Effects of Bacillus thuringiensis isolates and single nuclear polyhedrosis virus in combination and alone on Helicoverpa armigera

Archives of Phytopathology and Plant Protection, 2013 http://dx.doi.org/10.1080/03235408.2013.802460 Effects of Bacillus thuringiensis isolates and single nuclear polyhedrosis virus in combination and alone on Helicoverpa armigera Maryam Kalantari a, Rasoul Marzban a *, Sohrab Imani b and Hassan Askari a a Department of Biological Control, Iranian Research Institute of Plant Protection, Tehran, Iran; b Department of Entomology, Research and Science Azad University, Tehran, Iran (Received 15 April 2013; final version received 29 April 2013) Cotton bollworm is a key pest of cotton and other crops in many parts of Asia. Evaluation of three native Bacillus thuringiensis isolates and HaSNPV on cotton bollworm was studied. Results revealed that KD-2 isolate was the superior isolate, that its LC 50 was 1.2 10 7 spore ml 1 and LC 50 of HaSNPV equal to 9.2 10 3 OB ml 1. Interactions on the mortality and debilitating effects between B. thuringiensis KD-2 isolate (3 10 6, 6 10 6, 1.21 10 7 and 1.8 10 7 spore ml 1 ) and HaSNPV (2.3 10 3, 4.6 10 3 and 9.2 10 3 OB ml 1 ) on second instars larvae of Helicoverpa armigera were evaluated in laboratory. The larvae of cotton bollworm were fed for 48 h on the treated diets. The results revealed that the Bt at 1.2 10 7 spore ml 1, mixed with all above-mentioned concentrations of HaSNPV, had antagonistic effects. However, the Bt at 1.8 10 7 spore ml 1, mixed with HaSNPV at 9.2 10 3 OB ml 1, had additive effect. The Bt at 6 10 6 spore ml 1 mixed with all above-mentioned concentrations of HaSNPV demonstrated additive effects. The Bt at 3 10 6 spore ml 1 had additive effect with HaNPV at 2.3 10 3 and 4.6 10 3 OB ml 1. The mortality observed in larvae infected with the combination of both Bt (3 10 6 spore ml 1 ) and HaSNPV (9.2 10 3 OB ml 1 ) was interpreted as synergist effect. Therefore, it could be concluded that combination of the lowest Bt concentration and the highest HaSNPV concentration had synergist effect on H. armigera second instar larvae. Keywords: Bacillus thuringiensis; cotton bollworm; interaction; single nuclear polyhedrosis virus 1. Introduction The cotton bollworm, Helicoverpa armigera (Hübner). (Lepidoptera: Noctuidae), is an important pest of different crops, particularly on cotton and tomato, throughout Asia. The ability of this pest to adopt transient habitats in a short span of time accelerated the excessive use of pesticides resulting in the development of resistance to various classes of insecticides (Wu et al. 2008). Different methods, including application of chemical and microbial insecticides, are used to prevent the damage induced by this pest. Microbial control of agricultural and health-related insect pests has been considered as an alternative to synthetic insecticides. Microbial agents are generally highly specific against target insect pests, thus facilitating the survival of beneficial insects in treated crops (Lacey et al. 2001). Single Nucleocapsid nuclear polyhedrosis virus (SNPV) and Bacillus thuringiensis Berl. have been successfully used as a *Corresponding author. Email: ramarzban@yahoo.com Ó 2013 Taylor & Francis

