1623 or 16 y 2015 c B. thuringiensis on the basis of a,d* (Biobit 16% WP) have proved to be Biological insecticides and are used to control 14 18 , 17 kurstaki C. chalcites kurstaki 4% in treatments involving ± var. var. Aurelio Carnero, 10% to 96 100% infection after 7 days. Mortality of b 15 ∼ , ± 14 Bacillus thuringiensis Bacillus thuringiensis 3% in second-instar larvae fed for 2 days on and Primitivo Caballero ± , increasing to 1 d Correspondence to:CSIC-Gobierno Primitivo de Navarra, 31192 Caballero, Mutilva Baja, Spain. E-mail: Instituto [email protected] de Agrobiotecnología, Instituto de Agrobiotecnología, CSIC-Gobierno de Navarra, Mutilva Baja, Spain Instituto de Ecología AC, Xalapa, Veracruz, Mexico Instituto Canario de Investigaciones Agrarias, San Cristóbal de la Laguna, Spain Departamento de Producción Agraria, Universidadplona, Pública Spain de Navarra, Pam- − Attempts at biological control of 30% losses in bananas grown under greenhouse conditions in ∗ c a d b was 3–4-fold more effective than chemical or effective in greenhouse and field crops > the Canary Islands. predator or parasitoid releases have met with partial success, this pest on bananas. Similarly, the use of pheromone-based based on are presently in the process of evaluation. 1 − Trevor Williams, single nucleopolyhedrovirus (ChchSNPV-TF1) was evaluated in plant (: ) 10% to 94 This OBs L a 1 ± 9 10 × 5% pest mortality. All treatments significantly reduced plant defoliation 13 More impor- ± Delia Muñoz 15 , 14 c chal- For banana plants, In southern 14 2 (Esper) is a major pest of tomato in Mediterranean countries and attacks banana in the virus occlusion bodies (OBs) L Chrysodeixischalcites 6% and 32 and fodder crops such as 7 ± 5 10 × including greenhouses in north- 10 – and in banana crops in the Canary to 5 larvae of all instars. Alexandra Bernal, 7 3 6 is considered to be an emerging pest , whereas indoxacarb (Steward 30% WG) and 60 min of feeding. a artichokes 1 10 > − 4 , × 3 : 1623–1630 www.soci.org © 2014 Society of Chemical Industry SE) lethal infection varied from 77 It is one of the four most important pests of 71 6 ± OBs L C. chalcites Chrysodeixis chalcites Finally, this has recently been reported in 9 C. chalcites 12 2015; , 10 11 tomato looper; ChchSNPV; greenhouse trials; application rates ×

–1 8 (Esper) (Lepidoptera: Noctuidae), is regarded as a major pest

10 In Spain, major damage has been reported in horticultural crops × Canada and sporadically in parts of the United States. in Almería, southern Spain, Pest Manag Sci cites of greenhouse-grown crops, particularly in countriesthe surrounding Mediterranean, the Middle-East and sub-Saharan . 1 INTRODUCTION The golden twin-spot or tomato looper, Canary Islands (Spain). The efficacy of growth chambers and greenhouse trials performeda on compressed air tomato sprayer. and banana plants respectively. Treatments were applied using RESULTS: Mean ( Abstract BACKGROUND: Estrella Hernández-Suárez, Chrysodeixis chalcites Oihane Simón, biological insecticide for control of Efficacy of an alphabaculovirus-based (wileyonlinelibrary.com) DOI 10.1002/ps.3969 on tomato and banana crops Research Article Received: 24 October 2014 Revised: 19 December 2014 Accepted article published: 24 December 2014 Published online in Wiley Online Library: 21 Januar plants treated with 2 larvae collected from banana at different intervals post-application varied from 54 1 ern Europe. Islands, where that occurs throughout the production cycle. European greenhouse crops, highly polyphagous pest feeds on tomatoes,cifers peppers, and beans, ornamental cru- plants, among others. treatments produced betweencompared 22 with untreated controls. Application of 1 lucerne and clover. Keywords: CONCLUSION: This information should prove useful in theand registration Europe. of ChchSNPV-TF1 as a bioinsecticide in© the Canary 2014 Islands Society of Chemical Industry treatments. Larvae acquired lethaldisappeared infection following more rapidly when feeding on tomato than banana plants, but this difference injury mainly occurs on the young unfoldingor leaves, perforated which are by cut it also feeds on soya, tantly, late-instar larvae often feeding on banana the epidermis fruits, of producing develop- serious skin injuries that result in , C 3 ∘ 5% 10 et al. ± × values Larvae Solanum 35 ,4 50 32 4 and these C, 70 : 1623–1630 ∘ 10 Purified OBs 36 1 71 × 2.40 m height) 