Laboratory evaluation of chemical-biological control of the rice weevil (Sitophilus oryzae L.) in stored grains

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1 Journal of Stored Products Research 37 (2001) 77±84 Laboratory evaluation of chemical-biological control of the rice weevil (Sitophilus oryzae L.) in stored grains G. Dal Bello a, *, S. Padin a,c.loâ pez Lastra b, M. Fabrizio c a ComisioÂn de Investigaciones CientõÂ cas de la Provincia de Buenos Aires (CIC) y Departamento de Sanidad Vegetal de la Facultad de Ciencias Agrarias y Forestales de la UNLP, Calle 60 y 119. CC 31 (1900) La Plata, Buenos Aires, Argentina b Centro de Estudios ParasitoloÂgicos y de Vectores (CONICET), Calle 2 N o 584 (1900) La Plata, Buenos Aires, Argentina c CaÂtedra de EstadõÂstica de la Facultad de AgronomõÂa de la UBA, Avenida San MartõÂn 4453 (1417), Ciudad AutoÂnoma de Buenos Aires, Argentina Accepted 7 February 2000 Abstract The virulence of ten di erent fungal isolates of: Beauveria bassiana, Metarhizium anisopliae, Verticillium lecanii and Paecilomyces farinosus to the rice weevil Sitophilus oryzae was tested. A fungal mix of the most e cient isolates, B. bassiana ARSEF 5500+M. anisopliae ARSEF 2974, which caused the highest mortality, was assayed in combination with fenitrothion at a concentration lower (3 ppm) than the normal 6 ppm. Fungal inoculation of insects was done by spraying conidial suspensions of each fungus on wheat. Insecticide formulations were added by spraying wheat. Treated and untreated insects were incubated on durum wheat. Insects were kept in a climatized chamber for 30 days. Observations were performed at 7, 14 and 30 days to record insect mortality. Highly signi cant di erences were demonstrated for B. bassiana 5500 and 5501 and for M. anisopliae The level of mortality produced by treatments was: 6 ppm insecticide=97.50%, B. bassiana ARSEF 5500+M. anisopliae ARSEF ppm insecticide=74.17%, B. bassiana ARSEF 5500+M.anisopliae ARSEF 2974= 50% and 3 ppm insecticide=37.50%. There was a statistically signi cant di erence ( p < 0.05) among treatments Elsevier Science Ltd. All rights reserved. Keywords: Sitophilus oryzae; Entomopathogenic fungi; Beauveria bassiana; Metarhizium anisopliae; Fenitrothion; Integrated control * Corresponding author. Fax: address: gmdalbello@infovia.com.ar (G. Dal Bello) X/01/$ - see front matter Elsevier Science Ltd. All rights reserved. PII: S X(00)

2 78 G. Dal Bello et al. / Journal of Stored Products Research 37 (2001) 77±84 1. Introduction Stored grain insect pests can cause reductions in weight, quality, commercial value and seed viability. Seventy- ve percent of these insects are coleopterans (VinÄ uela et al., 1993) and the most damaging species of storage insects are in the genera Sitophilus and Tribolium (Marsans, 1987; Khan and Selman, 1988; Pinto et al., 1997). Sitophilus oryzae L. (Coleoptera: Curculionidae), an ubiquitous pest of economic importance, is an internal feeding insect that bores into stored grain. Adult weevils feed mainly on the endosperm, reducing the carbohydrate content and the larvae feed preferentially on the germ of the grain, thus removing a large percentage of the protein and vitamins. Insects that selectively attack the germ will cause a greater loss in germination than others. The control of arthropod pests on stored products has been primarily through the use of fumigants and residual chemical insecticides to augment the more obvious approach of hygiene (Brooker et al., 1992; Adane et al., 1996). The excessive use of conventional chemical insecticides has resulted in a number of serious problems, e.g. resistance to the chemical insecticides, elimination of economically bene cial insects, persistence in the environment, toxicity to humans and wildlife and higher cost of crop production (Khan and Selman, 1989). Many insects and mites are capable of tolerating virtually all pesticides available for their control as a result of cross and multiple resistance (Metcalf, 