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1 Advances in Environmental Biology, 6(1): , 2012 ISSN This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE The singular and combined effects of entomopathogenic fungi, Beauveria bassiana (Bals) and Lecanicillium muscarium (Petch) with insecticide imidacloprid on different nymphal stages of Trialeurodes vaporariorum in the laboratory conditions 1 Naser Malekan, 2 Bijan Hatami, 1 Rahim Ebadi, 1 Alireza Akhavan, 3 Rouhollah Radjabi 1 Department of Plant Protection, Isfahan University of Technology, Isfahan, Iran. 2 Department of Plant Protection, Islamic Azad University, Khorasgan Branch, Isfahan, Iran. 3 Plant Protection Department, Dezful Branch, Islamic Azad University, Dezful, Iran. Naser Malekan, Bijan Hatami, Rahim Ebadi, Alireza Akhavan, Rouhollah Radjabi: The singular and combined effects of entomopathogenic fungi, Beauveria bassiana (Bals) and Lecanicillium muscarium (Petch) with insecticide imidacloprid on different nymphal stages of Trialeurodes vaporariorum in the laboratory conditions ABSTRACT In biocontrol of whitefly, laboratory efficacy of Beauveria bassiana and Lecanicillium muscarium with imidacloprid has not been frequently assessed. In present study possible advantage of this combination treatment was assessed sequentially in laboratory bioassays on different nymphal stages of Trialeurodes vaporariorum. All nymphal stages of T. vaporariorum showed that B. bassiana and L. muscarium induced mycosis. Pathogenicity of B. bassiana and L. muscarium on the 3 rd and 4 th nymphs was higher than the 1 st and 2 nd nymphs. On young nymphs there was no significant difference between the mortality percentage of 10 6 conidia/ml of L. muscarium and 300mg/l of imidacloprid. Combination efficacy of the highest concentration of these two control agents was more than other treatments on old nymphs. Mortality in singular treatment of imidacloprid (300mg/l) was more than application of L. muscarium (10 6 conidia/ml) alone. There was no significant difference in application of 10 6 conidia/ml of B. bassiana and 300mg/l imidacloprid on young nymphs but the percentage of mortality was higher than 10 6 conidia/ml of B. bassiana alone. There was no significant difference between combinations of high concentration of two fungi with application of singular imidacloprid (300mg/l). Moreover, there was no significance different between co-application of 10 6 conidia/ml + 50 mg/l with 10 3 conidia/ml + 300mg/l (In two fungi). Key words: Bioassays, combination treatment, entomopathogenic fungi, Beauveria bassiana, Imidacloprid, Lecanicillium muscarium, Trialeurodes vaporariorum Introduction Many crops worldwide are affected by several genera of whiteflies among them greenhouse whitefly; Trialeurodes vaporariorum and tobacco whitefly; Bemisia tabaci are the most well-known [33,54,55]. T. vaporariorum discharge sticky honeydew which makes plants a site for a black, sooty mold and causes chlorosis and necrosis on leaves [2,17]. Difficulties caused as results of morphological characteristics and developed resistance to insecticides, whitefly insecticide phytotoxicity and their effects on natural enemies and pesticide residue problems on vegetables have caused increasing application of fungal pathogens for microbial control of aleyrodids pests [11,9,12]. To date, in various studies, the effectiveness of fungal pathogens for the control of T. vaporariorum has been studied and so far, more than 20 different Corresponding Author species of fungi have been recorded infecting T. vaporariorum [15]. The insect pathogenic fungus, Beauveria bassiana, can have good potential as a control agent for greenhouse insect pests such as thrips, aphids and whiteflies. It grows in soils and is pathogenic on some of the arthropods [52]. Beauveria bassiana attacks a wide host range and can infect insects at larval or adult stages [53]. Lecanicillium muscarium Gams & Zare is another well-known pathogen of arthropods. This fungus has a wide host range and has been isolated from a variety of insects [30,31] and was shown to be effective against different developmental stages of T. vaporariorum (Hall 1982). On the other hand, fungi cannot replace pesticides in commercial fields and thus, pesticides seem to remain a major element to rapid control of insect pest populations. To enhance efficiency and accelerate insect pest mortality, coapplication of pest pathogenic fungi with sub lethal Rouhollah Radjabi, Plant Protection Department, Dezful Branch, Islamic Azad University, Dezful, Iran. roholla_rajabi@yahoo.com

