ISSN X Raja Rishi et al., IJALS, Volume (5) Issue (1) November RESEARCH ARTICLE

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1 Evaluation of the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill., and Metarhizium anisopliae (Metsch.) for the control of Bamboo Leaf Roller Pyrausta coclesalis Wlk. (Pyralidae: Lepidoptera) R.Raja Rishi*, N.D.Barthakur and R.K.Borah Forest Protection Division, Rain Forest Research Institute, Jorhat (Assam) India. rajarishir@icfre.org Corresponding Author R. Raja Rishi Forest Protection Division, Rain Forest Research Institute, Jorhat (Assam) India. rajarishir@icfre.org Article History Received on 16 September, 2012; Revised in revised form 15 October, 2012; Accepted 24 October, 2012 Abstract Pyrausta coclesalis (Pyralidae: Lepidoptera) is a defoliating leaf roller and recorded as an important pest. It is reported to cause serious damage on bamboo nursery seedlings and young plants of bamboo species such as Bambusa tulda, B. nutans, B. balcooa and B.pallida. The larvae tie the leaves together as leaf cases and feed on the upper tissues of the leaves. The entomopathogenic fungi, Beauveria bassiana strain isolated from the field collected cadavers of Crocidophora sp. and Metarhizium anisopliae fungus were evaluated for its efficacy and effectiveness against the III instar larvae of P. coclesalis in laboratory as well in the field condition. Four different concentrations with five replications for each treatment were tested. Laboratory evaluation of the fungus B.bassiana showed that it was effective for the insect species by effecting desirable level of larval mortality in the 7 th day after treatment. Among the four different concentrations of B.bassiana and M. anisopliae tested, the concentrations of B. bassiana strain showed effective control. While M. anisopliae showed less than 36 percent of larval mortality. The details are discussed. Keywords: Beauveria bassiana, Metarhizium anisopliae, bamboo, fungi, efficacy and evaluation. Introduction The bamboo species like Bambusa tulda, B.balcooa, B.pallida, Dendrocalamus giganteus, B.vulgaris and B.nutans are getting importance in the recent past and are widely planted in most part of north-eastern states of India (Pandey et al., 2006) The defoliating leaf roller, Pyrausta coclesalis (Lepidoptera: Pyralidae) was recorded as an important pest and reported to cause severe damage on bamboo nursery seedlings and young plants of the above mentioned bamboo species in India (Seethalakshmi and Muktesh Kumar, 1998). It attacks many bamboo species including Phyllostachys pubescens, P. viridis, Bambusa vulgaris, D. latiflorus and D. strictus (Wang Haojie et al., 1998). The adult moth is 8-13 mm and yellowish brown in colour. The pest occurs from May to October in Assam region. The light green larvae feed in groups or individually. The larvae tie the leaves together as leaf cases and feed on the upper tissues of the leaves. The fully grown larvae pupate in cocoon made between the rolled leaves (Mathur, 1943). The over wintering larvae undergo hibernation from the end of the September to beginning of May and pupate for 10 to 15 days. The incidence and intensity of attack by the insect was severe level in the nursery and in the plantations. In this study, we attempt to use the entomopathogenic fungi, B.bassiana strain isolated from the field collected cadavers of Crocidophora sp. and M. anisopliae to control the pest P. coclesalis in laboratory as well in field condition. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 12