2 M. Kalantari et al. biopesticides on H. armigera (Hübner). However, resistance to the crystal proteins of B. thuringiensis has been documented for H. armigera Akhurst et al. (2003). Thus, study on the interactions of B. thuringiensis and HaSNPV is important for integrated pest management of cotton bollworm. The principal initial target tissue for B. thuringiensis and HaSNPV is midgut. B. thuringiensis toxins are ingested by the insect as protoxins, and they are activated by midgut proteases as a form of activated toxin to disrupting the midgut membrane wall, leading to insect death Washburn et al. (2003). HaSNPV infection is also accomplished following ingestion of virus particles. NPV is a natural microbial pathogen of Lepidoptera pests (Kumar et al. 2008) and it infects the insect midgut and causes acute and chronic infections Herz et al. (2000); Washburn et al. (2003). NPV products are commercially available for cotton pests in the USA and Australia and have been successfully used globally in various agricultural and horticultural settings in the development of IPM Raymond et al. (2005). While NPVs have limitations as insecticides, especially in their cost of production, they can, through the build-up of infectious particles and secondary cycles of infection in pest populations, be an effective means of long-term population control (Bonsall 2004). This would lead to increased spray intervals and reduced costs for growers (Moscardi 1999). Therefore the study use of B. thuringiensis and HaSNPV in integrated pest management programs has attracted attention for the biological control bollworm. However, B. thuringiensis and HaSNPV can interact either synergistically, antagonistically or additively owing to differences in their modes of action that in this study depends on their concentrations. The purpose of this study is to explore the interaction effects of HaSNPV and B. thuringiensis, both individually and combination on larval mortality and debilitating effects on H. armigera. This combination could resolve the resistance problem, decrease concentration requirement and costs in field, and increase efficacy. 2. Material and methods 2.1. Insect rearing The colony H. armigera was prepared by BioControl Department, Iranian Research Institute of Plant Protection. Eggs in each generation were soaked in formaldehyde 2% for approximately 15 min; rinsed thoroughly with running water, set out to dry and left to hatch in plates. To prevent cannibalism, after the third instar, individual larva was reared in a glass diet tube (2 10 cm) and was fed with artificial diet (Abbasi et al. 2007) at 26 C and 65% RH, with a 16:8 h photoperiod and adults were fed with 10% honey solution. 2.2. B. thuringiensis preparation The Iranian strains KD-2, 20 and 6R and a commercial product named Dipel were obtained from National Collection of Iranian Research Institute of Plant Protection. Isolates were kept in lyophilised form at 85 C. They were grown in LB medium (tryptone 1%, yeast extract 0.5%, NaCl 1%, ph 7 7.2) for 7 days at 27 C with shaking (260 rpm) at which time the separation of crystals from spores was confirmed by microscopic observation. The spore crystal mixture was centrifuged at 8000 g for 10 min at 4 C. Spores and crystals from stock culture were suspended in 10 ml Tween 80 0.4%. For experiments, bacterial suspensions ranging from 10 5 to 10 7 spore ml 1 were prepared by diluting with Tween 80 and stored at 4 C.

