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A laboratory assessment of the potential of Beauveria bassiana (Balsamo) Vuillemin as a biocontrol agent of Corcyra cephalonica Stainton (Lepidoptera: Pyralidae)
Journal of Stored Products Research 59 (2014) 185e189
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
A laboratory assessment of the potential of Beauveria bassiana (Balsamo) Vuillemin as a biocontrol agent of Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) Sanehdeep Kaur*, Abhinay Thakur, Mamta Rajput Department of Zoology, Guru Nanak Dev University, Amritsar, Punjab, 143005, India
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 14 August 2014 Available online
Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) is a wide spread pest of stored grains and responsible for considerable damage in godowns and warehouses. Beauveria bassiana (Balsamo) Vuillemin, the most common and ubiquitous fungal entomopathogen is known to have high potential for the control of insects belonging to various orders. To evaluate the pathogenicity of B. bassiana against C. cephalonica, sorghum grains were treated with three conidial concentrations of B. bassiana i.e. 1.49 105, 4.05 106, 2.02 108 spores/ml. The highest concentration was found to be the most effective. It induced significantly higher larval mortality. Adverse effects such as reduced adult emergence and longevity of C. cephalonica were also observed when the grains were treated with B. bassiana. Significantly lesser number of adults emerged from pupae, when shorghum grains were treated with 2.02 108 spores/ml. The adverse effects were also exhibited as malformed adults that lived for a short period. Overall study showed that B. bassiana gave effective control of C. cephalonica and thus may play a pivotal role in the integrated pest management program. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Corcyra cephalonica Beauveria bassiana Pathogenicity Biological control
1. Introduction Insect pests have been damaging and causing heavy losses to stored grains quantitatively and qualitatively throughout the world especially in tropical and semitropical countries (Madrid et al., 1990; Tripathi et al., 2009). In countries without modern storage technologies, insect pests cause 10e40% damage in stored grains (Shaaya et al., 1991).Traditionally, fumigants and other synthetic insecticides have been commonly used for the protection of stored products from attack by storage insects. However, the continuous use of these insecticides has led to the development of resistance in insect pests as well as residual problem in stored grains (Michalaki et al., 2006). These adverse effects of synthetic insecticide application have increased public awareness regarding the human safety and possible environmental damage and focused the research towards other alternative strategies for the management of stored grain insect pests. The use of entomopathogenic fungi (EPF) and other microbial control agents is known to be a promising strategy with good potential to minimize the adverse effects of insecticides
* Corresponding author. Tel.: þ91 0183-2258802-09; fax: þ91 0183-2258819, þ91 0183-2258820. E-mail address: [email protected] (S. Kaur). http://dx.doi.org/10.1016/j.jspr.2014.08.004 0022-474X/© 2014 Elsevier Ltd. All rights reserved.
(Padin et al., 1997). The fungal biocontrol agents can be used in close proximity to foods and feed (Cox et al., 2003, 2004). EPF can be employed to treat empty stores to control residual pests before the new harvest is brought in or may be applied as direct admixture of conidia to grains either as preventive or curative treatments to bulk grains provided it does not decrease the quality and marketability of grains (Steenberg, 2005). Out of 700 entomopathogenic species of fungi reported from insects, only10 have been widely known as biocontrol agents (Hajek and St. Leger, 1994). Among these, one of the most frequently used fungi is Beauveria bassiana (Balsamo) Vuillemin. Its potential as a mycoinsecticide has been demonstrated on a large number of different insect hosts (Inglis et al., 1993; Feng et al., 1994; Padin et al., 1995; Cherry et al., 2005). Beauveria bassiana has potential for the control of different stored product pests. It is registered by the U.S. Environmental Protection Agency (EPA) for a wide range of insect control applications (Lord, 2001). Most of the research has been done on formulations of this fungus for the management of stored product insect pests (Akbar et al., 2004; Lord, 2007; Vassilakos et al., 2006). Beauveria bassiana has been demonstrated to control Rhyzopertha dominica (Fabricius) (Mahdneshin et al., 2009; Wakil et al., 2011), Callosobruchus maculatus (Fab.) and Sitophilus granarius (Linnaeus) (Shams et al., 2011), Tribolium castaneum (Herbst) and Oryzaephilus surinamensis (L.) (Khashaveh et al., 2011). Several B. bassiana
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formulations (Boverosil, Mycotrol, ES, Mycotrol 22WP etc.) are commercially available and are registered for use in storage facilities (Khashaveh et al., 2011). Corcyra cephalonica is a serious pest of some important stored food commodities such as rice, wheat, maize, sorghum, groundnut, cotton seeds, coffee, spices and cocoa beans, especially in the tropics (Cox et al., 1981; Allotey and Kumar, 1985; Allotey, 1991). The larvae of C. cephalonica cause considerable damage to stored food commodities while feeding, leaving silken threads wherever they move. The present study was carried out to evaluate the potential of B. bassiana as a biological control agent against C. cephalonica. 