Proceedings of the National seminar on

Harmful/Beneficial Insects of Agricultural Importance with Special Reference to the Nuisance Pest Luprops tristis in Rubber Plantations
17th & 18th February 2011

Organised by P.G. and Research Department of Zoology St. Josephs College, Devagiri, Calicut

Co-sponsored by Department 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. Josephs College, 2011 ISBN 978-81-921589-0-7

Rs.100.00 Published by Principal St. Josephs College, Devagiri, Calicut - 673008, Kerala

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

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. Xaviers College (Autonomous), Palayamkottai-627002, Tamil Nadu E-mail: ksraj42@gmail.com*

Abstract Sea weeds/marine macroalgae are the renewable living resources which are rich source of structurally important novel and biologically active metabolites. The thalli extract [Hexane, Chloroform and Methanol] of Caulerpa scalpelliformis (R.Br.) Web. V. Bosse (Chlorophyceae) was tested against Dysdercus cingulatus (Fab.) third nymphal instar and fungal pathogen, Fusarium oxysporum f. sp. vasinfectum (Atk.) Snyd and Hans. (Fusarium Wilt of cotton) at different concentrations. The results of insecticidal activity of HE, CH and ME extracts 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 mortality gradually increased as the concentration increased. The Hexane (11.330.33mm zone of inhibition: ZI), Chloroform (10.330.33mm ZI) and Methanol (12.660.33mm ZI) extracts of C. scalpelliformis were found to inhibit the growth of F. oxysporum. Minimum inhibitory concentration (MIC) for Methanol was found to be 8mg/ml; for Hexane 8mg/ml and for Chloroform it was higher 11mg/ml. It is concluded that C. scalpelliformis possess Nymphicidal and fungicidal activity; hence this algal extract can be used as biocide in agriculture. Key words: algal seaweed, Caulerpa scalpelliformis, biocides. Short title: Biocidal activity of algal seaweed Caulerpa scalpelliformis. Introduction Marine algae are widely spread throughout the coastal areas around many
86

continents. Almost al investigations carried out on these materials focused on the different aspects concerned with their nature and growth. Little is known about its antimicrobial and insecticidal properties. These are rich source of structurally important novel and biologically active secondary metabolites with antimicrobial activities (Borowitzka & Borowitzka 1992, Del Val et al. 2001, Ely et al. 2004, Cordeiro et al. 2006) and also having pharmaceutical importance (Febles et al. 1995). Cotton is the most economically important natural fiber materials in the world. The major obstacles hindering cotton cultivation are insect pest and disease infestations. Dysdercus cingulatus (Fab.) commonly known as red cotton bug is an important pest of cotton. It is distributed all over the cotton producing regions of India (David & Ananthakrishnan 2004, Sahayaraj & Illayaraja 2008). These pests are difficult to control by insecticide application because they are highly mobile and have many alternate wild hosts belonging to Malvaceae (Iwata 1975, Kohno & Ngan, 2004). Nymphs and adults of D. cingulatus feed mainly on developing or mature cotton boll. Wilt of cotton (Gossypium spp) is a important vascular disease caused by the soil borne pathogen Fusarium oxysporum Schlechtend f.sp. vasinfectum (Atk.) Snyd and Hans. The disease is widespread and causes substantial crop losses in most of the major cotton-producing areas of the world (Assigbeste et al. 1994, Wang et al. 2004). Due to hazards associated with the increased use of synthetic pesticides the use of

biopesticides esp., from marine algae has gained considerable attention on the eco-friendly approaches for the management of insect pest and plant pathogens. Thus the exploitation of pesticides of marine algae-origin in agriculture has reduced the risk factor appreciably not only for the public health and human well-being but also for the environmental pollution. In present investigation, the bioefficacy of algal seaweed, Caulerpa scalpelliformis was evaluated against insect pest, D. cingulatus and fungal plant pathogen, F. oxysporum. Materials and methods 1 Collection, extraction and preparation of seaweed extract- The algae, Caulerpa scalpelliformis was collected by hand picking from the submerged marine rocks at Idinthakarai, Tirunelveli coast of Tamil Nadu, India during low tide. The seaweed was washed thoroughly thrice with tap water and once with sterile distilled ware to remove salt and epiphytes. They were then shade-dried for two weeks and partially powdered using domestic blender. 100 gm of powdered seaweed was packed in Soxhlet apparatus and refluxed with hexane (30C 40C), chloroform (30C 40C) and methanol (40C - 50C) individually for 24 hours continuously. Extraction solvent was evaporated and dried over Sodium Sulphate in dessicator under Vacuum. The crude extracts were stored at -20C until further use. 2 Phytochemical analysis- The qualitative phytochemical analyses were performed using standard procedures (Harbone 1983, Trease & Evans 1987). Tannins (Aparna Buzarbarua 2000), total phenols, ortho-dihydric phenols, bound phenols, alkaloids (Harbone 1973) and flavonoids (Suresh et al. 2010) were quantified. 3 Pest collection and rearing- Nymphs and adults of D. cingulatus were collected from cotton fields in the Tirunelveli district of Tamil Nadu, India. The collected pest were maintained in the insectory under laboratory conditions (282C; 70-75% RH; 11L&13D hours photoperiod) in plastic containers (300 ml capacity) containing a layer of sterile moist
87

