_sachet). no A. swirskii life stages were recovered on the host leaves, so an Duringaugmentative initial release sampling of phytoseiid weeks, no wasA. swirskiimade aftelifer week stages 3 were(Figure recovered 5c). Following on the the host population leaves, so an augmentativetrend of P. release latus, significantly of phytoseiid higher was numbers made after of A. week swirskii 3 (Figure were 5reportedc). Following in phytoseiid the population treatments trend of P. latus7039_40, significantly and 7141_40 higher than two numbers untreated of A.plots swirskii betweenwere weeks reported 4 and 8, in whereas phytoseiid it was treatments different from 7039_40 other treated plots on weeks 6–7. Overall, significantly higher A. swirskii counts were observed on and 7141_40 than two untreated plots between weeks 4 and 8, whereas it was different from other treatments 7039_40 and 7141_40 than other phytoseiid treatments (p < 0.05). treated plots on weeks 6–7. Overall, significantly higher A. swirskii counts were observed on treatments During the first fall trial, flower samples were collected from weeks 3–8. There was a significant 7039_40effect and of 7141_40time on the than F. occidentalis other phytoseiid population, treatments whereas, ( pA.< swirskii0.05). abundance was affected with both Duringthe main the effect first and fall their trial, interactio flowern samples (Supplementary were collected Table S1). from No sign weeksificant 3–8. difference There was in the a significantthrips effectpopulation of time on was the recordedF. occidentalis in anypopulation, phytoseiid whereas,treatment A.compared swirskii toabundance the two untreated was affected plots with both the(Supplementary main effect andFigure their S2). interaction A significantly (Supplementary higher number Table of S1).thrips No was significant observed di onfference week in4 the thripscompared population to the was rest recorded of the sampling in any phytoseiid period. The treatment overall thrips compared suppression to the in two the untreated phytoseiid plots (Supplementarytreatments of Figure two pepper S2). A significantlycultivars compared higher to number untreated of thripsplots of was respective observed cultivars on week were 4 compared low: to the17.1%, rest of39.9%, the samplingand 16.2% in period. treatments The 7039_20, overall 7039_40, thrips suppressionand 7039_sachet, in theand phytoseiid40.4%, 54%, and treatments 33.2% of in 7141_20, 7141_40, and 7141_sachet. A significantly higher count of A. swirskii was observed in the two pepper cultivars compared to untreated plots of respective cultivars were low: 17.1%, 39.9%, treatments that received direct application of phytoseiid compared to two untreated plots and 16.2% in treatments 7039_20, 7039_40, and 7039_sachet, and 40.4%, 54%, and 33.2% in 7141_20, 7141_40, and 7141_sachet. A significantly higher count of A. swirskii was observed in the treatments that received direct application of phytoseiid compared to two untreated plots (Supplementary Figure S2). Among phytoseiid treatments, 7141_40 exhibited higher A. swirskii than the two sachet treatments. Sustainability 2020, 12, 7816 12 of 20

3.2.2. Spring Field Study During the spring field season, the arthropod population was comparatively lower on the host leaves than the fall season. There was a significant effect of treatment, sampling period, and their interaction (treatment * week) observed on the B. tabaci and A. swirskii densities (Supplementary Table S1).Sustainability During 2020 weekly, 12, x FOR surveys, PEER REVIEW significantly lower counts of B. tabaci were observed on all treatments13 of 22 (except 7039_20) that received direct phytoseiid application on weeks 6 and 7, whereas sachet treatments (Supplementary Figure S2). Among phytoseiid treatments, 7141_40 exhibited higher A. swirskii than were effective only on week 7 (Figure6a). The overall B. tabaci suppression in the phytoseiid treatments the two sachet treatments. of two pepper cultivars compared to untreated plots were relatively higher (>85%) in the treatments 7039_40 and 7141_40 than rest of the treatments (7039_20 = 75.2%, 7039_40 = 87.6%, 7039_sachet = 79.3%, 3.2.2. Spring Field Study 7141_20 = 39.4%, 7141_40 = 85.9%, and 7141_sachet = 69.1%). UnlikeDuring in the the spring first field field assay, season, the theA. swirskiiarthropodpopulation population was was recovered comparatively during the lower initial on sampling the host weekleaves which than the was fall low season. at the beginningThere was and a significan peaked duringt effect week of treatment, 6, then maintained sampling aperiod, low population and their towardsinteraction the (treatment end of the * study week) (Figure observed6b). on Treatment the B. tabaci 7039_40 and A. exhibited swirskii adensities significantly (Supplementary higher A. swirskii Table populationS1). During comparedweekly surveys, to