Shah and Belozerova (2009) tested silica, palladium, gold and copper NPs and ...... Chakravarthy, A. K.; A. Bhattacharyya; P. R. Shashank; T. T. Epidi; B.
Kafrelsheikh University Faculty of Agriculture Economic Entomology Department
EFFICIENCY AND SAFETY OF SILICA NANOPARTICLES IN CONTROLLING THE MAIN INSECT PESTS ON FABA BEAN AND SOYBEAN
By
Ahmad Fouad Ahmad Thabet B.Sc. (Economic Entomology), Fac. of Agric., Kafrelsheikh Univ., 2009
THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE (M.Sc.)
In Agricultural sciences
(Economic Entomology) Department of Economic Entomology Faculty of Agriculture Kafrelsheikh University
2015
جامعة كفرالشيخ كلية الزراعة قسم الحشرات اإلقتصادية
فاعلية وآمان جزيئات النانوسيليكا في مكافحة اآلفات الحشرية الرئيسية للفول البلدي وفول الصويا رسالة مقدمة من
أحمـد فــؤاد أحـمـد ثـابــت بكالوريوس العلوم الزراعية (الحشرات اإلقتصادية) -كلية الزراعة -جامعة كفر الشيخ 9002
كجزء من متطلبات الحصول على درجة الماجستير في العلوم الزراعية (الحشرات اإلقتصادية)
قسم احلرشات الاقتصادية لكية الزراعة جامعة كفر الش يخ 5102
SUPERVISION COMMITTEE
Prof. Dr.
Hessien Abd El-Monem Boraei Professor Emeritus of Economic Entomology Faculty of Agriculture Kafrelsheikh University
Dr.
Magdy Farouk El-Samahy Associate Professor of Plant Protection Field Crop Pests Research Department Plant Protection Research Institute Agricultural Research Center
Dr.
Ola Abd El-Rahman Galal Associate Professor of Genetics Faculty of Agriculture Kafrelsheikh University
لجنة اإلشــراف أستاذ دكتور
حسين عبد المنعم برعي أستاذ الحشرات اإلقتصادية المتفرغ كلية الزراعة جامعة كفرالشيخ
دكتـــور
مجدي فاروق السماحي أستاذ وقاية النبات المساعد قسم بحوث آفات المحاصيل الحقلية معهد بحوث وقاية النباتات مركز البحوث الزراعية دكتـــور
عال عبد الرحمن جالل أستاذ الوراثة المساعد كلية الزراعة جامعة كفرالشيخ
ACKNOWLEDGEMENT Firstly, ultimate thanks and praise is due to mighty Allah for his continuous help through this study and all my life. I ought to express my sincere gratitude to late Prof. Dr. Hessien Abd ElMonem Boraei (rest in peace), professor emeritus of economic entomology, Faculty of Agriculture, Kafrelsheikh University for supervision, suggesting the problems, sincere guidance, valuable advice, highly appreciated efforts, carrying the present study continuous interest, precious guidance during the study and valuable revision of this thesis. I am indebted to Dr. Magdy Farouk El-Samahy, associate professor of plant protection, Field Crop Pests Research Department, Plant Protection Research Institute, Sakha Agricultural Research Station, Agricultural Research Center for supervision, valuable advice, constructive criticism and revision of this thesis. My respect gratitude and sincere thanks to Dr. Ola Abd El-Rahman Galal, associate professor of genetics, Faculty of Agriculture, Kafrelsheikh University for supervision, great help, guidance, suggesting solve the problems, sincere advice, valuable revision of this thesis and her encouragement during my work. My deep thanks to all staff of Economic Entomology and Genetics Departments, Faculty of Agriculture, Kafrelsheikh University. My deep thanks also to all staff at Field Crop Pests Research Department, Sakha Agricultural Research Station, Plant Protection Research Institute, Agricultural Research Center for their valuable help and support during this work. Thanks a lot for Prof. Dr. Mahmoud Ramzy Sherief, head of research, Rice Research and Training Center, Agricultural Research Center for his encouragement, help and valuable advice. My deep appreciation and thanks to my brother Dr. Osama Mohammed Rakha, lecturer of economic entomology, Faculty of Agriculture, Kafrelsheikh University for his encouragement and moral support during the course of this work. My thankfulness also is extended to Dr. Kareem Mohammed Mousa, lecturer of economic entomology, Faculty of Agriculture, Kafrelsheikh University for his help and valuable advice.
