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Crop Protection 42 (2012) 281e288

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Crop Protection

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Can spinosad-resistant Frankliniella occidentalis (Pergande) (Thysanoptera:Thripidae) be managed with spinosad and predatory mites (Acari)?

Touhidur Rahman a,b,*, Sonya Broughton b, Helen Spafford a,c

a School of Animal Biology, The University of Western Australia, 35 Stirling HWY, Crawley, WA 6009, AustraliabDepartment of Agriculture and Food Western Australia, 3 Baron-Hay Court, South Perth, WA 6151, AustraliacDepartment of Plant and Environmental Protection Sciences, The University of Hawaii, Manoa, 3050 Maile Way, Gilmore Hall 310, Honolulu, HI 96822, USA

a r t i c l e i n f o

Article history:Received 7 July 2011Received in revised form23 July 2012Accepted 26 July 2012

Keywords:Frankliniella occidentalisPredatory mitesSpinosad resistanceResidual toxicityIPM

* Corresponding author. Department of AgricultureEntomology Branch, 3 Baron-Hay Court, South Perth,(0)8 9368 3232; fax: þ61 (0)8 9368 2958.

E-mail addresses: mdtouhidur.rahman@agric.wa.gmail.com (T. Rahman), sonya.broughton@agric.hspafford@hawaii.edu (H. Spafford).

0261-2194/$ e see front matter Crown Copyright � 2http://dx.doi.org/10.1016/j.cropro.2012.07.020

a b s t r a c t

Frankliniella occidentalis (Pergande) is a serious pest of a wide range of horticultural and ornamentalcrops. Populations resistant to most conventional insecticides, includingespinosad, have been detected.To control spinosad-resistant thrips, growers could use a ‘high-rate’/biological control strategy. Theproposed strategy is based on a single application of spinosad at double the recommended applicationrate followed by releasing predatory mites (Acari), which are used as biological control agents ofF. occidentalis. This study compared two resistance management strategies on a spinosad-resistantF. occidentalis strain: applying spinosad at twice the recommended rate, and spraying at twice the ratethen releasing predatory mites, Typhlodromips montdorensis (Schicha), Neoseiulus cucumeris (Oudemans)and Hypoaspis miles (Berlese). Direct exposure to twice the recommended rate of spinosad killed 100% ofall adults of all species of predatory mites. Spinosad residues aged 2e48 h were also highly toxic to adultsof all three mite species, causing 96e100% mortality. Spinosad residues aged 48e168 h were less toxic toN. cucumeris than to T. montdorensis and H. miles. LT25 of double the recommended rate of spinosad forT. montdorensis, N. cucumeris and H. miles were calculated as 6.02, 5.3, and 7.08 days, respectively. Whenreleased after applying spinosad, T. montdorensis was the most successful species in reducing thripsnumbers, followed by N. cucumeris and H. miles. By releasing mites 6e7 days after a spinosad applica-tion, our results suggest that F. occidentalis can be effectively controlled. The practical implications ofimplementing a ’high-dose/biological control’ strategy are discussed.

Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Western flower thrips, Frankliniella occidentalis (Pergande)(Thysanoptera: Thripidae) is one of the most important pests ofagricultural and horticultural crops worldwide (Kirk and Terry,2003). In strawberry, Fragaria ananassa Duchesne (Rosaceae),F. occidentalis feed on fruit typically causing direct puncturedamage (Tommasini and Maini, 1995). Feeding damage can alsocontribute to the seediness of the fruit (Medhurst and Steiner,2001), which may be responsible for uneven ripening and yieldloss, reducing marketability and grower profits (Houlding andWoods, 1995). Feeding by F. occidentalis on blossoms may alsocause stigmas and anthers to turn brown and wither permanently

and Food Western Australia,WA 6151, Australia. Tel.: þ61

gov.au, m.touhidur.rahman@wa.gov.au (S. Broughton),

012 Published by Elsevier Ltd. All

(Zalom et al., 2008), and significantly reduce flower receptacle size(Coll et al., 2006).

To control F. occidentalis in strawberry and other horticulturalcrops, growers rely on pesticides. However, F. occidentalis hasdeveloped resistance to insecticides from different chemical classesincluding organophosphates, carbamates, synthetic pyrethroids,and to some newer chemistry insecticides (Brødsgaard, 1994;Broadbent and Pree, 1997; Jensen, 2000; Espinosa et al., 2002). Tomanage insecticide resistance in Australia, a national insecticideresistance management strategy (IRMS) was developed, based onrotation of insecticides from different chemical classes withdifferent modes of action (Herron and Gullick, 2001). However,chemical control failures against F. occidentalis in strawberries in2008 in Victoria, Australia, and in field and greenhouse capsicum inWestern Australia and Queensland in 2006 and 2007 (Herron,2010), have shown that growers are not using an IRMS. Forexample, analysis of strawberry grower spray diary records (S.Broughton, unpubl. data 2008) showed that commercial growerswere using the organophosphate methomyl to verify whether

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T. Rahman et al. / Crop Protection 42 (2012) 281e288282

F. occidentalis were present in the crop. Methomyl has efficacyagainst other thrips such as Thrips imaginis Bagnall, which alsooccur in strawberry (Houlding, 1997), but F. occidentalis has someresistance to this insecticide (Herron and James, 2005). If methomylis sprayed and thrips remain, the grower concludes thatF. occidentalis is present and sprays with spinosad which is highlyefficacious against F. occidentalis (Funderburk et al., 2000).