and coarse sand (4 cm thick). They were fed with its natural host cotton and artificial diet. The laboratory emerged third nymphal instars of D. cingulatus were used for the present experiment. 4 Nymphicidal activity bioassay- Bioassay studies were carried out using uniform sized third instar D. cingulatus, selected randomly from the stock culture. Six insects were placed in the plastic containers (300 ml capacity). Different concentrations of C. scalpelliformis extract, 100, 200, 400, 600 and 800 ppm were mixed in artificial diet. Insects were allowed to feed on artificial diet mixed with algae extract for 96 hours continuously. Five replications (n=30) were maintained for each concentration. Mortality was corrected using Abbotts formula (Abbott 1925) if any was recorded in control category. Then the data was subjected to Probit analysis (Finney 1971). After 96 hrs alive nymphs were provided with artificial diet. 5 Isolation and Identification of Fusarium oxysporum f.sp. vasinfectum - Fusarium oxysporum f.sp. vasinfectum was isolated from infected cotton plants (Melameignanapuram, Tenkasi district, Tamil Nadu, India) and were used for the experiment. The pathogen was isolated, sub-cultured on Potato Dextrose Agar (PDA) medium and identified using standard protocol (Burgess et al. 1994). 6 Antimicrobial bioassay6.1 Agar well diffusion assay: Antifungal activity was carried out using agar well diffusion method (Irobi et al. 1996). Petri plates were prepared with 20 ml of sterile PDA. Wells were made using sterile cork borer under aseptic condition. The C. scalpelliformis extract with various concentrations (0.05%, 0.1%, 0.2%, 0.4% and 0.8%) were prepared using Dimethyl Sulphoxide (DMSO) and were added to the respective wells. Carbendazim (Bavistin) (0.03%) was used as positive control and DMSO was maintained as negative control. They were incubated at 27C for 3 days. The

zone of inhibition was measured using a ruler and expressed in mm. 6.2 Minimum Inhibitory Concentration (MIC) determination (Rapid Susceptibility assay)- MIC was determined using glucose utilization by the fungi in microwell plate (Wetter et al., 2003). The medium, RPMI 1640 with 0.4% glucose were buffered with 0.165 M 3(N-morpholino)-Propanesulfonic acid. The C. scalpelliformis extract with various concentrations (1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg and 12 mg) were prepared using Dimethyl Sulphoxide (DMSO). The fungal conidial suspension was collected and adjusted with glucose deficient RPMI 1640 medium to an OD530 of 0.11. The assay was initiated by inoculating wells with 100 l of RPMI 1640 with 0.4% glucose and 50 l of extracts and 50 l of conidial suspension to the respective wells. The well with RPMI 1640 with 0.4% glucose served as glucose control. They were incubated at 27C for 3 days. Residual glucose level was detected by addition of 50 l of an enzyme color mix comprising of 0.6 M Sodium Phosphate buffer/ml (pH 6.0), 360 g of 4-amino antipyrine/ml, 490 g of Nethyl-N-Sulfopropyl-m-toluidine/ml, 0.68 U of horseradish peroxidase/ml and 0.4 U of Glucose oxidase/ml to each well. After 30 minutes the color intensity was measured at OD490 with a reference reading of OD630 in a microplate reader (Glaxo). The percent residual glucose of each test well was calculated by comparing the OD490 of the glucose control well based on the following equation (OD490 of test well/OD490 of glucose control well) x 100. MIC was determined by plotting the percent residual glucose level (y axis) against drug concentration (x axis). Results and discussion 1. Phytochemical analysis- The preliminary phytochemical analysis revealed the presence of steroids in all the three solvents extract. Phenols and flavanoids were present in polar solvent, methanol extract (Table 1). Flavonoid was found high in C. salpelliformis (11.920.72 mg/g) followed by total phenols (2.490.014 mg/g)

