Arvinth, S.

Kalaivani, A.

kumar, P. Mohan

Kumar, R. Raj

Sathyapriya, P.

English

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Bio-efficiency of lectin on red spider mite247Red spider mite (RSM) (Oligonychus coffeaeNietner), (Acarina: Tetranychidae) infestation is alimiting factor in tea productivity. Severe infestationleads to defoliation of maintenance leaves, whichultimately affects the bush physiology and bushhealth. Control measures of RSM are mainlyachieved by the use of synthetic acaricides(Amarasena et al., 2011). Emphasis is given for theapplication of eco-friendly botanical pesticides forthe pest management in tea. Number of botanicalpesticides was reported to control several insect pests(Prakash and Rao, 1997; Naveena et al., 2010). Bio-efficacy of plant lectin for insect control has alreadybeen reported (Czapla and Lang 1990; Roy et al.,2008). Pest resistant crops attract attention in a worldwhich is dominated by the use of chemicalinsecticides. Lectins are carbohydrate bindingproteins present in a range of organisms which arewell characterized in plants and posses defenseproperties like anti-fungal, anti-bacterial andinsecticidal (Lis and Sharon, 1998; Peumans andVan Damme, 1995). Bio-efficacy of garlic bulblectin on O. coffeae infesting tea has been provedby Roy et al. (2008). The present study was aimedat evaluating the bio-efficacy of chito-oligosaccharide/chitin binding lectin of Cucurbitamaxima (pumpkin) on RSM in the laboratory.Pumpkin fruit obtained from the local marketwas peeled off, deseeded and the edible part wasused in crude lectin isolation. Purification ofpumpkin phloem lectin (PPL) was accomplishedwith affinity chromatography on chitin columnfollowing Narahari and Swamy (2010). Mites werecollected from the infested tea fields of UPASIExperimental Farm, Valparai, Tamil Nadu, India.Bio-efficacy of pumpkin phloem lectin on red spider mite,Oligonychus coffeae (Nietner) infesting tea(Manuscript Received: 10-01-13, Revised: 18-06-13, Accepted: 01-07-13)Keywords: Camellia spp., lectin, mortality, Oligonychus coffeae, ovipositional deterrenceThe mites were maintained and mass cultured inlaboratory using the method described byRoobakkumar et al. (2010). Two laboratory bio-assays were conducted with purified protein on RSMmortality and ovipositional deterrence individually.Each treatment was replicated five times. Maturedtea leaves without any RSM infestation werecollected from the field and leaf discs of 2 cmdiameter were placed on moist cotton in a Petri dish(9 cm diameter). Different concentrations of PPL(10, 20, 30, 40 and 50 µg ml-1) was overlaid on theleaf discs and allowed to dry at room temperature.Leaf discs overlaid with distilled water served ascontrol. Ten adult RSM (less than 48 h old) weretransferred from the stock onto the PPL treated leafdiscs. Plates were maintained in the roomtemperature and the treated leaf discs were observedafter 24, 48, 72 and 96 hours under binocularmicroscope (Olympus No.1220). Ovipositionaldeterrence was studied by allowing the 10 adultfemales (individuals/treatment) to lay eggs both onthe PPL treated and untreated control leaf discs. Thedegree of deterrence was assessed in terms ofdifference in the number of eggs deposited by thefemales on control and treated leaves.For lectin purification pumpkin fruit (100 g)was homogenized with 100 ml of 0.1 M PBS-β-ME(phosphate buffer saline containing β- mercapto-ethanol) (1:1 v/w) contains 20 mM sodiumphosphate buffer, pH 7.4, 150 mM NaCl, 0.02 percent sodium azide and 10 mM β - mercaptoethanol.Purified PPL was estimated using Bradford method(1976) and about 10 µg of protein was analyzed on15 per cent SDS-PAGE for its subunit molecularweight in accordance with Laemmli (1970). NativeShort  Scientific  Report Journal of Plantation Crops, 2013, 41(2): 247-250Kalaivani et al.248PAGE was run without adding SDS in gel as wellas in running buffer. Haemagglutination and itsinhibition