Of the insecticides currently available for control ofF. occidentalis, spinosad is the only reduced-risk bioinsecticide(Sparks et al., 1998) considered to be soft on beneficials (Miles et al.,2003; Anh et al., 2004; Kim et al., 2005), and can be used in organicagriculture. However, resistance monitoring of AustralianF. occidentalis populations has shown that spinosad resistance isoccurring, and is increasing in level (Herron, 2010). In 2001/02, 1.8-fold spinosad resistance was detected in a single strain of F. occi-dentalis collected from lettuce (Herron and James, 2005). In 2007and 2008, of 14 strains collected from horticultural (capsicum,cucumber, lettuce, tomato) and ornamental crops across Australia,43% of strains contained 20% or more spinosad resistantF. occidentalis, and 28% of strains contained 30% or more spinosadresistant F. occidentalis, with a peak in resistance of 156-fold(Herron, 2010). In 2010/11 a single strain collected from orna-mentals was measured at 1402-fold (Herron and Langfield, 2011).However, spinosad resistance in Australia is much lower than theSpanish laboratory study of Bielza et al. (2008) who have docu-mented>23,000-fold resistance. No spinosad resistant strains havebeen collected from strawberry to date.

Since spinosad is the most widely used product in Australia tocontrol F. occidentalis (Infopest, 2008), it is important to exploreapproaches to extend its usefulness and that of the other spinosyns(Jensen, 2000). In F. occidentalis, spinosad resistance is thought tooccur through an altered target site, expressed as ‘an almostcompletely recessive trait (rr)’ (Bielza et al., 2007). One possiblemanagement strategy is to apply a single high dose of spinosad,followed by releasing natural enemies. This should theoreticallyremove most homozygous (RR and rr), and heterozygous (Rr)individuals. ‘High-dose’ strategies have been dismissed in theliterature since computer models have shown that they are morelikely to promote resistance if used repeatedly (Tabashnik andCroft, 1982). However, Gardner et al. (1999) suggest that whencrop or pesticide rotation is impossible, then low and high doses inalternate treatments can be used to delay resistance. A high-dose/refuge strategy has been applied to manage pests such as tobaccobudworm, Heliothis virescens Fabricius (Lepidoptera: Noctuidae)and pink bollworm, Pectinophora gossypiella (Saunders) (Lepidop-tera: Gelechiidae) for over 15 years in transgenic Bt (Bacillus thur-igiensis Berliner) cotton and maize crops (Vacher et al., 2003). Thestrategy requires that transgenic plants produce Bt toxins at levelshigh enough to kill 100% of the resistant genotype. This may becoupled with plantings of a non-crop refuge, such that anysurviving heterozygotes are more likely to mate with susceptiblehomozygotes (Fitt, 2000; Vacher et al., 2003). In our proposedstrategy for F. occidentalis, rather than maintain a refuge whichmaybe impractical, natural enemies are released to ‘mop up’ anysurvivors.

One other major criticism of the high-dose strategy is that it canseverely disrupt biological control (Tabashnik and Croft, 1982). Thepredatory mites Typhlodromips montdorensis (Schicha), Neoseiuluscucumeris (Oudemans) (Phytoseiidae) andHypoaspis miles (Berlese)(Laelapidae) are able to suppress F. occidentalis in laboratory andglasshouse (Rahman et al., 2011a, b, c), and low-tunnel strawberries(Rahman et al., 2012). For the proposed strategy to be effective,spinosad should also be compatible with mites. The aims of thisstudy were thus to evaluate the effect of a doubled spinosadapplication rate on survival of T. montdorensis, N. cucumeris, and

H. miles, and to compare biological control with a single high-doseapplication followed by predatory mite release to manage a spino-sad resistant F. occidentalis strain.

2. Materials and methods

Trials were conducted in a controlled temperature room(25 � 1 �C, 50e60% RH, 16:8 h L:D regime) from October 2008 toJanuary 2009 at the University of Western Australia (UWA).

2.1. Insect and plant rearing

Strawberry, Fragaria x ananassa Duchesne (Rosaceae) cv CaminoReal (short-day length cultivar), was used in this study. Strawberryrunners were obtained from a commercial grower and propagatedin plastic pots (405 mm � 325 mm � 325 mm) containing pottingmix (Baileys Fertilizers, Rockingham, WA) in glasshouses at theDepartment of Agriculture and FoodWA (DAFWA) and UWA. Plantswere irrigated every third day with drippers operated by an auto-matic timer. A liquid fertilizer (Thrive� Yates, Australia; NPK:12.4: 3: 6.2; rate: 5 mL/2 L water) was applied once a week.Individual pots were covered separately by thrips-proof cages(450 mm � 350 mm) made from 105 mm mesh (Sefar FilterSpecialists Pty Ltd., Malaga, WA) fitted over a steel-rod stand. Thebottom end of the cage was taped to the pot, and the top of the cagewas sealed with a rubber band.