the significantly two untreated lower plots counts in weeks of B. 4-7,tabaci whereas were observed 7141_20 on and all 7141_sachet treatments showed(except promise7039_20) on that weeks received 4–6 and direct 4, 6, andphytoseiid 7, respectively. application Among on treatedweeks 6 plots, and significantly7, whereas highersachet numberstreatments of wereA. swirskii effectivewere only recorded on week (over 7 8(Figure week period)6a). The on overall 7039_40 B. thantabaci 7039-20, suppression 7039_sachet, in the andphytoseiid 7141_40 treatments treatments. of two pepper cultivars compared to untreated plots were relatively higher (>85%)The in spring the treatments field planting 7039_40 season and 7141_40 was long than and re flowerst of the samples treatments were (7039_20 collected = from 75.2%, weeks 7039_40 4–10. = There87.6%, was7039_sachet a significant = 79.3%, effect 7141_20 of treatment, = 39.4%, time,7141_40 and = their85.9%, interaction and 7141_sachet on F. occidentalis = 69.1%). incidence, whereasUnlike the A.in swirskiithe firstpopulation field assay, was the aff ectedA. swirskii by sampling population time (Supplementarywas recovered during Table S1). the Exceptinitial forsampling treatment week 7039_40, which was none low of theat the phytoseiid beginning treatments and peaked were during efficacious week 6, in then regulating maintained the thripsa low populationpopulation intowards the flowers the end (Supplementary of the study Figure (Figure S3). 6b). The Treatment overall thrips 7039_40 suppression exhibited in a the significantly phytoseiid treatmentshigher A. swirskii of two population pepper cultivars compared compared to the to two the untreated untreated plots plots in of weeks respective 4-7, whereas cultivars 7141_20 were lower and than7141_sachet the previous showed season: promise 16%, 37%,on weeks and 0% 4–6 in treatmentsand 4, 6, and 7039_20, 7, respectively. 7039_40, and Among 7039_sachet, treated and plots, 2%, 7%,significantly and 0% inhigher 7141_20, numbers 7141_40, of A. andswirskii 7141_sachet. were recorded In the (over treated 8 week plots, period)A. swirskii on 7039_40numbers than 7039- were low20, 7039_sachet, throughout theand study 7141_40 period, treatments. and no significant differences in A. swirskii numbers in phytoseiid treatments were observed compared to untreated plots.

Figure 6. Cont. Sustainability 2020, 12, x FOR PEER REVIEW 14 of 22 Sustainability 2020, 12, 7816 13 of 20

Figure 6. Mean number ( SE) of (a) Bemisia tabaci and (b) Amblyseius swirskii life stages recorded per Figure 6. Mean number (±SE)± of (a) Bemisia tabaci and (b) Amblyseius swirskii life stages recorded per plot on pepper leaves in various sampling weeks of the second field study (spring season). The first part plot on pepper leaves in various sampling weeks of the second field study (spring season). The first of the treatment name represents pepper cultivar, and the second part shows the number of A. swirskii part of the treatment name represents pepper cultivar, and the second part shows the number of A. received per seedling before transplanting in the field. An augmentative release of A. swirskii was done swirskii received per seedling before transplanting in the field. An augmentative release of A. swirskii after week 3. Treatment bars within a sampling period with different letters are significantly different was done after week 3. Treatment bars within a sampling period with different letters are significantly (Fisher’s LSD test, p < 0.05). different (Fisher’s LSD test, p < 0.05). 3.2.3. Fall Field Study 2 The spring field planting season was long and flower samples were collected from weeks 4–10. During the third field season, both the main effects (treatment and time) and their interaction There was a significant effect of treatment, time, and their interaction on F. occidentalis incidence, (treatment * time) had a significant effect on the three arthropod populations recorded on the host whereas the A. swirskii population was affected by sampling time (Supplementary Table S1). Except leaves (Supplementary Table S1). None of the phytoseiid treatments were consistent in regulating the for treatment 7039_40, none of the phytoseiid treatments were efficacious in regulating the thrips B. tabaci population during various sampling weeks (Figure7a). The overall B. tabaci suppression population in the flowers (Supplementary Figure S3). The overall thrips suppression in the (over 8 weeks) in the phytoseiid treatments were comparatively lower than the spring season (37.8%, phytoseiid treatments of two pepper cultivars compared to the untreated plots of respective cultivars 64.2%, and 66.6% in treatments 7039_20, 7039_40, and 7039_sachet, and 63.2%, 56.8%, and 51.3% in were lower than the previous season: 16%, 37%, and 0% in treatments 7039_20, 7039_40, and 7141_20, 7141_40, and 7141_sachet), but followed