34 , ± × 23 ,2 5 using the droplet 2015; 10 × )orwater,asacontrol, for 5 min. 1 − g An OB-free solution was also 4.95 m length × 34 C. chalcites , × II, which had proved to be valu- Pest Manag Sci 24 , , previously calculated to result in OBs L 1 23 5% humidity, in constant darkness, Bgl − 7 ± For this, OBs were suspended in 10% 10 400. Purified OBs were stored at 4 100 mL of semi-synthetic diet. 33 × × ∼ OBs mL 2 Cand70 and 5 ∘ 10 × 7 C. OBs were extracted by thawing and homogenis- The biological activity of laboratory-produced OBs ∘ L., Solanaceae, var. Raf) maintained in a walk-in 10 from the laboratory colony were starved for 8–12 h 34 , × 20 24 − , ,1 and 1.6 23 6 C and then allowed to drink from an aqueous suspension 2 ∘ 10 10 × Groups of 25 larvae were collected from each branch at four dif- At 1 h post-application, when plants were completely dry, × humidity and a 16:8 h (light:dark) photoperiod. ferent intervals post-application: 2 h and 2,larvae 5 were and individually 7 placed days. in Collected 25 mLdiet, plastic reared cups at with 25 artificial between 95 and 5% mortality. 2.2 Efficacychambers of ChchSNPV-TF1 on tomato plants in growth A trial was performedlycopersicum using flowering tomatoplant growth plants room (3.38 ( m width illuminated with eight fluorescent(2 tubes (60 W). OB suspensions were applied until run-off(Matabi using 5; a Antzuola, compressed-air Guipúzcoa,0.1% hand (v/v) Spain). sprayer Agral 90 All (Syngenta Agro treatments S.A.,noxy included Madrid, polyethoxy ethanol, Spain), a nonylphe- non-ionic liquid wetting and spreading agent. tomato branches withof seven each leaves branchhydroponic were was nutrient removed. solution placed or The in Hoaglands solution, base a 25 mL glass jar containing a fed to a group of insectsuntil as a death control. or Mortality was pupation. recordedingested As daily by the each volume insect ofas was inoculum not lethal suspension quantified, concentrations, results rather weresubjected to treated than probit analysis lethal using the doses. POLO-PC program. Results were were resuspended in steriledetermined water, by and counting theirNeubauer concentration triplicate haemocytometer was samples (Hawksley, Lancing, using UK) undercontrast an phase microscopy improved at for up toassays. 1 The month identity prior ofendonuclease to analysis these use using OBs inable was laboratory for discrimination confirmed between and ChchSNPV by genotypes greenhouse instudies. restriction previous were checked daily formoribund signs of that polyhedrosis had disease.stored stopped at Dead moving and were collected and droplet feeding method. sucrose solution and 0.001% Fluorelladrank blue inoculum within food 10 dye. min were Larvae placed in that plastic groups of containers 150 in with 1.5 L was estimated infeeding second-instar bioassay technique and compared with the LC of the original stockfor inoculum this and virus. published For this, OBC. groups activity chalcites of values 25 newlyat moulted second-instar 26 containing 10% (w/v) sucrose,OBs 0.001% following a (w/v) fivefold Fluorella dilution blue series: and 1 8 branches were theners. placed Finally, individually eachsecond-instar in container larvae 2 L from wastime, the 25 glass artificially larvae laboratory from contain- infested thecontrols colony. laboratory to with colony At were determine individualised 200 the whether as an the same laboratory inapparent colony alphabaculovirus harboured infection.growth The chamber conditions of were the controlled as follows: 25 ing the cadaversmuslin and in centrifugation water, at 2000 and purified by filtration through 20 29 or , , 2 28 and 13 , 2 www.soci.org O Simón using the kurstaki 1 − larvae under single nucle- © 2014 Society of Chemical Industry var. monitoring OBs mL 8 23 Adults were fed 30% w/v C. chalcites 10 was started using pupae C. chalcites 32 × feeds on the parenchyma on we determined the influence and the feeding behaviour of 31 26 25 , 22 This isolate was named Tener- Previous studies indicated that an Chrysodeixis chalcites However, the low number of active Bacillus thuringiensis C. chalcites 24 22 , 21 C. chalcites , As the efficacy of a virus-based insecti- 23 larvae feeding on virus-treated tomato and 17 , 5% humidity and a photoperiod of 16:8 h 30 16 , ± 14 19 C, 70 ∘ C. chalcites Feeding in polyphagous lepidopteran larvae is often 27 ChchSNPV-TF1 OBs were produced by inoculating