2 Materials and Methods The fungal isolates of Beauveria bassiana and M. anisopliae were grown on Sabouraud Dextrose Agar medium (SDA) at room temperature C for 8 10 days, harvested and crude extract were prepared (stock solution) with distilled water. The concentration of spores in the final suspension was determined by haemocytometry. Estimation of Spores Dilution: 1 ml of the purified stock fungus suspension was made up to 10 or 100 ml with water containing 0.1 % wetting agent, teepol / liquid soap. The teepol / liquid soap solution ensured thorough mixing and uniform distribution of fungal spores (Shimuza, 2004). Counting : With the Thoma white cell pipette provided along with the haemocytometer, the fungal suspension was drawn to the 1 (One ) mark and made up to 11 mark with distilled water containing 0.1% teepol / liquid soap. Discarded the first 3 drops and introduced the fourth drop into the groove of the haemocytometer after placing the standard coverslip over the slide. The sufficient quantity of the liquid was introduced to fill up the calibrated area in the haemocytometer. Allowed the spores to settle down for 2 min. and then counted the spores in about 25 of the 1/400 sq. mm with the help of a research microscope under 40x magnification. This could be best done by counting the spores inside the as well as the spores on the top and left side lines only. The spore suspensions of known concentration were prepared from the stock solution by suitable dilution with distilled water. A series of four concentrations of 7.8 x 10 10, 7.8 x 10 8,7.8 x 10 6 and 7.8 x 10 4 Spores/ml of Beauveria bassiana and M. anisopliae spore suspensions were prepared and pathogenicity test were conducted on the test insect P. coclesalis. Healthy larvae of these species reared in the laboratory were surface sterilized with 1-5% sodium hypochlorite and sprayed with the fungal inoculum including the feeding material of B. nutans bamboo leaves. The freshly prepared spore suspensions of B. bassiana and M. anisopliae were sprayed on III instar larvae of P. coclesalis. Five treatments including control (T1-T4 s and T5 control) were tested on the targeted larvae. The inoculum sprayed larvae and the feeding materials were air dried on a filter paper. The inoculum sprayed larvae of P. coclesalis were released in the plastic containers with B. nutans leaves to feed. A control (T5) sprayed with 0.1% Tween 80 in sterilized distilled water was also maintained. Each treatment was replicated five times in completely randomized design with 10 insects in each replicate. The treated larvae were kept in petridishes lined with moist blotting paper for 24 hours and later provided leaves as usual. Observations were taken at every 24 hours interval. The mortality was observed up to 7 days after spraying of spores suspensions, until the higher concentration yield cent percent mortality. Likewise, in the field the inoculum of B. bassiana and M. anisopliae spore suspensions with all the four concentrations 7.8 x 10 10, 7.8 x 10 8,7.8 x 10 6 and 7.8 x 10 4 Spores/ml sprayed III instar larvae of P. coclesalis were released on the young seedlings of bamboo species B.nutans kept in the nursery. The mortality was observed up to 7 days after spraying of spores suspensions. The data on percent mortality were subjected to statistical analysis. Results and Discussion Pathogenicity of B. bassiana and M. anisopliae tested on the targeted insect P. coclesalis with four different concentrations 7.8 x 10 10, 7.8 x 10 8, 7.8 x 10 6 and 7.8 x 10 4 Spores/ml, B. bassiana showed that the concentrations 7.8 x and 7.8 x 10 8, and were found effective control for the pest in both laboratory and in field level experiments. However the concentration 7.8 x 10 6 Spores/ml showed 96% larval mortality only in Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 13

3 laboratory experiment (Table -1 and Table -3). Whereas M. anisopliae, in the concentration of 7.8X10 10 and 7.8X10 8 were resulting only 36 and 28% mortality respectively in the 7 th day after treatment in the laboratory condition. M. anisopliae, resulted very low level of effect on the targeted pest in the field experiment (Table- 5 and Table - 7). Statistical analysis was performed following completely randomized design (CRBD) with five replicates in each treatment. The data was subjected to analysis of variance (ANOVA) using statistical software MINITAB 11.2 and significance of various treatments was evaluated by F test (p<0.05) by calculating CD values (Table 2,4,6 and Table - 8). When III instar larvae were infected with different concentrations of B.bassiana caused larval mortality which increased with increase of dose concentration. The data shows linear relationship between dose concentration and mortality. Bioefficacy of fungal biopesticides have been studied by many workers. Arti Prasad (2010) reported that B.bassiana at the concentration 1.25 x10 8 conidia/ml causing 86.7% larval mortality on Helicoverpa armigera. Ferron (1981) reported the entomopathogenic fungi B. bassiana plays an important role in the regulation of pest population. Dherbare (2004) reported that B.bassiana was evaluated against gram pod borer H.armigera in the laboratory condition and found that after the treatment of the fungus on the 1st, 3rd, and 5th days revealed that as the spore intensities increased, the larval mortality also increased. B. bassiana was an aggressive pathogen of many different host species. Not only does it has a widehost range but insects were attacked at larval or adult stages both. When fungal spores come in contact with larval body it gets attached to the larval cuticle. The fungal conidia proliferate because larvae gave favorable conditions for conidia germination like high humidity and low temperature as an essential requirement for conidial germination. Hence, the infection was established further. The main route of the infection of B. bassiana was through the cuticle and very little through ingested conidia (Goettel and Inglis, 1997). Balu et al. (1998) reported B.bassiana used as a promising biocontrol agent on the bark feeding borer, Table 1. Evaluation of B. bassiana (natural strain) against defoliator Pyrausta coclesalis (III instar) in Laboratory condition. Evaluation of B.bassiana - Mortality % of Pyrausta coclesalis /conc. In (III instar) in lab. R1 R2 R3 R4 R5 Total 1 T1-7.8x T2-7.8x T3-7.8x T4-7.8x T5- Control Rep total (R) Grand Total 1638 Grand Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 14