2 doses of chemical pesticides can be employed [15,46]. We know factors such as slow action, poor effects on adult insect pests, potentially negative interactions with pesticides, relatively high costs and limited shelf life can lower the efficacy of pest pathogenic fungi [15]. Therefore, it is necessary to study the potential of combining fungi with other agents to develop Integrated Pest Management (IPM) strategies [15]. It seems that chemical pesticides can interfere the pest behavior and thus, enhance the efficacy of fungi. On the other hand, each species and strains are efficacious in a narrow spectrum of weather condition and different host insects. Extension of resistance to insecticides due to continuous use of these compounds in whitefly is present problems in control of pests [3]. These high levels of pesticide resistance in whiteflies and public demands for reducing pesticide use have promoted interest in the development of other control strategies [14]. Therefore, to increase efficiency and increase the pest mortality there is a need to investigate the potential of combining pest pathogenic fungi with other agents [1546]. If the entomopathogenic fungi complement an insecticide, or act synergistically, a beneficial effect can be obtained. Although chemical insecticides as stressors seem to interfere the insect behavior and therefore, enhance the efficacy of entomopathogenes but still limited data exist regarding the IPM employing these two factors [23]. The effectiveness of applications of sublethal doses of imidacloprid with fungi in control of termites has been demonstrated in several studies [38,39]. A few efforts have been made to utilize entomopathogenic fungi against whiteflies in Iran. In present study the use of singular and combined treatments of entomopathogenic fungi, B. bassiana, L. muscarium and commercially available imidacloprid on different nymphal instars of T. vaporariorum has been investigated on tomato plants and under laboratory conditions. The objective of this study was to investigate the combined effects of Beauveria bassiana and Lecanicillium muscarium with low and high concentrations of imidacloprod on the T. vaporariorum. Materials and Methods Collection and rear insect: T. vaporariorum adult specimens were collected using aspirator from a colony maintained in a greenhouse on campus of the Isfahan University of Technology (Isfahan, Iran) and were reared on whitefly-free tomato; Lycopersicum esculentum Miller (variety ch. Falat) at the 10 leaves stage in 25 C, and 16/8-hrs (light : dark) cycle. The subcolony was established from approximately 1000 male and female adults kept in cages (80H-80W-80D cm). Before transferring of greenhouse whitefly, each tomato leaves was covered with a clip cage that 424 modeled and described by Moniz and Nombela [32] (1 cage per leaf). These cages (8cm in diameter, 7.5cm in height) were constructed of plastic boxes with a 3.5 cm hole in order to allow aeration that covered with silk screening material in the center. 40 adults specimens randomly collected from a single population, were added into each individual clip cage. To obtain age-uniform nymphs, the adults were allowed to have an oviposition period on the healthy leaves at 24±1ºC; and 70±10R-H and 16/8-h (light/dark) cycle. After this time adults removed from the leaf. After 11 days all nymphal stages in the 1 st or 2 nd instars, and after 20 days, all nymphs in the 3 rd or 4 th nymphal instars appeared. Fungal isolations: The isolates DAOM of Lecanicillium muscarium and isolate Fashand of Beauveria bassiana which were obtained from the Research Institute of Forests and Rangelands (Iran) were grown on PDA in an incubator at 24±2ºC under a 16- h photoperiod. For bioassays freshly conidia (asexual spores) were harvested from 15 days