2. Materials and methods This experiment with B. bassiana as a biocontrol agent against C. cephalonica was done at the Biocontrol Laboratory, Department of Zoology, Guru Nanak Dev University, Amritsar (Punjab), India. The adults of C. cephalonica were collected from stored wheat in the godowns. Then the culture was maintained at 25 ± 2 C and 65 ± 5% RH in the laboratory on sorghum grains which is one of its preferred host (Muthukrishnan et al., 1996). Rearing was carried out on partially crushed sorghum grains in battery jars (15 cm 10 cm) covered with muslin cloth to ensure proper ventilation. Ten adults in the ratio of 4:6 (M: F) were released in each battery jars half filled with crushed sorghum. After oviposition the dead adults were removed and jars were kept undisturbed for further development of C. cephalonica. From this culture 2nd instar larvae were used for experimental purpose. Cultures of the fungus B. bassiana (NBAII-Bb-5a) were procured from National Bureau of Agriculturally Important Insects (NBAII), Bangalore and further maintained in the laboratory on Potato Dextrose Agar (PDA) at 30 C. Three weeks old culture was used for experimental studies. Fungal suspension was prepared by adding 10 ml of distilled water and a drop of 0.01% Tween 80 solution to the slants and filtered through muslin cloth to remove mycelia. From this concentrated spore suspension, serial dilutions were made and spore count was obtained with the help of hemocytometer. Three concentrations i.e. C 1 ¼ 1.49 105, C 2 ¼ 4.05 106 and C 3 ¼ 2.02 108 spores/ml were used for bioassay studies. The bioassay studies are described as follows. Thirty ml of each spore suspension was sprayed on 120 g of partially crushed sorghum grains with the help of hand sprayer followed by thorough mixing. In control, grains were sprayed with same quantity of water having a drop of 0.01% Tween 80 solution. After air drying the treated grains were equally distributed in 6 plastic containers (4 cm 6 cm) i.e. 20 g/container. Ten 2nd instar larvae were released in each container, which represents one replication. Similarly larvae were released in containers having untreated grains. The larvae were checked daily for larval mortality. Observations were also recorded on larval period, adult emergence, adult longevity and deformity in all the treatments and control. To verify that the larval death is due to fungus, the dead larvae were surface sterilized with 5% sodium hypochlorite and placed in autoclaved petri dishes having moist muslin cloth at the base. The petri dishes were kept in B.O.D. incubator at 25 ± 2 C temperature. Observations were made on the mycelial growth of fungus on the dead larvae. The larval counts showing fungal growth were only considered for percent mortality due to fungus. All the experiments were replicated six times with 10 larvae per replication and values were represented as their mean ± SE. To compare difference in the means, one way analysis of variance (ANOVA) with Tukey's test at P 0.05 was performed. SPSS software for windows version 16.0 (SPSS Inc., Chicago) and Microsoft office Excel 2007 (Microsoft Corp., USA) were used to perform the statistical analysis.
3. Results There was a significant difference in the mortality of C. cephalonica larvae when the highest concentration i.e. 2.02 108 spores/ml of B. bassiana was applied on sorghum grains (F ¼ 11.46, P < 0.001). Relative to control six times higher mortality was recorded at the highest concentration. However, the lower doses of fungus (4.05 106 and 1.49 105 spores/ml) failed to induce significant mortality in the larvae feeding on treated grains as compared to control (Fig. 1a). The larval mortality started after 24 h of feeding on grains treated with the highest concentration whereas the first larval death was recorded after 48e72 h of exposure at lower doses. After death, the larvae became hard and stiff. The white mycelial growth appeared 24 h after death at the highest concentration, but at lower concentrations it took 2e3 days to grow on dead larvae (Fig. 2aec). Larval mortality was also recorded on untreated grains. However, no fungal growth was observed on larvae died in control. Treatment of sorghum grains significantly delayed the larval development of C. cephalonica as compared to control (F ¼ 62.50, P < 0.001). The larvae took more time to pupate (6.79 days) when reared on grains treated with 2.02 108 spores/ml. Similarly the lower concentrations i.e. 4.05 106 and 1.49 105 spores/ml also significantly extended the development of larvae (Fig. 1b). Lesser number of adults emerged from pupae developed on treated sorghum as compared to control, but significant effect was observed only at 2.02 108 spores/ml of the fungus (F ¼ 11.46, P < 0.001) (Fig. 3a). Longevity of adult male decreased significantly at all the concentrations of B. bassiana (F ¼ 23.00, P < 0.001) (Fig. 3b). Significant influence of the fungus was also observed on female longevity at 4.05 106 and 2.02 108 spores/ml concentrations (F ¼ 9.77, P < 0.001) (Fig. 3c). Sublethal effects of B. bassiana on C. cephalonica were also manifested as emergence of deformed adults. As is evident from Fig. 3d, treatment of grains with 2.02 108 spores/ml of fungus induced morphological deformities in significantly higher number of adults (16.66%) as compared to adults emerged from larvae reared on untreated grains. The wings of these insects were underdeveloped and curled (Fig. 4aeb) as compared to normal adults (Fig. 4c). These malformed adults lived for only 1e2 days as compared to 5e6 days for normal adults. 4. Discussion Efficiency of biocontrol of insect pests with entomopathogenic fungi is mainly due to their capacity to produce epizootics from the infected cadavers (Leathers et al., 1993). These cadavers act as a source of inocula and thus further disseminate microbial agent in the environment (Gottwald and Tedders, 1982; Lacey et al., 1994). Earlier Samodra and Ibrahim (2006) evaluated the dried conidia of eight isolates of entomopathogenic fungi against C. cephalonica in rice grains. Among these two isolates of B. bassiana (BbGc and BbPs) and one isolate of Metarhizium anisopliae (MaPs) gave high mortality to C. cephalonica larvae. Isolates of BbGc and BbPs formulated in kaolin and BbPs formulation in tapioca flour provided 100% larval mortality by 15th day of introduction. Even more than 90% mortality was recorded when rice grains were stored for 4 months. However, the pathogenicity of M. anisopliae against C. cephalonica larvae decreased rapidly upon storage at room temperature affording less than 60% control by the 2nd month of storage. Adane et al. (1996) also reported 88% mortality in Sitophilus zeamais (Motschulsky) due to treatment with B. bassiana at 104 conidia/ml within 8 days. There are reports indicating the effectiveness of B. bassiana and M. anisopliae against many coleopteran pests of stored grains (Mahdneshin et al., 2009; Khashaveh et al., 2011).