sixth-instar The performance of a virus-based insecticide as a pest con- Chemical-based control measures usually involve applications Insect baculoviruses have an established record as the cide depends on the abilityfeeding to larval deliver stages a of lethal the quantity pest, of OBs to laboratory-reared larvae with 9.02 trol agent dependsbodies largely (OBs) applied on to the the crop quantity of virus occlusion of chlorpyrifos, fenamiphos or indoxacarb, among others. wileyonlinelibrary.com/journal/ps 22.1 EXPERIMENTAL METHODS Insects andA viruses laboratory colonyreceived from of the Instituto(ICIA), Canario Tenerife de (Spain), in Investigacionesodically 2007. Agrarias with pupae This from colony thereared Canary was Islands at refreshed (Spain). peri- 25 Insects were (light:dark) on a semi-synthetic diet. For example, in tomato, the underside of leavestogether and and then begins rollsappear to the skeletonised. eat edges of through the the leaves leaves, making them Synthetic insecticide treatmentswhich increase require production multiple costs,of hamper applications products the that commercialisation can occasionallyhave contain pesticide led residues, to and tionsofthispest. the development of resistance in certain popula- control measures can be effective for the pest. ife isolateof 1 kill (ChchSNPV-TF1). ofbaculovirus-based The insecticides. ChchSNPV-TF1 pathogenicity was and comparable with speed that of other of OB dose onexperiments virus-induced on tomato pest plants. mortality Using thesepared in results, growth we the then chamber com- performancewith of those of applications twoof of other this insecticides ChchSNPV-TF1 pest: frequently one OBs usedthe based for other, on control a modern syntheticrelationship between insecticide, acquisition indoxacarb. of Given infection the andprotection, in efficacy a of final crop study weinfection compared of the rate of acquisitionbanana of leaves to determinedifferences whether in leaf these structural or cropsdisease acquisition chemical significantly following application affect of ChchSNPV the OBs. probability of diluted honey. The ChchSNPV-TF1 isolate originated frominfected a larva single collected duringcrop in a Tenerife, as natural described previously. epizootic in a banana influenced by the species and phenology of the crop plant. mating disruption. opolyhedrovirus (ChchSNPV),(Spain), from was Tenerife, highly Canary pathogenic to Islands alphabaculovirus, named laboratory conditions. substances authorised by the European(http://www.agrocabildo.org/publica/Publicaciones/subt_48_ Union for use inAnexo%20II.pdf) banana limits the possible usestances, of thus increasing alternative the risks active associated sub- with theof development pesticide resistance to chemical control measures by this pest. basis for highlytain lepidopteran effective pests. biological insecticides against cer-

1624 1625 II 50 of Bgl 50 0.05), > P .The C. chalcites 1; = 2.40 m height) × value of the original II digestion of viral 1.56; df 0.05). C. chalcites (Fig. 1A), which con- 50 = ≤ Bgl 34 2 , P 𝜒 23 4.95 m length × ) and previously described LC 1 wileyonlinelibrary.com/journal/ps − , or water as a control, were applied 1 − OBs mL OBs L 6 , very similar to the LC 3 1 − 10 10 × × Concentration–mortality regression slopes were fit- 34 OBs mL , 3 24 , 23 10 × 0.05). ≤ P of larvae in eachwhereas treatment recent (taken foliar feeding as damage a wasthe estimated nominal final by number value counting of of foliar perforations 100%), feeding characteristic damage of on each leafthe initial in number each of treatment perforations and presenttreatments. prior subtracting to In the insecticidal themortality was second evaluated, using trial, the same methods onlythe as first described the for trial. The percentage percentages oftreatment of virus-induced were mortality larval for subjected each SPSS to repeated-measures v.12. ANOVA Within-subjectmeans using were determined comparisons by Tukey’s test among ( the estimated 2.4 Rate ofThe acquisition of rate viral infection of acquisitionhost plant of was a evaluated lethal usingcentrations tomato infection of 2 and on banana each leaves. Con- species of firmed the identityduring laboratory and production the of absence OBs. of Additionally, the cross-contamination LC DNA and insect bioassayprofile in of second-instar the virusidentical produced to in that laboratory-infected described larvae previously was values. 