Archives of Phytopathology and Plant Protection 3 2.3. HaSNPV preparation H. armigera single nuclear polyhedrosis virus (HaSNPV) was provided by the Iranian Research Institute of Plant Protection. Ten-day-old larvae of H. armigera were infected with a viral suspension of 1(10 7 OB ml 1 on the artificial diet and after four days, the infected cadavers were collected and homogenised by a homogeniser in Tris HCl 50 mm (ph 7.2). The viral suspension was filtered by a two-layer filter cloth to remove large debris before being filtered by a centrifuge at 700 rpm for 1 min. The supernatant was centrifuged at 3800 rpm for 10 min. Finally, pure polyhedral inclusion bodies were separated from the supernatant and resuspended in Tris HCl 50 mm (ph 7.2) and stored in a deep freezer. The number of OBs ml 1 was determined by using a Neubauer hemacytometer (Cory & Myers 2004). For treatments, viral suspensions ranging from 10 2 to 10 5 OBs ml 1 were prepared by diluting with Tris HCl buffer. 2.4. Bioassay procedures 2.4.1. Procedure 1 Bioassay tests were carried out to evaluate the virulence of Bt isolates in the host. Similarly, three native isolates coded as KD-2, 20 and 6R and a commercial product named Dipel were forcedly fed to cotton bollworm second instar larvae (four days old) under controlled conditions. At first, a primary bioassay of bacteria was carried out to obtain a minimum and maximum concentrations required for experiments. Afterwards, seven different concentrations were prepared based upon previous tests in a logarithmic fashion using Tween 80. Treatments were prepared by spraying concentrations on artificial diet by rate of 20 μl of concentration to 0.5 g of diet. After half an hour, 45 tubes (2 10 cm) were prepared each containing 0.5 g of artificial diet cubes. Then, four days old larvae with same size and colour were transferred by a fine brush into tubes individually (Kumar et al. 2008). Fifteen larvae were considered as a treatment and treatments were repeated three times. After 48 h, larvae were transferred into new tubes containing healthy diet. From day two onwards to day seven, the larval mortality was recorded. The data were analysed using SAS software and the LC 50 of each isolate was calculated. The bioassay of virus was studied by the same procedure of bacteria excepted using 50 mm Tris HCl for concentrations preparation. In the case of virus bioassay, the larval mortality was registered from day 4 to day 10 after treatments. 2.4.2. Procedure 2 The effects of pathogen combinations may depend on such factors as concentration, subspecies of bacteria or design of bioassay Farrar et al. (2004). Each bioassay consisted of 20 treatments and was replicated three times. The treatments were as follows: Control containing sterile Tween 80 (0.4%). Combination between four concentrations of Bt (1.8 10 7, 1.21 10 7, 6 10 6 and 3 10 6 spore ml 1 ) and three concentrations of HaSNPV (9.2 10 3, 4.6 10 3 and 2.3 10 3 OB ml 1 ) Individual Bt at four concentrations and individual HaSNPV at three concentrations.

4 M. Kalantari et al. The artificial diets surfaces were treated with the appropriate concentrations of Bt, HaSNPV or a combined solution of them. One bioassay procedure was adopted for the experiment. Newly moulted second instar (four days old) larvae were placed individually in glass diet tubes (2 10 cm) containing 0.2 g artificial diet. The artificial diets were treated with 20 μl of the appropriate concentration of Bt, HaSNPV or a combined solution of them. Larvae fed on the treated artificial diets for 48 h. Surviving H. armigera larvae were transferred to individual glass containing 1 cm 3 artificial diet. Bioassay was conducted at 25 C in 60 70% relative humidity, with a 14:10 h photoperiod and larvae mortality was recorded every 24 h until the larvae had either died or pupated. In addition each treatment was replicated three times using 15 larvae for each replicate. 