A glasshouse colony of F. occidentalis was established fromindividuals collected from calendula, Calendula officianalis L.(Asteraceae), at DAFWA and reared on calendula plants, planted inplastic pots (50 mm � 100 mm) and kept in insect proof Perspexcages (500 mm � 420 mm � 400 mm). The sides of the cage werefitted with 105 mm mesh net, and the front of the cage was fittedwith a removable cover held in place withmagnetic strips. The cagewas fitted over a Nylex tote box (cage tray:432mm� 320mm� 127mm; Blyth Enterprises Pty Ltd, Australia).Cages were kept in tunnel houses (insect proof net house) at UWA.Every second week, adults were collected from caged plants andreleased onto new caged plants to ensure the continuous avail-ability of thrips.

A population of spinosad eresistant strain of F. occidentalis wasobtained from NSW Department of Primary Industries (NSW DPI)(S. Broughton, pers. Comm.), which are pressured on an ad hocbasis by NSW DPI every two months. The strain had been bio-assayed (Capsicum WA, 02/2007), and found to have a resistancefactor of 110 compared to the New Zealand susceptible strain (NZ2strain), with 43% of individuals dying when treated at the recom-mended rate (80 ml/100 l) (Herron, 2010). The colony was main-tained on calendula plants in Perspex cages as described abovesince July 2007 at UWA. To obtain uniformly aged F. occidentalis foruse in the experiments, adult females (20 individuals) werecollected from the colony, released onto fresh plants, and allowedto lay eggs for 24 h. After 24 h, females were removed with anaspirator. The plants were then checked daily for larval emergence.Larvae that emerged on the same day were collected and releasedonto fresh plants. Larvae/adults emerged in the same day wereused in trials.

Predatory mites (T. montdorensis, N. cucumeris and H. miles)were obtained from commercial suppliers (Biological Services, SA;Chilman IPM Services, WA; and Beneficial Bug Company, NSW).Single batches of each mite (1e2 days old adults with even sexratio) were provided in plastic buckets as a mix of adults andnymphs in vermiculite. Trials were conducted immediately uponreceipt of mites.

T. Rahman et al. / Crop Protection 42 (2012) 281e288 283

2.2. Experiment 1: direct toxicity of spinosad to western flowerthrips and predatory mites

2.2.1. Western flower thripsAdults of the spinosad resistant strain (5e6 d old) were aspi-

rated from calendula and sexed. Only gravid females were thentransferred to a glass Petri dish (150 mm � 15 mm) containinga strawberry leaf 24 h pre-trial. The top of the Petri dish wascovered with mesh with 105 mm sieve opening, and the side sealedwith paraffin film (Parafilm M�, Micro Analytix Pty Ltd, Australia).At the start of the experiment, 20 cold e anaesthetized adults wereplaced on a paper towel and sprayed once with either 5 mL ofspinosad (Success�, 120 g/l EC, Dow AgroSciences Australia Ltd)(diluted in distilled water) at one of the three treatment rates ordistilled water (control). The three rates were (1) the recommendedrate at 80 ml/100 l, 0.096 g a.i./l), (ii) 2� recommended rate(160 ml/100 l) and (iii) 3� recommended rate (240 ml/100 l).Treatments were applied with separate hand-held atomizers (HillsSprayers, BH220063). After spraying, any excess liquid wasremoved with a tissue paper and adults were transferred to a Petridish containing a strawberry leaf (adaxial side up) on a moistenedfilter paper. The edge of the leaf was glued to filter paper so thatthrips could not hide underneath the leaf and the petiole wascovered with cotton soaked in 10% sugar solution to extend the leaflife. The Petri dish was covered as described above (¼ 1 replicate).There were 20 replicates (20 � 20 ¼ 400 individuals) of eachtreatment and a control. Petri dishes were placed (randomly) ona laboratory bench in the CT room. The experiment was repeatedwith first instars.

Spinosad is a slow-acting insecticide (Bret et al., 1997) andcumulative mortality of a test organism usually plateaus at 2e6days after exposure (Viñuela et al., 2001; Cisneros et al., 2002).Adults were examined at 2, 6, 24, 48, 72, and 96 h post-treatmentunder a stereomicroscope. Larvae were checked at 2, 6, 24, 48and 72 h post treatment exposure period after 72 h post-treatment,all the surviving larvae had pupated. Adults and larvae wererecorded as dead if they did not respond to probing with a finepaintbrush.

2.2.2. Predatory mitesBased on the thrips mortality in previous trial (see above),

double the recommended rate of spinosad was evaluated againstthree species of predatory mites. The bioassay method describedabove was used, except a thin barrier of Tac Gel (Stickem�, TheOlive Centre, Australia) was applied to the edge of the leaf toconfine mites on the leaf surface. Adult mite individuals, 2e3 daysold (even sex ratio) were used in this bioassay. First or second instarthrips were added to provide food for the mites. In each Petri dish,40, 120 or 200 untreated thrips larvae were added as food forH. miles (Berndt et al., 2004), N. cucumeris (Zilahl-Balogh et al.,2007), and T. montdorensis (Steiner et al., 2003), respectively.During the trial period, additional thrips larvae were added to thePetri dishes as required. Mite mortality was assessed at 6 h, 24 h,48 h, 72 and 96 h post-treatment. Trial was replicated 20 times(20 � 20 ¼ 400 individuals) for each species of predatory mite.

