Efficacy of Olive Stones and Corncobs Crystalline Silica Nanoparticles (SiO2, NPs) Treatments on Potato Tuber Moths (Phthorimaea Operculella)

Morphological and microstructural properties of silica (SiO2) are essential factors that need to be considered during the experimental applications. In this report, SiO2 nanoparticles (NPs) have been prepared at different concentrations from SiO2 powder derived from olive stones and corncobs by an alkali leaching extraction method. Thermal treatment has been used to modify the morphological and microstructural properties of the extracted SiO2. X-ray diffraction (XRD) shows that the extracted amorphous SiO2 has been transformed into a crystalline phase after the thermal treatment. Nitrogen adsorption–desorption measurements revealed significant reduction in both the specific surface area (SABET) and the total pores volume (Vtotal) of the derived SiO2 samples after the thermal treatment. The acquired thermal treatment properties of SiO2 found to have high impacts on the influence of the SiO2 NPs on the survival and development of larvae and pupae of P. opercullela. SiO2 NPs prepared from the crystalline silica samples exhibited less efficiency on suppressing potato tuber moth comparing to SiO2 NPs of the amorphous silica powders. Varied effects on biological parameters including larvae mortality, pupae weight, larval and pupal development time, fecundity and fertility are also linked to the SiO2 NPs source type and showed concentration depending manner effects. SiO2 NPs are conditionally effective as an alternative pesticide against P. opercullela, based on their sources, mesoporous structures and concentrations.


Introduction
Syria cultivates 24.789 thousand hectares and produces around 486.605 thousand tons of potato each year. Therefore, after wheat (Triticum aesitvum L.), potato is the Syrian second most widely cultivated crop that adds economical and nutritional value to the country [1,2]. In addition, potato crop has been recognized as a key component that is ensuring national food security and it contributes to decrease poverty levels. However, the diseases and insects are jeopardizing potato crop either directly by feeding on tubers, or indirectly by feeding on leaves and stems or during the storage period. Potato tuber moth (Phthorimaea operculella Zeller (Lepidoptera: Gelatiidae)) is one of the catastrophic pests that can destroy potato crops by 50% to 100% and it is currently affecting over 90 countries in tropical and subtropical regions [3,4].
To date, pesticides are the most popular, effective and dominant method that has been used in controlling P. operculella. However, pesticides are still a major health and environment pollution hazards concerns as they eventually end up in consumers' bodies and destroy the nature elements. Moreover, insects are becoming resistant to insecticides over time and help to produce superbugs [5]. Hence, finding control methods for P. operculella becomes increasingly important to continue in growth and development of the potato industry [3,6].
In the recent years, agricultural nanoparticles applications generally became more affordable, simpler to manufacture, and have less or non-environmental. Agricultural nanoparticles such as TiO 2 , Al 2 O 3 , and SiO 2 nanoparticles, are generally preferred [7][8][9]. In plant protection, SiO 2 NPs have been widely studied and used to reduce the effects of salinity, metal toxicity, and nutrient deficiency in plants [10]. SiO 2 NPs have promising properties that makes them usable and applicable such as simplicity, efficiency, nonrecorded environmental pollution and cost efficiency [11]. SiO 2 NPs are increasingly prepared from plants and agriculture byproducts. Researchers reported that SiO 2 NPs can effectively control Spodoptera littoralis, a polyphagous plant pest [8,9,12]. In a recent study of our lab (To be published), SiO 2 NPs prepared from amorphous silica powder derived from olive stones and corncobs were effectively impacted in larval mortality, pupal weight, larval and pupal development times of P. operculella. Moreover, SiO2 NPs derived from olive stones at 1500 ppm concentration showed the ultimate toxicity against potato tuber moths [13]. Hence, the present study is a continuing investigation by our team to gain a better understanding of the effect of morphological and microstructural aspects of SiO 2 NPs on potato tuber moths. For this purpose, SiO 2 NPs are prepared at different concentrations from silica powder samples of different microstructural and morphological properties. The amorphous silica powder samples are derived from two different agricultural waste sources; olive stones and corncobs, and then they were transformed into a crystalline phase by a proper thermal treatment. The microstructure and morphological changes of the studied silica samples are characterized by X-ray diffraction (XRD) and nitrogen absorption and desorption measurements respectively. The influence of SiO 2 NPs against potato tuber moth pest including fecundity, fertility, and sex ratio were investigated.