2.5. Statistical analysis One-way ANOVA was performed using SPSS (1998). The larvae that were unable to move or feed were confirmed dead. Pupation rate, larval period, pupal period and pupal weight were calculated based on the initial number of larvae used in each treatment. The mortality was corrected by the equation: M [%] = [(t c)/(100 c)] 100, where M is corrected mortality, c is the mortality in controls and t is mortality in treatments (Abbott 1925; Duffield & Jordan 2000). Corrected mortality, pupation rate, larval period, pupal period and pupal weight were separated and compared using a one-way Duncan test among treatments. All tests were conducted with α = 0.05. The equation CTF = (Oc Oe)/Oe 100 was used to determine the type of interaction between different concentrations, where CTF is the cotoxicity factor, Oc is the observed mortality occurred by the combination and Oe, the expected mortality, is the sum of mortalities caused by each pathogen used in the combination Mansour et al. (1966). This factor was used to differentiate the results into three categories. A positive factor of 20 or more meant synergism, while a negative factor of 20 or more stood for antagonism and any intermediate value (i.e. between 20 and +20) was considered as additive. In order to calculate LC 50 to compare the effect of combinations of NPV and B. thuringiensis, the data were analysed by SAS statistical software. 3. Results Results of B. thuringiensis, isolates bioassay, proved that KD-2 isolate was selected as the superior isolate compared with others. KD-2 isolate LC 50 was equal to 1.2 10 7 spore ml 1. Following that, the bioassay of HaSNPV revealed its LC 50 equal to 9.2 10 3 OB ml 1. (Table 2). Combination of B. thuringiensis and HaSNPV showed that significant differences were detected in larval mortality and development when second instars larvae of H. Table 1. B. thuringiensis isolates bioassay on cotton bollworm second instar larvae. Isolates LC 50 spore ml 1 95% limit lower-upper Slope Intercept χ 2 Df Pr > chisq 20 8.8 10 7 1.4 10 7 5.5 10 8 0.77 6.16 2.53 5 0.9892 Dipel 6.9 10 7 4.5 10 7 1.09 10 8 0.96 2.56 1.43 5 0.9792 KD-2 1.2 10 7 7.6 10 6 1.8 10 7 1.18 3.37 2.84 5 0.9985 6R 1.3 10 7 8.7 10 6 1.8 10 7 1.22 3.73 1.28 5 0.9763

Archives of Phytopathology and Plant Protection 5 Table 2. HaSNPV bioassay on cotton bollworm second instar larvae. Virus LC 50 OB ml 1 95% limit Lower-Upper Slope Intercept χ 2 Df Pr > chisq HaSNPV 9.2 10 3 2.4 10 3 1.4 10 4 0.68 2.29 1.28 5 0.9892 armigera were forcedly fed diets containing Bt-HaSNPV. Mortality: F (19, 40) = 32.75; p < 0/000, larval period: F (19, 40) = 15.01; p < 0/000, pupal period: F (19, 40) = 16.56; p <0/ 000; pupation rate: F (19, 40) = 32.75; p < 0/000, Pupal weight: F (19, 40) = 15.44; p < 0/000 and adult emergence: F (19, 40 = 35.33; p < 0/000) (Table 1). Antagonism, additive and synergism effects on mortality of H. armigera were observed in combination of Bt and HaSNPV depending on pathogen concentrations. Exposure of H. armigera larvae to Bt, HaSNPV or combination of them have negative impact on growth and development of it, and resulted in reduced pupation, pupal weight and adult emergence and an extended development also showed significant variation in the combination of Bt and HaSNPV among concentrations. The results revealed that the Bt concentration (1.2 10 7 and 1.8 10 7 spore ml 1 ) mixed with all mentioned concentration of HaSNPV had antagonistic effect. However, the Bt concentration (1.8 10 7 spore ml 1 ) mixed with HaSNPV concentration (9.2 10 3 OB ml 1 ) had additive effect. Moreover, the Bt concentration (6 10 6 spore ml 1 ) mixed with mentioned concentration of HaNPV demonstrated additive effects. The Bt concentration (3 10 6 spore ml 1 ) had also additive effect with HaSNPV concentration (2.3 10 3, 4.6 10 3 OB ml 1 ). Finally, The mortality observed in larvae infected with the combination both Bt (3 10 6 spore ml 1 ) and HaNPV (9.2 10 3 OB ml 1 ) was interpreted as synergist effect (Tables 3 and 4). 