the similar trend as in the first fall season in the 7039_sachet, and 2%, 7%, and 0% in 7141_20, 7141_40, and 7141_sachet. In the treated plots, A. swirskii presence of P. latus. numbers were low throughout the study period, and no significant differences in A. swirskii numbers The P. latus population started appearing on week 3 and peaked towards the middle of the study in phytoseiid treatments were observed compared to untreated plots. period (weeks 4–5) (Figure7b). All phytoseiid treatments showed a significant e ffect on the P. latus population3.2.3. Fall Field on weeks Study 5 and2 6, where no differences among treatments with direct phytoseiid applications were observed. The overall P. latus suppression (over 8 weeks) in the phytoseiid treatments compared to untreatedDuring plotsthe third was field>70% season, in the directboth the application main effects method (treatment with maximum and time) reduction and their observed interaction in the(treatment treatment * time) 7039_40 had (7039_20a significant= 73.5%, effect 7039_40 on the three= 85.4%, arthropod 7141_20 populations= 82.4%, and recorded 7141_40 on= the74.7%). host Inleaves two (Supplementary sachet treatments, TableP. latus S1).suppression None of the wasphytos 74.7%eiid (7039_sachet)treatments were and consistent 63.7% (7141_sachet). in regulating the B. tabaciUnlike population the previous during fall season,various thesamplingA. swirskii weekspopulation (Figure was7a). recoveredThe overall during B. tabaci initial suppression sampling weeks(over 8 which weeks) peaked in the phytoseiid on weeks 5–6. treatments Although were all comparatively the phytoseiid lower treatments than the sustained spring low-moderateseason (37.8%, counts64.2%, ofandA. 66.6% swirskii inthroughout treatments the 7039_20, study period,7039_40, significantly and 7039_sachet, higher numbersand 63.2%, of 56.8%,A. swirskii and(than 51.3% two in untreated7141_20, 7141_40, plots) were and reported 7141_sachet), in treatments but followed 7039_40 onthe weeks similar 3–8, trend and as weeks in the 3–7 first in 7141_40 fall season (Figure in 7thec). Overall,presence significantly of P. latus. higher A. swirskii counts were observed on treatments 7039_40 and 7141_40 than otherThe phytoseiid P. latus population treatments (startedp < 0.05). appearing on week 3 and peaked towards the middle of the study periodDuring (weeks the 4–5) second (Figure fall 7b). field All phytoseiid study, flower treatments samples showed were a collectedsignificant from effect weeks on the 3P. tolatus 8, andpopulation comparatively on weeks higher 5 thripsand 6, abundance where no was differen recordedces thanamong the treatments first season. with There direct was a phytoseiid significant eapplicationsffect of treatment were andobserved. time on The the A.overall swirskii P.population, latus suppression whereas (overF. occidentalis 8 weeks)abundance in the phytoseiid was only atreatmentsffected by compared sampling time.to untreated No significant plots was eff >70%ect of in treatment the direct * application week interaction method was with observed maximum on reduction observed in the treatment 7039_40 (7039_20 = 73.5%, 7039_40 = 85.4%, 7141_20 = 82.4%, and Sustainability 2020, 12, 7816 14 of 20

Sustainability 2020, 12, x FOR PEER REVIEW 15 of 22 any of the arthropod recovered in flowers (Supplementary Table S1). No significant difference in the thrips7141_40 population = 74.7%). wasIn two recorded sachet treatments, in any phytoseiid P. latus treatments suppression compared was 74.7% to (7039_sachet) the two untreated and 63.7% plots (Supplementary(7141_sachet). Figure S4). A significantly higher number of thrips was observed on week 3 than rest of theUnlike sampling the previous period. Thefall season, overall thripsthe A. swirskii suppression population in the was phytoseiid recovered treatments during initial of two sampling pepper cultivarsweeks which compared peaked to on untreated weeks 5–6. plots Although of respective all th cultivarse phytoseiid were treatments lower than sustained the first fall low-moderate season: 0%, 27%,counts and of 0%A. swirskii in treatments throughout 7039_20, the 7039_40,study period, and 7039_sachet, significantlyand higher 20%, numbers 36%, and of 6.7%A. swirskii in 7141_20, (than 7141_40,two untreated and 7141_sachet. plots) were Areported significantly in treatments higher count 7039_40 of A. on swirskii weekswas 3–8, observed and weeks in the3–7 treatmentsin 7141_40 7039_40(Figure 7c). and Overall, 7141_40 significantly compared to higher two untreated A. swirskii plots counts (Supplementary were observed Figure on treatments S4). Among 7039_40 phytoseiid and treatments,7141_40 than no other significant phytoseiid diff erencetreatments in A. (p swirskii< 0.05). abundance was observed. Significantly higher numbers of A. swirskii were recorded on weeks 4 and 5 than 6–8.