3RESULTS 3.1 Identityin and efficacy biological trials activity of ChchSNPV OBs forFollowing use the production of ChchSNPV-TF1in OBs in plastic larvae reared semi-purified containers, OBs the were identity confirmed and by biological activity of ted in parallel (test for non-parallelism: laboratory-produced ChchSNPV-TF1 OBs for1.23 second instars was using a compressed-aircations handheld were sprayer performed (Solo untilcriterion 402). run-off for All application, to appli- reflecting provideperformed the a on high-volume these standardised crops. applications All treatments included 0.1%90 (v/v) as Agral wetter-sticker. When plants were completely dry,each the branch stem (tomato) of or each leafglass (banana) jar was containing placed Hoagland’s solution, in and a these 25then branches mL individually were placed in 2 Ltainer glass was containers. artificially Finally, infested each withthe con- laboratory 500 colony. second-instar The containers larvae were maintained from inplant a growth walk-in room (3.38 m width illuminated by eight fluorescent tubes (60 W).larvae At the from same time, the 25 laboratory colonyon were semi-synthetic individualised diet and reared as controlsoratory to colony determine harboured whether the an lab- tion. inapparent Groups of alphabaculovirus 25–30 infec- larvae were randomly collectedplants from at treated 5, 10, 15, 20,and 40, 24 60, h 80, 100 following and the 120individually infestation min placed process. and in at Collected 4, 25 larvae mL 6, were 9, plasticand 12 cups checked with daily semi-synthetic until diet performed death on or five pupation. The occasions,formed experiment each at a was representing different moment a in time. replicatein Percentage each mortality per- treatment values were normally distributed and were subjectedrepeated-measures to ANOVA using SPSS v.12 (SPSS Inc.).icance The of signif- treatment effects atby each within-subject sample comparisons time among was( means determined using Tukey’s test inoculum (1.45 9 ® 37 10 var. .All 1 × − .Thefirst 2 eggs placed ,var.Dwarf treatments were Bt B. thuringiensis 7 m), totalling 28 young C. chalcites × ; (iv) ChchSNPV-TF1 1 1 − Musa acuminata egg masses, but the total number 80–120 ). Five days after the infestation, 1 ∼ − 0.05). OBs L ≤ 8 P 10 × C. chalcites : 1623–1630 © 2014 Society of Chemical Industry 71 20–30 on each of the four youngest leaves of 2800 eggs plot ∼ 2015; ∼ larvae from each plot at time point 0 (immediately (Biobit 16% WP; Aragro, Madrid, Spain) as a bioinsecticide; ; (v) water control. Indoxacarb and 1 − All treatments included 0.1% (v/v) Agral 90 wetter-sticker and In the first trial, sampling from border plants was used to esti- Pest Manag Sci OBs L kurstaki (iii) ChchSNPV-TF1 1 The significance of treatmentdetermined effects by within-subject at comparisons each among themeans sample estimated using time Tukey’s test ( were 2.3 Efficacyin of greenhouses ChchSNPV-TF1 in banana plants grown Experiments on the efficacyusing of young ChchSNPV-TF1 wereCavendish) performed banana grown in plantsTenerife, mesh-structure Canary ( greenhousesfive Islands in treatments: Güimar, (Spain).WG; (i) The DuPont, 0.004% Wilmington, experiments (w/v) DE); involved indoxacarb (ii) 0.05% (Steward 30% Virus insecticide for Chrysodeixis chalcites controland checked daily untilformed death on or four occasions. pupation. Asfrom Bioassays containers groups on were four of occasions, per- samples 25 werein larvae not independent were time, sampled so thatrepeated-measures analysis percentage of variance (ANOVA) mortality in SPSS v.12 results (SPSS Inc., were Chicago, subjected IL). to Thematrix characteristics were examined of by the applying variance-covariance Maunchly’s sphericity test. www.soci.org when larvae had reachedwith the a second 1 L instar, volume plots of each were treatment, sprayed equivalent to 357 L ha banana plants, ofcentral which plants. 