1980). Recognition of the deleterious e ects of pesticides has prompted the development of alternative, less obtrusive management strategies, such as the use of microbial control agents. The biggest impetus for the growth of biopesticides comes from the growing awareness by farmers of the value of integrated pest management as a more environmentally sound, economical, safer and a selective approach to crop protection (Menn, 1996). Many e orts with biopesticides have been successfully conducted using entomopathogenic fungi. Experience indicates that e ciency and consistency of the biological control is improved by combining di erent antagonistic modes of action that are expressed by di erent antagonists (Lemanceau and Alabouvette, 1993). The successful development of Beauveria bassiana (Bals.) Vuill within hosts is based on simply overcoming the host hemocyte response (Hou and Chang, 1985); numbers of granulocytes are dramatically reduced 3 days after fungal challenge (Hung and Boucias, 1992). Hung et al. (1993) proposed that the cellular-defense response is the initial target of metabolites produced by B. bassiana. These metabolites block the recruitment of hemocytes required for nodule formation, although the initial recognition and phagocytosis responses remain functional. Metarhizium anisopliae (Metschn.) Sorok. produces immunodepressive substances, such as the toxin destruxin E, that inhibits the formation of nodules by paralyzing hemocytes (Huxham et al., 1989; Cerenius et al., 1990). Mycopesticides have been shown to have considerable potential for the management of insects and they have been used worldwide to control various coleopteran pests (Gottwald and Tedders, 1983; Khan and Selman, 1984, 1987, 1988; Rodrigues and Pratissoli, 1990; Adane, 1994; PadõÂ n et al., 1997). Although several stored product insects su er high mortality as a result of pathogenic disease, their use as biological control agents on stored grain insects has received little attention. The objective of this study was to determine the e ects of 10 di erent isolates of B. bassiana;

3 G. Dal Bello et al. / Journal of Stored Products Research 37 (2001) 77±84 79 M. anisopliae var. anisopliae; Verticillium lecanii (Zimm.) Viegas, and Paecilomyces farinosus (Holm) Brown and Smith against S. oryzae. Mixtures of the most e cient isolates were also evaluated in combination with fenitrothion to assess the use of an integrated measure to improve biological control of rice weevil. 2. Material and methods 2.1. Source and rearing of S. oryzae The initial stock of S. oryzae was obtained from infested wheat. Insects were mass produced in a climatized chamber at 27218C and 7025% r.h. with alternating light±dark cycles of 12 h. Insects were maintained in glass jars (0.25 l capacity) with 200 g of wheat free from pesticides. The jars were covered with a muslin cloth and after 2 weeks the original adults were removed by sieving. Once new adult weevils started to emerge, each jar was observed daily to collect the progeny which were kept in separate jars, according to their age group Source of fungal isolates Four isolates of B. bassiana, three isolates of M. anisopliae, two isolates of P. farinosus, and one isolate of V. lecanii were used (Table 1). Cultures of M. anisopliae (ARSEF 2575), P. farinosus (ARSEF 2937), and V. lecanii (ARSEF 1328) were obtained from R. Humber (Entomopathogenic Culture Collection, Plant Soil and Nutrition Lab, USDA-ARS, Ithaca, NY, USA). The B. bassiana (LPS 169, ARSEF 5500, 5501, 5502) and M. ansiopliae (LPS 84, ARSEF 2974) isolates were originally collected from di erent geographic regions in Argentina and di erent hosts/sources. The strains are registered at the USDA-ARS Collection in Ithaca, NY (Humber, 1992; Humber, 1998). One strain of P. farinosus was kindly provided by A. Hajek (Entomology Department, University of Cornell, Ithaca, NY, USA). Cultures of the ten isolates were grown and stored at 258C in PDA (potato dextrose agar). Table 1 Fungal isolates tested