Proceedings of the National seminar on

Harmful/Beneficial Insects of Agricultural Importance withSpecial Reference to the Nuisance Pest Luprops tristis in

Rubber Plantations

17th & 18th February 2011

Organised by

P.G. and Research Department of ZoologySt. Joseph’s College, Devagiri, Calicut

Co-sponsored byDepartment of Science and Technology (DST), University Grants Commission

(UGC) and Kerala State Council for Science, Technology & Environment(KSCSTE)

© P.G. and Research Dept. of Zoology, St. Joseph’s College, 2011

ISBN 978-81-921589-0-7

Rs.100.00

Published byPrincipalSt. Joseph’s College, Devagiri,Calicut - 673008, Kerala

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Biocidal activity of algal seaweed on insect pest and fungal plant pathogen

Rajesh S., Asha A., Kombiah P. and Sahayaraj K.*Crop Protection Research Centre, Department of Advanced Zoology and Biotechnology, St.Xavier�s College (Autonomous), Palayamkottai-627002, Tamil NaduE-mail: ksraj42@gmail.com*

Abstract

Sea weeds/marine macroalgae are therenewable living resources which are rich sourceof structurally important novel and biologicallyactive metabolites. The thalli extract [Hexane,Chloroform and Methanol] of Caulerpascalpelliformis (R.Br.) Web. V. Bosse(Chlorophyceae) was tested against Dysdercuscingulatus (Fab.) third nymphal instar and fungalpathogen, Fusarium oxysporum f. sp.vasinfectum (Atk.) Snyd and Hans. (FusariumWilt of cotton) at different concentrations. Theresults of insecticidal activity of HE, CH and MEextracts caused 92.59, 88.89, 84.62 %mortality at 96 hours of exposure respectively(LC50 = 180.80, 232.40, 276.10 ppm for CH,ME and HE extracts, respectively). The mortalitygradually increased as the concentrationincreased. The Hexane (11.33±0.33mm zoneof inhibition: ZI), Chloroform (10.33±0.33mmZI) and Methanol (12.66±0.33mm ZI) extractsof C. scalpelliformis were found to inhibit thegrowth of F. oxysporum. Minimum inhibitoryconcentration (MIC) for Methanol was foundto be 8mg/ml; for Hexane � 8mg/ml and forChloroform it was higher � 11mg/ml. It isconcluded that C. scalpelliformis possessNymphicidal and fungicidal activity; hence thisalgal extract can be used as biocide in agriculture.

Key words: algal seaweed, Caulerpascalpelliformis, biocides.Short title: Biocidal activity of algal seaweedCaulerpa scalpelliformis.

Introduction

Marine algae are widely spreadthroughout the coastal areas around many

continents. Almost al investigations carried outon these materials focused on the differentaspects concerned with their nature and growth.Little is known about its antimicrobial andinsecticidal properties. These are rich source ofstructurally important novel and biologicallyactive secondary metabolites with antimicrobialactivities (Borowitzka & Borowitzka 1992, DelVal et al. 2001, Ely et al. 2004, Cordeiro et al.2006) and also having pharmaceuticalimportance (Febles et al. 1995).

Cotton is the most economicallyimportant natural fiber materials in the world.The major obstacles hindering cotton cultivationare insect pest and disease infestations.Dysdercus cingulatus (Fab.) commonly knownas red cotton bug is an important pest of cotton.It is distributed all over the cotton producingregions of India (David & Ananthakrishnan2004, Sahayaraj & Illayaraja 2008). Thesepests are difficult to control by insecticideapplication because they are highly mobile andhave many alternate wild hosts belonging toMalvaceae (Iwata 1975, Kohno & Ngan,2004). Nymphs and adults of D. cingulatusfeed mainly on developing or mature cotton boll.

Wilt of cotton (Gossypium spp) is aimportant vascular disease caused by the soilborne pathogen Fusarium oxysporumSchlechtend f.sp. vasinfectum (Atk.) Snyd andHans. The disease is widespread and causessubstantial crop losses in most of the majorcotton-producing areas of the world (Assigbesteet al. 1994, Wang et al. 2004).

Due to hazards associated with theincreased use of synthetic pesticides the use of

National Seminar on Harmful/Beneficial Insects of Agricultural Importance, 17- 18 February 2011,P.G. & Research Dept. of Zoology, St. Joseph’s College, Devagiri, Calicut-673008, Kerala

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biopesticides esp., from marine algae has gainedconsiderable attention on the eco-friendlyapproaches for the management of insect pestand plant pathogens. Thus the exploitation ofpesticides of marine algae-origin in agriculturehas reduced the risk factor appreciably not onlyfor the public health and human well-being butalso for the environmental pollution. In presentinvestigation, the bioefficacy of algal seaweed,Caulerpa scalpelliformis was evaluatedagainst insect pest, D. cingulatus and fungalplant pathogen, F. oxysporum.