4 Table 2. Test for significance of differences among the treatments of B. bassiana against Pyrausta coclesalis in Laboratory condition through Analysis of variance. Degree of freedom Sum of Tabular F Source of variation F calculated 5% Significance Replication NS Sig** Error Total CV 6.34 SED 1.66 CD 2.9 ** significant at p=0.05 level Table 3. Evaluation of B. bassiana (natural strain) against defoliator Pyrausta coclesalis (III instar) in Field condition. /conc. In Evaluation of B.bassiana - Mortality % of Pyrausta coclesalis (III instar) in field R1 R2 R3 R4 R5 Total 1 T1-7.8x T2-7.8x T3-7.8x T4-7.8x T5- Control Rep total (R) Grand total 1272 Grand mean Table - 4. Test for significance of differences among the treatments of B. bassiana against Pyrausta coclesalis in Field condition through Analysis of variance Source of variation Degree of freedom Sum of F calculated Tabular F 5% Significance Replication NS Sig** Error Total CV 7.02 SED 1.43 CD 2.5 ** significant at p = 0.05 level Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 15

5 Table 5. Evaluation of M. anisopliae against defoliator Pyrausta coclesalis (III instar) in Laboratory condition. /conc. In Evaluation of M. anisopliae - Mortality % of Pyrausta cocles (III instar) in lab. R1 R2 R3 R4 R5 Total 1 T1-7.8x T2-7.8x T3-7.8x T4-7.8x T5- Control Rep total (R) Grand total 428 Grand mean Table 6. Test for significance of differences among the treatments of M. anisopliae against Pyrausta coclesalis in Laboratory condition through Analysis of variance. Source of variation Degree of freedom Sum of F calculated Tabular F 5% Significance Replication NS Sig** Error Total CV SED 0.98 CD 1.7 ** significant at p=0.05 level Table - 7. Evaluation of M. anisopliae against defoliator Pyrausta coclesalis (III instar) in field condition. /conc. In Evaluation of M. anisopliae - Mortality % of Pyrausta coclesalis (III instar) in field R1 R2 R3 R4 R5 Total 1 T T T T T Rep total (R) Grand total 240 Grand mean 9.60 Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 16

6 Table - 8. Test for significance of differences among the treatments of M. anisopliae against Pyrausta coclesalis in field condition through Analysis of variance. Source of Degree of Sum of variation freedom F calculated Tabular F Replication % Significance NS Error Sig** Total CV SED 0.77 CD 1.3 ** significant at p=0.05 level Indarbela quadrinotata. Vanderberg et al. (1998) reported the efficacy and persistence of B.bassiana on the diamond back moth larvae Plutella xylostella. Boas et al. (1983) and Alves et.al. (1985) reported the infection of B. bassiana on giant sugar cane borers, Castnia licus and Diatraea. Sinay and Rizk (2007) reports that after inoculation with B. bassiana fungul isolations, the hyphae penetrated the integument inside the whole body, cavities then reached to all the cells like fat, neural cells and muscle tissues and damaged them. Quesada-Moraga (2006) reports that in addition to larval pupal intermediate formation at 0.2x 10 8 ml dose level, the infected larvae revealed the formation of weak and fragile cuticle that generally rupture at midgut, where it brought the oozing out of fungal bodies from the larval skin. The death of Spodoptera littoralies larval instars by fungul species Beauveria and Metarhizium anisopliae was due to the crude toxic protein extracted by fungi and that was dose related because in high doses may reach 100 percent of treated larvae. The mode of entry of fungi may be through the cuticle or mouth parts into the haemocoel. Infection might have resulted from contact between a virulent infection inoculums and susceptible insect cuticle, germination, penetration of the germ tubes through the integument and finally spread of the pathogen through the host tissues. Entomopathogenic fungi, produce mycotoxin which kills the host by inducing progressive degeneration of host tissues, due to loss of structural integrity of membranes followed by dehydration of cells as a result of fluid loss (Hafez et al., 1994). Therefore the microbial pesticide fungi have demonstrated considerable potential in microbial control of arthropods especially within Integrated Pest Management programs. Their restricted host ranges allow for control of insect pests with limited harm to non target organisms including predators, parasites, and other pathogens. They can be successfully employed for the pest management program due to their non hazardous and environmental friendly nature. Acknowledgement The authors are thankful to the Director, Rain Forest Research Institute, Jorhat for providing the laboratory facilities. References Alves, S.B., Botelho, P.S.M., Macedo, N. and Riseado, G.M The occurrence of some pathogens on sugarcane borer, Diatraea saccharalis in field and laboratory conditions. Ent. Newl., Int. Soc. Sug. cane Technol., 17 : 4-5. Arti Prasad and Nilofer Syed, Evaluating prospects of fungal biobesticide Beauveria bassiana (Balsamo) against Helicoverpa armigera (Hubner): An ecosafe strategy for pesticidal pollution. Society of Applied Sciences, 1(3): Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 17