S. Kaur et al. / Journal of Stored Products Research 59 (2014) 185e189
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Fig. 1. (a, b) Effect of different concentrations of B. bassiana (C 1 ¼ 1.49 105, C 2 ¼ 4.05 106 and C 3 ¼ 2.02 108 spores/ml) on mortality and development of 2nd instar larvae of C. cephalonica. Means and SE are given. Means followed by the same letter are not significantly different (P 0.05) based on Tukey's test.
Fig. 2. (aec) Different stages of mycelial growth on dead larvae.
Post-mortem mycelia and conidial growth demonstrated that the insects had died due to fungal infection. Beauveria bassiana treated sorghum grains induced significantly higher mortality in C. cephalonica as compared to untreated grains. Besides causing mortality the other sublethal effects were prolonged development period, reduced adult longevity and morphological deformities.
The present studies indicated a dose dependent mortality but significant effects of B. bassiana on survival of C. cephalonica were observed only at the highest concentration. Similarly Padin et al. (2002) documented that wheat grains infested with Sitophilus oryzae without the conidia was significantly more damaged by weevils than grains treated with B. bassiana. Present study revealed
Fig. 3. (aed) Effect of different concentrations of B. bassiana (C 1 ¼ 1.49 105, C 2 ¼ 4.05 106 and C 3 ¼ 2.02 108 spores/ml) on adults of C. cephalonica. Means and SE are given. Means followed by the same letter are not significantly different (P 0.05) based on Tukey's test.
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Fig. 4. (aeb) Morphologically deformed adults and (c) normal adults.
that exposure of C. cephalonica larvae to B. bassiana treated sorghum grains resulted in significant reduction of adult emergence as compared to control. Treatment of grains with 2.02 108 spores/ml of fungus induced morphological deformities in significantly higher number of adults (16.66%) as compared to adults emerged from larvae reared on untreated grains. Beauveria bassiana at concentration of 1 108 conidia/ml showed highest oviposition reduction (60.58%), and adult mortality against the pulse beetle, C. maculatus (99.44%) at 92 h (Vanmathi et al., 2011). Similarly Cherry et al. (2005) found that B. bassiana 0362 at 1 107 and 1 108 conidia g1 grains led to significant adult mortality and reduced F1 emergence relative to untreated population of C. maculatus. The efficiency of B. bassiana has been reported against T. castaneum larvae with 62% mortality @ 2700 mg/kg concentration (Akbar et al., 2004). Athanassiou and Steenberg (2007) also demonstrated the insecticidal effect of B. bassiana against adults of the granary weevil, S. granarius. The present investigations indicated that B. bassiana not only caused mortality in C. cephalonica but also prolonged its development, decreased adult emergence and induced morphological deformities in adults which ultimately lead to suppression of pest population. One advantage of pathogen based protection systems is that disease cycling occurs. At death the cadaver produces many infective agents from internal and external sporulation renewing inoculum wherever the insects have died (Hidalgo et al., 1998). The storage insects can absorb lethal doses of the entomopathogen from sporulating cadavers. Use of this fungus can potentially benefit the environment and lead to more effective protection of stored grains. So from these results it is evident that B. bassiana may become an integral part of safe IPM program of stored grain pests. To assess the practical value of these results further experiments are required to evaluate the efficacy of fungal treatment on a large scale. References Adane, K., Moore, D., Archer, S.A., 1996. Preliminary studies on the use of Beauveria bassiana to control Sitophilus zeamais (Coleoptera: Curculionidae) in the laboratory. J. Stored Prod. Res. 32, 105e113. Akbar, W., Lord, J.C., Nechols, J.R., Howard, R.W., 2004. Diatomaceous earth increases the efficacy of Beauveria bassiana against Tribolium castaneum larvae and increases conidia attachment. J. Econ. Entomol. 97, 273e280. Allotey, J., 1991. Development and fecundity of the rice moth Corcyra cephalonica (Pyralidae). Discov. Innov. 3, 123e126. Allotey, J., Kumar, R., 1985. Competition between Corcyra cephalonica (Stainton) and Ephestia cautella (Walker) in cocoa beans. Insect Sci. Appl. 6, 627e632. Athanassiou, C.G., Steenberg, T., 2007. Insecticidal effect of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) in combination with three diatomaceous earth formulations against Sitophilus granarius (L.) (Coleoptera: Curculionidae). Biol. Control 40, 411e416. Cherry, A.J., Abalo, P., Hell, K., 2005. A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea. J. Stored Prod. Res. 41, 295e309.