against Sitophilus oryzae with their origin and source of isolation No. Fungal isolates Isolate number Source of isolation Country 1 B. bassiana LPS 169 Diatraea saccharalis Argentina 2 M. anisopliae ARSEF 2974 Aedes crinifer Argentina 3 M. anisopliae LPS 84 Soil Argentina 4 B. bassiana ARSEF 5500 D. saccharalis Argentina 5 B. bassiana ARSEF 5501 Xanthogaleruca luteola Argentina 6 B. bassiana ARSEF 5502 D. saccharalis Argentina 7 P. farinosus Unknown Unknown Unknown 8 M. anisopliae ARSEF 2575 Curculio caryae USA 9 V. lecanii ARSEF 1328 Hypera postica France 10 P. farinosus ARSEF 2937 Otiorhynchus sulcatus USA

4 80 G. Dal Bello et al. / Journal of Stored Products Research 37 (2001) 77±84 Conidia were harvested from 10-day-old cultures by gently rinsing colonies with sterile distilled water. Conidial suspensions were ltered through a 60-mesh sieve to remove debris and resuspended in sterile Tween 20 (0.01% v/v aqueous solution) in distilled water. The suspensions were vortexed for 2 min, to break up the conidial clumps, the number of conidia were estimated with an hemocytometer and adjusted to conidia ml Bioassays Laboratory-reared adult insects (8±15-day- old) were used for this study. The weevils were collected from rearing jars, placed in a Petri dish and mixed thoroughly to facilitate random selection of the insects. To test the virulence of each fungus, three Petri dishes containing lter paper (9 cm dia) were assembled and 20 insects were placed in each Petri dish. Using a perfume sprayer, each of three replicates of S. oryzae were sprayed directly with 1 ml of conidial suspension containing spores ml 1 of fungus. Controls were sprayed with 0.01% Tween 20 in distilled water. Each treated replicate was kept for 24 h without food at C and 70% 2 5% r.h. After 24 h, insects from each replicate were transferred to a plastic cup (100 ml capacity) containing 50 g of undamaged wheat kernels and each cup was sealed using muslin cloth and a perforated lid. All the insects were maintained under controlled conditions as described above. Observations of each of the cups were done at 7, 14 and 30 days by emptying the contents of each cup onto white paper to identify dead individuals. The dead adults collected in the indicated periods were immediately submerged in 95% ethanol for 1 min, washed in sterile distilled water for 5 min, allowed to dry and then placed on moistened lter paper. Cadavers were kept at 258C for 3±5 days in the dark and those that showed hyphal growth characteristic of the entomopathogenic fungi were recorded as infected. A second series of bioassays involved the three most e cient isolates. Insects were inoculated with one fungus and in di erent fungal combinations and pathogenicity was evaluated. The test was conducted as described above but observations were done at 7 and 14 days. A nal spore concentration of conidia ml 1 of each fungus or fungal mixture was used. Finally, the fungal mixture which caused the highest mortality was tested in combination with fenitrothion at low concentration (3 ppm). Twenty rice weevils were mixed together for random selection and were placed in 100 ml plastic cups containing 50 g of undamaged wheat kernels. Inoculation of each batch of 40 insects was made by spraying 1 ml of the mix onto the grain. Integrated treatment was compared with the following controls: 6 ppm fenitrothion (normal concentration), 3 ppm fenitrothion and distilled water. The treated grain was shaken thoroughly and maintained at 278C228C and 70%25% r.h. for 30 days. There were three replicates for each treatment and insects were checked daily to record mortality. Dead insects in all treatments were removed and held under high humidity in Petri dishes containing damp lter paper to determine the number of sporulating cadavers. The mortality responses across the whole assessment period were analyzed using analysis of variance test (ANOVA) for a completely randomized design and the means were compared using a Tukey's test ( p < 0.05).

