Insecticidal effects of zinc oxide nanoparticles and Beauveria bassiana TS 11 on Trialeurodes vaporariorum ( Westwood , 1856 ) ( Hemiptera : Aleyrodidae )

Greenhouse whitefly, Trialeurodes vaporariorum is a major pest of horticultural and ornamental plants and is usually controlled with insecticides or biological control agents. In the current study, we examined the effects of synthesized zinc oxide nanoparticles (ZnO NPs) and Beauveria bassiana TS11 on T. vaporariorum adults. ZnO NPs were synthesized by precipitation method. Field emission scanning electron microscope images indicated that ZnO NPs were noncompacted uniformly. X-ray diffraction results confirmed the hexagonal wurtzite structure of ZnO NPs. Fourier transform infrared analysis showed an intense absorption peak at a range of 434-555 cm related to Zn-O bond. In bioassays, adults were exposed to different concentrations of ZnO NPs (3, 5, 10, 15, 20 mg l) and fungi (10, 10, 10, 10, 10 spores ml). LC50 values for ZnO NPs and fungi were 7.35 mg land 3.28×10 spores ml, respectively. Mortality rates obtained with ZnO NPs and fungi at the highest concentration were 91.6 % and 88.8 %, respectively. The results indicate a positive effect of ZnO NPs and B. bassiana TS11on adults. The current study was conducted under laboratory conditions, therefore, more studies are needed in field.


INTRODUCTION
The greenhouse whitefly, Trialeurodes vaporariorum (Westwood, 1856) (Hemiptera, Aleyrodidae), is a globally distributed pest because of its quick multiplication, virus transmission, sap puncture, secretion of honeydew and promotion of sooty molds on the host leaves (Guzman et al., 1997).Excessive use of chemical pesticides and increased resistance to insecticides (Whalon et al., 2008), environmental pollutions, impacts on human health and other organisms and finally pesticides residues in nature and agricultural products have provided great impetus to the development of alternative techniques of pest control (Van Lenteren et al., 1996;Laznik et al., 2011;Sandhu et al., 2012).
Recent progress in nano-technology has provided new opportunities in the fields of science such as agriculture (Chaudhry et al., 2008).Application of nanomaterials can revolutionize agriculture by developing potential and effective methods for pest management (Rai and Ingle, 2012).Some studies have reported the toxic effects of metal nanoparticles (such as silver, zinc, aluminum, and titanium oxide) on plants, crustaceans, bacteria, fungi, pathogens and pests (Elchiguerra et al., 2005;Goswami et al., 2010;Kairyte et al., 2013;Kirthi et al., 2011;Manzo et al., 2011;Morones et al., 2005;Reddy et al., 2007;Rouhani et al., 2012;Samuel and Guggenbichler 2004).Among them, zinc oxide (ZnO) nanoparticle is best-known compound (Mitra et al., 2012) commonly used as agricultural fungicide (Waxman 1998).Furthermore, cytotoxicity in eukaryotic cells (Gupta and Gupta, 2005;Magrez et al., 2006), preventing growth in prokaryotic cells (Brayner et al., 2006), bactericidal and fungicidal activity and low effects of ZnO NPs on human cells have also been reported (Kairyte et al., 2013).Low cost synthesis, protective effect against ultraviolet (UV) rays, biocompatibility and non-toxicity has attracted the attention of many researchers (Brayner et al., 2006;Zhang et al., 2007;Ahmad Umar et al., 2009).In spite of the studies performed on NPs to control limiting factors in ecosystems, little research has been carried out to investigate the toxicity effect of ZnO nanoparticles on agricultural pests.
In addition to nanotechnology, biological control is another alternative method to chemical pesticides due to its non-toxicity for human and other nontarget organisms, decreasing toxic residue in nature and food (Lacey et al., 2001).Aleyrodidae entomopathogens are limited to fungi since they are capable to penetrate the insect cuticle of sucking insects such as Aphis gossypii (Glover, 1877) (Gurulingappa et al., 2011), Rhynchophorus ferrugineus (Olivier, 1790) (Sewify et al., 2009), Laniifera cyclades (Druce, 1895) (Lozano-Gutierrz and Espana-Luna, 2008) and Galleria melonella (Linnaeus, 1758) (El-Sinary and Rizk, 2007).Among entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.)Vuill.with broad host range is known as an effective organism to control medical and agricultural pests (Inglis et al., 2001).The aim of this research was to investigate the insecticidal activity of synthesized ZnO nanoparticles and B. bassiana TS11 on Trialeurodes vaporariorum under greenhouse conditions.