4. Discussion B. thuringiensis is the most widely used biocontrol agents for plant pests. Native isolates of Bt may affect more efficient than commercial products. In this research, the effect of B. thuringiensis pathogens on cotton bollworm was investigated. Fifty per cent lethal concentration (LC 50 ) of native strains of KD-2, 20, 6R and commercial Dipel Bt bacteria on the second larval instars of H. armigera were 1.2 10 7, 8.8 10 7, 1.31 10 7 and 6.9 10 7 spore ml 1, respectively. This is the part of results. The highest mortality of Bt bacterial strain belongs to isolate KD-2 that was used in combination with the virus. KD-2 isolate toxins are Cry1A and Cry2 (Kalantari et al. 2013). LC 50 of HaSNPV was determined equal to 9.2 10 3 OB ml 1. Bt toxins bind to specific receptors of the targeted insect midgut epithelium where they disrupt the cytoplasmic membrane, causing the cells to lyse. As a result, the insect quickly stops feeding and dies (Thiem 1997). As such, the feeding refusal rate is positively correlated with Bt toxin concentration. HaNPV infection, on the other hand, is accomplished following ingestion of virus particles. As the virus replicates itself within the host s cells, the host s tissues disintegrate, leading to death. Combinations of two microbial insecticides, such as Bt and HaNPV, have been suggested as a means of increasing the spectrum of insect pathogens and thus managing multiple pests (Pingel & Lewis 1999). It is also possible that the pathogens may interact to increase virulence compared with either alone. An additive effect was obtained after treatment of Spodoptera littoralis (Boisd.) larvae with a combination of Bt and NPV and reported that mortality from a combination of HaNPV and Dipel (Bt product) was higher than application of HaNPV alone (Reddy & Manjunatha 2000). An additive and antagonism effect was observed after treatment of H. armigera larvae with HaNPV and

6 M. Kalantari et al. Table 3. Mortality of H. armigera second instar larvae exposed to B. thuringiensis and HaSNPV simultaneously a. Bt b spore ml 1 HaNPV OB (ml 1 ) Actual mortality c (%) Expected mortality (%) Cotoxity factor Type of Interaction d 2.3 10 3 32.0 ± 2.0 bc 39.3 18.5 add 3 10 6 4.6 10 3 55.0 ± 5.9 d 50.3 9.34 add 9.2 10 3 86.0 ± 0.0 e 64.0 34.3 sin 2.3 10 3 39.3 ± 4.7 c 48.3 18.6 add 6 10 6 4.6 10 3 61.7 ± 4.3 d 59.3 4.0 add 9.2 10 3 82.0 ± 5.9 e 73.0 12.3 add 2.3 10 3 50.7 ± 7.9 d 74.7 29.3 ant 1.2 10 7 4.6 10 3 55.3 ± 4.7 d 85.7 35.4 ant 9.2 10 3 79.7 ± 3.8 e 99.3 20.7 ant 2.3 10 3 59.7 ± 3.8 d 81.3 26.5 ant 1.8 10 7 4.6 10 3 61.7 ± 8.1 d 92.3 33.1 ant 9.2 10 3 95.7 ± 0.0 f 106.0 9.7 add 3 10 6 0 15.3 ± 2.3 ab 6 10 6 0 24.3 ± 4.3 bc 1.2 10 7 0 50.7 ± 2.3 d 1.8 10 7 0 57.3 ± 4.3 d 0 2.3 10 3 24.0 ± 2.0 bc 0 4.6 10 3 35.0 ± 5.9 c 0 9.2 10 3 48.7 ± 4.3 d 0 0.05 ± 0.0 a DF 59 F 32.8 P <0.000 a The second instar larvae of H. armigera were fed by infected leaf discs by B. thuringiensis and HaSNPV. b Native isolate. c The data in the table are means (±SE). Means within the same column followed by a different letter are significant at p < 0.05, Duncan test. d Abbreviation: add = additive, sin = synergism, ant = antagonism. Cry1Ac that concentrations closed together (Liu et al. 2006). In the present study, interactions between Bt (native isolate) and HaSNPV varied depending on the