Egyptian Journal of Biological Pest Control, 24(1), 2014, 247-253 Efficacy of Entomopathogenic Nematodes and Fungi as Biological Control Agents against the Cotton Leaf Worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) Souad A. Shairra and Gehan M. Noah Biological Control Dept., Plant Protection Res. Institute Agricultural Res. Center, Giza, Egypt. (Received: May 25, 2014 and Accepted: June 25, 2014) ABSTRACT Pathogenicity of the two entomopathogenic nematodes, Heterorhabditis bacteriophora Poinar (HP88 strain) and Steinernema riobrave and two fungi, Metarhizium anisopliae and Beauveria bassiana as well the combined effect of both against 3rd instar larvae of the cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) was studied under laboratory conditions. Data revealed that the tested nematodes differed in their efficacy against 3 rd instar larvae of S. littoralis. The LD50 values for S. riobrave reached 438.8 and 180.1/ IJs ml/100g diet indicated the most potent nematodes at 48 and 72 h post treatment, respectively. On the contrary, LC50 reached 584.5 and199.2/ IJs ml/100 g diet, for H. bacteriophora, respectively. The LC50 values of fungus, B. bassiana revealed least mortality percentage (7.84 x 1011spores ml-1), while M. anisopolie was the most potent (2.89x 108spores ml-1) after 7 days of treatment. Probit regression lines for the combination of the tested concentration of fungi and nematodes (B. bassiana+ HP88) and (M. ansoplae + S. riobrave) against 3rd instar of S. littoralis showed that highest larval mortality increased with increasing fungal spores and/ or nematodes juvenile's concentrations. Key words: Spodoptera littoralis, entomopathogenic nematodes, entomopathogenic fungi, Efficacy. INTRODUCTION Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) is one of the most notorious chewing insect pests that causes heavy losses in cotton, thus, deprives the farmers from getting high yield. Insecticides of synthetic origin have been used to manage insect pests for more than 50 years (Charnley and Collins, 2007). However, due to adverse effects of insecticides on environment, their rational use is being advocated. Development of an effective control method against the cotton leaf worm has been urgently needed since it does serious damage to many important agricultural crops in Egypt. There is a serious interest in the use of microbial insecticides for biological control of insect pests, as alternatives to chemical control, since they neither leave toxic chemical residues in the environment nor do they induce resistance in their insect hosts (Evans, 1999). And hence, the public awareness and concern for environmental quality, has led to more focused attention on research aiming at developing biological agents (Hidalgo et al., 1998 and Shairra, 2010). A promising strategy with good potential to control insect pests and at the same time, to minimize adverse effects of chemical insecticides is the use of entomopathogenic microbial agents such as nematodes and fungi. The entomopathogenic nematodes (EPNs) belong to both families Steinernematidae and Heterorhabditidae associated with their symbiotic bacteria Xenorhabdus, and Photorhabdus, respectively, have been used commercially as biocontrol agents of economic insect pests (Gaugler, 2002). After penetration of the infective juveniles (IJs) into the selected host insects, they release their symbiotic bacteria into the insect hemocoel where they grow causing septicemia and/ or toxemia leading to host death (Georgis, 1992). Biological control methods offer alternative choices to use the unsuccessful pollutant chemicals. Among biological control agents, EPNs and the entomopathogenic fungi (EPFs) seemed to be the most appropriate weapon for controlling this serious pest in soil (Ibrahim and Shairra, 2011). EPFs are classified as fungi that infect, invade, and eventually kill their host insects (Singkaravanit et al., 2010). Because of low availability and cultivation difficulties, there