18 Plants werespace were border between present rows, plants at giving and each 1 m plot ten an intervals were area with of 28 a m 1 m applied to banana crops at the(http://www.agrocabildo.org/publica/Publicaciones/subt_48_ product label recommended rates Anexo%20II.pdf). were applied using402; a compressed-air Solo handheld NZwere sprayer Ltd, (Solo arranged Hamilton, New infour Zealand). a rows with Experimental fully seven plots plants randomised per row design. (4 Plots comprised trial was performedin using October–November eight 2011 replicateperformed (2011 plots using trial). a per2012 The fully treatment (2012 randomised second trial), plot with trial threewere design was replicate artificially in plots infested September per with treatment. Plants applications were made between 07:00 and 10:00 a.m. mate the prevalenceused of to infection, determine whereaspercentage larval central of survival plants larval and were mortalityC. defoliation. was chalcites For determined this, byprior collecting the to 25 the applicationpost-application. of Larvae treatments), were reared and individually at in thein 1, laboratory 3, 25 mL 5 plastic and cupsNumbers 7 with days of artificial surviving diet larvaemated until and death for foliar or the feeding pupation. ten injurylarvae on were central central esti- plants plants were oftion not of each affected larvae by plot. performed the The for periodicplant border collec- densities damage plants. were Survival of estimated of by larvaeof direct and larvae counting and of the theapplication foliar number (time feeding 0) and damage at 7 immediately dayssurviving before post-application. larvae Percentages the were of calculated on the basis of the initial number in groups of each plant. Thisvariation variation in the in size of eggof number reflected eggs the placedtreatments natural on ( plants in each plot was similar among all 1 − et al. oassay OBs L 8 5% lethal : 1623–1630 ± 10 × 71 7% lethal infection 0.001). Prior to the 2015; ± < P 11% lethal infection in ± 0.05). > P 41.323; = Pest Manag Sci resulted in 86 and ChchSNPV-TF1 1 1 4,35 − F Bt OBs mL SE) lethal infection in larvae sampled at 1 day 9 ± SE) percentage mortality of larvae collected from 10 ± × Mean ( treatments resulted in 22–33% mortality and 13–32% 10% (mean ± Bt Significant reductions in larval densities on experimental plants post-application, which increased to 81 larvae sampled at 5application of days 1 post-application (Fig. 3A). In contrast, tomato plants at different timesratory post-application until and death reared or inthree pupation. different the Experimental OB labo- treatments concentrations. involved Valuesand above one the those of bars followed indicatecomparisons means, by of identical time(repeated-measures letters points ANOVA; Tukey’s test, did within each not virus differ concentration significantly treatment for Figure 2. in 54 in larvae collectedinfection at in larvae 1 sampled at day 3and days post-application post-application. Indoxacarb to 92 mortality respectively, depending on sample time. were observed in all treatmentscompared relative with to the initial control larval ( densities, application of treatments, the meanin number the of control, larvae indoxacarb, registered and time * 7 www.soci.org O Simón 10 resulted SE) lethal 1 × − 0.001). The ± < P © 2014 Society of Chemical Industry time interaction: * OBs mL II) following digestion of genomic DNA of the original inoculum (Orig.) and the laboratory-amplified 8 0.05) indicated that > 9% (mean 54.203; 10 Bgl P ± = × 2; 3,9 F = larvae. 3.23; df 0.210). = = 2 P 𝜒 larvae that acquired a lethal virus infec- 1.581; (A) (B) 0.015). Application of 1 = treatment resulted in 55 = population used to infest plants was healthy. The , indicating no significant effect of OB concentra- Chrysodeixis chalcites 1 1 P − − 6,18 F C. chalcites (A) Restriction endonuclease analysis ( OBs L OBs L 2.306; 7 6 = 3% in larvae collected at 7 days post-application (Fig. 2). Sim- C. chalcites 10 10 ± In the first trial, treatment effects on larval mortality differed × × 12,72 3.3 Efficacyin of greenhouses ChchSNPV-TF1 in banana plants grown Lethal polyhedrosis disease was notat observed time point in zero larvae (pretreatment sample) collected in theHowever, first following or OB second treatment, trial. virus mortality2% varied in from control 1 larvae to in bothpossibly trials, owing depending to low on levels the of sample cross-contamination time, virus from adjacent treatments. significantly during the experiment (treatment 3.2 Efficacychambers of ChchSNPV-TF1 on tomato plants in growth No virus mortality was registered in control larvae rearedoratory in the to lab- determine the prevalencethe of infection, indicating that the potency of thesignificantly laboratory-produced from inoculum that of did the not source inoculum differ (Fig. 1B). number