Figure 7. Cont. Sustainability 2020, 12, x FOR PEER REVIEW 16 of 22 Sustainability 2020, 12, 7816 15 of 20

Figure 7. Mean number ( SE) of (a) Bemisia tabaci,(b) Polyphagotarsonemus latus, and (c) Amblyseius swirskii Figure 7. Mean number± (±SE) of (a) Bemisia tabaci, (b) Polyphagotarsonemus latus, and (c) Amblyseius life stages recorded per plot on pepper leaves in various sampling weeks of the third field study (fall season). swirskii life stages recorded per plot on pepper leaves in various sampling weeks of the third field The first part of the treatment name represents pepper cultivar, and the second part shows the number of study (fall season). The first part of the treatment name represents pepper cultivar, and the second A. swirskii received per seedling before transplanting in the field. An augmentative release of A. swirskii part shows the number of A. swirskii received per seedling before transplanting in the field. An was done after week 3. Treatment bars within a sampling period with different letters are significantly augmentative release of A. swirskii was done after week 3. Treatment bars within a sampling period different (Fisher’s LSD test, p < 0.05). with different letters are significantly different (Fisher’s LSD test, p < 0.05). 3.3. Pepper Yield during Two Fall Seasons During the second fall field study, flower samples were collected from weeks 3 to 8, and During the first fall field season, there was a significant effect of treatment on pepper fruit count comparatively higher thrips abundance was recorded than the first season. There was a significant (F7,35 = 3.73; p = 0.0041), but not on marketable yield (F7,35 = 2.01; p = 0.0816). Significantly lower effect of treatment and time on the A. swirskii population, whereas F. occidentalis abundance was only fruit counts were observed in treatment 7141_sachet and 7141_0 than other treatments (Table1). affected by sampling time. No significant effect of treatment * week interaction was observed on any Although the extrapolated yield difference in the treatments with a high rate of direct phytoseiid of the arthropod recovered in flowers (Supplementary Table S1). No significant difference in the application 7039_40 and 7141_40, compared to untreated control plots 7039_0 and 7141_0 of respective thrips population was recorded in any phytoseiid treatments compared to the two untreated plots cultivars were >7000 and >14,000 kg/ha, a significant yield difference was observed only in cultivar 7141 (Supplementary Figure S4). A significantly higher number of thrips was observed on week 3 than (Table1). The overall yield di fference in the phytoseiid treatments of two pepper cultivars compared rest of the sampling period. The overall thrips suppression in the phytoseiid treatments of two pepper to untreated plots of respective cultivars were 11%, 13%, and 3% in treatments 7039_20, 7039_40, cultivars compared to untreated plots of respective cultivars were lower than the first fall season: 0%, and 7039_sachet, and 16%, 24%, and 15% in 7141_20, 7141_40, and 7141_sachet. 