assays were carried out in 96 well microtiter plates using 4 per cent rabbit erythrocytesdescribed by Narahari and Swamy (2010) and theagglutination was observed visually. Data generatedfrom bioassay were subjected to statistical analysis(ANOVA) and results presented in accordance withDuncan’s multiple range test (DMRT). TheDiscrimination Quotient (DQ) was calculated usingthe formula, DQ = C-T/C+T, where C = number ofeggs on control leaves; T = number of eggs on treatedleaves.Whole pumpkin fruit was used for theisolation of crude protein, since the lectin in phloempart has the tendency to get bound to the column.All active fractions were lyophilized and used forfurther studies. In native PAGE (Fig. 1), lectin wasresolved as a single band which confirmed thehomogeneity of purified lectin (free from otherproteins of pumpkin phloem). Pumpkin lectins werereported to be existing as a dimer (Bostwick et al.,1994). Presence of ~23 kDa single band in SDS-PAGE (Fig. 2) indicating that subunits of the PPLare having the same molecular weight homodimer(Narahari and Swamy, 2010). Lectin from snakegourd (Cucurbitaceae) was reported as a hetero-dimer by Narahari and Swamy (2011) whichdemonstrated the diversity of lectin structure withinthe family. Biological and bio-ecological studies onRSM have already been reported (Vitarana, 2000;Babu et al., 2010). In the present study, PPL bioassayagainst RSM resulted in total mortality in 96 h at 50µg ml-1 concentration (Table 1). Survival ofindividual mites was observed by touching each mitewith a single hair brush. Mites that were unable tomove were considered dead and those which wereFig. 1. Native PAGE analysis of PPLNote: Presence of single band indicates the homogeneity of lectinpreparationFig. 2. SDS-PAGE analysis of PPLLane 1, 2, 3- purified PPL (~23kDa), Lane 4- protein marker,Lane 5, 6- unbound pumpkin phloem proteins of chitin columnTable 1. Influence of PPL on RSM mortality with reference to incubation timePPL (µg ml-1) Mortality (%) after24 h 48 h 72 h 96 hControl 00 ± 00 (1.00)a 00 ± 00 (1.00)a 00 ± 00 (1.00)a 00 ± 00 (1.00)a10 24 ± 2.1(5.00)b 34 ± 2.4 (5.92)b 46 ± 2.4 (6.86)b 52 ± 2 (7.28)b20 36 ± 2.4(6.08)c 50 ± 3.1 (7.14)c 54 ± 2.4 (7.42)c 60 ± 3.1 (7.81)c30 44 ± 2.4(6.71)d 56 ± 2.4(7.55)cd 60 ± 3.1 (7.81)c 70 ± 3.1 (8.43)d40 54 ± 2.4(7.42)e 62 ± 2.0 (7.94)d 72 ± 3.7 (8.54)d 82 ± 3.7 (9.11)e50 66 ± 2.4(8.19)f 74 ± 2.4 (8.66)e 82 ± 2.0 (9.11)e 100 ± 0 (10.04)fNote: Figures denote mean of five replicates followed by ± standard error. Figures in the parenthesis are transformed values of (x+1)0.5. Figures followed bythe same alphabet in a column do not differ significantly at P = 0.05 according to DMRTBio-efficiency of lectin on red spider mite249alive were counted. Slow movement of RSM wasnoticed after 24 h in test plates when compared tothe control. Dose and time dependant mortality wasobserved in the present study. As the concentrationof lectin increased, mortality rate of RSM enhancedconcurrently (Table 1). Incubation period also playedan important role on RSM mortality where ~60 percent mortality was observed even at lowerconcentration. Results of this study showed thepotential of PPL as insecticidal by causingsignificant mortality on RSM. In a similar kind ofstudy, this statement was accordance withRoobakkumar et al. (2010) where he tested the garlicaqueous extract, neem kernel aqueous extract andpongam kernel aqueous extract which wereeffectively used for the control of RSM.Lectins are different in their specificity; forinstance, Roy et al. (2008) reported the toxicity ofmannose binding garlic bulb lectin against RSM.Specificity of the garlic bulb lectin and pumpkinphloem lectin is different because the former ismannose specific and the latter is specific to chito-oligosaccharides. Mannose specific lectin target sitein the insect is glycoproteins present in brush bordermembrane vesicles while chitin binding lectins havetarget site in the glycoproteins