of tion increased significantly over time ( infection in larvae collected atthe 2 h laboratory post-application until and death rearedtion or in of pupation, infection. indicating The rapid percentage97 acquisi- of lethal infection increasedilar to results were obtained with OB concentrations of 1 2 wileyonlinelibrary.com/journal/ps and a test for equality ( Figure 1. (Amplif.) OBs of ChchSNPV-TF1. The DNA 1the kb original Marker inoculum Ladder (Original, (Nippon, triangular Tokyo, Japan) points)in was and second-instar used that as obtained a after molecular laboratory size production marker (M). (Amplified, (B) square Probit points) regression were lines determined of by bi tion over the time course of the experiment (concentration F 5 interaction:

1626 1627 , 1 1 Bt − − 5% ± C. chal- SE) and OBs L ± 6 10 × 3% and 62 ± ), sampled at 7 days 3% (mean 1 3% in larvae sampled − ± ± larvae as OB applications OBs L 0.001). During the first hour 9 < P 6% during the same period. 10 × ± 2% and 70 wileyonlinelibrary.com/journal/ps ± C. chalcites 17.654; = population was healthy. The percentage 7% to 7 (and two ChchSNPV-TF1 concentrations, TF1-low ± 1,13 0.05). By 12 h exposure, 62 Bt F > SE) percentage of surviving larvae (A) and recent P ± 4% during the trial, whereas mortality in the ) and TF1-high (1 1 larvae that acquired a lethal infection increased sig- ± − C. chalcites Mean ( OBs L 8 2% of larvae had already acquired a lethal infection on tomato 5% to 8 larvae on tomato plants in growth chambers and on banana 10 (A) 0.05). ± ± C. chalcites × (B) > of exposure, a significantlylethal higher infection when proportion feeding on of tomato compared larvae with thosefed that acquired on banana plants (Fig. 5). The greatestin difference was the observed sample taken at13 40 min, in which 23 and banana plants respectively. However, larvae fromsample subsequent times showed no significant host-plant-related differences in virus mortality ( of larvae from either tomato or bananation, had acquired and a this lethal increased infec- to 68 following a 24 h periodplants of respectively (Fig. feeding 5). on treated banana or tomato (1 post-application. Values abovelowed the by bars identical letters indicate didP means, not differ and significantly (ANOVA; those Tukey’s test, fol- cites plants cultivated in greenhouses. Application of 2 The present study aimedconcentrations to of determine ChchSNPV-TF1 the OBs efficacy for the of control different of to tomato plantsducing in lethal growth infection chambers in that was as were effective fivefoldto in or tomato pro- 25-fold plants higher. usually As involve pesticide volumes applications of 600–1000 L ha 23 nificantly over time ( 4 DISCUSSION Figure 4. foliar feeding damage (B)chemical (indoxacarb), in banana plants treated with water (control), treatment fell from 30 3.4 Rate ofNo acquisition of lethal viral infection virus diseasethe was laboratory observed to in determine control theing larvae prevalence that reared of the in infection, indicat- of depending on plant phenology, the growth chamber treatments , 1 1 − − 5% and OBs ± 8 9.254; Bt kurstaki 10 = 7% lethal × var. OBs mL ± 12,24 (TF1-low) and 8 F 1 10 − 0.05). × > P resulted in 71 (shown as TF1-high OBs L 1 1 8 − − 10 × Bacillus thuringiensis had similar densities of larvae . Levels of defoliation in treat- OBs mL OBs mL 1 1 time interaction: 9 − 9 − * and ChchSNPV-TF1 at 1 10 10 SE) lethal infection in larvae sam- × Bt × ± OBs mL OBs mL 0.001), likely reflecting the differences in 9 8 < : 1623–1630 © 2014 Society of Chemical Industry 10 10 P plots was 30.2, 18.7, 26.4, 28.6 and 33.5 larvae × × 71 SE) percentage mortality of larvae collected from 1 4% lethal infection in larvae sampled at 5 days − 6% (mean 0.05) (Fig. 4B). ± ± ± > (TF1-high), in 2011 (A) and in 2012 (B) in greenhouse trials. 