Preparation and Characterization of SiO 2 NPs
A previous report described in detail the alkaline leaching extraction technique for obtaining silica powder from corncobs and olive stones [11]. Briefly, the cleaned and dried corncobs or olive stones powders were heated in an electric Labotherm furnace at 500 ºC for 2 h. 20 g of the resultant, corncobs/olive stones, ash was mixed with 2.5 N NaOH (200 mL) solution and then heated at 90 °C for 3 h in a covered Erlenmeyer flask with continuous magnetic stirring. At room temperature conditions (~ 20 °C), the pH of the filtrated solution (Na 2 SiO 3 ) was adjusted to pH 7.4 using 20% H 2 SO 4 under vigorous stirring, shaken for 24 h at a rotation speed of 200 rpm, then it was aged for 48 h. The precipitated silica was filtered, washed with distilled water several times in order to be sodium sulphate-free and then dried.
The thermal treatment was carried out by heating the as-extracted silica powder in an electric furnace for 2 h in the air at 1000 °C. A STOE Powder diffractometer was used to record the x-ray diffraction (XRD) patterns of the extracted silica before and after the thermal treatment. Using nitrogen adsorption-desorption measurements, the specific surface area and total pore volume of the studied silica samples were estimated by the Brunauer-Emmett-Teller (BET) method, while their pores size distribution were determined by Barrett-Joyner-Halenda (BJH) method. A Quantachrome NOVA 2200 BET surface area analyzer was used for the measurements at 77 K, and nitrogen adsorption-desorption isotherms were studied in the range of relative pressure [P/P0] from 0.05 to 1 atm.

Application of SiO 2 NPs on P. operculella
Potato tuber moth has been raised from our laboratory stock cultures on wax coated potato slices, kept at a constant temperature of 25 ± 1 °C with 70 ± 5% relative humidity, and a 12-h light/dark cycle.
SiO 2 NPs were prepared by milling the extracted silica powder in a porcelain mortar, dispersing it in double distilled water (1.5 mg/ml), and then ultrasonicated for 30 min at a frequency of 30 kHz using an ultrasonic homogenizer with a tip in order to break big cluster agglomerates. Five groups of fresh potato leaves were treated with three concentrations of SiO 2 NPs (1500 ppm, 1000 ppm, 500 ppm), along with a control treatment and Deltamethrin as an insecticide. In each treatment, 200 larvae were fed on potato leaves in 10 plastic boxes (18 × 12 × 8 cm), resealed with parafilm to prevent larvae from escape until pupation. Incubation was performed under 25 ± 1 o C with daylight 12 h and relative humidity of 70% in boxes. A percentage of larval mortality was calculated by counting pupae that emerged from the tested larvae: Percentage of mortality = (number of larvae tested-number of pupae emerging) / number of larvae tested × 100, the experiment was repeated three times. Pupae and larvae (N = 30) were selected per treatment, and their development periods were recorded. Each treatment consisted of collecting 25 pupae aged four days placing them in smaller plastic tubes for weight calculations and to be sexed (male / female). Within each treatment (N = 15), newly emerged males (0-18 h) paired with 1-day-old normal females (N = 15) were placed in 350 ml transparent plastic boxes with an oviposition site (filter paper) and food source (10% sucrose solution). All pairs were kept together until death, eggs were removed every day, counted, and left to hatch. We measured the mean number of laid eggs and their hatchability ratio for each pair.

Statistical Analysis
Statistical significance is defined as P < 0.05. StatView statistical software (version 5.0; SAS Institute, Cary, North Carolina, USA) was used to perform all statistical analyses. the analysis of variance was performed on the data of mortality and pupae weight to determine the statistical significance of the mean and percentage difference according to the ANOVA-Tukey HSD test. The chi-squared tests were used to compare the ratio of females to males with the expected ratio of 1:1 (F:M).