Preparation of ZnO nanoparticles by precipitation
To prepare ZnO nanoparticles, first, 1 g of zinc oxide powder was dissolved in 100 ml of 1 % acetic acid oxidizing zinc-to-zinc cations.Then, the mixture was sonicated for 30 minutes.After 5 minutes, sodium hydroxide solution (1 M) was added drop wise to the above solution until the pH of solution reached to 10.The solution was heated in a water bath at 40-80 °C for 3 hours.Then, it was filtered through a filter paper and the precipitate was washed twice with distilled water.Finally, it was placed in an oven at 50 °C for 1 hour to form ZnO nanoparticles (Abdelhady, 2012).

Characterization of the synthesized ZnO nanoparticles
An analysis of nanoparticles was performed using Fourier transform infrared (FTIR) spectrometer (Bruker Optics Ft Tensor 27, Germany) by KBr (potassium bromide).The XRD analyses were performed using Bruker D8 X-ray diffractometer.FESEM (Hitachi S4160) analysis was carried out to observe the morphology of ZnO nanoparticles.

Entomopathogenic fungus culture and suspension preparation
Isolate TS11 of fungus B. bassiana isolate TS11 was obtained from Department of Plant Protection, University of Tehran, Iran.The fungus was inoculated on Sabouraud dextrose agar (SDA) medium in 8 cm Petri dishes and grown at 27 °C.
After 12 days, to prepare inoculum, 10 ml of sterile distilled water containing 0.2 % of aqueous Tween 20 solution (Sigma Alrdich, Spain) was spread over the petri dishes using a suitable tool.The suspension was filtered through cheesecloth to separate conidia from remnants of the mycelium.
The suspension was vortexed to separate spores from each other and to prevent mass formation during counting.Haemocytometer was used to count spores and prepare various concentrations of spores per unit volume.

Insect rearing
Adults of T. vaporariorum were collected from the surface of cucumber leaves (Cucumis sativus L) attacked with this pest from Sistan region (31.0256°N, 61.5011 °E, and average of 480 meters above the sea level) located in the east of Iran.The adults were reared on young green bean (Phaseolus vulgaris L.) plants in a laboratory greenhouse (University of Zabol, Zabol, Iran) under controlled conditions (27±2 °C, 60±10 % RH and a photoperiod of 16:8 (L: D) h).In order to perform bioassays, Muniz and Nombela`s method (2001) was followed.

Bioassay and determination of lethal concentration of ZnO nanoparticles
To find concentrations with 10-90 % mortality, primary tests were done with concentration ranges between 1-30 mg l -1 of the synthesized ZnO NPs.20 adults of T. vaporariorum with same age were considered for each concentration.Following method was carried out in both primary and final tests.To diminish the activity of greenhouse whiteflies, the numbers of insects per concentration were released into the plastic tubes using an aspirator.The tubes were then put in the incubator at 5 °C for 2 minutes (personal observations).Then, the specific numbers of insects were transferred to glass petri dishes already with filter paper-covered floor, but before getting started, the desired concentrations of 5 ml were sprayed on them using a 5 ml handy sprayer.
To avoid precipitation of nanoparticles in solutions, before spraying, all the prepared concentrations were sonicated for 10 minutes (Velayutham et al., 2013).After finishing, the insects were transferred to leaf cages installed over green bean leaves.The numbers of dead insects were counted after 24 hours and mortality was computed after three replications.In this experiment, distilled water was used for control.