concentrations of them. When H. armigera larvae were simultaneously infected with HaNPV and Bt for 48 h, an additive, synergism, antagonism effects were observed in the combinations. A synergism effect was observed in the combination of the highest concentration of HaNPV (9.2 10 3 OB ml 1 ) and the lowest concentration of Bt (3 10 6 spore ml 1 ). Marzban et al. (2009) reported that mortality from combination Cry1Ac and Cpv on H. armigera have synergist and additive effects. These results agree with the finding of Marzban 2012. The mode of action of Bt is to damage the cells of the targeted insect gut; Bt toxin in lower concentration may cause bacteriuminduced retardation in larvae development, thus giving the opportunity for the virus to propagate and cause its lethal effects. This may explain why Bt and HaNPV showed synergism in some of the present tests. Matter & Zohdy 1981 reported synergistic effects of the two pathogens, Bt and NPV, on H. armigera. The results revealed that the Bt concentrations (1.2 10 7 and 1.8 10 7 cfu ml 1 ) mixed with all mentioned concentrations of HaNPV had antagonistic effect. The mode of action of Bt toxin is to damage the cells of the targeted insect gut, and this may inhibit passage of HaNPV into the midgut cells. This may explain why Bt and HaNPV

Archives of Phytopathology and Plant Protection 7 Table 4. The debilitating effects of B. thuringiensis HaSNPV combination, and in alone on H. armigera a. Bt b Cfu ml 1 HaNPV OB ml 1 Larval period c (days) Pupal period (days) Pupation rate (%) Pupal weight(mg) Emergence rate 2.3 10 3 16.0 ± 0.6 fgh 11.3 ± 0.3 abc 68.0 ± 2.0 de 26.4 ± 0.6 bcd 68.0 ± 6.8 de 3 10 6 4.6 10 3 16.6 ± 6.3 efg 11.0 ± 0.6 abc 45.0 ± 5.8 c 26.1 ± 0.2 bcde 37.3 ± 0.7 c 9.2 10 3 17.7 ± 0.3 cdef 11.7 ± 0.3 abc 26.7 ± 0.0 b 26.7 ± 0.4 bcd 8.3 ± 4.7 bc 2.3 10 3 16.3 ± 0.7 ef 11.3 ± 0.3 abc 60. 7 ± 4.7 c 26.0 ± 0.6 bcde 60.7 ± 4.3 c 6 10 6 4.6 10 3 16.7 ± 0.3 efg 11.3 ± 0.3 abc 38.3 ± 4.3 c 27.4 ± 0.6 b 38.3 ± 4.6 c 9.2 10 3 18.6 ± 0.3 bcd 11.0 ± 0.3 bc 25. 6 ± 0.9 b 25.6 ± 0.9 cdef 12.3 ± 7.9 bc 2.3 10 3 16.0 ± 0.6 fgh 12.0 ± 0.6 abc 49.3 ± 7.9 c 24.0 ± 0.6 ghi 49.3 ± 4. 7 c 1.2 10 7 4.6 10 3 19.3 ± 0.3 bc 12.3 ± 0.3 ab 44.7 ± 4.8 c 24.3 ± 0.6 fgh 44.7 ± 3.8 c 9.2 10 3 21.3 ± 0.3 a 12.7 ± 0.3 ab 23.2 ± 3.8 b 23.0 ± 0.5 hi 23.2 ± 3.8 b 2.3 10 3 18.3 ± 0.7 bcde 12.0 ± 0.0 abc 40.3 ± 3.8 c 22.9 ± 0.2 hi 40.3 ± 8.1 c 1.8 10 7 4.6 10 3 20.0 ± 0.3 ab 12.7 ± 0.3 ab 38.3 ± 8.1 c 22.7 ± 0.2 i 38.3 ± 0.0 c 9.2 10 3 21.3 ± 0.3 a 13.0 ± 0.6 a 0.1 ± 0.0 a 21.0 ± 0.5 J 0.0 ± 2.3 a 3 10 6 0 14.0 ± 0.6 i 11.7 ± 0.3 abc 84.7 ± 2.3 e 26.9 ± 0.4 bc 84.7 ± 4.3 e 6 10 6 0 15.3 ± 0.9 ghi 11.7 ± 0.7 abc 75.7 ± 4.3 d 26.9 ± 0.9 bcd 75.7 ± 2.3 de 1.2 10 7 0 17.3 ± 1.2 def 12.3 ± 0.3 ab 49.3 ± 2.3 c 25.3 ± 0.7 defg 49.3 ± 4.3 c 1.8 10 7 0 19.0 ± 0. 9 bcd 12.0 ± 1.0 abc 42.7 ± 4.3 c 24.9 ± 0.2 efg 42.7 ± 2.0 c 0 2.3 10 3 14.3 ± 0.6 hi 12.0 ± 0.6 abc 76.0 ± 2.0 de 27.4 ± 0.2 b 76.0 ± 5.9 de 0 4.6 10 3 15.3 ± 0.3 ghi 12.0 ± 0.6 abc 65.0 ± 5.9 d 27.4 ± 0.1 b 65.0 ± 4.3 d 0 9.2 10 3 17.3 ± 0.6 fgh 11.7 ± 0.7 abc 51.3 ± 4.3 c 26.7 ± 0.4 bcd 51.3 ± 0.2 c 0 0 13.7 ± 0.3 fgh 10.3 ± 0.3 c 100 ± 0.0 f 30.5 ± 1.1a 100 ± 15.5 f DF 59 59 59 59 59 F 15.0 16.6 32.8 15.4 35.3 P >0.000 >0.000 >0.000 >0.000 >0.000 a Larvae of H. armigera were fed normal artificial diets until the second instar, infected by a combination of HaSNPV-Bt, HaSNPV and Bt in alone for 48 h, then fed a normal artificial diet. b Native isolate. c The data in the table are means (±SE). Means within the same column followed by a different letter are significant at p<0.05, Duncan test.