have been only a few studies on the characterization of the products of EPFs. It is estimated that over 800 fungal species belong to more than 90 genera have been described as pathogenic against different insect species (Thackar, 2002) but only a dozen of EPFs species are available for pest management at grower level (Hajek and St. Leger, 1994). Fungal biological control agents have demonstrated efficacy against a wide range of insect pests including Spodoptera species (Purwar and Sachan, 2005; Lin et al., 2007; Asi et al., 2013; Gabarty et al, 2014 and Husnain et al., 2014). The present study aimed to evaluate the infectivity of different isolates of two nematode species, two fungi species and combined effect of both against larvae of S. littoralis under laboratory conditions. MATERIALS AND METHODS Host insect: Spodoptera littoralis larvae were reared on castor leaves, (Ricinus communis) according to Ibrahim (1974(. Newly molted 3rd instar larvae were utilized 248 in all experiments. Nematode species: Imported nematode species, Steinernema riobrave; Heterorhabditis bacteriophora Poinar (HP88 strain), supplied by Dr. El-Sadawy, National Research Center, Giza, Egypt were used in experimentation. For culturing, last instar larvae of both the greater wax moth, Galleria mellonella L. and S. littoralis were used as insect hosts according to Shamseldean (1994). Infective juveniles (Ijs) were harvested with the White's traps (White, 1927). The Ijs were kept under sterile conditions at 20±2°C and used in all experiments. Fungus species: Tow entomopathogenic fungi isolates, Metarhizium anisopliae and Beauveria bassiana, imported by Gaara Establishment, Import and Export Company. The Manufacture Company was M/S.T. Stares Company Limit- India. The fungi were kept at 4°C as stock culture. Conidia developed at 25°C from the stock culture were used for the bioassays. Susceptibility of S. littoralis larvae to the nematode species Third instar larvae, in five groups, were chilled at 4°C for 5 min. About 18 larvae were then infected with S. riobrave and H. bacteriophora Poinar (HP88) strains at each dose of the suspensions (16, 32, 64, 128 or 256 IJs /larva / ml distilled water). Plastic cups (5 x 6 x 6 cm), each contained 120 g of dried beach sand added with larvae diet (100g leaf of R. communis) and moistened with 1ml of distilled water containing the IJs of the nematodes. R.H. of the soil was (80%) throughout the bioassay. The host insects were kept in the cups which covered and placed in an incubator at 27+2oC. Control experiment was conducted by placing the host larvae on sand moistened with 1ml distilled sterilized water. Number of dead larvae in each cup was recorded at 24, 48, 72, 96 & 120 hours post infection. Each dose was repeated 3 times for each nematode isolate or strain. Values of LC50 and Slope were calculated. Dose-mortality effect of EPFs against 3rd instar larvae of S. littoralis Third instar larvae of S. littoralis were sprayed individually by different spore concentrations (1x104, 1x106 and 1x108 conidia ml-1) of the fungal isolates, M. anisopliae and B. bassiana. For the control treatment, larvae were sprayed with 0.1% Tween 80 solution. Each treatment, had a batch of 18 larvae, was replicated three times. Insect mortality was recorded daily up to ten days. The median lethal concentrations (LC50) were determined. Combined effect of entomopathogenic fungi and nematodes on S. littoralis larvae Individuals in three groups of 3rd instar larvae of S. littoralis were placed in a Petri dish of 9 cm diameter, lined with a filter paper disk. Larvae of the first group were fed for 2 days on castor leaves contaminated with M. anisopliae or B. bassiana, suspensions (1x108, 1x106, 1x104 spores/ml-1), respectively. Second group larvae were co-infected with the nematodes S. riobrave or H. bacteriophora Poinar (HP88 strain) suspensions (104 +64; 106+32 and 108+16, spores/ IJs / larva / ml distilled water), respectively. Larvae of the third group