Materials and methods

1 Collection, extraction and preparation ofseaweed extract- The algae, Caulerpascalpelliformis was collected by hand pickingfrom the submerged marine rocks atIdinthakarai, Tirunelveli coast of Tamil Nadu,India during low tide. The seaweed was washedthoroughly thrice with tap water and once withsterile distilled ware to remove salt andepiphytes. They were then shade-dried for twoweeks and partially powdered using domesticblender. 100 gm of powdered seaweed waspacked in Soxhlet apparatus and refluxed withhexane (30ÚC � 40ÚC), chloroform (30ÚC �40ÚC) and methanol (40ÚC - 50ÚC)individually for 24 hours continuously. Extractionsolvent was evaporated and dried over SodiumSulphate in dessicator under Vacuum. The crudeextracts were stored at -20ÚC until further use.

2 Phytochemical analysis- The qualitativephytochemical analyses were performed usingstandard procedures (Harbone 1983, Trease& Evans 1987). Tannins (Aparna Buzarbarua2000), total phenols, ortho-dihydric phenols,bound phenols, alkaloids (Harbone 1973) andflavonoids (Suresh et al. 2010) were quantified.

3 Pest collection and rearing- Nymphs andadults of D. cingulatus were collected fromcotton fields in the Tirunelveli district of TamilNadu, India. The collected pest weremaintained in the insectory under laboratoryconditions (28±2ÚC; 70-75% RH; 11L&13Dhours photoperiod) in plastic containers (300ml capacity) containing a layer of sterile moist

and coarse sand (4 cm thick). They were fedwith its natural host cotton and artificial diet. Thelaboratory emerged third nymphal instars of D.cingulatus were used for the presentexperiment.

4 Nymphicidal activity bioassay- Bioassaystudies were carried out using uniform sized thirdinstar D. cingulatus, selected randomly fromthe stock culture. Six insects were placed in theplastic containers (300 ml capacity). Differentconcentrations of C. scalpelliformis extract,100, 200, 400, 600 and 800 ppm were mixedin artificial diet. Insects were allowed to feed onartificial diet mixed with algae extract for 96hours continuously. Five replications (n=30)were maintained for each concentration.Mortality was corrected using Abbott�s formula(Abbott 1925) if any was recorded in controlcategory. Then the data was subjected to Probitanalysis (Finney 1971). After 96 hrs alivenymphs were provided with artificial diet.

5 Isolation and Identification of Fusariumoxysporum f.sp. vasinfectum- Fusariumoxysporum f.sp. vasinfectum was isolatedfrom infected cotton plants(Melameignanapuram, Tenkasi district, TamilNadu, India) and were used for the experiment.The pathogen was isolated, sub-cultured onPotato Dextrose Agar (PDA) medium andidentified using standard protocol (Burgess etal. 1994).

6 Antimicrobial bioassay-

6.1 Agar well diffusion assay: Antifungalactivity was carried out using agar well diffusionmethod (Irobi et al. 1996). Petri plates wereprepared with 20 ml of sterile PDA. Wells weremade using sterile cork borer under asepticcondition. The C. scalpelliformis extract withvarious concentrations (0.05%, 0.1%, 0.2%,0.4% and 0.8%) were prepared using DimethylSulphoxide (DMSO) and were added to therespective wells. Carbendazim (Bavistin)(0.03%) was used as positive control andDMSO was maintained as negative control.They were incubated at 27ÚC for 3 days. The

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zone of inhibition was measured using a rulerand expressed in mm.

6.2 Minimum Inhibitory Concentration(MIC) determination (Rapid Susceptibilityassay)- MIC was determined using glucoseutilization by the fungi in microwell plate (Wetteret al., 2003). The medium, RPMI 1640 with0.4% glucose were buffered with 0.165 M 3-(N-morpholino)-Propanesulfonic acid. The C.scalpelliformis extract with variousconcentrations (1 mg, 2 mg, 3 mg, 4 mg, 5 mg,6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg and 12mg) were prepared using Dimethyl Sulphoxide(DMSO). The fungal conidial suspension wascollected and adjusted with glucose deficientRPMI 1640 medium to an OD530 of 0.11. Theassay was initiated by inoculating wells with100 l of RPMI 1640 with 0.4% glucose and50 l of extracts and 50 l of conidial suspensionto the respective wells. The well with RPMI 1640with 0.4% glucose served as glucose control.They were incubated at 27ÚC for 3 days.Residual glucose level was detected by additionof 50 l of an enzyme color mix comprising of0.6 M Sodium Phosphate buffer/ml (pH 6.0),360 g of 4-amino antipyrine/ml, 490 g of N-ethyl-N-Sulfopropyl-m-toluidine/ml, 0.68 U ofhorseradish peroxidase/ml and 0.4 U of Glucoseoxidase/ml to each well. After 30 minutes thecolor intensity was measured at OD490 with areference reading of OD630 in a microplatereader (Glaxo). The percent residual glucoseof each test well was calculated by comparingthe OD490 of the glucose control well based onthe following equation (OD490 of test well/OD490of glucose control well) x 100. MIC wasdetermined by plotting the percent residualglucose level (y axis) against drug concentration(x axis).