2.3. Experiment 2: bioassay of residual toxicity of spinosad topredatory mites

Mortality of adult mites via consumption of treated thrips larvaeand simultaneous exposure (contact) to spinosad residues wasassessed in this experiment (Zilahl-Balogh et al., 2007). Spinosadwas applied at 160 ml/100 l (twice recommended rate) to straw-berry leaves, as this dose effectively controlled >50% of spinosadresistant F. occidentalis in Experiment 1. Strawberry plants (3e4

weeks old) were split into eight groups: 2, 24, 48, 72, 96, 120,144, and 168 h old residue and control (water) and both sides of theleaves were sprayed with a handeheld atomizer until run-off (Egeret al., 1998). Plants were sprayed at intervals such that plants fromall treatment groups could be exposed to mites at the same time.The control groupwas sprayedwith distilled water 24 h prior to thestart of the experiment. To avoid contamination, separate atomizerswere used for the water and spinosad spray. After spraying, eachplant was coveredwith amodified thrips proof-cage as described inExperiment 1 and kept in a glasshouse (25 � 1 �C, 50e60% RH,16:8 h L:D regime).

Spinosad was also applied to Petri dishes (150 mm � 15 mm) toobtain dishes of the same exposure age. Both halves of the Petridish were sprayed with spinosad or water (control), and then driedin the same glasshouse where sprayed-plants were kept for oneand half hours. Thereafter, dried Petri dishes were stored in sepa-rate plastic trays in the same glasshouse until required. A spinosadtreated strawberry leaf was placed adaxial side up on a Petri dish,and a thin barrier of Tac Gel applied to the edge of the leaf toprevent escape of thrips larvae. First instars following the ratio asdescribed above were then released onto the treated leaf andallowed to feed for 12 h. Control was prepared from leaves and Petridishes treated with distilled water only.

Testing arenas were prepared using the same age residues ofleaf, Petri dish and intoxicated thrips larvae. For each Petri dish, 20adult mites (2e3 days old, with even sex ratio) of one species wereplaced on the leaf in a Petri dish. Each treatment (residue ages) wasreplicated 20 times for each mite species. Additional thrips larvaewere added to the Petri dish if required followed the ratio asdescribed above. Testing arenas were arranged randomly ona laboratory bench and mortality was checked under a stereomi-croscope at 24, 48, 72 and 96 h post-release exposure.

2.4. Experiment 3: efficacy of predatory mites with spinosad againsta spinosad-resistant western flower thrips resistant strain

Thrips adults (spinosad-resistant) that emerged on the sameday were collected from the stock colony and released onto freshlycaged strawberry plants for 24 h. Forty potted strawberry plants,2e3 weeks old with 3e4 leaves (excess leaves were pruned) wererandomly divided into two groups (n ¼ 20) and sprayed with160 ml/100 l (twice recommended rate) or distilled water (control).Plants were covered with separate thrips-proof cages and allowedto dry for two hours. Fifteen spinosad-resistant thrips adults werereleased onto each plant. Each group was further divided into 4groups and received: (i) No mites, (ii) 6 T. montdorensis, (iii) 6N. cucumeris, and (iv) 6 H. miles. Adult mite individuals of 2e3 daysold with even sex ratio were used in the study. Based on theresidual threshold estimated from the previous experiment(Rahman et al., 2011b), N. cucumeris, T. montdorensis or H. mileswere released on to plants after five, six, or seven days. After 24 h,plants were checked with a battery-powered, hand-held magni-fying glass [50 mm (200) illuminated round 2-� power with 4�bifocal magnifier] for live thrips, then every fifth day for fiveweeks.The number of live thrips adults and larvae per plant were recordedat each inspection. Plants were watered manually as required.

2.5. Data analysis

The number of individuals that died at each observation wascounted and expressed as a percentage of the total number ofindividuals in the arena. Mortality was corrected using Abbott’sformula (Abbott, 1925). Control mortality of thrips or mites did notexceed 5%.

T. Rahman et al. / Crop Protection 42 (2012) 281e288284

The differences in corrected cumulative mortality (mortalityafter 96 h post-release exposure period) of each residue age amongpredatory mites were analyzed by one-way ANOVA (Proc Mixed),except cumulative mortality of mites exposed to 2 and 24 h oldresidues, which caused 100% mortality to all three-mite species. Todetermine the difference in toxicity between spinosad residue ages,corrected mortality of T. montdorensis, N. cucumeris, and H. miles ateach post-release exposure period was subjected to separate one-way ANOVAs. If ANOVA showed significant differences, mortalitymeans were separated by least square mean differences (LS mean)at 5% probability. Data were subjected to arcsine transformation(Healy and Taylor, 1962) to normalize before analysis, though actualmeans are reported.