were fed normally and served as control. Control and experimental larvae were anesthetized on ice for 5 min before used under laboratory temperature ranged between 25 and 27±2°C. Number of cadavers was recorded at 3,7 and 10 days post-infection. Accumulative mortality percentages of the host individuals were calculated. Each treatment was repeated three times. Statistical analysis Percentages of mortalities were corrected according to Abbott's formula (Abbott, 1925). The data were then subjected to probit analysis through software Computer program to obtain the LC50 and slope values. Or using a software package “Ldp-line” a copyright by Ehab Bakr, Plant Protection Res. Institute, Giza, Egypt and Finney (1971). Analysis of variance (ANOVA) was conducted on all data using “COSTAT”, computer program software. Means were compared by Duncan’s multiple range test (Duncan, 1955). RESULTS AND DISCUSSION Susceptibility of S. littoralis larvae to nematodes Results in Table (1) show the corrected mortality percentages of S. littoralis larvae after feeding on castor leaves treated with different concentrations of nematodes Ijs (Ijs/ml/100 g diet). Obtained data clearly revealed that the tested nematodes differed in their efficacies against 3rd instar larvae of S. littoralis. Results indicated that the mortality rates increased with increasing the concentration and period after treatment. Corrected mortality percentages after 24 and 48 hours of treatment with nematodes, H. bacteriophora (HP88) and S. riborave ranged between 4.49-15.07% for both and ranged between 4.49-35.24 and 4.49-39.11% at the lowest (16 ijs/ml/100g diet) and the highest doses (256 ijs/ml/100g diet) concentrations, respectively (Table, 1). While the corresponding figures after 72; 96 and 120 hours of treatment with the respective nematodes ranged between (7.17-54.88 and 6.30-58.79%); (14.71-72.10 and 7.97-71.02%) and (20.73-86.32 and 249 Table (1): Corrected mortality % of S. littoralis larvae treated with H. bacteriophora (HP 88) & S. riborav at successive days post treatment under laboratory conditions Nematode strain / concentration ( Ijs/ml/ 100g diet ) 16 H. bacterio-phora 32 (HP88) 64 128 256 16 32 S. riborave 64 128 256 24h 4.49 6.29 8.62 11.53 15.07 4.49 6.29 8.62 11.53 15.07 Corrected mortality (%) after 48h 72h 96h 4.94 7.17 14.71 9.13 18.56 26.11 15.51 28.93 40.85 24.30 41.44 57.03 35.24 54.88 72.10 4.47 6.30 7.97 8.96 13.75 17.96 16.17 25.66 33.48 26.37 41.46 52.54 39.11 58.79 71.02 120h 20.73 36.77 55.55 73.14 86.32 31.19 40.93 51.26 61.51 71.02 Table (2): LC50 (ml/100 g diet) and slope values for H. bacteriophora (HP88) and S. riborav against 3rd instar larvae of S. littoralis at confidence limits (95%) Treatment H. bacteriophora (HP88) S. riborave H. bacteriophora (HP88) S. riborave H. bacteriophora (HP88) S. riborave H. bacteriophora (HP88) S. riborave H. bacteriophora (HP88) S. riborave LC50 Confidence limits (95%) 24h after treatment 19271.86 (1974.96 - 2.89 E+09) 19271.86 (1974.96 - 2.89 E+09) 48h after treatment 584.47 (345.22 - 1523.03) 438.78 (195.86 - 13950.09) 72h after treatment 199.18 (147.88 - 303.80) 180.11 (143.13 - 244.70) 96h after treatment 94.76 (77.55 - 119.18) 116.99 (97.85 -114.30) 120h after treatment 52.27 (43.50-62.15) 58.85 (42.57-80.04) Slope 0.551 0.551 1.056 1.181 1.126 1.455 1.358 1.629 1.587 0.867 Fig. (1): Probit regression lines of combination of the tested concentrations of fungi and nematodes (B. bassiana+ HP88) against 3rd instar of S. littoralis. 