of peritrophicmembrane which contains chitin like structures(Macedo et al., 2008). Huesing et al. (1991) havedemonstrated the toxicity of chitin binding wheatgerm agglutinin (WGA), rice lectins and nettlelectins against Callosobruchus maculates. It hasbeen assumed that PPL may bind to the chitin orchitinacious structures (digestive enzymes or theproteins) present inside the body of RSM. However,further studies are required in this line. Since RSMhas a short life span, the population density attainsits peak in short duration which leads to severe croploss. Hence, control of their population demandsimmediate attention. Ovipositional deterrence whichis considered as significant characteristic feature inRSM control and discrimination quotient value ofdifferent concentrations of PPL on RSM is given inTable 2. DQ ranging from 0 to 1 is an index fordetermination of the effect of PPL on RSM’sovipositional behaviour. DQ value of 0.52 wasachieved at 50 µg ml-1 of PPL which means that theadult mites showed discrimination among the treatedleaves with respect to number of eggs laid. RSMlaid lesser number of eggs on PPL treated leaves thanthe untreated control. After 96 h 100 per cent mortalitywas achieved hence the eggs were laid before 96 hindicating the moderate interference of lectin in itsovipositional behavior. In a previous study, seed andleaf extracts of Datura stramonium L. decreased theoviposition in two-spotted red spider mite, T. urticae(Koch) (Kumral et al., 2010). Application of acetoneextract of garlic bulb on leaves of Phaseolus vulgarisreduced fecundity of T. urticae (Barakat et al., 1986).Roobakkumar et al. (2010) has reported ovipositionldeterrence by garlic bulb aqueous extract and neemkernel aqueous extract when sprayed on RSM. Theneed of the hour is to identify an alternative methodfor chemical control of pest and an eco-friendly wayof pest management. In addition, the fact that a lectinpresent in an edible plant may contribute towards aneasier acceptance as an insect resistance factor intransgenic crop plants (Sadeghi et al., 2008). Fromthe results, PPL may be considered to be a potentcontrol component of integrated pest management(IPM) due its propensity to control the survival rateand fecundity of the RSM and its usefulness inaffecting the normal metabolism of RSM. Detailedstudy of the mechanism of lectin action in mite willstrengthen its application in biotechnology.AcknowledgementsAuthors acknowledge National Tea ResearchFoundation (NTRF) for their financial assistance.Authors are grateful to K.N. Chandrashekara, SeniorBiotechnologist for his suggestions in preparing themanuscript and A. Roobakkumar for his help inlaboratory bio-assay. We extend our thanks to KovaiMedical Center and Hospital (KMCH), College ofPharmacy for providing rabbit erythrocytes.Table 2. Ovipositional deterrence and DQ value of PPL on RSMPPL Dose No. of eggs DQ value(µg ml-1) observed at 96 h*10 85.6 ± 6.94 (9.31)e 0.1220 73.8 ± 2.15 (8.65)d 0.1930 63.8 ± 2.80 (8.05)c 0.2640 43.2 ± 2.17 (6.65)b 0.4350 34.0 ± 1.64 (5.92)a 0.52Control 109.4 ± 4.41 (10.51)f 0.00Note: *Values represent mean of five replications ± SE. Values in theparenthesis are transformed values of (x+1) 0.5. Figures followed by thesame alphabet in a column do not differ significantly at P = 0.05 accordingto DMRTKalaivani et al.250UPASI Tea Research FoundationTea Research Institute, Valparai 642 127,Coimbatore District, Tamil Nadu, India1Vanavarayar Institute of Agriculture, Pollachi 642 123,Coimbatore District, Tamil Nadu, IndiaA. KalaivaniP. SathyapriyaS. Arvinth1R. Raj Kumar*P. 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Bio-efficacy of pumpkin phloem lectin on red spider mite, Oligonychus coffeae (Nietner) infesting tea

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Bio-efficacy of pumpkin phloem lectin on red spider mite, Oligonychus coffeae (Nietner) infesting tea

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