Theeffect of spinosad andpredatorymite releases on thenumberof the spinosad-resistant F. occidentalis adults and larvae over timeswere compared by two-way ANOVA (Proc Mixed) (independentvariables: spray treatment and predatory mite releases; responsevariables: thrips adults or larvae). Separateanalyseswerecarriedoutfor each observation between 10 and 35 DAS (days after spray). Asthere was a significant interaction between spray (spinosad andwater) and predatory mite releases (no mites, T. montdorensis,N. cucumeris and H. miles), additional one-way ANOVAs were per-formed (QuinnandKeough, 2002). Thripsnumbers (adults or larvae)at each observation were analyzed with two separate one-wayANOVAs (one for each spray treatment). As multiple comparisonswere used, an adjustment to the significance level was made,a ¼ 0.025 (0.05/2). To determine the difference in the numbers ofthrips adults and larvae between groups prior to mite release, one-way ANOVAs (Proc Mixed) were performed for each spray treat-ment at 1 and 5 DAS. If significant, means were separated with LSmeans. Datawere transformedusingO(xþ 0.5) transformationpriorto analysis (Healy and Taylor, 1962).

The residual toxicity of spinosad to each species of predatorymite at each post e release exposure period was classified into fourcategories following the International Organization of BiologicalControl (IOBC) guidelines for laboratory trials and ranked as:1 ¼ harmless (<30% mortality), 2 ¼ slightly harmful (30e79%mortality), 3 ¼ moderately harmful (80e99% mortality), and4¼ harmful (>99%mortality) (Sterk et al., 1999). The persistence ofspinosad for each species of predatorymitewas classified accordingto the time taken to lose toxicity (<30% mortality, IOBC persistenceclass): A ¼ short-lived (<5 d) and B ¼ slightly persistent (5e15 d)(Hassan et al., 1994; Sterk et al., 1999). The residual toxicitythreshold of spinosad for T. montdorensis, N. cucumeris and H. mileswas estimated with Probit analysis (Finney, 1971) by Proc ProbitProcedure. The LT25 (lethal time of 25% mortality) was used, whichis considered an acceptable level for non-target organisms (Shippet al., 2000).

All analyses were computed using Statistical Package SAS 9.1.3(SAS Institute, Cary, NC, USA).

3. Results

3.1. Experiment 1: direct toxicity of spinosad to western flowerthrips and predatory mites

The recommended rate of spinosad was no more toxic tospinosad-resistant adults (0.50 � 0.46%) or larvae (0.00%) than thecontrol (distilled water spray). However, when the application ratewas doubled, >70% of thrips adults (87.94 � 2.36%) and larvae(78.57 � 3.45%) were killed. Direct exposure to triple the recom-mended rate of spinosad killed 100% of spinosad-resistant thripsadults and larvae. In the control treatment (sprayed with distilledwater), 0.5% thrips adults and no larvae died.

Spinosad applied at twice the recommended rate resulted in100% mortality of all three predatory mite species. Mite mortalitynever exceeded 5% when sprayed with water only.

3.2. Experiment 2: bioassay of residual toxicity of spinosad topredatory mites

Spinosad residues of all ages were toxic to T. montdorensis,N. cucumeris andH. miles (Table 1), although toxicity declined as theresidual period increased. Spinosad residues aged 2 h and 24 hwere very toxic to all three predatory mite species, causing 100%mortality within 24 h of exposure. Similarly, the cumulativemortality of mites to 48 h old residues was close to 100% and didnot differ among predatory mite species (F2, 57 ¼ 3.01, P ¼ 0.057).However, different mortality rates were recorded when mites wereexposed to 72e168 h old residues over a 96 h exposure period(72 h: F2, 57 ¼ 35.08, P < 0.0001; 96 h: F2, 57 ¼ 5.64, P ¼ 0.0058;120 h: F2, 57 ¼ 9.61, P ¼ 0.0002; 144 h: F2, 57 ¼ 25.41, P < 0.0001;168 h: F2, 57 ¼ 35.43, P < 0.0001; Table 1). For each residue age(72 he168 h), mortality was the highest for H. miles (89.7 � 1.9 d)and the lowest for N. cucumeris 68.9 � 1.8). Mortality ofT. montdorensis (71.1 � 1.9) and N. cucumeris did not differ whenexposed to 72 h old residues. Similarly, mortality ofT. montdorensis and H. miles did not differ when exposed to 96 hold residues (Table 1).

As residue age increased, mortality decreased over each post-release exposure period (Table 1). According to the IOBC toxicityclassification, spinosad residues aged 2 he24 h were harmful(>99% mortality) to all three species (Table 1). Moreover, spino-sad residues aged 48 h were also harmful to T. montdorensis at 72and 96 h post-release exposure. Similarly, 48 h old spinosad resi-dues were harmful to H. miles at 48, 72 and 96 h, though onlymoderately harmful to N. cucumeris. H. mileswas themost sensitivespecies, with 144 h old residue classified as slightly harmful. Spi-nosad caused no harmful effect to N. cucumeris five days post-release (rating 1 ¼ <25% mortality), and was considered to beshort-lived (persisting for less than five days). For T. montdorensisand H. miles, spinosad was classified as slightly persistent with120 h (5 days) old residues for T. montdorensis and 144 h (6 days)old residues for H. miles classified as slightly harmful. The LT25indicates the period after a spinosad application, which wouldallow 75% survival of predatory mites. The LT25 spinosad wasestimated as 6.02 days [144.54 h (c2 ¼ 12.14, df ¼ 6, P ¼ 0.058; 95%fiducial limits: 140.54e152.02 h); Antilog102.16] for T. montdorensis,5.3 days [127.85 h (c2 ¼ 10.20, df ¼ 6, P ¼ 0.12; 95% fiduciallimits: 124.01e132.12 h); Antilog102.11] for N. cucumeris and 7.08days [169.82 h (c2 ¼ 11.06, df ¼ 6, P ¼ 0.069; 95% fiducial limits:157.89e171.33 h); Antilog102.23] for H. miles.