250 Fig. (2): Probit regression lines of combination of the tested concentration of fungi and nematodes (M. ansoplae + S. riobrave) against 3rd instar of S. littoralis. Table (3): Efficacy (corrected mortality %) of the fungi, B. bassiana and M. ansoplae against 3rd instar larvae of S. littoralis at different days post treatment Fungi Concentration ( spores/ml-1/100g diet) 104 B. bassiana 106 108 104 M. ansoplae 106 108 Corrected mortality (%) after 3-days 7-days 10-days 4.27 8.93 9.95 21.75 19.80 41.44 6.15 11.97 19.37 10.67 25.81 49.80 17.18 45.15 80.36 31.19-71.02%) at the lowest (16ijs/ml/100g diet) and the highest doses (256ijs/ ml/100g diet) concentrations, respectively. It could be noted that mortality percentages were distributed in first two intervals of treatments (i.e. about 50% of the recorded mortality occurred within 72 hours). On the other hand, higher concentrations of nematodes caused an acute effect while latent effect was observed in the case of lower ones. Statistical analysis of the accumulative percentage mortalities of S. littoralis larvae revealed a significant increase by increasing the doses in a comparable manner for the tested nematode isolates (Table 1). Data showed that death of the insect larvae started within 2-3 days after application with the nematode, H. bacteriophora (HP88) at the concentrations 16; 32; 64, 120 and 256, respectively and increased within 3-5 days after application. Obtained results agree with those of Shairra (2007) who found a positive relationship between doses and host mortality, mainly due to the numbers of infecting nematodes. However, the defense reactions against the nematodes and their associated bacteria may play an important role. ElBishry et al. (2002) demonstrated that nematode dose, infective juvenile age, exposure period, host species, host size, larval diet and starvation were the factors affected penetration of three isolates Heterorhabdits (AS1, RM1 and HP88). of Ricci et al. (1996) reported that complete mortality was achieved when the insects were exposed to S. riboravis or S. feltiae for only 2h and the number of individuals that penetrated into the cadavers increased gradually as the time of exposure lengthened. As shown in table (1), statistical analysis revealed no differences in LC50 values for both nematode species. The LC50 values showed S. riobrave as the most potent nematode after 48 & 72h post treatment, as LC50 reached 438.779 and 180.113/ IJs ml/100g diet, respectively. On the contrary, for H. bacteriophora the respective values were 584.4752 and 199.182/ IJs ml/100 g diet. H. bacteriophora had the lowest LC50 value (94.764 and 52.267/IJs ml/100 g diet) (Table 2). While, S. riobrave recorded a moderate effect against S. littoralis, with LC50 reached 116.990 and 58.850, after 96 and 120h, respectively. Infectivity of Heterorhabditis to its insect hosts considerably differs depending on the nematode species and insect host. Also, the range in number of invading nematodes varied. These results may imply that variable insect susceptibility to heterorhabditids is not due to numbers of infecting nematodes but it is mainly due to the defense reactions against the nematodes and their associated bacteria. Shairra (2000) and Shamseldean et al., 2008 demonstrated that in case of G. mellonella, S. littoralis or S. litura, for example, 100 %mortality was achieved with 10, 200 and 1000 IJs/caterpillar, respectively 48h after exposure to the tested nematodes. In the present study, the two tested nematode species grew faster in S. littoralis larvae. Low susceptibility of insect pest to the nematode isolates observed may be attributed to physical or behavioral host reaction which prevents IJs from penetrating the target host. This finding agrees with El-Bishry et al., )2002) and Ibrahim and 251 Table (4): LC50 (spores/ml-1/100 g diet) and slope values of B. bassiana & M. ansoplae against 3rd instar larvae of S. littoralis at confidence limits (95%) Treatment B. bassiana M. ansoplae B. bassiana M. ansoplae B. bassiana M. ansoplae LC50 Confidence limits (95%) 3days after treatment 14 2.330E x 10 (4.819E x 1010-1.000E x 1038) 7-days after treatment 7.839E x 1011 (6.571E x 109-1.830E x 1019) 2.897E x 108 (4.763E x 107-9.023E x 109) 10-days after treatment 5.835E x 108 (9.057E x 107-2.079E x 1010) 1.027E x 106 (4.384E x 105-2.410E x 106) Shairra, (2011). Susceptibility of S. littoralis larvae to fungi Corrected mortality percentages, 3 days after treatment with the