Spring 2003 Resistant Pest Management Newsletter Vol. 12, <strong>No.2</strong>was 0.90, which indicated that resistance to spinosaddid not develop in P. xylostella after continuousselection at least up to the 7th generation. Crossresistanceto commonly used insecticides was alsostudied. The toxicity of commonly used insecticideswas also calculated against spinosad-selected P.xylostella. Comparisons of LC50 values indicated thatmonocrotophos (1.21278%) was the least toxic of allthe insecticides whereas cypermethrin (0.0276%) wasfound to be the most toxic. The relative toxicity ofcypermethrin, dichlorvos, malathion, endosulfan, andcarbaryl reveal that these insecticides were 43.94,20.80, 5.85, 4.24, and 4.17 times more toxic thanmonocrotophos.INTRODUCTION The diamondback moth (DBM),Plutella xylostella L. (Lepidoptera: Plutellidae) is oneof the major constraints in the profitable cultivation ofcole crops. The pest occurs in endemic form with highpopulation densities on early and late sowncauliflower. In case of severe infestation, the growinghearts are also damaged, affecting the production ofmarketable curds. The control of P. xylostella hasdepended primarily and extensively on the use ofinsecticides recommended for the last over forty years(Syed, 1992). However, the promiscuous use of anumber of commercial insecticides lead to thedevelopment of resistance in this pest in most countriesof Southeast Asia (Georghiou, 1981). Many factors likepronounced cultivation of early and late varieties ofcauliflower, intensive use of conventional insecticides,and prospects of the higher value of the crop duringoff-season have been outlined for its extraordinarypropensity to develop resistance to all classes ofcompounds. By and large one of the countermeasuressuggested to combat the menace of resistance is theintroduction of, and switching over to, newer,ecofriendly, and more potent insecticides.Spinosad is a new class of polyketide-derivedmacrolide effective against a broad range of pestsbelonging to orders lepidoptera, diptera, andhymenoptera (Sparks et al., 2001). It contains a mixtureof two very active principles: Spinosyn A andSpinosyn D, and is derived from a new species of soilbacterium Saccharopolyspora spinosa which acts bothas a contact and a stomach poison.Electrophysiological evidences have demonstrated thatit alters nicotinic currents in neuronal cell bodies andalso disrupts the functions of GABA receptors of smallneurons in the central nervous system (Salgado et al.,1997).Keeping in view the introduction of this chemicalin India and the potential of P. xylostella to developresistance to all classes of compounds, a need wasrealized to generate base line susceptibility data andquantify resistance to spinosad as well as crossresistancewith other chemicals with the aim to detectshifts in susceptibility following its commercial useand to formulate future strategies to manage this pest.MATERIALS and METHODSInsect Rearing: The culture of P. xylostella wasinitiated by collecting about 200 larvae from farmers'cauliflower fields and brought to the laboratory forfurther multiplication at a regularly maintainedtemperature of 24±20C. The larvae were reared oncauliflower leaves till pupation. The adult moths wereallowed to lay eggs in oven-dried glass jars havingcauliflower leaves to serve as substrate for oviposition.They were provided with 10% honey solution fortifiedwith multivitamins for feeding on a cotton swab. Theneonates were provided with fresh cauliflower leavesto feed upon. At every successive instar, the larvaewere shifted to clean jars containing fresh leaves. Thewhole stock was divided into two lots. One lot wasnamed as parental stock and the other was used forexposure to spinosad.Preparation of Insecticidal Concentration: Theproprietary products of all the insecticides were used toprepare one percent stock solution in acetone fromwhich further dilutions were prepared subsequently.Bioassay and <strong>Lab</strong>oratory Selection: FAO method<strong>No.2</strong>1 (Busvine, 1980) for topical application ofinsecticide to the larvae with slight modification wasfollowed. Instead of directly treating the larvae withoutany substrate, the larvae released on cut discs ofcauliflower leaves of the size of a petri dish (2.5 cmdiameter) were treated to simulate the application ofinsecticide in the field. To facilitate the movement ofthe larvae on both sides of the leaf disc, the leavesbearing slightly thick midribs and veins were selectedfor cutting the leaf discs. Before spraying, ten 3rdinstar larvae were released on the upper side of the leafdisc as one replication. 