3.3. Experiment 3: efficacy of a combination of predatory mites withspinosad against a spinosad-resistant strain of western flower thrips

Predatory mites were more effective at reducing thrips adultsafter spinosad had been applied compared to plants treated withwater (control) (Fig. 1). Since there was a significant interactionbetween spray treatment (spinosad, water) and mite releases (nomites, T. montdorensis, N. cucumeris, H. miles) (all P < 0.0001), spraytreatments (spinosad, water) were separately analyzed at eachobservation (DAS ¼ days after spraying). There were no differencesin adult thrips numbers (plants assigned tomites release) for eitherthe spinosad or control plants, prior to mite releases at 1 and 5 DAS(P > 0.05) (Fig. 1). For spinosad-treated plants from 10 to 35 DAS,themean numbers of adult thrips differed (10 DAS: F3, 16¼ 21.70; 15DAS: F3, 16¼ 554.34; 20 DAS: F3, 16¼ 51.72; 25 DAS: F3, 16¼ 30.85; 30DAS: F3, 16 ¼ 56.80; 35 DAS: F3, 16 ¼ 112.26; all P < 0.0001) among

Table 1Residual toxicity of spinosad (twice recommended rate) to predatory mites at 24 h, 48 h, 72 h and 96 h post-release exposure periods. Mites were fed spinosad intoxicatedF. occidentalis larvae and simultaneously exposed to residue. Residual toxicity was classified: 1 ¼ harmless (<30% mortality), 2 ¼ slightly harmful (30e79% mortality),3 ¼ moderately harmful (80e99% mortality), and 4 ¼ harmful (>99% mortality). Persistence class: A ¼ short-lived (<5 d), B ¼ slightly persistent (5e15 d).

Residue age (h) Corrected mortality (%) (mean � SE) at post-release periods Toxicity class Persist. class

24 h 48 h 72 h 96 h 24 h 48 h 72 h 96 h

T. montdorensis2 100e 100f 100f 100f 4 4 4 4 B24 100e 100f 100f 100f 4 4 4 448 91.3 � 2.3e 96.0 � 1.7f 100f 100f 3 3 4 472 50.3 � 1.4d 60.5 � 1.5e 67.5 � 1.6e 71.1 � 1.9e 2 2 2 296 33.8 � 1.6c 45.8 � 1.7d 54.1 � 1.9d 56.2 � 1.5d 2 2 2 2120 10.0 � 0.9b 22.4 � 1.3c 30.5 � 1.6 31.0 � 1.8c 1 1 2 2144 9.5 � 0.8b 17.6 � 1.3b 24.6 � 1.7b 24.6 � 1.7b 1 1 1 1168 3.0 � 0.9a 8.3 � 0.9a 16.5 � 1.5a 16.8 � 1.4a 1 1 1 1F7, 152 709.26 669.08 409.94 372.11P <0.0001 <0.0001 <0.0001 <0.0001N. cucumeris2 100g 100g 100g 100f 4 4 4 4 A24 100g 100g 100g 100f 4 4 4 448 91.5 � 2.2f 94.4 � 1.8f 96.4 � 1.3f 96.7 � 1.3f 3 3 3 372 52.5 � 1.5e 58.5 � 1.7e 66.1 � 1.6e 68.9 � 1.8e 2 2 2 296 29.8 � 1.4d 38.5 � 1.9d 45.2 � 2.0d 48.7 � 2.1d 2 2 2 2120 14.8 � 1.0c 21.0 � 1.5c 26.8 � 1.4c 26.8 � 1.4c 1 1 1 1144 3.3 � 1.1b 10.7 � 1.2b 16.3 � 1.2b 16.3 � 1.2b 1 1 1 1168 0.0 � 0.0a 5.8 � 0.6a 7.4 � 0.9a 7.4 � 0.9a 1 1 1 1F7, 152 881.76 647.50 636.77 621.14P <0.0001 <0.0001 <0.0001 <0.0001H. miles2 100f 100f 100e 100e 4 4 4 4 B24 100f 100f 100e 100e 4 4 4 448 95.8 � 1.8f 100f 100e 100e 3 4 4 472 79.8 � 2.1e 86.5 � 2.4e 86.9 � 2.4d 89.7 � 1.9d 3 3 3 396 40.8 � 1.5d 49.4 � 1.4d 51.8 � 1.5c 57.6 � 2.5c 2 2 2 2120 27.3 � 2.4c 35.9 � 2.2c 37.4 � 2.0b 37.4 � 2.0b 1 2 2 2144 10.5 � 0.6b 23.2 � 1.3b 32.3 � 1.8b 32.3 � 1.8b 1 1 2 2168 3.8 � 1.0a 11.5 � 1.2a 22.3 � 1.8a 22.3 � 1.8a 1 1 1 1F7, 152 537.45 471.23 348.82 373.99P <0.0001 <0.0001 <0.0001 <0.0001

For each species, within column, means with different letters differed significantly (a ¼ 0.05).