fungi, ranged between (0 - 6.15 %) at the lowest (5x104 spores/ml-1/100g diet) and (017.18%) at the highest (5x108 spores/ml-1/100g diet) concentrations for B. bassiana and M. ansoplae,, respectively (Table 3). Corrected mortality percentages increased after 7 days of treatment and ranged between 4.27-11.97 and 19.80-45.15% at the lowest (5x104 spores/ml1 /100g diet) and the highest (5x108 spores/ml-1/100g diet) concentrations of fungi, B. bassiana & M. ansoplae, respectively (Table 3). Results indicated that mortality rates of both fungi increased with increasing the concentration and the period after treatment. Corrected mortality percentages, 10 days after treatment with fungi, ranged between 8.93-19.37 and 41.44-80.36%, at the lowest (5x104 spores/ml1 /100g diet) and the highest (5x108 spores/ml-1/100g diet) concentrations of fungi, B. bassiana & M. ansoplae, respectively. Virulence of EPFs varied from species to species and strain to strain against S. littoralis (Lin et al., 2007). Susceptibility of the insect to EPFs decreases with advancement in age of larvae of the target host (Purwar and Sachan, 2005). Cumulative mortality indicated that the strains of M. anisoplaie and B. bassiana affected the 3rd instar larvae of S. littoralis. B. bassiana revealed least mortality percentage while M. anisopolie showed the highest mortality rate against S. littoralis larvae. These results agree with those of Godonou et al. (2009), and Husnain et al. (2014). About 50% of the recorded mortality recorded within the 7 days at tested M. anisoplaie concentrations, except the lowest one, while in case of B. bassiana, the most of mortality occurred after 10 days following treatment (Table 3). Dose-mortality effect of tested fungi against larvae of S. littoralis Third instar larvae of S. littoralis were susceptible Slope 0.149 0.218 0.264 0.282 0.430 to all EPF isolates used in the bioassay in a dose dependent manner (Table 4). Mortality caused by each fungus was low at lower spores’ concentrations. It increased with increase of spores’ concentration. The highest mortality was induced by M. anisopliae, followed by B. bassiana. The median lethal concentration (LC50) value for 3rd instar larvae was (LC50 = 0; 7.839E+11& 5.835E+08 conidia ml-1) with B. bassiana at 3; 7 and 10 days post treatment (Table 4). On the other hand, the fungus M. anisopliae showed (LC50= 2.330E+14; 2.897E+08 and 1.027E+06 conidia ml-1) at 3; 7 & 10-days after treatment. M. anisopliae was the most potent fungi after 7 days of treatment (LC50 reached 2.897E+08 conidia ml-1). On the contrary, B. bassiana fungi (LC50= 7.839E+11 conidia ml-1) was the least effective fungi (Table 4). Ten days after treatment, M. anisopliae became the most effective fungus, as it had the lowest LC50 value (1.027E+06 conidia ml-1). This might be due to defense mechanisms of target insect. It is well documented that older instars of the cotton leaf worm are able to tolerate toxic effect of this fungus. EPFs that infect insects have received considerable attention by scientists for their potential use in biological control of pests. Some pathogenic fungi have restricted host ranges while others have a wide host range, e.g., B. bassiana and M. anisopliae. Many researchers have focused on the selection of virulent strains for target pests and their development as biological control agents (Angel-Sahagum et al., 2005 and Godonou et al., 2009). Similarly, Anand and Tiwary (2009) observed highest morality of 2nd instar larvae of S. litura at the highest spores’ concentration of fungal isolates. The growth indicated by white spores of B. bassiana on the dead Agrotis ipsilon larvae treated with the LC50 (2 x 108spore/ml) after 7, 10, 13 and 16 days. The mycelium started to grow after 7 days from death of infected larvae, and then the insect cadaver was covered by mycelium after 10 days. The formation and discharge of spores were found after 13 and 16 days; respectively (Gabarty et al., 2014). 