8 M. Kalantari et al. showed antagonism in some of the present tests. Salama et al. s (1993) larval feeding ceased once Bt was ingested, thereby preventing ingestion of an adequate NPV dose. In some of the present tests, when second instar larvae were force fed on lowest concentration of Bt with often concentrates containing HaNPV, combinations showed additive effects. In this study, the combination of low concentration of Bt 3 10 6 spore ml 1 and a low concentration of HaSNPV 2.3 10 3 OB ml 1 and up concentration of Bt 1.8 10 7 spore ml 1 and a up concentration of HaNPV 9.2 10 3 OB ml 1 showed an additive effect; these result agree with the finding of Liu et al. (2006) and Marzban et al. (2009). Developmental time is an important aspect of the biology of an insect. In this study, Bt and SNPV together adversely affect the growth and development of cotton bollworm more than each would have done alone. Prolonged developmental time at any stage would mean greater exposure to natural enemies and environmental stresses, which could reduce the rate of population build-up of the insect. Furthermore, a longer generation time could result in fewer generations per season. When evaluating the use of these pathogens as microbial control agents, time to death (speed of killing the pathogen) should be considered. In the present study, larvae of H. armigera started to die from the 2nd to 3rd days after infection with HaNPV-Bt, that it was similar to Bt has done alone, but treated larvae with HaNPV alone of H. armigera started to die from the fourth day after infection, that it was later than combination HaNPV-Bt. The symptoms of these two microbial agents on the host larvae were observed as delay in growth and development of larvae and pupae and decrease in pupal weight, and the adults emergence, compared with those of control. To conclude, suitable combination of Bt and HaSNPV may enhance the efficiency of both Bt and HaSNPV and also delay the resistance process to solve application of either. Acknowledgements This research was carried out in the BioControl Department, Iranian Research Institute of Plant Protection, Tehran, Iran. The authors thank Dr David Mota-Sanchez for helpful comments and revision of the manuscript. References Abbasi BH, Khalique K, Khalique F, Ayub N, Liu HJ, Kazemi SAR, Naumak M. 2007. Rearing the cotton bollworm, Helicoverpa armigera on a tapioca based artificial diet. J Insect Sci. 7:35 41. Abbott WS. 1925. A method of computing the effectiveness of insecticides. J Econ Entomol. 18:265 267. Akhurst RJ, James W, Bird LJ, Beard C. 2003. Resistance to the Cry1Ac δ-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J Econ Entomol. 96:1290 1299. Bonsall MB. 2004. The impact of diseases and pathogens on insect population dynamics. J Physiol Entomol. 29:223 236. Cory AD, Myers JH. 2004. Adaptation in an insect host-plant pathogen interaction. Econ Lett. 7:632 639. Duffield S, Jordan SL. 2000. Evaluation of insecticides for the control of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) on soybean, and the implications for field adoption. J Aust Entomol. 39:322 327. Farrar RF, Shapiro M, Shepard BM. 2004. Activity of the nucleopolyhedrovirus of the Fall Armyworm (Lepidoptera: Noctuidae) on foliage of transgenic sweet corn expressing a CryIA (b) toxin. Environ Entomol. 33:982 989.

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