A.F. Thabet
CONTENTS INTRODUCTION REVIEW OF LITERATURE 1. Nanoparticles in insect pest control 2. Toxicity studies 2.1. Effects of nanoparticles on germination and plant growth 2.2. Genotoxicity effects 2.2.1. Genotoxicity of nanoparticles on plant 2.2.2. Genotoxicity of SiNPs on other biological systems 2.2.2.a. Effect of SiNPs on chromosomal aberrations 2.2.2.b. Effect of SiNPs on genomic stability and integrity 2.2.2.c. Effect of SiNPs on gene expression
MATERIALS AND METHODS 1.Seed material 2. Silica nanoparticles 3. SiNPs efficiency assays 3.1. Efficiency of SiNPs against faba bean main insect pests 3.1.1. Field procedures 3.1.2. Treatment against Aphis craccivora infestation 3.1.3. Treatment against Liriomyza trifolii infestation 3.2. Efficiency of SiNPs against soybean main insect pest; Spodoptera littoralis 3.2.1 Experimental design and treatments under field conditions 3.2.2. Biological studies on S. littoralis under laboratory conditions 4. Collecting of predators associated with insect pests 5. SiNPs toxicity assays 5.1. Pollen vitality 5.2. Growth experiment 5.3. Cytological analysis 5.4. Molecular analysis 5.4.1. Genomic DNA extraction 5.4.2. Polymerase Chain Reaction (PCR) 5.4.3. Genomic template stability 5.5. Biochemical analysis 5.5.1. Preparation of reagents
1 3 3 10 10 13 13 15 15 17 19 21 21 21 21 22 22 22 23 23 23 24 25 25 26 26 26 27 27 27 29 30 30
5.5.2. Preparation of samples 5.5.3. Preparation of gel 5.5.4. Electrophoresis 5.5.5. Staining 6. Statistical analysis
RESULTS AND DISCUSSION 1. Efficiency of SiNPs against insect pests 1.1. Efficiency of SiNPs against faba bean main insect pests 1.1.1. Effect of SiNPs on A. craccivora 1.1.2. Effect of SiNPs on L. trifolii 1.2. Efficiency of SiNPs against soybean main insect pest; S. littoralis 1.2.1. Effect of SiNPs on S. littoralis under field conditions 1.2.2. Effect of SiNPs on S. littoralis under laboratory conditions 2. Effect of SiNPs on predators associated with insect pests 2.1. Effect of SiNPs on predators in faba bean field 2.2. Effect of SiNPs on predators in soybean field 3. Toxicity of SiNPs on faba bean plants 3.1. Effect of SiNPs on pollen vitality 3.2. Effect of SiNPs on seed germination and shoot length 3.3. Cytological effects of SiNPs on mitosis 3.3.1. Mitotic index and frequency of mitotic phases 3.3.2. Percentage and types of abnormalities 3.4. Effect of SiNPs on genomic DNA 3.4.1. RAPD profile 3.4.2. Genomic template stability 3.5. Effect of SiNPs on seed storage proteins
SUMMARY REFERENCES ARABIC SUMMARY
32 32 33 33 33 35 35 35 35 37 39 39 42 45 45 47 50 50 50 53 53 56 63 63 66 68 74 80
LIST OF TABLES Table
Title
Page
Table (1)
The used primers and their nucleotide sequences
28
Table (2)
Reduction percentages of Aphis craccivora populations after SiNPs application during 2012/13 and 2013/14 seasons
36
Table (3)
Reduction percentages of Liriomiza trifolii larvae after SiNPs application during 2012/13 and 2013/14 seasons
38
Table (4)
Reduction percentages of Spodoptera littoralis larvae after SiNPs application under field conditions during 2013 and 2014 seasons
40
Table (5)
Biological aspects of Spodoptera littoralis reared on soybean leaves treated with SiNPs under laboratory conditions
43
Table (6)
Reduction percentages of predators in faba bean field after SiNPs application during 2012/13 and 2013/14 seasons
46
Table (7)
Reduction percentages of predators in soybean field after SiNPs application during 2013 and 2014 seasons
48
Table (8)
Effect of SiNPs on seed germination and shoot length of faba bean
51
Table (9)
Mitotic index and percentage of mitotic phases (relative to dividing cells) of faba bean root tip cells treated with SiNPs
55
Table (10)
Percentage of abnormal mitotic cells (relative to dividing cells) of faba bean root tip cells treated with SiNPs
57
Table (11)
Types and percentage of abnormalities (relative to dividing cells) of faba bean root tip cells treated with SiNPs
60
Table (12)
Total number of bands, polymorphic bands and percentage of polymorphism in DNA-RAPD profiles of faba bean treated with SiNPs
65
Table (13)
Changes in DNA-RAPD profile of faba bean treated with different concentrations of SiNPs
67
Table (14)
Seed storage protein banding patterns of faba bean treated with SiNPs
70
LIST OF FIGURES Figure
Title
Page
Fig. (1)
The carton box used for predators collecting
25
Fig. (2)
Mortality percentages of Spodoptera littoralis larvae after 15 days of feeding on soybean plants treated with SiNPs
43
Fig. (3)
Effect of SiNPs on faba bean pollen vitality
51
Fig. (4)
Mitotic phases in faba bean root tip cells under normal conditions
54
Fig. (5)
Types of chromosomal aberrations induced in faba bean root tip cells treated with SiNPs
59
RAPD profiles of genomic DNA extracted from M2 faba bean seedlings treated with different concentrations of Fig. (6) SiNPs using primers OPA20, OPB01, OPB06, OPB07, OPB12 and OPB14
64
Gel image (a) and its diagram (b) of faba bean seed storage proteins treated with SiNPs
69
Fig. (7)