cultures by flooding the Petri dishes with sterile distilled 0.01% Tween80 supplemented water. Aqueous spore suspensions were shaken by horizontal shaker for 5min at 3000rpm and the suspension filtered by 2 layers of sterile filter paper. Bioassay of entomopathogenic fungi: A mixture of conidia and hyphae were harvested from days-old cultures after adding 10ml of sterile distilled water supplemented with 0.01 Tween80. Conidia were filtered by 2 layers of sterile filter paper. The concentration of conidial suspension was determined with a haemocytometer. According to the method of Hall [22], spore germination rate was checked a day prior to the bioassays and always was 95.5%. For each treatment serial dilutions of conidia/ml of spores were applied to the underside of each leaf using a sterilized hand sprayers with fine droplet spray nozzle from a distance of 20cm. All experiments were performed in a complete randomized block design with 6 replications (each replication consisted of a single leaf). The leaves were transferred individually into Petri dishes that lined with two 90 mm moist filter paper in order to maintain high humidity for fungal infection. The stalk of the leaf was covered with a piece of sterile cotton to keep the leaf fresh with a nutrient solution containing 1%NPK ( ) fertilizer. A piece of plastic was placed on the filter papers to ensure the leaves did not contact with the wet surface. The control leaves were sprayed in a similar way with 0.01% Tween80 supplemented water. Leaves were allowed to air dry for a few minutes. All assay containers were held in an incubator at 24±1ºC and 70±10 ambient relative

3 humidity under 16/8-h (light/dark) cycle. The percentage mortality (number of either nymphs that exhibited hyphal coverage on exocuticle or color changed from transparent greenish to opaque white) was registered post treatment after seven days by using a binocular [20]. Bioassay of combination treatment: The combination treatments were done in a sequential treatments approach, in which fungal pathogen followed by imidacloprid. To achieve acceptable results this experiment was done in growth chamber. The treatments were 4 concentration of imidacloprid (300, 200, 100 and 50mg/lit), 2 concentration of fungi (10 6 and 10 3 conidia/ml) and all possible combinations of control agents. After reaching to a certain nymphal stages, the leaves on the plants sprayed with different solution of imidacloprid from a 20cm distances by hand sprayer with fine nozzle. Fungi were sprayed on infested leaves 8days after exposure to imidacloprid. The treated nymphal stages were monitored 7days after the incubated leaves were sprayed using design described in Table 1 and 2. Statistical analysis: A regular transform of data was calculated by following formula [1]: % corrected mortality = %T - %C %C %T= percentage of treated T. vaporariorum % C= percentage of untreated T. vaporariorum 425 The corrected mortality data of dead nymphs at each concentration analyzed by variance (ANOVA) and mean separation was conducted by the least significance difference (LSD) test (p 0.05). The SAS procedures (GLM) (version 8) were used in all statistical analyses [41]. Results: Pathogenicity of B. bassiana on nymphal instars: The results revealed that mortality percentage, as a consequence of application of B. bassiana treatment, was significant (p=0.05) among T. vaporariorum nymphal instars. Infection levels were generally higher for the 3 rd and 4 th instars. In 10 6 conidia/ml this value was 63.74% (F = , df = 9, P < 0.001) and 71/68% (F = 97.01, df = 12, P < 0.001) for young and old nymphs respectively. This means that T. vaporariorum 3 rd and 4 th nymphs were highly susceptible to fungal treatment compared to the 1 st and 2 nd nymphs. Mortality percentages averaged 2.87, 15.87, and 63.74% in young nymphs and 14.89, 29.25, and 71.68% in old nymphs with fungal concentration of 10 3, 10 4, 10 5 and 10 6 conidia/ml, respectively (Fig1). Probit analysis test showed that the medium lethal conidial concentrations (Lc 50 s) for young and old nymphs were (r=1.46, p= 0.90) and conidia/ml (r=1.52, p= 0.90) (Table1). mortality percentage d d concentarion(conidia/ml) c