T. Rahman et al. / Crop Protection 42 (2012) 281e288 285

predatory mite treatments. Similarly, for the control treatment, themean numbers of adult thrips per plant differed significantlyamong predatory mite treatments (15 DAS: F3, 16 ¼ 70.70: 20 DAS:F3, 16 ¼ 58.51; 25 DAS: F3, 16 ¼ 48.61; 30 DAS: F3, 16 ¼ 38.88; 35 DAS:F3, 16 ¼ 104.33, all P < 0.0001), except at 10 DAS (P > 0.05). In bothspinosad and control treatments, plants with no predatory miteshad higher numbers of adult thrips compared to plants that hadbeen treated with predatory mites (Fig. 1). T. montdorensis was themost effective species at reducing thrips adult numbers for bothspinosad and control treatments, except at 10 and 15 DAS. H. mileswas most effective at 10 and 15 DAS in both the spinosad andcontrol treatments, but the least effective species from 20 to 35 DAScompared to T. montdorensis and N. cucumeris.

Similarly, mites were most successful at reducing larval thripsnumbers after spinosad was applied (Fig. 1). Since there wasa significant interaction between spray and mite releases (allP < 0.0001), spray treatments were separately analyzed at eachDAS. When plants were examined at 1 DAS, no thrips larvae werefound. At 5 DAS, prior to the release of predatory mites, there wereno differences in the numbers of thrips larvae per plant betweentreatments (plants assigned to predatory mites release; P > 0.05)(Fig. 1). From 10 to 35 DAS, mean numbers of thrips larvae per plantdiffered significantly amongst predatory mite treatments for boththe spinosad (10 DAS: F3, 16 ¼ 137.59; 15 DAS: F3, 16 ¼ 31.69; 20 DAS:F3, 16¼ 25.89; 25 DAS: F3, 16¼ 213.26; 30 DAS: F3, 16¼ 61.49; 35 DAS:F3, 16 ¼ 136.26, all P < 0.0001) and the control (10 DAS: F3,16 ¼ 25.64; 15 DAS: F3, 16 ¼ 47.78; 20 DAS: F3, 16 ¼ 59.92; 25 DAS: F3,16 ¼ 69.59; 30 DAS: F3, 16 ¼ 51.21; 35 DAS: F3, 16 ¼ 89.75, allP < 0.0001) treatments. For both the spinosad and control

treatments, plants that did not receive any mites had the highestnumbers of thrips larvae, compared to plants treated with preda-tory mites (Fig. 1). For both the spinosad and control treatments,T. montdorensis was the most effective species in reducing thripslarvae, except at 15 DAS for the spinosad treatment and 10 DAS forthe control. N. cucumeris was the most effective in reducing thripslarvae at 15 DAS and 10 DAS for the spinosad and control treat-ments, respectively. H. miles was the least effective species inreducing the number of thrips larvae compared to T. montdorensisand N. cucumeris.

4. Discussion

To control a spinosad-resistant strain of F. occidentalis, a strategyof not spraying was less effective that a single ‘high-dose’ of spi-nosad followed by releasing predatory mites. Spinosad reduced thenumber of resistant F. occidentalis adults and larvae by 70%compared to the untreated control. In comparison, when predatorymites were released after applying spinosad, the number ofspinosad-resistant F. occidentalis adults and larvae was reduced by50% compared to releasing predatory mites alone.

Spinosad is considered to be a short-lived insecticide thatrapidly degrades in nature (Thompson et al., 2000; Williams et al.,2003). Our previous study suggested that fresh to relatively freshresidues (2e72 h old) were harmful to all three predatory mitespecies (Rahman et al., 2011b). Under laboratory conditions, spi-nosad is considered to be highly stable and capable of causing highmortality (Bernando and Viggiani, 2000). However, semi-field andfield studies suggest that spinosad residues pose little or no toxicity

0

10

20

30

40

No mites TM NC HM

Spinosad

Adults

0

10

20

30Spinosad

Larva

1* 5* 10 15 20 25 30 350

10

20

30

40 Water

1* 5* 10 15 20 25 30 350

10

20

30Water

Num

ber o

f thr

ips

per p

lant

(Mea

n±

SE)

Days after spray (DAS)

Fig. 1. Mean numbers of thrips per plant sprayed with spinosad or water in the presence or absence of predatory mite. DAS ¼ days after spray, TM ¼ T. montdorensis,NC ¼ N. cucumeris, HM ¼ H. miles. *Thrips number per plant before mites were released.