5 3. Results and discussion G. Dal Bello et al. / Journal of Stored Products Research 37 (2001) 77±84 81 Post-mortem mycelial and conidial growth demonstrated that most of the insects had died due to the pathogens. Observations of cadavers indicated that the external mycelium appears within 24±48 h after placing on damp lter paper. Although all isolates tested caused mortality in S. oryzae, fungal species showed great variability in virulence against the insects (Table 2). In general, all ten isolates caused low mortality in the weevils. However, highly signi cant di erences ( p < 0.05) in bioinsecticide e ects were demonstrated for B. bassiana ARSEF 5500, B. bassiana ARSEF 5501 and M. anisopliae ARSEF 2974, which caused the largest mortality. B. bassiana 5500 isolate showed the greatest virulence toward S. oryzae. The isolates of B. bassiana were originally obtained from Diatraea saccharalis Fabricius and the isolate of M. anisopliae was isolated from Aedes crinifer Theobald. Other reports (Rodrigues and Pratissoli, 1990; Moino and Alves, 1997; Moino et al., 1998) also indicated greater mortalities of storedgrain pests inoculated with Beauveria spp than with M. anisopliae. Those isolates resulted in mortality rates ranging from 20 to 50% within the test period (1±30 days) and mortality increased with the time. Based on the results obtained in the initial experiments, the three isolates were selected for further studies in which the fungi were applied in di erent combinations (Table 3). The mix B. bassiana ARSEF 5500+M. anisopliae ARSEF 2974 caused the highest mortality (51.66%) of S. oryzae towards the end of the experiment. According to Baker and Cook (1982) it is a general principle that complex associations in nature are more stable and there is a better chance of attaining successful biological control with a mixture of several antagonists than with a single one. Adane et al. (1996) demonstrated that several isolates of B. bassiana tested against S. zeamais (Motsch.) adults showed pathogenicity to insects, but there were Table 2 Mean percentage mortality by days 7, 14 and 30 of Sitophilus oryzae adults treated with ten isolates of entomopathogens (values are means of three replications) Percent mortality a Isolates tested Day 7 Day 14 Day 30 Means B. bassiana LPS b 30 c 30 c 26.7 cd M. anisopliae ARSEF a 40 b 50 a 40 a M. anisopliae LPS b 20 d 20 d 20 ef B. bassiana ARSEF a 50 a 50 a 43 a B. bassiana ARSEF b 40 b 40 b 33 b B. bassiana ARSEF b 30 c 40 b 30 bc P. farinosus 10 c 20 d 20 d 17 f M. anisopliae ARSEF b 20 d 20 d 20 ef V. lecanii ARSEF b 20 d 20 d 20 ef P. farinosus ARSEF c 30 c 27 cd 22 de DATE/Overall mean for day 20 b 30 a 32 a a Values followed by the same letter in the same column do not di er signi cantly ( p < 0.05) according to Tukey's studentized range test.

6 82 G. Dal Bello et al. / Journal of Stored Products Research 37 (2001) 77±84 highly signi cant di erences among the isolates with respect to virulence. These isolates caused 37±100% mortality and had median lethal times ranged from 2.7 to 8.8 days. Rodrigues and Pratissoli (1990) evaluated in laboratory experiments the pathogenicity of B. brongniartii and M. anisopliae isolates against S. zeamais and Acanthoscelidnes obtectus Say. B. brongniartii (Sacc.) Petch caused 89% mortality to S. zeamais and 47% to A. obtectus while M. anisopliae produced less than 50% mortality for both insects. Our results are similar in percentage mortality with B. bassiana and M. anisopliae to what has been previously demonstrated with other stored insect pests (Rodrigues and Pratissoli, 1990; Adane et al., 1996; Moino et al., 1998). Improvement in weevil control over that produced by each fungus alone was shown by mixtures of B. bassiana and M. anisopliae in this study. Mixtures may be advantageous for di erent reasons, including control of multiple insect species, more consistent control, or control over a wider range of environmental conditions, which were not evaluated in this assay. B. bassiana 5501 and M. anisopliae 2974 together produced a low lethal e ect, but incorporation of B. bassiana 5500 to that mix enhanced the