b a mortality percentage e d c concentration (conidia/ml) b a Fig. 1: Means corrected survival percentage of the 1 st and 2 nd (left) and the 3 rd and 4 th instar nymphs (right) of Trialeurodes vaporariorum 7 days following treatment with different conidia suspension of Beauveria bassiana. The different letters a, b, c and d on top of each column indicate significant differences for mortality percentage after counting. Table 1: Values of Lc 50s for Beauveria bassiana against different nymphal instars of Trialeurodes vaporariorum 7 days after inoculation T. vaporariorum Lc 50 (conidia/ml) (95% CL) Y Slope±SE X 2 1 th and 2 nd 0/ (0/ / ) 1/46x+0/23 1/46± rd and 4 th 3/ (2/ / ) 1/52 x+0/64 1/52± SE: Standard Error

4 Pathogenicity of L. Muscarium on nymphal instars: The results as presented in Fig2 show that based on whitefly nymph counts after 7 days from inoculation date by L. muscarium the survival of nymphs had a mean value ranging from 62.49± 0.47% (F = , df = 11, P < 0.001) to 87.13±0.45 % (F = , df = 11, P < 0.001) for the 1 st and 2 nd instar nymphs and the 3 rd and 4 th instar nymphs, respectively (Fig2). Overall there was a significant 426 difference in percentage mortality among instars. The 3 rd and the 4 th instar nymphs were significantly more susceptible than the 1 st and 3 rd instars. Likewise, the average mortality percentages in young nymphs were 13.82, 22.67, and 62.49% and 13.41, 23.75, and 87.13% with fungal concentration of 10 3, 10 4, 10 5 and 10 6 conidia/ml, respectively (Fig2). Amounts of Lc 50 s in young and old nymphs were (r=1.3, p= 0.90) and 3/ (r=1.69, p= 0.90) conidia/ml, respectively (Table2). mortality percentage e d concentration (conidia/ml) c b a mortality percentage e d concentration (conidia/ml) c b a Fig. 2: Means corrected survival percentage of the 1 st and 2 nd (left picture) and 3 rd and 4 th instar nymphs (right picture) instar nymphs of Trialeurodes vaporariorum 7 days following treatment with different conidia suspension of Lecanicillium muscarium. The different letters a, b, c and d on top of each column indicate significant differences for mortality percentage after counting Table 2: Values of Lc 50s for Lecanicillium muscarium against different nymphal instars of Trialeurodes vaporariorum 7 days after inoculation T. vaporariorum Lc 50 (conidia/ml) (95% CL) Y Slope±SE X 2 1 th and 2 nd ( ) 1.3x ± rd and 4 th ( ) 1.69x+1/ ± SE: Standard Error Combination of B. bassiana with imidacloprid on the 1 st and the 2 nd instar nymphs of T. vaporariorum: As data in table 3 indicate there was no significant difference in treatment of 10 6 conidia/ml B. bassiana and singular application of 300mg/l imidacloprid but the percentage of mortality was higher than 10 6 conidia/ml of B. bassiana alone (F=174.34, df=13, P<0.001). In contrast the percentage mortality in treatment of 10 6 conidia/ml+50mg/l imidacloprid was more than 10 3 conidia/ml±300mg/l. Furthermore the efficacy of 50mg/l of imidacloprid was more than 10 3 conidia/ml of B. bassiana alone (Table3). Table 3: The effect combination of imidacloprid with Beauveria bassiana treatments on the 1 st and 2 nd nymphs of T. vaporariorum Treatment Number of nymphs infested (%)± SE 10 6 conidia/ml (B. bassiana)+300mg/l (imidacloprid) 89.91±0.97a 300mg/l 89.65±0.73a 10 6 conidia/ml+50mg/l 79.93±1.17b 10 3 conidia/ml+300mg/l 63.74±0.71c 10 6 conidia/ml 50.52±0.55d 10 3 conidia/ml+50mg/l 37.33±.076e 50mg/l 33.79±0.28e 10 3 conidia/ml 7.91±0.54f control 2.87±0.45f LSD: 5.59 Combination of B. bassiana with imidacloprid on the 3 rd and 4 th nymphs of T. vaporariorum: There was no significant difference between combination of high concentration of two agents with application of singular 300mg/l imidacloprid treatment (F=216.47, df=13, P<0.001). Moreover there was no significance different between coapplication of 10 6 conidia/ml + 50 mg/l and 10 3 conidia/ml + 300mg/l and. On the other hand,