2015; 11.118; P 1 OBs L − = 9 Mean ( respectively, whereas at 7 days post-application these 10 4,35 OBs L F 1 × were intermediate and were similar among these treatments 9 − 1 0.001). Application of ChchSNPV-TF1 at 1 10 − ) and two concentrations of ChchSNPV, 1 In the second trial, all treatments resulted in a significant Foliar feeding damage values were significantly affected by treat- < × (B) (A) Bt increase in mortality compareding with the the control experiment treatment dur- (treatment banana plants atthe four laboratory different until death intervals orapplication pupation. of after water Experimental (control), treatment treatments indoxacarb, involved and( reared in post-application. In the indoxacarb treatment, mortality fell from pest densities in eachdefoliation treatment were (Fig. registered 4B). inest The control highest levels plants, levels of of whereasChchSNPV-TF1 defoliation at the 1 were low- observed in plants treated with 1 Values above the barsletters did not indicate differ significantly means, for comparisons of and treatmentstime within point each those (repeated-measures ANOVA; followed Tukey’s test, by identical (Tukey’s test, P Pest Manag Sci pled at 1 day post-application,infection which increased in to larvae 86 sampledIn at contrast, 5 application days of post-application 1 (Fig. 3B). resulted in 60 and 1 Figure 3. Virus insecticide for Chrysodeixis chalcites control www.soci.org densities had fallen to 20.8, 4.7, 7.9, 7.0 and 3.1 larvae plant (Fig. 4A). ment ( plant lethal infection in larvae sampled atincreased 1 day to post-application, 96 which ChchSNPV-TF1 at 1 in Fig. 4A), whereas the treatments involving indoxacarb, which represents 69, 25, 30,larvae 24 respectively and (Fig. 9% 4A).plot survival Survival of plants values experimental were for independent larvaetimes of of post-application sampling central performed onplots. at plants The earlier greatest at reduction in thein numbers the edge of treatment larvae of involving was 1 experimental observed mL ments involving indoxacarb, 42 11 In Bt 10 et al. 45 , How- × 46 larvae. 38 , 44 , 44 39 : 1623–1630 but also the larvae from Nonetheless, 71 42 44 , S. exigua 43 2015; C. chalcites , depending on plant phe- 1 . Higher OB application rates 1 − − Previous studies with SeMNPV would be equivalent to approx- 1 47 Pest Manag Sci , − 44 OBs ha feed on the undersides and internal plot. This is equivalent to 3.57 12 OBs L 2 9 10 × 10 × –2.0 larvae collected at 6 h post-application. larvae on tomato plants than on banana plants, C. chalcites 12 10 × OBs was necessary efficiently to protect banana young S. exigua 9 C. chalcites 10 Effective spray targeting of the feeding sites of the pest is In the present study, an application volume of 1 L containing Infection acquisition experiments revealed that larvae that fed ChchSNPV-TF1 appeared to be slightly more effective in control- The highest concentration of ChchSNPV-TF1 OBs was more × plants present in a 28 m also likely greatlyinsecticides, to the influence effectiveness of the which dependsbehaviour performance on and larval the of probability feeding of consuming baculovirus a lethal dose of OBs. 1 OBs for the treatment ofstage. However, 1 given ha that pesticides of are usually plants applied to at banana crops the at same volumes phenological of 1600–2000 L ha Early instars of parts of young leaves.underside Given of banana leaves this would be behaviour, expected to OBs providecipal the prin- source applied of inoculum, to especially the under greenhousewhere conditions, UV protection conferred by the leaves, infiltering addition activity to of the UV the greenhouseimprove structure, the is persistence of likely OBs markedly on plant to surfaces. recorded 15–31% lethal infection,tion, in depending on OB concentra- imately 1.6 ever, larvae that feed on plants contaminatedacquired with lethal ChchSNPV-TF1 infection farAfter more 20 rapidly min, than betweenbanana 8 and tomato andinfection plants, 16% and respectively, of died had duringthe acquired prevalence subsequent of a lethal rearing. infection reached lethal Similarly, 45–48%.underline by These the findings 6 h, importance of pest feeding behaviour during the phenological stage of banana cropsexplain grown in the the higher open concentration field ofefficient may OB pest applications control required compared for withprotection of those young required banana for plants grown effective in greenhouses. on tomato plants acquired aas lethal infection rapidly approximately as twice thoseonly that observed fed to on be banana. significantto However, during this virus-treated the effect plants, first was hour whereasferences of no were exposure significant observed at plant-relatedble subsequent dif- that sample the times. hardness Ittomato of is may have banana possi- reduced larval leaves feeding rates compared duringof with the exposure. first those hour The first of 48 hacquisition of of feeding a are considered viral crucial infection. in the