27%, and 0% in treatments 7039_20, 7039_40, and 7039_sachet, and 20%, 36%, and 6.7% in 7141_20, 7141_40,Table and 1. Mean 7141_sachet. ( SE) number A significantly of pepper fruit higher and marketablecount of A. yield swirskii harvested was observed per plot during in the the treatments two ± 7039_40fall fieldand studies.7141_40 compared to two untreated plots (Supplementary Figure S4). Among phytoseiid treatments, no significant difference in A. swirskii abundance was observed. Significantly higher Fall Season 1 Fall Season 2 numbers of A. swirskii were recorded on weeks 4 and 5 than 6–8. Fruit Yield/Plot Extrapolated Fruit Yield/Plot Extrapolated Treatments Count/Plot (kg) Yield (kg/ha) Count/Plot (kg) Yield (kg/ha) 3.3. Pepper Yield during Two Fall Seasons 7039_0 625.3 41.8 ab 125.2 10.3 ab 54,659 514.0 28.4 c 112.0 5.4 c 48,861 ± ± ± ± 7039_20 695.8 35.9 a 140.3 7.7 a 61,190 604.5 23.4 ab 131.0 4.8 b 57,153 During the first± fall field season,± there was a significant effect± of treatment± on pepper fruit count 7039_40 705.5 34.7 a 143.7 6.2 a 62,714 628.2 29.9 ab 140.2 6.4 ab 61,170 ± ± ± ± (F7039_Sachet7,35 = 3.73; p = 623.0 0.0041),32.7 but ab not 129.1 on marketable6.7 ab 56,322yield (F7,35 585.2= 2.01;20.3 p = bc0.0816). 134.1 Significantly6.1 ab lower 58,518 fruit ± ± ± ± counts7141_0 were observed 489.8 51.4 in treatment c 107.9 7141_sachet11.7 b 47,060 and 7141_0 406.2 than other15.9 d treatments 90.0 3.9 (Table d 1). 39,283 Although ± ± ± ± 7141_20 587.2 37.8 b 128.9 9.7 ab 56,242 619.7 23.2 ab 139.3 5.1 ab 60,754 the extrapolated yield± difference in± the treatments with a high± rate of direct± phytoseiid application 7141_40 644.2 16.0 ab 142.6 4.4 a 62,199 666.0 41.7 a 148.9 7.0 a 64,970 ± ± ± ± 7039_407141_Sachet and 7141_40, 574.8 29.3 compared c 126.2 to untreated7.6 ab control 55,075 plots 626.7 7039_018.2 and ab 7141_0 136.2 4.31of respective ab 59,409 cultivars ± ± ± ± were >7000Means and within >14,000 a column kg/ha, followed a significant by different yield letters diff are significantlyerence was di observedfferent (Fisher’s only LSD in test,cultivarp < 0.05). 7141 (Table 1). The overall yield difference in the phytoseiid treatments of two pepper cultivars compared to untreatedDuring plots the secondof respective fall field cultivars season, were a significant 11%, 13%, effect and of 3% treatment in treatments was observed 7039_20, on 7039_40, both pepper and 7039_sachet, and 16%, 24%, and 15% in 7141_20, 7141_40, and 7141_sachet. fruit count (F7,35 = 11.34; p < 0.0001) and marketable yield (F7,35 = 12.11; p < 0.0001). All the phytoseiid Sustainability 2020, 12, 7816 16 of 20 treated plots had a significantly higher fruit count and yield compared to two untreated plots (Table1). The extrapolated yield difference in the treatments 7039_40 and 7141_40, compared to irrespective untreated control plots were higher than the previous season i.e., >12,000 kg/ha and >25,000 kg/ha (Table1). The overall yield di fference in the phytoseiid treatments of two pepper cultivars compared to untreated plots of respective cultivars were: 15%, 20%, and 17% in treatments 7039_20, 7039_40, and 7039_sachet, and 35%, 40%, and 34% in 7141_20, 7141_40, and 7141_sachet.