5 efficacy of 50mg/l of pesticide was more than 10 3 conidia/ml of B. bassiana. The mortality in singular application of 10 6 conidia/ml of B. bassiana 427 was lower than combination of two agents except the treatment of 10 3 conidia/ml+50mg/l (Table4). Table 4: The effect of different imidacloprid and Beauveria bassiana treatments used to control old nymphs on Trialeurodes vaporariorum Treatment Number of nymphs infested (%)± SE 10 6 conidia/ml (B.bassiana)+300mg/l(imidacloprid) 98.61±0.5a 300mg/l 88.27±0.65b 10 6 conidia/ml+50mg/l 79.78±1.08c 10 3 conidia/ml+300mg/l 77.72±0.63c 10 6 conidia/ml 70.43±1.09d 10 3 conidia/ml+50mg/l 33.12±0.78e 50mg/l 26.73±0.69f 10 3 conidia/ml 13.26±0.55g control 5.46±0.23h LSD: 5.26 Combination of L. muscarium with imidacloprid on the 1 st and 2 nd instar nymphs of T. vaporariorum: There was no significant difference between the percentage mortality of 10 6 conidia/ml of L. muscarium with 300mg/l of imidacloprid (F=117.08, Combination of L. muscarium with imidacloprid on the 3 rd and the 4 th instar nymphs of T. vaporariorum: Efficacy of combination of the highest concentration of two agents was more than other treatments (F=162.72, df=13, P<0.001). Mortality in df=13, P<0.001). While combined 300mg/l of imidocloprid with 10 6 conidia/ml the percentage of mortality was lower than 10 6 conidia/ml of L. muscarium with 50mg/l. The percentage of mortality in the highest concentration of each of the two agents was more than their combined application (Table5). singular treatment of 300mg/l of imidacloprid was higher than 10 6 conidia/ml L. muscarium treatment. The mortality percentage of 300mg/l of imidacloprid was not significantly different compared to 10 6 conidia/ml L. muscarium treatment. In all experiments, mortality in control treatment was lower than 8% (Table 6). Table 5: The effect of different imidacloprid and Lecanicillium muscarium treatments used to control young nymphs on Trialeurodes vaporariorum Treatment Number of nymphs infested (%)± SE 300mg/l (imidacloprid) 88.27±0.65a 10 6 conidia/ml (L. muscarium) 86.63±0.64a 10 6 conidia/ml+50mg/l 71.83±0.79b 10 6 conidia/ml+300mg/l 65.24±0.76c 10 3 conidia/ml+50mg/l 58.28±1.11d 10 3 conidia/ml+300mg/l 51.45±0.76e 50mg/l 29.73±0.69f 10 3 conidia/ml 15.79±1.05g control 6.95±0.4h LSD: 6.18 Table 6: The effect of different imidacloprid and Lecanicillium muscarium treatments used to control the 3 rd and 4 th nymphs on Trialeurodes vaporariorum Treatment Number of nymphs infested (%)± SE 10 6 conidia/ml+300mg/l 96.86±0.74a 300mg/l(imidacloprid) 88.27±0.63b 10 6 conidia/ml(l. muscarium) 86.63±0.64b 10 6 conidia/ml+50mg/l 77.77±0.75c 10 3 conidia/ml+300mg/l 73.99±0.82c 10 3 conidia/ml+50mg/l 48.89±0.81d 50mg/l 26.73±0.96e 10 3 conidia/ml 15.79±1.17f control 7.67±0.39g LSD: 5.98 Discussion: Generally our results are in agreement with findings of previous studies that show these entomopathogenic fungi were effective against whiteflies [36,45,20,26,10,35]. Our results also showed that for both young and old nymphal stages, in comparison with B. bassiana alone, the mortality rate was significantly higher when a combination of imidacloprid and B. bassiana was used. The mortality caused by a combination of L. muscarium with imidacloprid for old nymphal stages was also

6 higher than separate treatments. These results show that the interaction between pests and fungi can be complicated and be dependent on pest life stage. Moreover, with adding a pesticide to this interaction, all effects of the pesticide on the interaction components should also be considered. Our results revealed that old instars were significantly more susceptible than young instars. This difference in mortality was probably due to occurrence of moulting shortly after fungal inoculation in young nymphs and moreover, conidial persistence on old nymphal stages [50]. In other studies low mortality caused by a fungus compared others fungi and also antifungal effects of some plant secondary metabolites such as tomatine and gossypol on insect pathogens demonstrated [13]. For example it was observed that immature stages of B. tabaci are more susceptible to Aschersonia spp. than B. bassiana [29] and L. muscarium rather than B. bassiana [19]. Pathogenicity of the entomopathogenic fungi is related to the ability of the fungus to germinate on the insect cuticle and overcome the secondary metabolites and to the defense mechanisms of the host to prevent fungal infection and growth [40,8]. Lacey and Mercadier determined a severe reduction of germination and growth in P. fumosoroseus and Nomuraea rileyi due to tomatine and Alpha-tomatine respectively [27]. In this study nymphal stages of T. vaporariorum showed different susceptiblty to entomopathogenic fungi used and the 3 rd and 4 th nymphs of T. vaporariorum are were more susceptible to B. bassiana and L. muscarium compared with the 1 st and 2 nd nymphs. Likewise different susceptibility of nymphal instars of whiteflies has determined in other studies. In contrast to our results, Gridin et al showed that the older nymph is less susceptible to infection by L. muscarium [19]. According to Poprawski et al the T. vaporariorum nymph III showed the highest mortality percentages for Paecilomyces fumosoroseus and B. bassiana. Siongers and Coosemans found that BotaniGard had the greatest impact on the 1 st instars and sensitivity was decreased with age [43]. A number of factors (such as developmental stage) can influence the efficacy of entomopathogenic fungi. Movement of crawler (1 st nymph) caused conidia to fall out of the cuticle and subsequently, penetration can not be carried out. Most host factors, such as insect developmental rates, can not be considered independent of environment factores (e.g. temperature). In 24±1ºC and on tomato after hatching the time required for juvenile appearance for the 1 st and the 2 nd stages was 11 days and 20 days for the 3 rd and the 4 th stages. Therefore in young nymphs time between molts was shorter. High temperatures increase insect stage development and will reduce the time between molts, which can decrease the infection rate due to loss of inoculum on exuviae and before penetration since, conidia are separated from exuviae. Amounts of cuticle lipids 428 can also influence the infection potential [24]. These lipids inhibit penetration of fungal conidia to cuticle layers [28]. From the current study, this would appear that L. muscarium and B. bassiana are potent against the instar nymphs of T. vaporariorum, thus using of IPM systems incorporating L. muscarium and B. bassiana for control of T. vaporariorum in greenhouses may be developed but further work is required to reach the pest mortality to commercially acceptable levels. We believe that in pest fungal control, stressed insects are more susceptible to entomopathogens. Our results showed that the mortality percentage of insect pests treated with imidacloprid and subsequently infected by B. bassiana was approximately about 20% higher than the fungus B. bassiana alone. Therefore, Imidacloprid might increase the susceptibility of the insect pests to fungal infection. Moreover, our results demonstrated that the association of imidacloprid and B. bassiana and L. muscarium on the old nymphs revealed compatibility between them. We conclude that the association of these chemical and biological agents may be used as a tool for integrated control of the greenhouse whitefly T. vaporariorum. In our study, in almost all associations of B. bassiana, L. muscarium and imidacloprid, the mortality rates were higher than those treatments using either fungus or imidacloprid alone at equivalent doses. This association can suggest that both components could be used at reduced concentrations, increasing safety and efficacy. As a conclusion, this investigation has points to the successful integration of imidacloprid with the pest pathogenic fungi, L. muscarium and B. bassiana. However, inhibitory effects of pesticides on entomopathogenic fungi cannot be ignored. In the other words, variations in toxicity response of entomopathogenic fungi to pesticides have been observed [47]. In one way, fungal control agents and selective pesticides may act synergistically increasing the efficiency of the control program, allowing the lower doses of pesticides, more preservation of natural enemies and minimizing environmental concerns [48]. For example it has been reported that the coaplication of Metarhizium anisopliae with Imidacloprid increased virulence against Aedes aegypti (Diptera: Culicidae), the dengue vector [56]. Several experiments have carried out to investigate effects of chemicals used to control of aleyrodid pests [45,49] but few have studied the effect of these chemical insecticides on L. muscarium and B. bassiana. Malekan [50] showed lack of negative influence of imidacloprid on B. bassiana and recommended the combination of these two factors in integrated pest management programs. Alves and Leucona stated that pesticides may have a variety of effects on entomopathogenic fungi such as inhibition of both germination and germ tube development and even mutations that may alter virulence. It is seen that Imidacloprid increase the