nology, the rate of 1 are likely to be necessary to protectciently, fully given grown banana the plants high effi- volumecover applications the needed foliage effectively of large-sized to plants. ling as a lower concentration ofhigh prevalence OBs of was pest required mortality.may to In environmental produce factors banana a such field very as trials, temperature,dates not leaf and surface sunlight only have exu- reduced treatment efficacy, the persistence of ChchSNPV OBscrops in on the greenhouse-grown Canary banana Islands needs to be confirmed experimentally. effective in reducing plantinsecticides defoliation in than banana crops. the In chemical manytreatments instances, and alphabaculovirus can be as effective as chemical insecticides. contrast, when pestsynthetic populations insecticides, virus-based have insecticides represent developed aopportunity unique resistance for pest to control, as cross-resistance between synthetic insecticides and virus insecticides has neverto been the reported markedly owing different mode of action of these agents. 6 1 21 − , 10 the 17 × 40 In this www.soci.org O Simón 24 OBs ha 9 survived in treatments, 10 15 , Bt Bt © 2014 Society of Chemical Industry × 14 –2 Spodoptera exigua 8 control in field- or Helicoverpa armigera 0.05). 10 The apparently low effi- ≤ × P 41 treatments. Application of Bt resulted in exceptionally high 39 , 1 38 C. chalcites − insecticide currently used in banana )resultedinlarvalmortalitythatwas 1 − Bt OBs mL 90%) in larvae collected at various inter- 9 ∼ 10 × OBs mL 8 SE) percentage mortality of larvae collected at different early instars on banana plants does not usually 10 ± × of ChchSNPV-TF1 to banana and tomato leaves. Values above Mean ( 1 − C. chalcites In greenhouse trials on young banana plants, the low-rate virus The potential of ChchSNPV-TF1 as a bioinsecticide is likely to be Surprisingly, larvae treated with indoxacarb or crops in the Canary Islands (Spain) alsotence suggests of the resistance possible exis- to these products in the pest population, high numbers during the first trial,ments and appeared the to efficacy of decrease these overincreasingly treat- time. resistant Although to larvae become baculovirus infection as they age, whereas larval survival onwere similar to treated those of plants chemical and and defoliation levels thehigherrateof1 treatment (1 wileyonlinelibrary.com/journal/ps although this was not determined in the present study. affected by numerous factors, including the timingin of applications combination withas the susceptibility age to structure infection decreases of with the larval stage. pest population, of ChchSNPV-TF1. Thisto appeared to define be application a rates useful for starting point that we used would be equivalent to 1.2 greenhouse-grown tomatoes. Crucially, this rate is1000-fold approximately lower than OBcommercial-virus-based application insecticides rates determined such formultiple as other nucleopolyhedrovirus (SeMNPV) or nucleopolyhedrovirus (HearNPV). Figure 5. OBs mL time points and reared in the laboratory following application of 2 cacy of indoxacarb or the translate into yield losses or cosmeticcontrast, damage late to instars banana often feed fruits. on In fruits,significant resulting losses in when economically infestation levels are high. the bars indicate means. Horizontal linesdifferences with between asterisks host indicate plants significant (Tukey’s test, levels of pest mortality ( vals post-application, and significantlydamage reduced compared levels with of the other feeding treatments. significantly higher than those of chemical and lower susceptibility offor later through larval increased stages consumptionNPV-infected larvae can dying of earlier or be OB-contaminated OBs leaf surfaces compensated producedleading to a by higher ingested other dose of OBs. study, OBs were appliedthe to pest banana population plants was in whenare the the second highly majority instar. At of susceptible this stage,plants to larvae compared infection with and thatby of cause later little instars. Indeed, damage foliar to feeding

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We and also thank Itxaso Teresa González, IbáñezModesto Ernesto (UPNA) G Del Fuentes for Pino and technical (ICIA)This for study collaboration received in financial support greenhousede Investigaciones from assays. 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