1mL each of the insecticidalconcentrations were sprayed on each side of the leafdisc with Potter's tower at 5lb/inch^2 pressure. Allthree replications were maintained for eachconcentration. After the treatment the petri dishes wereshifted to BOD at 24±10C with 60-70% relativehumidity. Larval mortality was recorded after every 72hours of the exposure by counting the larvae as deadwhen they did not resume activity after repeatedproddings. The survivals from the experiments,affording around 85 percent mortality, were reared tothe next generation. The progeny of the first survivinglot was termed the F1 generation and the exposures andselections were conducted up to 7 generations. Theparental strain was also maintained through withoutexposure.Statistical Analysis: Data on mortality was subjected toAbbott's formula for correction wherever required28
Spring 2003 Resistant Pest Management Newsletter Vol. 12, <strong>No.2</strong>(Abbott, 1925). LC50 values of allinsecticides were determined by Probitanalysis (Finney, 1971).Quantification of Insecticidal Resistance:The degree of development of resistancethrough different generations wasdetermined by working out LC50 values ineach generation and thus computing theresistance ratio by dividing the LC50 valuefor that generation with LC50 value of theF1.Cross-resistance with Other Insecticides:The studies on cross-resistance ofspinosad-selected strains of P. xylostella tocommonly used insecticides viz.monocrotophos, malathion, endosulfandichlorvos, cypermethrin, and carbarylwere also made as per the proceduredescribed.RESULTS and DISCUSSION Selection pressureof spinosad and responses produced indifferent generations has been presented inTable 1. Significant variations in the LC50values of all seven generations indicatethat there was no consistency in thetoxicity of spinosad to P. xylostella larvae.The LC50 in the 2nd generation(0.000299%) decreased slightly comparedto the 1st generation with a furtherdecrease in the 3rd, 4th, and 5thgenerations. Thereafter, in the 6th and 7thgenerations, it increased slightly but nevercame at par with the F1. Thus, thedecrease in LC50 values in the subsequentgenerations showed slightly increasedsusceptibility. The resistance ratios also showed asimilar trend. The resistance ratio for the 7th generationas compared to the 1st generation was 0.90, whichindicated that resistance to spinosad did not develop inP. xylostella after continuous selection at least up to 7generations. From this, it can be construed thatspinosad can be used commercially as an alternative toparticularly those insecticides against which P.xylostella has developed resistance. As per theavailable literature, such studies have not beenundertaken on the development of resistance tospinosad in P. xylostella. However, the bioefficacy ofspinosad to 10 different strains of Pseudoplusiaincludens (Walker) revealed that LC50 values variedfrom 4.19 to 13.46 ppm (Mascarenhas and Boethal,1997). The results of topical bioassays conducted bySparks et al. (1998) with spinosyn A and D againstHeliothis virescens larvae showed LC50 values rangingbetween 1.28 to 2.56 µg/g.The toxicity of commonly used insecticides wasalso calculated against spinosad-selected P. xylostella(Table 2). The comparisons of LC50 values indicatedthat monocrotophos (1.21278%) was the least toxic ofall the insecticides used against this strain whereascypermethrin (0.0276%) was found to be the mosttoxic. Data pertaining to the relative toxicity ofcypermethrin, dichlorvos, malathion, endosulfan, andcarbaryl revealed that these insecticides were 43.94,20.80, 5.85, 4.24, and 4.17 times more toxic thanmonocrotophos. It could be concluded that spinosadselectedstrains of P. xylostella did not show any crossresistance to these insecticides. As per literaturescreened, no such studies have been carried out so faron the quantification of cross resistance to thespinosad-selected strain of P. xylostella. Since themode of action of spinosad is altogether different fromother insecticides, the chances of any cross-resistancein this case seem to be quite dim (Salgado et al.1997).29
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