T. Rahman et al. / Crop Protection 42 (2012) 281e288286

to natural enemies after three to seven days depending on species(Boyd and Boethel, 1998; Ruberson and Tillman, 1999; Crouse et al.,2001; Rahman et al., 2011a, b, c). Though the present study wasconducted in a controlled environment, the effect on predatorymites tested here supports the premise that spinosad residuesbecome less toxic over time. Based on residual threshold (LT25)calculations and confirmed with laboratory trials, mites can besafely released 5e7 days after applying spinosad at double therecommended rate. N. cucumeris can be released after 5.3 days,T. montdorensis after 6.02 days, and H. miles after 7.07 days.

The variable response of beneficials to spinosad residues is notunexpected. Our previous study reported that spinosad applied atthe recommended rate (80 ml/100 l) was short-lived toT. montdorensis and N. cucumeris, but slightly persistent to H. miles(Rahman et al., 2011c). It has been reported that LC50 values ofspinosad exceeded 960 ppm for the predatory bug Podius nig-rispinus (Dallas) (Hemiptera: Pentatomidae) (Torres et al., 1999),while the LC50 was only 50 ppm for Podius maculaiventris (Say)(Hemiptera: Pentatomidae) (Viñuela et al., 1998). Khan and Morse(2006) tested the impact of four pesticides on the predatory miteEuseius tularensis Congdon, and found residual effects if mites werereleased five to six days after spinosad was applied, but no residualeffects if mites were released seven days after the spinosadapplication.

The efficacy of the different mite species in reducingF. occidentalis numbers varied. T. montdorensis appeared to be themost effective species in suppressing F. occidentalis, followed byN. cucumeris and H. miles. There are a number of possible reasonsfor such variation (Brødsgaard, 1989; van Houten et al., 1995;Steiner and Goodwin, 1998, 2001; Berndt et al., 2004; Messelinket al., 2006; Skirvin et al., 2007). Regardless, all three speciestested in this study provided better control of a resistant F. occi-dentalis strain when released after spinosad was applied.

Though we have shown that a high-dose/biological controlstrategy may be used to control a spinosad resistant F. occidentalisstrain, at least under laboratory conditions, it requires furtherassessment in the field. Practical implications of implementinga ’high-dose/biological control’ strategy also need addressing. First,the method should only be considered for crops where biologicalcontrol of F. occidentalis is effective, and growers may be unable torotate pesticides. At present, IPM of F. occidentalis is confined tofield and greenhouse-grown strawberries, and greenhousecucumber and sweet pepper in Australia (L. Chilman, pers. comm2012). These growers are limited in their choice of pesticides thatare compatible with IPM and efficacious against F. occidentalis totwo: spinosad and spinetoram. Second, would other pests ornatural enemies present in the crop be affected by a higher dose ofspinosad? In strawberry, aphids (e.g. Myzus persicae (Sulzer)), themoths Helicoverpa armigera (Hubner) and H. punctigeraWallengren(Lepidoptera: Noctuidae), and two-spotted mite (Tetranychus urti-cae Koch) may also be present. Of these, resistance of H. armigera tospinosad may be ascerbated, but unlike F. occidentalis, otherpesticides such as Bt and nuclear polyhedrosis virus (NPV) areavailable for its control. Phytoseiulus persimilis Athias-Henriot(Acarina: Phytoseiidae) is released for biological control ofT. urticae, and is reported to be compatible for use with spinosad(Cote et al., 2002), but would need to be verified at the higher rate.Third, how effective do the natural enemies need to be to ’mop up’any survivors? This is unknown and would most easily be tested byconsidering different scenarios with computer models. Fourth,resistance monitoring for F. occidentalis populations to spinosadand other spinosyns are needed to verify the proposed manage-ment technique. Indeed ongoing resistance monitoring would berequired to underpin this, and any other management strategy.Finally, doubling the recommended rate of spinosad (80 ml/100 l,0.096 g a.i./l) may increase residues and extend the withholding

T. Rahman et al. / Crop Protection 42 (2012) 281e288 287

period, depending when spinosad is applied. Ideally, a single highdose could be applied at the beginning of the growing season tominimize risk to consumers. However, residue testing is required toverify that the proposed rate is acceptable to the Australian Pesti-cides and Veterinary Medicines Authority (APVMA). In summary,the challenge for managing pesticide resistant populations ofF. occidentalis and other insecticide resistant species is to conserveuseful insecticides. Thoughwe have suggested that resistance couldbe managed by a one-off application of double the current rec-ommended rate, the effects on the natural enemy may be such thatthe two tactics cannot be used together. Since predatory mitespredate on either first instar or pupal stages but not the adult stage,the adult F. occidentalis population needs to be reduced beforepredatory mites are released. Subsequent to our study, a nativepirate bug, Orius armatus Gross (Heteroptera: Anthocoridae) hasbecome commercialized in Australia and feeds on adults and larvae(Broughton and Chilman, 2011). Further studies should examinethe effect of combining O. armatus with predatory mites andspinosad.

Acknowledgments

We thank Anthony Yewers (Berry Sweet, Bullsbrook, WA,Australia) for providing strawberry runners. David Cousins(Department of Agriculture and Food, Western Australia) helpedraise and maintain strawberry runners. Grant Herron (NSWDepartment of Primary Industries) kindly provided spinosad-resistant thrips. We also thank Kevin Murray (School of Mathe-matics and Statistics, The University Western Australia) for exper-tise and assistance with statistical analyses used in this study.

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