4. Discussion A PIF strategy can be a sustainable pest management option for conventional and organic vegetable growers, specifically engaged in pepper productions. A single application of A. swirskii directly applied on seedling transplants (pre-planting) of 7039 and 7141 pepper cultivars was sufficient for a significant reduction in the B. tabaci population during fall and spring greenhouse studies compared to the untreated control. Further, a >65% reduction in the F. occidentalis larval population was observed in the treatments with the high rate of A. swirskii mite during both study seasons. With a few exceptions, no significant difference in the performance of two rates of phytoseiid mites was observed indicating an initial release of 20 mites/plant could help reduce pest populations; however, the 40 mites/plant may result in efficient management of multiple pepper pests under controlled conditions. The carrier material provided initial refuge and supplement necessary for the mite survival and establishment on seedlings. In their study on assessing the effect of pollen in the pre-establishment of A. swirskii on pepper plants, Kutuk and Yigit [27] showed significant suppression in the F. occidentalis with >30 A. swirskii released per plant. They suggested the initial release of plant pollen (pre or at the time of predator release) would provide nourishment and improve the establishment of the A. swirskii population in the absence of their prey. This would also prove more cost-effective rather than making multiple applications of the predator during the growing season. Consistent with the current results, Ramakers [13] indicated that the early establishment of phytoseiid mites on pepper seedlings could enhance thrips management under protected cultivation. Other researchers also showed promise in the utility of plant supplement for supporting the phytoseiid mite and/or achieving thrips or whitefly suppression [14,17,26,33,34] on the host, which could prove beneficial in developing a PIF approach targeting pests of different vegetables. Based on these results, PIF has the potential to serve as an essential tool for nursery growers with low input costs for pest management of a single release of the phytoseiid mite. The abundance of A. swirskii in the greenhouse studies was greatly influenced by the availability of food sources. In both seasons, A. swirskii populations were low during trial initiation, but with the increased flowering and pest buildup, predatory mite numbers increased over time, peaking towards the end of the experiments. In their previous study, Kumar et al. [26] reported similar population dynamics of A. swirskii when applied on hot and sweet peppers. A minimal contamination occurred in the fall (0.7% of high rate seasonal mean) and slightly higher in the spring (3–6% of high rate) which had three times the predatory mite populations. Plausible reasons contributing to contamination include (1) handling error in managing growing plants, (2) A. swirskii hitchhiking on adult or people, and (3) dropping of pepper flowers containing motile stages from adjacent plots. Although extreme caution was maintained in minimizing any handling error during the second season, dispersal of A. swirskii in the untreated control plots could not be avoided in the spring greenhouse trial. In the current study, the primary source of inter-plot dispersal of A. swirskii could not be ascertained; however, we assume both direct and/or indirect movement of this tenacious phytoseiid mite would prove a boon for nursery pepper growers. The field application of the PIF approach was assessed over three pepper growing seasons and is one of the few studies where the biological control potential of A. swirskii was evaluated on any vegetable in multi-pest situations in Florida agroecosystems. Results from the field studies showed the impact of planting seasons on the arthropods’ abundance and the performance of the PIF approach. Unlike the greenhouse assays, in the field conditions, two applications of A. swirskii may be necessary (weather permitting) to ensure their establishment and achieve significant suppression in the pest Sustainability 2020, 12, 7816 17 of 20 populations inhabiting different plant parts. As discussed earlier, in the current study, we chose to make an augmentative application of A. swirskii after the first season for the fairness in trial comparisons. However, it would be valuable to know if the single application of phytoseiid mite (40, 60, or 80/plant) could deliver similar results to reduce the associated input costs. Among three phytoseiid mite treatments, plots with the high rate of A. swirskii (40 per plant) were consistent in addressing foliage pests, B. tabaci, and P. latus on two pepper cultivars. However, their efficacy in regulating F. occidentalis larvae inhabiting flowers was questionable. This phenomenon could be explained based on the low abundance of A. swirskii during the spring season (compared to fall seasons) due to relatively colder weather in mid-Florida affecting their population on pepper transplants. Amblyseius swirskii is native to the Mediterranean region and adapted to survive in temperatures between 20 and 32 ◦C[35]. Another possible reason could be their tendency to prey on readily available food. Such differential predation behavior of A. swirskii has been previously reported on field in Florida [28]. In their study, significant suppression in a Thrips palmi Karny population inhabiting cucumber foliage was observed, but A. swirskii failed to regulate Frankliniella schultzei Trybom present in flowers. The low rate of phytoseiid mites and combination of sachet and banker plant treatments did show some promise, but two treatments were not as consistent in regulating pest populations and are not as field compatible as the direct application of the high rate. The results of the two fall seasons indicated that the initial application of A. swirskii using the PIF approach not only delivered significant suppression in the pest population but also translated to an increased pepper yield. During the first fall field season, there was an increase in marketable yield of 12.8% and 24.3% in the treatment with a high rate of A. swirskii for cultivars 7039 and 7141, respectively. In the second season, the yield difference between the two treatments was 20.1% and 39.5%, respectively. Very heavy rains and tropical force winds in the first fall season delayed A. swirskii population increases and pest population levels were lower than the second fall season which may have resulted in the lack of differences seen in pepper yield. The higher yield difference during the second fall season could be due to the high infestation of P. latus (>3 ) and F. occidentalis (>5 ) populations. Although it is × × hard to substantiate with the current data, cultivar 7039 appeared to have a higher intrinsic ability to resist P. latus damage, which resulted in lower yield differences between two of its treatments for both fall seasons compared to 7141. When the extrapolated yield of two cultivars with the high rate of A. swirskii was compared to the average bell pepper yield of Florida in 2018, a >40% yield increase was reported in two pepper cultivars during two fall seasons [36]. Pepper yield (both number and weight) increased in treatments receiving direct application method of A. swirskii (especially plants receiving 40 mites); however, numerical measurements do not accurately reflect the increase in fruit quality. Polyphagotarsonemus latus damage to fruit was extensive in the untreated control plots and, although some fruit were marketable, the quality was poor (Supplementary Figure S5). Although the results clearly demonstrate the yield benefits of using the PIF approach during fall seasons for the Florida pepper growers, further testing of this technique in fields separated by greater geographical distance is needed.