Cox, P.D., Crawford, L.A., Gjestrud, G., Bell, C.H., Bowley, C.R., 1981. The influence of temperature and humidity on the life cycle of Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae). Bull. Entomol. Res. 71, 171e181. Cox, P.D., Wakefield, M.E., Price, N.R., Wildey, K.B., Moore, D., Aquino de Muro, M., Bell, B.A., 2003. Entomopathogenic fungi for the control of invertebrate pests in storage structures: advances in stored product protection. In: Proceedings of the 8th International Working Conference on Stored Product Protection, NewYork, UK, pp. 87e94. Cox, P.D., Wakefield, M.E., Price, N., Wildey, K.B., Chambers, J., Moore, D., Aquino de Muro, M., Bell, B.A., 2004. The Potential Use of Insect Specific Fungi to Control Grain Storage Pests in Empty Grain Stores. HGCA Project Report No. 341, 49 pp. Feng, M.G., Poprawski, T.J., Khachatourians, G.G., 1994. Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: current status. Biocontrol Sci. Technol. 4, 3e34. Gottwald, I.R., Tedders, W.L., 1982. Studies on conidia release by the entomogenous fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) from adult pecan weevil (Coleoptera: Curculionidae) cadavers. Environ. Entomol. 11, 1274e1279. Hajek, A.E., St. Leger, R.J., 1994. Interactions between fungal pathogens and insect hosts. Annu. Rev. Entomol. 39, 293e322. Hidalgo, E., Moore, D., Le Patourel, G., 1998. The effect of different formulations of Beauveria bassiana on Sitophilus zeamais in stored maize. J. Stored Prod. Res. 34, 171e179. Inglis, D.G., Goettel, M.S., Johnson, D.L., 1993. Persistence of the entomopathogenic fungus, Beauveria bassiana, on phylloplanes of crested wheatgrass and alfalfa. Biol. Control 3, 258e270. Khashaveh, A., Ghosta, Y., Safaralizadeh, M.H., Ziaee, M., 2011. The use of entomopathogenic fungus, Beauveria bassiana in assays with storage grain beetles. J. Agric. Sci. Tech. 13, 35e43. Lacey, L.A., Martins, A., Ribeiro, C., 1994. The pathogenicity of Metarhizium anisopliae and Beauveria bassiana for adults of the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae). Eur. J. Entomol. 91, 313e319. Leathers, T.D., Gupta, S.C., Alexander, N.J., 1993. Mycopesticides: status, challenges and potential. J. Ind. Microbiol. 12, 69e75. Lord, J.C., 2001. Desiccant dusts synergize the effect of Beauveria bassiana (Hyphomycetes: Moniliales) on stored-grain beetles. J. Econ. Entomol. 94, 367e372. Lord, J.C., 2007. Desiccation increases the efficacy of Beauveria bassiana for stored grain pest insect control. J. Stored Prod. Res. 43 (4), 535e539. Madrid, F.J., White, N.D.G., Loschiavo, S.R., 1990. Insects in stored cereals and their association with farming practices in Southern Manitoba. Can. Entomol. 122, 515e523. Mahdneshin, Z., Safaralizadah, M.H., Ghosta, Y., 2009. Study on the efficacy of Iranian isolates of Beauveria bassiana (balsamo) vuillemin and Metarhizium anisopliae (metsch.) sorokin against Rhyzopertha dominica F. (Coleoptera: Bostrichidae). J. Biol. Sci. 9, 170e174. Michalaki, M.P., Athanassiou, C.G., Kavallieratos, N.G., Batta, Y.A., Balotis, G.N., 2006. Effectiveness of Metarhizium anisopliae (Metschinkof) (Deuteromycotina: Hyphomycetes) applied alone or in combination with diatomaceous earth against Trilobium confusum larvae (Coleoptera: Tenebrionidae): influence of temperature, relative humidity and type of commodity. Crop Prot. 25, 418e425. Muthukrishnan, N., Venugopal, M.S., Ilamurugu, K., Janarthanan, R., 1996. Recycling of spent larval food of Corcyra cephalonica for fuel gas production. Int. Biodeterior. Biodegrad. 38, 123e124. Padin, S.B., Dal Bello, G.M., Vasicek, A.L., 1997. Pathogenicity of Beauberia bassiana for adults of Tribolium castaneum in stored grains. Entomophaga 42, 569e574. Padin, S.B., Dal Bello, G.M., Vasicek, A.L., 1995. Potential bioinsecticida de hongos entomopathgenos de plagas en granos almacenados. Rev. Fac. Agron. UBA 15, 1e7. Padin, S., Dal Bello, G., Fabrizio, M., 2002. Grain losses caused by Tribolium castaneum, Sitophilus oryzae and Acanthoscelides obtectus in stored durum wheat and beans treated with Beauveria bassiana. J. Stored Prod. Res. 38, 69e74. Samodra, H., Ibrahim, Y.B., 2006. Effectiveness of selected entomopathogenic fungi in packed rice grain at room temperature against Corcyra cephalonica stainton. AJSTD 23, 183e192. Shaaya, E., Ravid, U., Paster, N., Juven, B., Zisman, U., Pissarev, V., 1991. Fumigant toxicity of essential oils against four major stored product insects. J. Chem. Ecol. 17, 499e504.
S. Kaur et al. / Journal of Stored Products Research 59 (2014) 185e189 Shams, G., Safaralizadeh, M.H., Imani, S., Shojai, M., Aramideh, S., 2011. A laboratory assessment of the potential of the entomopathogenic fungi Beauveria bassiana (Beauvarin®) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and Sitophilus granarius (L.) (Coleoptera: Curculionidae). Afr. J. Microbiol. Res. 5, 1192e1196. Steenberg, T., 2005. Safety aspects related to the use of entomopathogenic fungi in stored products. In: Proceedings of the 6th Meeting of COST Action 842 Working Group IV Biocontrol of Arthropod Pests in Stored Products. 10-11 June, 2005, Locorotondo, Italy. Tripathi, A.K., Upadhyay, S., Bhuiyan, M., Bhattacharya, P.R., 2009. A review on prospects of essential oils as biopesticides in insect pest management. J. Pharmacogn. Phytother. 1, 52e63.
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Vanmathi, J.S., Padma Latha, C., Ranjit Singh, A.J.A., 2011. Impact of entomopathogenic fungus, Beauveria bassiana on stored grains pest, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J. Biopestic. 4, 194e197. Vassilakos, T.N., Athanassiou, C.G., Kavallieratos, N.G., Vayias, B.J., 2006. Influence of temperature on the insecticidal effect of Beauveria bassiana in combination with diatomaceous earth against Rhyzopertha dominica and Sitophilus oryzae on stored wheat. Biol. Control 38, 270e281. Wakil, W., Riasat, T., Ghazanfer, M.U., Kwon, Y.J., Shaheen, F.A., 2011. Aptness of Beauveria bassiana and enhanced diatomaceous earth (DEBBM) for control of Rhyzopertha dominica. Entomol. Res. 41, 233e241.