mortality. The more lethal e ects were always related to the presence of strain Nevertheless its combination with strain 5501 did not cause mortality as high as the mix containing M. anisopliae There were no signi cant di erences ( p < 0.05) between these two last treatments (Table 3). Insecticide incorporation bioassays demonstrated that S. oryzae was relatively resistant to 3 ppm fenitrothion. The highest insect mortality was obtained for 6 ppm fenitrothion treated grain. The level of mortality produced by treatments was: 6 ppm insecticide > B. bassiana ARSEF 5500+M. anisopliae ARSEF ppm insecticide >B. bassiana ARSEF 5500+M. anisopliae ARSEF 2974 > 3 ppm insecticide (Table 4). There was a highly ( p < 0.05) signi cant di erence between the various treatments. With 6 ppm fenitrothion, 100% mortality was achieved within 14 days. There was no signi cant di erence among the treatments with respect to time, since there was little increase in mortality after 7 days. Treatment with the fungal mix+3 ppm fenitrothion gave 80% mortality by day 14. The fungal mix+insecticide achieved the second highest mortality (80%). This integrated Table 3 Mean percentage cumulative mortality by day 14 of Sitophilus oryzae adults treated with combinations of Beauveria bassiana ARSEF 5500, B. bassiana ARSEF 5501 and Metarhizium anisopliae ARSEF 2974 (values are means of three replications) Isolates tested Percent mortality (corrected) a Control 1.7 a ab bc bc c a Values followed by the same letter in the same column are not signi cantly di erent at p < 0.05 (Tukey's studentized multiple range test).

7 G. Dal Bello et al. / Journal of Stored Products Research 37 (2001) 77±84 83 Table 4 Mean percentage mortality of Sitophilus oryzae adults treated with Beauveria bassiana, Metarhizium anisopliae and di erent fenitrothion concentrations (values are means of three replications) Percent mortality a Treatment Day 7 Day 14 Means 6 ppm fenitrothion 95.0 a a 97.5 a c 51.7 c 50.0 c ppm fenitrothion 68.3 b 80.0 b 74.2 b 3 ppm fenitrothion 35.0 d 40.0 d 37.5 d Means 61.7 b 67.9 a a Means followed by the same letter in the same column are not signi cantly di erent at p < 0.05 (Tukey's studentized multiple range test). control killed a higher number of insects than the use of either the fungal mix or a low insecticide concentration alone. Biological control could be an alternative method to control stored grain insects. However progress must still be made to improve the e cacy and the reliability of biological control. Although this is only an initial investigation into the use of combinations of fungal isolates and insecticides, interesting results had been obtained and need to be further developed. It seems that the biological control should be compatible with other practices like the use of pesticides. Acknowledgements We thank to Marõ a Virginia Lo pez for help with the statistical analysis. References Adane, K., Microbial control of storage pests using the entomopathogenic fungus, Beauveria bassiana with special reference to Sitophilus zeamais and Callosobruchus chinensis. MSc Thesis. University of London, p. 93. Adane, K., Moore, D., Archer, S.A., Preliminary studies on the use of Beauveria bassiana to control Sitophilus zeamais (Coleoptera: Curculionidae) in the laboratory. Journal of Stored Products Research 32, 105±113. Baker, K., Cook, R.J., Biological Control of Plant Pathogens. The American Phytopathological Society, St. Paul, MN, USA. Brooker, D.B., Bakker-Arkema, F.W., Hall, C.W., Drying and Storage of Grains and Oilseeds. Van Nostrand Reinhold, New York, USA. Cerenius, L., Thornquist, P.O., Vey, A., Johansson, M.W., SoderhaÈ ll, K., The e ect of the fungal toxin destruxin E on isolated cray sh haemocytes. Journal of Insect Physiology 36, 785±789. Gottwald, T.R., Tedders, W.L., Suppression of pecan weevil (Coleoptera: Curculionidae) populations with entomopathogenic fungi. Environmental Entomology 12, 471±474. Hou, R.F., Chang, J., Cellular defense response to Beauveria bassiana in the silkworm, Bombyx mori. Applied Entomological Zoology 20, 118±125.

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