Bioassays of B. bassiana TS11
Preliminary bioassays were conducted on adult insects with 10 2 -10 10 spores ml -1 concentrations of B. bassiana TS11.Concentrations of 10 4 and 10 8 spores/ml caused 30 and 80 % mortality were selected as minimum and maximum concentrations, respectively and between them three logarithmic concentrations of 10 5 , 10 6 and 10 7 spores/ml were selected for final bioassays.In each treatment, 0.2 % Tween 20 (Sigma Aldrich, Spain) as an emulsifying agent was added to suspension of fungal conidia.Distilled water containing 0.2 % Tween 20 was used as control treatment.The experiments were conducted in a completely randomized design with three replicates of treatments.
Bioassays in these experiments started with control treatment; next, they were continued from lowest level of concentration to the highest level.In order to infect the insects, similar as in bioassay procedure with ZnO nanoparticles, the insects were first deactivated at 5 °C (2 min).30 adult whiteflies were considered for each concentration.The specific numbers of the insects were individually placed in glass petri dishes with filter papercovered floor; the desired spore concentrations were then sprayed on insects (5 ml of each concentration using a 5 ml handy sprayer).After application of fungal suspension, whiteflies were placed inside glasses containing water-agar and green bean leaf.In order to provide moisture for primary germination of spores, the glasses were put in the germinator (27 °C, 80 % RH) for 24 hours.After 24 hours, whiteflies were released into leaf cages installed over young green bean leaves outside germinator and mortality of insects was recorded under controlled condition (27±2 °C, 60±10 % RH, a photoperiod of 16: 8 ([L: D] h) for 10 days.

Statistical analysis
To determine LC 50 and LC 25 , SPSS 21 software (IBM, New York, US) with confidence limits of 95 % and Probit analysis were used.

RESULTS AND DISCUSSION
The current study presents the results of insecticidal activity of the synthesized ZnO nanoparticles after 24 hours and pathogenicity of entomopathogenic B.bassiana TS11 after 10 days.