Characterization of SiO 2 NPs by XRD Technique
The as-extracted silica samples obtained from both olive stones and corncobs are characterized with a very broad XRD peak which is typically associated with amorphous silica [11,13]. Figure 1 shows the XRD patterns of thermally treated silica samples that extracted from olive stones (a) and corncobs (b). The XRD patterns for both samples show sharp and well-defined diffraction peaks corresponding to crystalline tetragonal SiO 2 (Cristobalite low, syn) phase [PDF 00-077-1317]. However, unlike the silica extracted from olive stones, the XRD pattern of the silica extracted from corncobs has clearly showed three weak diffraction peaks at 2θ = 21.7, 27.58 and 37.7°, indicating the presence of small amount of crystalline Tridymite [PDF 00-042-1401], in this sample. In addition, the type of plant source has a clear impact on the width of the XRD peaks, which in turn affects the particles size of the treated silica. The average particles size of the treated silica extracted from olive stones and corncobs is estimated from these patterns using the famous Scherer's formula from measuring the position of XRD peak and its full width at half maximum (FWHM) [14]. It is found to be around 38.2 nm and 45.8 nm for the silica sample derived from olive stones and corncobs respectively.
The XRD results were assisted by nitrogen adsorption-desorption measurements using BET method. Figure 2 shows the nitrogen adsorption-desorption isotherm of the studied silica samples. Based on, the isotherms in case of both as-extracted samples show characteristics that according to IUPAC classification of porous materials correspond to IV type of mesoporous material [15]. After the thermal treatment, the shape of the isotherm becomes more similar to type (VI) of nonporous or macroporous materials. However, the shape of the observed of hysteresis loop in the isotherms of these two samples indicates the existence of non-rigid aggregates of plate-like particles or assemblages of slit-shaped pores, which is not expected to provide a reliable assessment of either the pore size distribution or the total pore volume [16]. The specific Surface Area (SABET) and the total Pore Volume (Vtotal) of studied silica samples are measured from these isotherms using BET method, and the results are summarized in Table 1. As compared to as-extracted silica (amorphous) samples, the treated silica (crystalline) samples show drastic decrease in both SA BET and V total values. This, as explained above, is attributed to the change in the pore morphology and formation of nonrigid aggregates on the thermal treatment process. In addition, the obtained specific surface area results in a good agreement with our above XRD results, where it is observed that the silica extracted from olive stones has particles size lower that extracted from corncobs. Figure 3 shows the pores size distribution of the studied silica samples obtained from the nitrogen adsorption-desorption measurements by the Barrett-Joyner-Halenda   (BJH) method. Both as-extracted samples show pores diameter distribution consists with mesoporous materials. However, the treated samples do not show any specific pores distribution. This is coordinate with the above isotherms results, because it is a well-known that the pores distribution of macroporous materials cannot be determined using BJH gas adsorption method [17]. These are in a good constancy with the above BET results.