7 susceptibility of the insects to fungal infection. In a study of the possible synergistic effects of combinations of entomopathogenic fungi and the insecticide. Almeida et al. observed a reduction of field populations of Heterotermes tenuis when using traps baited with imidaclopriod and B. bassiana [4]. It has also been proved that the synergistic effect of imidaclopriod and fungus on Diaprepes abbreviatus, was due to the reduction in motility, increased adhesion of the spores that would normally be removed by friction as the larvae move through their tunnels in the soil [37,6]. Almeida and Alves found no synergism when termites were treated with conidia of B. bassiana + imidacloprid compared to application of conidia alone [4]. Based on data analyzed here, in the treatments with each fungus alone the lower mortality rate (<10%) was observed in the 1 st and 2 nd nymphs treated with B. bassiana at a concentration of 10 3 conidia/ml and the highest mortality rate (>80%) was observed with L. muscarium at a concentration of 10 6 conidia/ml. The association of imidacloprid and B. bassiana and so L. muscarium on the 3 rd and 4 th nymphs revealed compatibility between them. We conclude that the association of this chemical and biological agent may be used as a tool for integrated control of the greenhouse whitefly T. vaporariorum. The results reported by Paiâo et al. are in agreement with the results presented in current study which agrees on the compatibility between imidacloprid and B. bassiana [34]. In all associations of B. bassiana, L. muscarium and imidacloprid, the mortality rates were higher than those treatments using either fungus or imidacloprid alone at lower or equivalent doses in association. In present study the results showed that treatment of 10 6 conidia/ml of L. muscarium+imidacloprid at 300mg/l on the 1 st and 2 nd nymphs, mortality rate was lower than in that of only the fungus L. muscarium in concentration 10 6 conidia/ml (Table 5). But even the highest concentrations of imidacloprid which was 300 mg/l did not influenced the germination of L. muscarium on 3 rd and 4 th nymphs. In addition, we can consider that insecticides may act in a combined form with entomopathogenic agents. For instance, sub-lethal doses facilitate the infectious process due to debility or stress of the arthropod which becomes more susceptible to the action of entomopathogens. This association can ensure that both components could be used at reduced concentrations, reducing costs, optimizing benefits and improving safety and efficacy. As a conclusion this study has identified two approaches to co-application imidacloprid with L. muscarium and B. bassiana. Firstly, increasing the mortality percentage of greenhouse whitefly in combination of imidacloprid with B. bassiana compared to B. bassiana alone, and secondly higher mortality achieved by L. muscarium compared to B. bassiana on different nymphal stages of T. 429 vaporariorum. Therefore, this investigation can help to establish a successful integration of imidacloprid with entomopathogenic fungi, L. muscarium and B. bassiana. Acknowledgements This work was supported by a grant from the graduate school of Isfahan University of Technology, Iran. The authors would also like to thank Dr. H. Askary (Forests and Rangelands Institute, Tehran, Iran) for supplying the isolate DAOM of L. muscarium and isolate Fashand of B. bassiana. References 1. Abbott, W.S., A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18: Agoitia, G.J., Jitomate: tendenciasy perspectivas de exportaci n. BANCOMEX. Mexico., pp: Ahmad, M., M.I. Arif, Z. 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