Contents lists available at ScienceDirect
Journal of Stored Products Research journal homepage: www.elsevier.com/locate/jspr
A laboratory assessment of the potential of Beauveria bassiana (Balsamo) Vuillemin as a biocontrol agent of Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) Sanehdeep Kaur*, Abhinay Thakur, Mamta Rajput Department of Zoology, Guru Nanak Dev University, Amritsar, Punjab, 143005, India
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 14 August 2014 Available online
Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) is a wide spread pest of stored grains and responsible for considerable damage in godowns and warehouses. Beauveria bassiana (Balsamo) Vuillemin, the most common and ubiquitous fungal entomopathogen is known to have high potential for the control of insects belonging to various orders. To evaluate the pathogenicity of B. bassiana against C. cephalonica, sorghum grains were treated with three conidial concentrations of B. bassiana i.e. 1.49 105, 4.05 106, 2.02 108 spores/ml. The highest concentration was found to be the most effective. It induced significantly higher larval mortality. Adverse effects such as reduced adult emergence and longevity of C. cephalonica were also observed when the grains were treated with B. bassiana. Significantly lesser number of adults emerged from pupae, when shorghum grains were treated with 2.02 108 spores/ml. The adverse effects were also exhibited as malformed adults that lived for a short period. Overall study showed that B. bassiana gave effective control of C. cephalonica and thus may play a pivotal role in the integrated pest management program. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Corcyra cephalonica Beauveria bassiana Pathogenicity Biological control
1. Introduction Insect pests have been damaging and causing heavy losses to stored grains quantitatively and qualitatively throughout the world especially in tropical and semitropical countries (Madrid et al., 1990; Tripathi et al., 2009). In countries without modern storage technologies, insect pests cause 10e40% damage in stored grains (Shaaya et al., 1991).Traditionally, fumigants and other synthetic insecticides have been commonly used for the protection of stored products from attack by storage insects. However, the continuous use of these insecticides has led to the development of resistance in insect pests as well as residual problem in stored grains (Michalaki et al., 2006). These adverse effects of synthetic insecticide application have increased public awareness regarding the human safety and possible environmental damage and focused the research towards other alternative strategies for the management of stored grain insect pests. The use of entomopathogenic fungi (EPF) and other microbial control agents is known to be a promising strategy with good potential to minimize the adverse effects of insecticides
* Corresponding author. Tel.: þ91 0183-2258802-09; fax: þ91 0183-2258819, þ91 0183-2258820. E-mail address: [email protected] (S. Kaur). http://dx.doi.org/10.1016/j.jspr.2014.08.004 0022-474X/© 2014 Elsevier Ltd. All rights reserved.
(Padin et al., 1997). The fungal biocontrol agents can be used in close proximity to foods and feed (Cox et al., 2003, 2004). EPF can be employed to treat empty stores to control residual pests before the new harvest is brought in or may be applied as direct admixture of conidia to grains either as preventive or curative treatments to bulk grains provided it does not decrease the quality and marketability of grains (Steenberg, 2005). Out of 700 entomopathogenic species of fungi reported from insects, only10 have been widely known as biocontrol agents (Hajek and St. Leger, 1994). Among these, one of the most frequently used fungi is Beauveria bassiana (Balsamo) Vuillemin. Its potential as a mycoinsecticide has been demonstrated on a large number of different insect hosts (Inglis et al., 1993; Feng et al., 1994; Padin et al., 1995; Cherry et al., 2005). Beauveria bassiana has potential for the control of different stored product pests. It is registered by the U.S. Environmental Protection Agency (EPA) for a wide range of insect control applications (Lord, 2001). Most of the research has been done on formulations of this fungus for the management of stored product insect pests (Akbar et al., 2004; Lord, 2007; Vassilakos et al., 2006). Beauveria bassiana has been demonstrated to control Rhyzopertha dominica (Fabricius) (Mahdneshin et al., 2009; Wakil et al., 2011), Callosobruchus maculatus (Fab.) and Sitophilus granarius (Linnaeus) (Shams et al., 2011), Tribolium castaneum (Herbst) and Oryzaephilus surinamensis (L.) (Khashaveh et al., 2011). Several B. bassiana
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formulations (Boverosil, Mycotrol, ES, Mycotrol 22WP etc.) are commercially available and are registered for use in storage facilities (Khashaveh et al., 2011). Corcyra cephalonica is a serious pest of some important stored food commodities such as rice, wheat, maize, sorghum, groundnut, cotton seeds, coffee, spices and cocoa beans, especially in the tropics (Cox et al., 1981; Allotey and Kumar, 1985; Allotey, 1991). The larvae of C. cephalonica cause considerable damage to stored food commodities while feeding, leaving silken threads wherever they move. The present study was carried out to evaluate the potential of B. bassiana as a biological control agent against C. cephalonica. 