and bound phenol was found minimum in C. salpelliformis (7.710.01 g/g) whereas alkaloids was absent. 2. Nymphicidal bioassay- The toxicity of algal extracts was evaluated against D. cingulatus to suggest a safe method for their control. The percentage of mortality increased when the concentration level increased. C. scalpelliformis chloroform extract was highly toxic (92.59 %) followed by methanol (88.89 %) and hexane (84.62 %) at 96 hours to D. cingulatus. The LC50 value of C. scalpelliformis chloroform extract to D. cingulatus was 364.6 ppm at 24h; and it was decreased to 343.8, 294.3 and 256.4 ppm at 48, 72 and 96 hrs treatment period respectively (Figure 1). Similar trend was also recorded for LC30 and LC90. The algal extracts mixed with artificial diet might enter into the alimentary canal while feeding and caused mortality. Cetin et al. (2010) reported the larvicidal efficacy of the acetone extract of the thalli of Caulerpa scalpelliformis var. denticulata against late 2nd to early 3rd instars of Culex pipiens at 1,200 ppm, the extract caused >70% larval mortality at 24-h, 48-h, and 72-h exposure. The LC50 and LC90 values of C. scalpelliformis were 338.91 and 1,891.31 ppm, respectively. Bai and Koshy (2004) reported that 40% leaf and 10% seed ethanolic extracts of Thevetia neriifolia Juvenomimetic 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 the three solvent extracts of C. scalpelliformis the chloroform extract has potential at sub lethal concentration followed by the methanol and hexane extracts. 3. Antifungal bioassay- The result of agar well diffusion assay revealed that methanolic extract (12.660.33 mm) was potential followed by hexane (11.330.33 mm) and chloroform (10.330.33 mm) extracts in inhibiting the growth of Fusarium oxysporum. MIC for methanol extract was found to be 8mg/ml; for hexane 8mg/ml and for chloroform it was
88

higher- 11mg/ml. Alam et al. (2002) showed that different parts of Vinca rosea and Azadirachta indica showed potential effect against F. oxysporum f.sp. vasinfectum. Suwitchayanon and Kunasakdakul (2009) reported that the clove extract at the concentration of 2600 ppm was required for MIC to control Fusarium oxysoporum, in our experiment the C. scalpelliformis extract at the concentration of 8mg/ml was required to inhibit this pathogen. Afifah et al. (2010) investigated the antifungal activity of Halimeda discoidea and they reported that the algae was found to inhibit phytopathogenic fungus such as Aspergillus niger , Penicillium sp. and Rhizopus sp. Obongoya et al. (2010) investigated water based crude plant extracts of Neem ( Azadirachta indica), Mexican marigold (Tagetes minuta), Tobacco (Nicotiana tobacum) and Peri-winkle (Vinca rosea) in controlling soil-borne fungi (Fusarium oxysporum Schl. f. sp. phaseoli) of common bean (Phaseolus vulgaris L.). They found that Neem extract was the most effective, while Periwinkle was the least in inhibiting F. oxysporum. Acknowledgements We gratefully acknowledge the MOES (Ref. No. MRDF/1/33/P/07), New Delhi for the financial assistance. We thank Rev. Fr. Dr. Alphonse Manickam, S. J. Principal, St. Xaviers College, Palayamkottai for the laboratory facilities and encouragements. References Abbott WS. 1925. Methods for comparing the effectiveness of an insecticide. Journal of Economic Entomology 18: 265267. Afifah SN, Daah I, Fariza SS, Nordin MKMJ, Ali ZN. 2010. Antimicrobial Activity of Various Extracts of a Tropical Chlorophyta Macroalgae, Halimeda discoidea. Journal of Applied Sciences 10(23): 30073013. Alam S, Banu MS, Ali MF, Akhter N, Islam MR, Alam MS. 2002. In vitro inhibition of
89