5. Conclusions In the past decades, the Florida vegetable industry has been faced with considerable challenges because of the influx and establishment of new invasive pest species in the region. Some of which, such as F. occidentalis, B. tabaci, F. schultzei, Scirtothrips dorsalis Hood, and T. palmi, pose a continuous risk to vegetable growers [37–43]. Due to their invasiveness, an essential question before pest management professionals is how to manage these pests simultaneously in a sustainable and eco-friendly manner. Thus, to receive broader acceptance of any alternate control strategies, one of the essential requirements is to make it economical and competitive to chemical control strategies. The PIF approach offers one such alternative to conventional and organic vegetable growers of Florida. Results suggested that a single application of A. swirskii at pre-planting can help regulate multiple pests affecting pepper under controlled production. In the field, one application at the pre-planting and another release Sustainability 2020, 12, 7816 18 of 20 during early growth stages to augment the existing population of phytoseiid mite can help reduce pests and improve crop yield. The PIF is a sustainable management strategy for regulating multiple pests (like broad-spectrum insecticides) without any chemicals. We believe it to be an economically viable option for pepper growers as it would reduce the cost of labor and chemical applications. However, in Florida, its utility for the organic pepper growers could be limited to only fall production seasons due to the pepper weevil A. eugenii’s presence in the spring season, and the cooler spring temperatures at transplant are not conducive for A. swirskii sustainability. Future studies should focus on implementing the PIF approach in other horticultural crops and integrating it with conventional chemical-based management programs.

Supplementary Materials: The following are available online at http://www.mdpi.com/2071-1050/12/18/7816/s1, Table S1 Generalized linear mixed model statistics for abundance of arthropods in the greenhouse and field trials; Figure S1: Anthonomus eugenii damage observed in different pepper plots during spring field production season; Figure S2: Mean number ( SE) of Frankliniella occidentalis and Amblyseius swirskii observed per plot in pepper flowers during first fall field± study. Bars with different lower-case letters shows significant difference among treatments for abundance of F. occidentalis, and upper-case letters represents difference between treatments for A. swirskii; Figure S3: Mean number ( SE) of Frankliniella occidentalis and Amblyseius swirskii observed per plot in pepper flowers during spring field study.± Treatment bars with different letters are significantly different; Figure S4: Mean number ( SE) of Frankliniella occidentalis and Amblyseius swirskii observed per plot in pepper flowers during second fall field± study. Bars with different lower-case letters shows significant difference among treatments for abundance of F. occidentalis, and upper-case letters represents difference between treatments for A. swirskii; Figure S5: Pepper fruits from control plot (on top) exhibiting heavy Polyphagotarsonemus latus damage, and the treatment with the high rate of 40 Amblyseius swirskii per pepper plant (on bottom) during second fall harvest. Author Contributions: Conceptualization, L.S.O. and C.L.M.; Methodology, C.L.M., L.S.O., and V.K.; Validation, C.L.M. and V.K.; Formal Analysis, L.M.; Investigation, V.K. and C.L.M.; Resources, C.L.M., and L.S.O.; Writing—Original Draft Preparation, V.K.; Writing—Review & Editing, V.K., L.M., and C.L.M.; Visualization, V.K.; Project Administration, C.L.M. and L.S.O.; Funding Acquisition, C.L.M. and L.S.O. All authors have read and agreed to the published version of the manuscript. Funding: Funding for this study was supported by the Florida Department of Agriculture and Consumer Services—Specialty Crop Block Grant Program (Project # 00098877). Acknowledgments: We would like to thank Aaron Dickey, Steven Arthurs, Yingfang Xiao, Katherine Houben, John Prokop, Florian Grant, and Jennifer Wildonger for their technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

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