2. Materials and methods This experiment with B. bassiana as a biocontrol agent against C. cephalonica was done at the Biocontrol Laboratory, Department of Zoology, Guru Nanak Dev University, Amritsar (Punjab), India. The adults of C. cephalonica were collected from stored wheat in the godowns. Then the culture was maintained at 25 ± 2 C and 65 ± 5% RH in the laboratory on sorghum grains which is one of its preferred host (Muthukrishnan et al., 1996). Rearing was carried out on partially crushed sorghum grains in battery jars (15 cm 10 cm) covered with muslin cloth to ensure proper ventilation. Ten adults in the ratio of 4:6 (M: F) were released in each battery jars half filled with crushed sorghum. After oviposition the dead adults were removed and jars were kept undisturbed for further development of C. cephalonica. From this culture 2nd instar larvae were used for experimental purpose. Cultures of the fungus B. bassiana (NBAII-Bb-5a) were procured from National Bureau of Agriculturally Important Insects (NBAII), Bangalore and further maintained in the laboratory on Potato Dextrose Agar (PDA) at 30 C. Three weeks old culture was used for experimental studies. Fungal suspension was prepared by adding 10 ml of distilled water and a drop of 0.01% Tween 80 solution to the slants and filtered through muslin cloth to remove mycelia. From this concentrated spore suspension, serial dilutions were made and spore count was obtained with the help of hemocytometer. Three concentrations i.e. C 1 ¼ 1.49 105, C 2 ¼ 4.05 106 and C 3 ¼ 2.02 108 spores/ml were used for bioassay studies. The bioassay studies are described as follows. Thirty ml of each spore suspension was sprayed on 120 g of partially crushed sorghum grains with the help of hand sprayer followed by thorough mixing. In control, grains were sprayed with same quantity of water having a drop of 0.01% Tween 80 solution. After air drying the treated grains were equally distributed in 6 plastic containers (4 cm 6 cm) i.e. 20 g/container. Ten 2nd instar larvae were released in each container, which represents one replication. Similarly larvae were released in containers having untreated grains. The larvae were checked daily for larval mortality. Observations were also recorded on larval period, adult emergence, adult longevity and deformity in all the treatments and control. To verify that the larval death is due to fungus, the dead larvae were surface sterilized with 5% sodium hypochlorite and placed in autoclaved petri dishes having moist muslin cloth at the base. The petri dishes were kept in B.O.D. incubator at 25 ± 2 C temperature. Observations were made on the mycelial growth of fungus on the dead larvae. The larval counts showing fungal growth were only considered for percent mortality due to fungus. All the experiments were replicated six times with 10 larvae per replication and values were represented as their mean ± SE. To compare difference in the means, one way analysis of variance (ANOVA) with Tukey's test at P 0.05 was performed. SPSS software for windows version 16.0 (SPSS Inc., Chicago) and Microsoft office Excel 2007 (Microsoft Corp., USA) were used to perform the statistical analysis.
3. Results There was a significant difference in the mortality of C. cephalonica larvae when the highest concentration i.e. 2.02 108 spores/ml of B. bassiana was applied on sorghum grains (F ¼ 11.46, P < 0.001). Relative to control six times higher mortality was recorded at the highest concentration. However, the lower doses of fungus (4.05 106 and 1.49 105 spores/ml) failed to induce significant mortality in the larvae feeding on treated grains as compared to control (Fig. 1a). The larval mortality started after 24 h of feeding on grains treated with the highest concentration whereas the first larval death was recorded after 48e72 h of exposure at lower doses. After death, the larvae became hard and stiff. The white mycelial growth appeared 24 h after death at the highest concentration, but at lower concentrations it took 2e3 days to grow on dead larvae (Fig. 2aec). Larval mortality was also recorded on untreated grains. However, no fungal growth was observed on larvae died in control. Treatment of sorghum grains significantly delayed the larval development of C. cephalonica as compared to control (F ¼ 62.50, P < 0.001). The larvae took more time to pupate (6.79 days) when reared on grains treated with 2.02 108 spores/ml. Similarly the lower concentrations i.e. 4.05 106 and 1.49 105 spores/ml also significantly extended the development of larvae (Fig. 1b). Lesser number of adults emerged from pupae developed on treated sorghum as compared to control, but significant effect was observed only at 2.02 108 spores/ml of the fungus (F ¼ 11.46, P < 0.001) (Fig. 3a). Longevity of adult male decreased significantly at all the concentrations of B. bassiana (F ¼ 23.00, P < 0.001) (Fig. 3b). Significant influence of the fungus was also observed on female longevity at 4.05 106 and 2.02 108 spores/ml concentrations (F ¼ 9.77, P < 0.001) (Fig. 3c). Sublethal effects of B. bassiana on C. cephalonica were also manifested as emergence of deformed adults. As is evident from Fig. 3d, treatment of grains with 2.02 108 spores/ml of fungus induced morphological deformities in significantly higher number of adults (16.66%) as compared to adults emerged from larvae reared on untreated grains. The wings of these insects were underdeveloped and curled (Fig. 4aeb) as compared to normal adults (Fig. 4c). These malformed adults lived for only 1e2 days as compared to 5e6 days for normal adults. 4. Discussion Efficiency of biocontrol of insect pests with entomopathogenic fungi is mainly due to their capacity to produce epizootics from the infected cadavers (Leathers et al., 1993). These cadavers act as a source of inocula and thus further disseminate microbial agent in the environment (Gottwald and Tedders, 1982; Lacey et al., 1994). Earlier Samodra and Ibrahim (2006) evaluated the dried conidia of eight isolates of entomopathogenic fungi against C. cephalonica in rice grains. Among these two isolates of B. bassiana (BbGc and BbPs) and one isolate of Metarhizium anisopliae (MaPs) gave high mortality to C. cephalonica larvae. Isolates of BbGc and BbPs formulated in kaolin and BbPs formulation in tapioca flour provided 100% larval mortality by 15th day of introduction. Even more than 90% mortality was recorded when rice grains were stored for 4 months. However, the pathogenicity of M. anisopliae against C. cephalonica larvae decreased rapidly upon storage at room temperature affording less than 60% control by the 2nd month of storage. Adane et al. (1996) also reported 88% mortality in Sitophilus zeamais (Motschulsky) due to treatment with B. bassiana at 104 conidia/ml within 8 days. There are reports indicating the effectiveness of B. bassiana and M. anisopliae against many coleopteran pests of stored grains (Mahdneshin et al., 2009; Khashaveh et al., 2011).