7 Balu, A., Sasidharan, K.R. and Raja Rishi, R A new record of the promising biocontrol agent Beauveria bassiana on the bark feeder/stem borer, Indarbela quadrinotata. Sylva plus 6 (1), 6. Boas, A. M., Vilas, E.J., Marques and Ribeiro, S.M.A Pathogenicity of the fungus, Beauveria bassiana (Bal.) Vuill., to larvae of Castnia licus (Drury) (Lep. Castniidae) the giant Sugarcane borer. Anais da sociedade Entomologica do brazi., (12): Dhembare, A.J. and Siddique, N.H Evaluation of mycoinsecticide, Beauveria bassiana (Balsamo) formulation against gram pod borer, Helicoverpa armigera. Journal of Experimental Zoology., 7: Ferron, P Pest control by the fungi Beauveria and Metarhizium, In: Microbiol Control of pest plant diseases, H.D Burges (Eds). Academic press,london, pp: Goettel, M.S. and Inglis, G. D Fungi: Hyphomycetes, In : Manual of Techniques in Insect Pathology, (Ed. Lacey L.A), Academic Press, London, UK, pp Hafez, M., Zaki, F.N., Moursy, A. and Sabbour, M Biological Effects of the Entomopathogenic Fungus, Beauveria bassiana on the potato tuber moth Phthorimaea operculella (Seller). J. of Islamic Academic of Sciences., 7(4): Mathur, R.N Bamboo defoliators. Indian journal of Entomology., 5(1) : Pandey, H.N., Barik, S.K. and Tripathi, O.P Ecology,diversity and conservation of plants and ecosystems in India, Regency Publications, New Delhi.8., pp.414. Quesada -Moraga, E., Carrasco-Diaz, J.A. and Santiago-Alvarez, C Insecticidal and antifeedant activities of proteins secreted by entomopathogenic fungi against Spodoptera littoralis (Lep., Noctuidae). J. Applied Entomol., 130 : Seethalakshmi, K.K. and Muktesh Kumar, M.S Bamboos of India a compendium, Pub. KFRI, Kerala, India and INBAR, Beijing, China., p.345. Shimuza, M A novel technique to inoculate conidia of entomopathogenic fungi and its application for investigation of susceptibility of the Japanese pine sawyer, Monochamus alternatus, to Beauveria bassiana. Appl. Entomol. Zool., 39: Sinay, N.H. and Rizk, S.A Entomopathogenic Fungus, Beauveria bassiana (Bals.) and Gamma Irradiation Efficiency against the Greater Wax Moth, Galleria melonella (L.) J. American- Eurasian journal of Scientific Research., 2(1): Vanderberg, J.D., Shelton, A.M., Wilsey, W.T. and Ramos, M Assessment of Beauveria bassiana sprays for control of diamond back moth on Crucifers. J. Econ. Ent., 91(3): Wang Haojie, Varma, R.V. and XuTiansen Insect pests of Bamboos in Asia, An illustrated manual, INBAR. pp Corresponding Author : R. Raja Rishi, Forest Protection Division, Rain Forest Research Institute, Jorhat (Assam) India. rajarishir@icfre.org. 2012, IJALS. All Rights Reserved. Int. J. Adv. Lif. Sci., Available online on at www. ijals.com Page 18