Results and discussion

1. Phytochemical analysis- The preliminaryphytochemical analysis revealed the presence ofsteroids in all the three solvents extract. Phenolsand flavanoids were present in polar solvent,methanol extract (Table 1). Flavonoid was foundhigh in C. salpelliformis (11.92±0.72 mg/g)followed by total phenols (2.49±0.014 mg/g)

and bound phenol was found minimum in C.salpelliformis (7.71±0.01 g/g) whereasalkaloids was absent.

2. Nymphicidal bioassay-The toxicity of algalextracts was evaluated against D. cingulatusto suggest a safe method for their control. Thepercentage of mortality increased when theconcentration level increased. C. scalpelliformischloroform extract was highly toxic (92.59 %)followed by methanol (88.89 %) and hexane(84.62 %) at 96 hours to D. cingulatus. TheLC50 value of C. scalpelliformis chloroformextract to D. cingulatus was 364.6 ppm at 24h;and it was decreased to 343.8, 294.3 and 256.4ppm at 48, 72 and 96 hrs treatment periodrespectively (Figure 1). Similar trend was alsorecorded for LC30 and LC90. The algal extractsmixed with artificial diet might enter into thealimentary canal while feeding and causedmortality. Cetin et al. (2010) reported thelarvicidal efficacy of the acetone extract of thethalli of Caulerpa scalpelliformis var.denticulata against late 2nd to early 3rd instarsof Culex pipiens at 1,200 ppm, the extractcaused >70% larval mortality at 24-h, 48-h, and72-h exposure. The LC50 and LC90 values ofC. scalpelliformis were 338.91 and1,891.31 ppm, respectively. Bai and Koshy(2004) reported that 40% leaf and 10% seedethanolic extracts of Thevetia neriifoliaJuvenomimetic activity on D. cingulatus.Sharma et al. (2010) reported that the 1.0%concentration of A. indica (Neem Seed Kernel)caused about 75% mortality in D. cingulatus.In conclusion, it can be stated that among thethree solvent extracts of C. scalpelliformis thechloroform extract has potential at sub lethalconcentration followed by the methanol andhexane extracts.

3. Antifungal bioassay-The result of agar welldiffusion assay revealed that methanolic extract(12.66±0.33 mm) was potential followed byhexane (11.33±0.33 mm) and chloroform(10.33±0.33 mm) extracts in inhibiting thegrowth of Fusarium oxysporum. MIC formethanol extract was found to be 8mg/ml; forhexane � 8mg/ml and for chloroform it was

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higher- 11mg/ml. Alam et al. (2002) showedthat different parts of Vinca rosea andAzadirachta indica showed potential effectagainst F. oxysporum f.sp. vasinfectum.Suwitchayanon and Kunasakdakul (2009)reported that the clove extract at theconcentration of 2600 ppm was required forMIC to control Fusarium oxysoporum, in ourexperiment the C. scalpelliformis extract atthe concentration of 8mg/ml was required toinhibit this pathogen. Afifah et al. (2010)investigated the antifungal activity of Halimedadiscoidea and they reported that the algae wasfound to inhibit phytopathogenic fungus such asAspergillus niger, Penicillium sp. andRhizopus sp. Obongoya et al. (2010)investigated water based crude plant extractsof Neem (Azadirachta indica), Mexicanmarigold (Tagetes minuta), Tobacco(Nicotiana tobacum) and Peri-winkle (Vincarosea) in controlling soil-borne fungi (Fusariumoxysporum Schl. f. sp. phaseoli) of commonbean (Phaseolus vulgaris L.). They found thatNeem extract was the most effective, while Peri-winkle was the least in inhibiting F. oxysporum.

Acknowledgements

We gratefully acknowledge the MOES(Ref. No. MRDF/1/33/P/07), New Delhi forthe financial assistance. We thank Rev. Fr. Dr.Alphonse Manickam, S. J. Principal, St. Xavier�sCollege, Palayamkottai for the laboratoryfacilities and encouragements.

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