conidial germination of Colletotrichum gloeosporioides Penz. by fungicides, plant extracts and phytohormones. Pakistan Journal of Biological Sciences 5:303306. Aparna Buzarbarua. 2000. A text book of practical plant chemistry. Chand and company Ltd. New Delhi, pp 100108. Assigbeste KB, Fernandoz D, Dubois MP, Gerger J,P. 1994. Differentiation of Fusarium oxysporum f.sp. vasinfectum races on cotton by RAPD analysis. Phytopathology 84: 622 626. Bai H, Koshy G. 2004. Juvenomimetic activity of extracts of Thevetia neriifolia Juss. to Dysdercus cingulatus F. (Hemiptera: Pyrrhocoreidae) Jorunal of Tropical Agriculture 42(1-2): 4547. Borowitzka MA, Borowitzka LJ. 1992. Vitamins and fine chemicals from micro algae. In: Microalgal Biotechnology. Cambridge University Press. Great Britain p 179. Burgess LW, Summerell BA, Bullock S, Gott KP, Backhouse D. 1994. Laboratory manual for Fusarium research. 3rd edn, University of Sydney, Sydney. Cetin H, Gokoglu M, Oz E. 2010. Larvicidal Activity of the Extract of Seaweed, Caulerpa scalpelliformis , Against Culex pipiens. Journal of American Mosquito Control Association 26(4):433435. Cordeiro RA, Gomes VM, Carvalho AFU, Melo VMM. 2006. Effect of proteins from the red seaweed Hypnea musciformis (Wulfer) Lamouraux as the growth of human pathogen yeasts. Brazilian Archieves of Biology and Technology. 49(6): 915921. David BV, Ananthakrishnan TN. 2004. General and applied entomology, Tata Mc Graw Hill Publishing Compant Ltd. New Delhi.

Del Val AG, Platas G, Basilio A. 2001. Screening of antimicrobial activities in red, green and brown macroalgae from Gran Canaria (Canary Islands, Spain). International Journal of Microbiology. 4: 3540. Ely R, Supriya T, Naik CG. 2004. Antimicrobial activity of marine organisms collected off the coast of South East India. Journal of Experimental Biology.and Ecology . 309: 121127. Febles CI, Arias A, Gil-Rodriguez MC. 1995. In vitro study of antimicrobial activity in algae (Chlorophyta, Phaeophyta and Rhodophyta) collected from the coast of Tenerife (in Spanish). Anuario del Estudios Canarios. 34: 181192. Finney DJ. Probit analysis Cambridge university Press (1971), London. Harbone JG. 1973. Phyochemical methods, London, Chapman and Hall, Ltd. New York. Harbone JG. 1983. Phyochemical methods, London, Chapman and Hall, Ltd. 49-188 pp. Irobi ON, Moo-Young M, Daramola SD. 1996. Antimicrobial activity of Annato Bixoovellana Extra. Journal of pharmacognosy. 34:8790. Iwata K. 1975. Shizen kansatsusha no shuli (Memoirs on Nature by an observer). Asahi Shimbun Co., Tokyo. pp 584. Kohno K, Ngan BT. 2004. Effects of host plant species on the development of Dysdercus cingulatus (Heteroptera: Pyrrhocoridae). Applied Entomology and Zoology . 39(1): 183187.

Obongoya BO, Wagai SO, Odhiambo. 2010. Phytotoxic effect of selected crude plant extracts on soil-borne fungi of common bean. African Crop Science Journal. 18(1): 1522. Sahayaraj K, Illayaraja R. 2008. Ecology of Dysdercus cingulatus (Fab.). Egyptian Journal of Biology. 10: 122125. Sharma T, Qamar A, Khan AM. 2010. Evaluation of Neem (Azadirachta indica) extracts against the eggs and adults of Dysdercus cingulatus (Fab.). World Applied Science Journal. 9(4): 398402. Suresh CD, Muralirangan MC. 2010. Influence of flavanoids and phenolics on the food consumption by Oxya nitidula (Walker). Journal of Applied Zoological Research. 21(1): 1923. Suwitchayanon P, Kunasakdakul K. 2009. In Vitro effects of clove and turmeric extracts controlling crucifer pathogens. Journal of Agriculture and Technology 5(1): 193199. Trease E, Evans WC.1987. Pharmacognosy, Billiare Tindall, London. Wang B, Brubaker CL, Burdon J. 2004. Fusarium species and Fusarium Wilt pathogens associated with native Gossypium populations in Australia. Mycological Research. 108(1): 35 44. Wetter TJ, Hazen KC, Cutler JE. 2003. Modification of rapid susceptibility assay for antifungal susceptibility testing of Aspegillus fumigatus. Journal of Clinial Microbiology 41(90): 42524258.

90

91