Effect of SiO 2 NPs Concentrations Treatment on Larval Mortality and Pupal Weight of P. operculella
Significant differences have been observed in larval mortality between control, SiO 2 NPs treatments, and Deltamethrin (Df = 7, f value = 28.46, P < 0.0001), as it is shown in Fig. 4. The toxicity values on larvae stage treated with olive  stone and corncob amorphous SiO 2 NPs in our recent study [13] recorded higher significant values of larval mortality than crystalline SiO 2 NPs.. It is observed from Table 1, the values of SA BET and Vtotal are ~ 299.7 m 2 /g-0.327 cm 3 /g and ~ 269.6 m 2 /g-0.678 cm 3 /g for silica derived from olive stones and corncobs of amorphous silica phase (as-extracted) respectively. Meanwhile, SA BET and V total value are shown to be much higher than the corresponding values of treated crystalline silica samples (2.02 m 2 /g-0.0045 cm 3 /g and 1.23 m 2 /g-0.0021 cm 3 /g). The differences in SA BET and Vtotal properties between crystalline and amorphous SiO 2 NPs have been associated with a significant difference in toxicity on larvae stage in favor of amorphous SiO 2 NPs prepared from the thermally treated samples. According to previous research, silica NPs size has a considerable factor that play effect roles in cytotoxicity. Also, there was an inverse relationship between NPs size and cytotoxicity as it has been found that relatively larger NPs size give fewer toxic effects comparing to smaller NPs [18]. Additionally, the surfaceactive properties of SiO 2 NPs enable them to interact with biological surfaces, such as cell membranes. Therefore, the specific particle surface area increases linearly as particle size decreases [19]. As result of exposure to SiO 2 NPs, a higher mortality rate has been observed in larvae and pupae in three mosquito species, and SiO 2 NPs increased the toxicity of two insect pest species when combined with chemical compounds or with essential oils during the control of two grain storage beetles [20][21][22]. A significant reduction in pupae weight was observed in both of the SiO 2 NPs (crystalline phase) treatments when compared with the control. The noticeable weight reduction was recorded at 1500 ppm in olive stone SiO 2 NPs treatment (df = 7, f -value = 10.623, P < 0.0001). The weight reduction in pupae seems to be greater with SiO 2 NPs that have been obtained from amorphous phase [13] than the weight loss when SiO 2 NPs in crystalline phase have been used (Fig. 5), presumably because of the size of the silica nanoparticles. There are a variety of ways in which SiO 2 NPs can damage and eventually kill the insects such as desiccation, cuticle abrasion, and spiracle obstruction [20,23,24]. As a result of physiological disruption by SiO 2 NPs, these factors may contribute to pupae mortality and weight reduction. Table 2 shows a significant difference between SiO 2 NPs types and concentrations treatments versus the development times of larval and pupal stages. The maximum increase in time of development was observed in the olive stone SiO 2 NPs (1500 ppm and 1000 ppm), and maize nanoparticle (1500 ppm) treatments as compared to the control (df = 7, f -value = 4.87, P < 0.0001; df = 7, f -value = 6.75, P < 0.0001, respectively). A recent study in our laboratories has obtained the same results when amorphous phase of SiO 2 NPs was applied [13]. However, the results also confirm that the larval and pupal development times were prolonged when amorphous phase of SiO 2 NPs was applied comparing to crystalline SiO 2 NPs type ( Table 2). The size of SiO 2 NPs may cause difficulties in the larvae feeding that eventually makes larvae develop more slowly. Spodoptera littoralis larval and pupal development times are prolonged when larvae ingest nanoparticles silica [8]. This finding is in line with previous studies that indicate that larvae treated with toxicity agents have longer development times [25][26][27].

Effect of SiO 2 NPs Concentration Treatment on the Development Time of Larval and Pupae, Fecundity, and Fertility of P. operculella
As it is shown from Table 3, significant differences have been also found in fecundity between treatments and control, the most fecundity decreasing was in olive stone amorphous phase SiO 2 NPs treatment at 1500 ppm concertation (df = 13, f -value = 121.401, P < 0.0001) ( Table 3). El-Bendary et al. [8] found that the number of eggs has not been affected by low weight of pupae of Spodoptera littoralis when it has been treated with low concentrations of SiO 2 NPs (200-500 ppm). In addition, early studies showed that normal small size pupae of potato tuber moths did not affect the number of eggs [28]. Although our results were partially in agreement with previous research, however at height levels of SiO 2 NPs treatment show clear impacts on the formation of eggs in the ovaries after emerging from the pupae. Moreover, a significant difference in the percentage of eggs hatching between treated and control samples has been observed as Table 3 shows. The most eggs hatching decreasing was recorded when olive stone and maize amorphous phase SiO 2 NPs applied at 1500 ppm concertation (df = 13, f -value = 56.222, P < 0.0001). Egg hatching has been reduced due to the reduction in insect size and weight, as well as the reduction in sperm transfer and fertilization [26]. According to previous studies, there is a substantial correlation between body size and fertility in lepidopterans [29][30][31]. silica NPs treatment in this study revealed the absence of sex ratio alteration (Table 3).

Conclusions
We investigated the influence of morphological and microstructural factors on potato tuber moth's reproduction, such as porosity and crystallinity. The effectiveness of SiO 2 NPs prepared from silica powder of olive stones and corncobs on suppressing potato tuber moth pest was lower in the SiO 2 NPs prepared from crystalline silica than those prepared from amorphous silica. This is correlated to the induced microstructural and morphological changes by the thermal treatment, which are revealed by XRD and BET measurements.