252 Combined effect of fungi strains and nematodes isolates on larvae of S. littoralis The combined effects of fungi strains based on B. bassiana or M. anisopliae and entomopathogenic nematode, H. bacteriophora (HP88) and S. riborave differed than the applications of each pathogen alone (Figs. 1 and 2). In the present work, most combinations showed increased of host mortality. Combined applications of entomopathogens and sublethel dosages of synthetic insecticides or other biological control agents have been proposed as a strategy to improve the efficacy of microbial control agents (Anderson et al., 1989). Several studies have reported additive or synergistic effects from the combination of entomopathogens, mostly fungi, with insecticides (Ericsson et al., 2007). Rao et al., (1990) reported that more attention should be paid to the use of bioinsecticides based on bacteria, fungi, and viruses. These groups have unique modes of action (Thompson et al., 1999) and their properties may differ considerably from the conventional agents with which growers are familiar. In the present study, it was observed that highest larval mortality of S. littoralis increased with increase in fungi spores and nematodes infective juveniles’ concentrations and time elapsed after treatment. The interaction between fungi and nematodes may allow reducing chemical application rates. Additionally, the nematodes may become established and begin to offer a long term reduction in the larval populations (Klein and Georgis, 1992). This study gives additional support to the importance of combinations between the entomopathogenic fungi and nematodes for the control of insect pests. REFERENCES Abbott, W. S. 1925. A method of computing the effectiveness of an Insecticide. J. Econ. Entomol., 18:265-267. Anand, R. and B. N. Tiwary. 2009. Pathogenicity of entomopathogenic fungi to eggs and larvae of Spodoptera litura, the common cutworm. Biocontrol Sci. Technol., 19(9): 919-929. Anderson, T. E.; Hajek, A. E.; Roberts, D. W.; Priesler, H. K. and Robertson, J. L. 1989. Colorado potato beetle (Coleoptera: Chrysomelidae) effects of combinations of Beauveria bassiana with insecticides. J. Econ. Entomol., 82: 83-89. Angel-Sahagum, C. A., Lezama-Guierre` z, A., Molina-ochoa, J., Galindo-velasco, E., LopezEdwards, M., Rebolledo-Dominguez, O. 2005. Susceptibility of biological stages of the horn fly, Haematobio irritans to entomopathogenic fungi (Hyhphomycetes). J. Insect Sci,. ISSN: 15362442. Asi, M. R., Bashir, M. H., Afzal, M., Zia K. and Akram M. 2013. Potential of entomopathogenic fungi for biological of Spodoptera litura fabricus (Lepidoptera: Noctuidae). J. Anim. Plant Sci. 23(3): 913-918. Charnley, A. K. and S. A. Collins. 2007. Entomopathogenic fungi and their role in pest control. In: Kubicek, C.P. and I.S. Druzhinina (eds.), Environment and microbial relationships. 2nd Edition, The Mycota iv. Springer-Verlag Berlin Heidelberg. pp.159-185. Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics, 11: 1-41. El-Bishry, M. H.; El-Assal, F. M. and Abd ElRahman, R. M. 2002. Factors affecting penetration rate of the entomopathogenic nematodes Heterorhabditis spp. Egypt. J. Biol. Pest Cont, 12 (1): 25-38. Ericsson, J. D.; Kabaluk, J. T.; Goettel, M. S. and Myers, J. H. 2007. Spinosad interacts synergistically with the insect pathogen, Metarhizium anisopliae against the exotic wire worms; Agriotes lineatus and Agriotes obscurus (Coleoptera: Elateridea). J. Econ. Entomol., 100 (1): 31-38. Evans, H. C. 1999. Biological control of weed and insect pests using fungal pathogens, with particular reference to. Biocontrol News and Information. 