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Fig. 1. (a, b) Effect of different concentrations of B. bassiana (C 1 ¼ 1.49 105, C 2 ¼ 4.05 106 and C 3 ¼ 2.02 108 spores/ml) on mortality and development of 2nd instar larvae of C. cephalonica. Means and SE are given. Means followed by the same letter are not significantly different (P 0.05) based on Tukey's test.
Fig. 2. (aec) Different stages of mycelial growth on dead larvae.
Post-mortem mycelia and conidial growth demonstrated that the insects had died due to fungal infection. Beauveria bassiana treated sorghum grains induced significantly higher mortality in C. cephalonica as compared to untreated grains. Besides causing mortality the other sublethal effects were prolonged development period, reduced adult longevity and morphological deformities.
The present studies indicated a dose dependent mortality but significant effects of B. bassiana on survival of C. cephalonica were observed only at the highest concentration. Similarly Padin et al. (2002) documented that wheat grains infested with Sitophilus oryzae without the conidia was significantly more damaged by weevils than grains treated with B. bassiana. Present study revealed
Fig. 3. (aed) Effect of different concentrations of B. bassiana (C 1 ¼ 1.49 105, C 2 ¼ 4.05 106 and C 3 ¼ 2.02 108 spores/ml) on adults of C. cephalonica. Means and SE are given. Means followed by the same letter are not significantly different (P 0.05) based on Tukey's test.
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Fig. 4. (aeb) Morphologically deformed adults and (c) normal adults.
that exposure of C. cephalonica larvae to B. bassiana treated sorghum grains resulted in significant reduction of adult emergence as compared to control. Treatment of grains with 2.02 108 spores/ml of fungus induced morphological deformities in significantly higher number of adults (16.66%) as compared to adults emerged from larvae reared on untreated grains. Beauveria bassiana at concentration of 1 108 conidia/ml showed highest oviposition reduction (60.58%), and adult mortality against the pulse beetle, C. maculatus (99.44%) at 92 h (Vanmathi et al., 2011). Similarly Cherry et al. (2005) found that B. bassiana 0362 at 1 107 and 1 108 conidia g1 grains led to significant adult mortality and reduced F1 emergence relative to untreated population of C. maculatus. The efficiency of B. bassiana has been reported against T. castaneum larvae with 62% mortality @ 2700 mg/kg concentration (Akbar et al., 2004). Athanassiou and Steenberg (2007) also demonstrated the insecticidal effect of B. bassiana against adults of the granary weevil, S. granarius. The present investigations indicated that B. bassiana not only caused mortality in C. cephalonica but also prolonged its development, decreased adult emergence and induced morphological deformities in adults which ultimately lead to suppression of pest population. One advantage of pathogen based protection systems is that disease cycling occurs. At death the cadaver produces many infective agents from internal and external sporulation renewing inoculum wherever the insects have died (Hidalgo et al., 1998). The storage insects can absorb lethal doses of the entomopathogen from sporulating cadavers. Use of this fungus can potentially benefit the environment and lead to more effective protection of stored grains. So from these results it is evident that B. bassiana may become an integral part of safe IPM program of stored grain pests. To assess the practical value of these results further experiments are required to evaluate the efficacy of fungal treatment on a large scale. References Adane, K., Moore, D., Archer, S.A., 1996. Preliminary studies on the use of Beauveria bassiana to control Sitophilus zeamais (Coleoptera: Curculionidae) in the laboratory. J. Stored Prod. Res. 32, 105e113. Akbar, W., Lord, J.C., Nechols, J.R., Howard, R.W., 2004. Diatomaceous earth increases the efficacy of Beauveria bassiana against Tribolium castaneum larvae and increases conidia attachment. J. Econ. Entomol. 97, 273e280. Allotey, J., 1991. Development and fecundity of the rice moth Corcyra cephalonica (Pyralidae). Discov. Innov. 3, 123e126. Allotey, J., Kumar, R., 1985. Competition between Corcyra cephalonica (Stainton) and Ephestia cautella (Walker) in cocoa beans. Insect Sci. Appl. 6, 627e632. Athanassiou, C.G., Steenberg, T., 2007. Insecticidal effect of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) in combination with three diatomaceous earth formulations against Sitophilus granarius (L.) (Coleoptera: Curculionidae). Biol. Control 40, 411e416. Cherry, A.J., Abalo, P., Hell, K., 2005. A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea. J. Stored Prod. Res. 41, 295e309.