ZnO nanoparticles
Insecticidal activity of the synthesized ZnO nanoparticles revealed that values of LC 50 and LC 25 (7.35 and 3.76 mg l -1 , respectively) had significant lethal effects on T. vaporariorum adults.Statistical results showed a significant difference between concentrations of ZnO nanoparticles at 5 % level.
Where k is equal to 0.89; λ is X-ray wavelength (1.54 A), β is peak width at half maximum height in radian and θ is bragg diffraction angle of the maximum peak (Zhu et al., 2005;Suwanboon et al., 2013).In FTIR spectrum of ZnO nanoparticles sample, two strong absorptions are seen at 503 cm -1 and 432 cm -1 in which 432 cm -1 peak represents tensile bond of ZnO and 503 cm -1 peak represents oxygen vacancies in ZnO (Figure 4). ) revealed an increase in mortality when concentration increased; moreover, considering optimal time for lethal effects of ZnO nanoparticles after 24 hours, it was 100 % proved.In the comparison made between acaricidal, licicidal and larvicidal activities of zinc oxides and the synthesized ZnO nanoparticles, the mortality effect of ZnO nanoparticles was significant (Kirthi et al., 2011).Rouhani et al. (2012) studied the insecticidal effects of imidacloprid, Ag and Ag-Zn nanoparticles on Aphis nerii (Boyer de Fonscolombe, 1841) and LC 50 values after 24 hours were seen to be 0.13 µl ml -1 , 424.67 and 539.46 mg ml -1 , respectively.In a similar study, Samih et al. (2011) investigated the insecticidal effect of Amitraz, ZnO nanoparticles and ZnAl 2 O 3 against Pistachio psylly (Agonoscena pistaciae (Burckhardt and Lauterer, 1989)) and found out a greater insecticidal effect of Amitraz than the above nanoparticles.It should be taken into consideration that nanoparticles, especially the synthesized zinc nanopartilces, besides their insecticidal activity and also their slower effect than imidacloprid and Amitraz insecticides, have a lower risk of resistance to these nanoparticles in comparison to commercial insecticides (Rouhani et al., 2011).Clausen et al., (2011) investigated the efficiency of ZnSO 4 and ZnO nanoparticles on mortality of Reticulitermes flavipes (Kollar, 1837) (Isoptera, Rhinotermitidae).Their results confirmed that R. flavipes feeding on wood impregnated with ZnO nanoparticles, decreased to less than 4 % in comparison to control treatment.Despite low usage of wood impregnated with ZnO nanoparticles, 94-99 % of mortality rate was seen in termites.In contrast, using wood impregnated with ZnSO 4 , 10-12 % was consumed and low mortality rate (10-29 %) was seen in these termites.Thus, ZnO nanoparticles have the necessary potential for application in wood preservatives for protecting woods against termites.
LC 50 value of the synthesized ZnO nanoparticles on T. vaporariorum were consistent with the results of the above researches.Therefore, it can be said that ZnO nanoparticles have the necessary potential for insecticidal activity on T. vaporariorum and causes maximum lethality (91.6 %) at the highest concentration.
Nowadays, the only successful control strategy for the greenhouse whitefly is the combined use of pesticides and natural enemies (Hayashi, 1996); therefore, applications of some concentrations of pesticides with minimal impacts on natural enemies seem quite necessary (Laznik and Trdan, 2014).In the current study, through calculation of LC 25 , we can obtain a concentration of the synthesized ZnO nanoparticles to control T. vaporariorum, which is likely to distort its physiology.Since no research has been done on the impact of ZnO nanoparticles on natural enemies of the greenhouse whitefly, therefore, we conclude that using LC 25 , survival rate of T. vaporariorum decreases; however, this concentration would have minimal impact on natural enemies.With application of LC 25 (3.76 mg l -1 ), which is quite less compared to LC 50 (7.35mg l -1 ), we can prevent the occurrence of adverse effects on natural enemies of whiteflies.
Based on new and significant properties of nanoparticles, these materials are widely used in industrial and agricultural sectors; therefore, assessment of their potential toxic effects on human health and environment seems quite necessary (Auffan et al., 2009).As a discussion, the best approach to avoid adverse effects on the environment and ecotoxicological effects of nanoparticles on beneficial insects such as parasitoids wasps is using low concentrations of nanoparticles to control pest insects such as T. vaporariorum.
In the present study, B. bassiana TS11 had a significant pathogenic effect on T. vaporariorum adults, because it had a low LC 50 value and led to high mortality rate (88.8 %) in concentration of 10 8 spore ml -1 .Mortality in control treatment was so low and no fungal growth was seen on dead adult whiteflies.In terms of pathogenicity of B. bassiana for T. vaporariorum, similar to the effects of synthesized ZnO nanoparticles, calculation of lethal concentrations such as LC 25 (0.106×10 5 spore ml -1 ) is in turn particularly important.
We should pay attention to the interactions of the entomopathogenic fungi and whiteflies natural enemies (i.e.Encarsia spp., Eretmocerus spp., etc.) to minimize adverse effects on them.Therefore, despite the best effect of B. bassiana on pest insects such as T. vaporariorum, we should also take into account the low lethal effects of this entomopathogenic fungus so that they have minimal impact on natural enemies.

CONCLUSION
The current study was performed to demonstrate the insecticidal effects of ZnO nanoparticles and B. bassiana on the greenhouse whitefly.The obtained results proved the efficiency of synthetic nanoparticles and entomopathogenic fungi as effective control agents, which can lead also to the delay in pest resistance mechanisms to chemical insecticides.It is possible that by adding nanoparticles and entomopathogenic fungi to formulations of insecticides, toxicity of chemical insecticides for humans and other non-target organisms would be mitigated.Further study will need to focus on methods to increase stability and on physiological mechanisms of nanoparticles and entomopathogenic fungi interactions to increase their effects in integrated pest management programs at large greenhouse and field levels.

Figure 1 :
Figure 1: Mortality of T. vaporariorum adults, 24 hours after application of ZnO nanoparticles.Values followed by same letter indicate there is no overlap in 95 % confidence interval.

Figure 3 :
Figure 3: XRD spectrum of synthesized ZnO nanoparticles (Star symbols indicated the presence of ZnO NPs).

Figure 5 :
Figure 5: Mean of mortality of T.vaporariorum adults 10 days after treatment by B.bassiana

Table 1 :
Table 1 illustrates values of LC 25 and LC 50 , confidence limits (CL), slope and Chi-square (χ 2 ) for ZnO nanoparticles and B.bassiana TS11.Mean values of T. vaporariorum mortality by lethal concentrations of ZnO nanoparticles and B. bassiana TS11