20(2): 63N – 68N. Finney, D.J. (1971). Probit-analysis, 3rd Ed., Cambridge University Press, London. Gabarty, A., Salem, H. M., Fouda, M. A., Abass, A. and Ibrahim, A. A. 2014. Pathogenicity induced by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in Agrotis ipsilon (Hufn.). J. Radiat. Res. and Appl. Sci., 7(1): 95 -100. Gaugler, R. (ed.) 2002. Entomopathogenic Nematology. CAB International, Wallingford, UK, 388 pp. Georgis, R. 1992. Present and future prospects for entomopathogenic nematode products. Biocontrol Sci. Tech., 2: 83-89. Godonou, I., James, B., Atcha-Ahowe, C., Vodouhe, S., Kooyman, C., Ahanche´de , A. 2009. Potential of Beauveria bassiana and Metarhizium anisopliae isolates from Benin to control Plutella xylostella L. (Lepidoptera: Plutellidae). Crop Protection, 28, 220-224. Hajek, A. E. and R. J. St. Leger. 1994. Interactions between fungal pathogens and insect hosts. Ann. Rev. Entomol., 39: 293-322. Hidalgo, E., Moore, D. and Le Patourel, G. 1998. The effect of different formulations of Beauveria bassiana on Sitophilus zeamais in stored maize. J. Stor. Prod. Res. 34: 171–179. Husnain, H., Shahid, A. A., Ul haq, M. I., Ali, A., Muhammed, U. and Anam, U. 2014. Efficacy of entomopathogenic fungi as biological control agent against insect pests of Gossypium hirsutum. 253 J. Nat. Sci. Res., 4 (5): 68-72. Ibrahim A. A. and Shairra S. A. 2011. Effect of eicosanoid biosynthesis inhibitors on the immune response of the cotton leafworm, Spodoptera littoralis (Boisd.) infected with the nematode, Steinernema glaseri (Rhabditida: Steinernematidae). Egypt. J. Biol. Pest Cont. 21 (2): 197-202. Ibrahim, A. Amira 1974. Studies on biological control of Spodoptera littoralis (Boisd.( in A.R.E . Ph .D . Thesis, Fac .of Agric., Cairo Univ., Egypt, 282 pp. Klein, M. A. and Georgis, R. 1992. Persistence of control of Japanese beetle (Coleoptera: Scarabaeidae) larvae with steinernematid and heterorhabditid nematodes. J. Econ. Entomol., 85: 727–730. Lin, H. P., X. J. Yang, Y. B. Gao and S. G. Li. 2007. Pathogenicity of several fungal species on Spodoptera litura. Chinse J. Appl. Ecol., 18(4): 937-940. Purwar, J. P. and G. C. Sachan. 2005. Biotoxicity of Beauveria bassiana and Metarhizium anisopliae against Spodoptera litura and Spilarctia oblique. Ann. Pl. Protec. Sci., 13(2): 360-364. Rao, N. V., A. S. Reddy and P. S. Reddy 1990. Relative efficacy of some new insecticides on insect pests of cotton. Indian J. Plant Prot., 18: 5358. Ricci, M., Glazer, I. and Gaugler, R., 1996. Entomopathogenic nematode infectivity assay: Comparison of laboratory bioassays. Biocontrol Sci. and Technol., 6: 235-245. Shairra, S. A. 2000. Studies on the effects of some entomopathogenic nematode isolates on different host species. M. Sc. Thesis, Fac. Sci., Cairo Univ., Egypt, 108. Shairra, S. A. 2007. Effects of entomopathogenic nematodes and some pharmaceutical inhibitors of eicosanoid biosynthesis on the desert locust Schistocerca gregaria )Forskal.( Ph. D. Thesis, Fac. Sci., Cairo Univ., Egypt, 65. Shairra, S. A. 2010. Improving the biological control of locust Schistocerca gregaria using cyclooxygenase inhibitor with the entomopathogenic nematode Steinernema glaseri. Bull. ent. Soc. Egypt, Econ. Ser., 36, 139-154. Shamseldean, M. M. 1994. Effects of temperature on survival and infectivity of Egyptian heterorhabditid nematode isolates . Egypt .J . Appl .Sci., 9 :53-59. Shamseldean, M. M.; A. A. Ibrahim; N. M. Zohdi; S. A. Shairra and T. H. Ayaad 2008. Effect of the Egyptian entomopathogenic nematode isolates on controlling some economic insect pests. Egypt. J. Biol. Pest Cont. 18(1), 81-89. Proceeding of 2nd Arab Conference of Applied Biological Pest Control, Cairo, Egypt, 7-10 April 2008. Singkaravanit, S., Kinoshita, H., Ihara, F. and Nihir,a T. 2010. Geranylgeranyl diphosphate synthase genes in entomopathogenic fungi, Appl. Microbiol. Biotechnol., 85: 1463-1472. Thackar, J. R. M. 2002. An introduction to arthropod pest control. Cambridge University Press, The Edinburg Building Cambridge CB2 2RU, UK, pp. 144. Thompson, G. D., S. H. Hutchins and T. C. Sparks. 1999. Development of spinosad and attributes of a new class of insect control products. Univ. of Minnesota, available at: http://www.ipmworld umn.edu/chapters/hutchins2.htm. White, G. F. 1927. A method for obtaining infective nematode larvae from cultures. Science, 66:3 02-303.