Cox, P.D., Crawford, L.A., Gjestrud, G., Bell, C.H., Bowley, C.R., 1981. The influence of temperature and humidity on the life cycle of Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae). Bull. Entomol. Res. 71, 171e181. Cox, P.D., Wakefield, M.E., Price, N.R., Wildey, K.B., Moore, D., Aquino de Muro, M., Bell, B.A., 2003. Entomopathogenic fungi for the control of invertebrate pests in storage structures: advances in stored product protection. In: Proceedings of the 8th International Working Conference on Stored Product Protection, NewYork, UK, pp. 87e94. Cox, P.D., Wakefield, M.E., Price, N., Wildey, K.B., Chambers, J., Moore, D., Aquino de Muro, M., Bell, B.A., 2004. The Potential Use of Insect Specific Fungi to Control Grain Storage Pests in Empty Grain Stores. HGCA Project Report No. 341, 49 pp. Feng, M.G., Poprawski, T.J., Khachatourians, G.G., 1994. Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control: current status. Biocontrol Sci. Technol. 4, 3e34. Gottwald, I.R., Tedders, W.L., 1982. Studies on conidia release by the entomogenous fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) from adult pecan weevil (Coleoptera: Curculionidae) cadavers. Environ. Entomol. 11, 1274e1279. Hajek, A.E., St. Leger, R.J., 1994. Interactions between fungal pathogens and insect hosts. Annu. Rev. Entomol. 39, 293e322. Hidalgo, E., Moore, D., Le Patourel, G., 1998. The effect of different formulations of Beauveria bassiana on Sitophilus zeamais in stored maize. J. Stored Prod. Res. 34, 171e179. Inglis, D.G., Goettel, M.S., Johnson, D.L., 1993. Persistence of the entomopathogenic fungus, Beauveria bassiana, on phylloplanes of crested wheatgrass and alfalfa. Biol. Control 3, 258e270. Khashaveh, A., Ghosta, Y., Safaralizadeh, M.H., Ziaee, M., 2011. The use of entomopathogenic fungus, Beauveria bassiana in assays with storage grain beetles. J. Agric. Sci. Tech. 13, 35e43. Lacey, L.A., Martins, A., Ribeiro, C., 1994. The pathogenicity of Metarhizium anisopliae and Beauveria bassiana for adults of the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae). Eur. J. Entomol. 91, 313e319. Leathers, T.D., Gupta, S.C., Alexander, N.J., 1993. Mycopesticides: status, challenges and potential. J. Ind. Microbiol. 12, 69e75. Lord, J.C., 2001. Desiccant dusts synergize the effect of Beauveria bassiana (Hyphomycetes: Moniliales) on stored-grain beetles. J. Econ. Entomol. 94, 367e372. Lord, J.C., 2007. Desiccation increases the efficacy of Beauveria bassiana for stored grain pest insect control. J. Stored Prod. Res. 43 (4), 535e539. Madrid, F.J., White, N.D.G., Loschiavo, S.R., 1990. Insects in stored cereals and their association with farming practices in Southern Manitoba. Can. Entomol. 122, 515e523. Mahdneshin, Z., Safaralizadah, M.H., Ghosta, Y., 2009. Study on the efficacy of Iranian isolates of Beauveria bassiana (balsamo) vuillemin and Metarhizium anisopliae (metsch.) sorokin against Rhyzopertha dominica F. (Coleoptera: Bostrichidae). J. Biol. Sci. 9, 170e174. Michalaki, M.P., Athanassiou, C.G., Kavallieratos, N.G., Batta, Y.A., Balotis, G.N., 2006. Effectiveness of Metarhizium anisopliae (Metschinkof) (Deuteromycotina: Hyphomycetes) applied alone or in combination with diatomaceous earth against Trilobium confusum larvae (Coleoptera: Tenebrionidae): influence of temperature, relative humidity and type of commodity. Crop Prot. 25, 418e425. Muthukrishnan, N., Venugopal, M.S., Ilamurugu, K., Janarthanan, R., 1996. Recycling of spent larval food of Corcyra cephalonica for fuel gas production. Int. Biodeterior. Biodegrad. 38, 123e124. Padin, S.B., Dal Bello, G.M., Vasicek, A.L., 1997. Pathogenicity of Beauberia bassiana for adults of Tribolium castaneum in stored grains. Entomophaga 42, 569e574. Padin, S.B., Dal Bello, G.M., Vasicek, A.L., 1995. Potential bioinsecticida de hongos entomopathgenos de plagas en granos almacenados. Rev. Fac. Agron. UBA 15, 1e7. Padin, S., Dal Bello, G., Fabrizio, M., 2002. Grain losses caused by Tribolium castaneum, Sitophilus oryzae and Acanthoscelides obtectus in stored durum wheat and beans treated with Beauveria bassiana. J. Stored Prod. Res. 38, 69e74. Samodra, H., Ibrahim, Y.B., 2006. Effectiveness of selected entomopathogenic fungi in packed rice grain at room temperature against Corcyra cephalonica stainton. AJSTD 23, 183e192. Shaaya, E., Ravid, U., Paster, N., Juven, B., Zisman, U., Pissarev, V., 1991. Fumigant toxicity of essential oils against four major stored product insects. J. Chem. Ecol. 17, 499e504.
S. Kaur et al. / Journal of Stored Products Research 59 (2014) 185e189 Shams, G., Safaralizadeh, M.H., Imani, S., Shojai, M., Aramideh, S., 2011. A laboratory assessment of the potential of the entomopathogenic fungi Beauveria bassiana (Beauvarin®) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and Sitophilus granarius (L.) (Coleoptera: Curculionidae). Afr. J. Microbiol. Res. 5, 1192e1196. Steenberg, T., 2005. Safety aspects related to the use of entomopathogenic fungi in stored products. In: Proceedings of the 6th Meeting of COST Action 842 Working Group IV Biocontrol of Arthropod Pests in Stored Products. 10-11 June, 2005, Locorotondo, Italy. Tripathi, A.K., Upadhyay, S., Bhuiyan, M., Bhattacharya, P.R., 2009. A review on prospects of essential oils as biopesticides in insect pest management. J. Pharmacogn. Phytother. 1, 52e63.
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Vanmathi, J.S., Padma Latha, C., Ranjit Singh, A.J.A., 2011. Impact of entomopathogenic fungus, Beauveria bassiana on stored grains pest, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). J. Biopestic. 4, 194e197. Vassilakos, T.N., Athanassiou, C.G., Kavallieratos, N.G., Vayias, B.J., 2006. Influence of temperature on the insecticidal effect of Beauveria bassiana in combination with diatomaceous earth against Rhyzopertha dominica and Sitophilus oryzae on stored wheat. Biol. Control 38, 270e281. Wakil, W., Riasat, T., Ghazanfer, M.U., Kwon, Y.J., Shaheen, F.A., 2011. Aptness of Beauveria bassiana and enhanced diatomaceous earth (DEBBM) for control of Rhyzopertha dominica. Entomol. Res. 41, 233e241.