INTRODUCTION Grain legumes are plants belonging to the family Leguminosae which are grown primarily for their edible grains or seeds. These seeds are harvested mature and marketed dry to be used as food or feed or processed into various products. Among the common legume seeds in Egypt, faba bean (Vicia faba L.) and soybean (Glycine max L.) are included. Production of faba bean and soybean crops has been constrained by the limitations imposed by insect pests and diseases (Metwally et al., 1997). Aphids; Aphis craccivora Koch. (Hemiptera: Aphididae), are considered the major insect pests in Egypt which play an important role in transmitting virus diseases that cause a considerable loss in the quantity and quality of the faba bean yield (Mohamed and Salman, 2001; El-Samahy, 2008). The faba bean leafminer; Liriomyza trifolii (Burgess) (Diptera: Agromyzidae), is considered also as a serious insect pest infesting vegetable, ornamental plants and broad bean (Mesbah and Sherif, 1994). Cotton leaf worm; Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae), is considered one of the major pest in Egypt in a wide range of cultivation including cotton, corn, soybeans, peanuts and vegetables. This pest is not only widely spread in Egypt but also in other Middle East countries in addition to temperate zones in Asia and Africa (Salama et al., 1990). Great efforts have been made to control the insect pests chemically. Because of hazards of pesticides on public health and environmental balance in addition the harmful effects on beneficial insects such as natural enemies, relatively recent direction of using nanomaterials (NMs) in pest control management was introduced. Nanomaterials consist of one or more components present in various forms that possess at least one dimensional structure of diameters in the range of 1 to 100 nm (Warheit et al., 2008). Among NMs, silica nanoparticles (SiNPs) have received considerable attention due to their unique properties. Silica NPs can be good
alternative to the popular insecticides which are hazardous to human health and because of huge environmental concerns associated with them. The use of amorphous nanosilica as biopesticide has been reported (Barik et al., 2008). Due to their small size and great surface area coupled with physicochemical characteristics such as metal contaminations and charged surfaces, NPs may exhibit unpredictable genotoxic effects. The genotoxic effects are the most dangerous negative consequences (Tarasov, 1998) because; in contrast to toxic injuries, genotoxic damages can be preserved in the population and transmitted from generation to generation (Pesnya and Romanovsky, 2013). The genotoxic effect of SiNPs have been evaluated in a variety of studies on different biological systems (Lin et al., 2006; Barnes et al., 2008; Park and Park, 2009; Galal and El-Samahy, 2012).
The present study was undertaken to achieve the following objectives: 1. Evaluate SiNPs as a new alternative insecticide on the main insect pests of faba bean (A. craccivora and L. trifolii) and soybean (S. littoralis). 2. Study the effect of SiNPs on the associated predators. 3. Investigate the biosafety and genetoxicity of SiNPs through cytological, molecular and biochemical analyses which were studied for faba bean plants of M2 generation.
REVIEW OF LITERATURE 1. Nanoparticles in insect pest control: Nanotechnology is one of the most important tools in modern agriculture. It is currently used as the most important and promising technology for protection of host plants against insect pests. Thus nanotechnology will revolutionize agriculture including pest management in the near future (Bhattacharya et al., 2010). Several studies confirmed that metal nanoparticles can be effective against plant pathogens, insects and pests. Hence, nanoparticles can be used in the preparation of new formulations such as pesticides, insecticides and insect repellants (Barik et al., 2008; Gajbhiye et al., 2009; Goswami et al., 2010). Ulrichs et al. (2005) reported that surface charged, modified, hydrophobic SiNPs (~3-5 nm) can be successfully used to control a range of agricultural insect pests and animal ectoparasites of veterinary importance. Barik et al. (2008) revealed that SiNPs; when applied on leaves and stem surface, get absorbed into the culticular lipids by physisorption and caused death of insects purely by physical means. Patil (2009) reported that aluminosilicate filled nanotube can stick to plant surfaces while nano ingredients of nanotube have the ability to stick to the surface hair of insect pests and ultimately enters the body and influences certain physiological functions. Yang et al. (2009) demonstrated that polyethylene glycol-coated nanoparticles loaded with garlic essential oil were efficacious against adult Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) found in stored products. They observed that the control efficacy was about 80 % mortality.
Debnath et al. (2010) studied the toxic effects of silica (SiO2), zinc oxide (ZnO), titanium dioxide (TiO2), aluminium oxide (Al2O3) nanoparticles (a
variety of 15–50 nm size range) on Lipaphis pseudobrassicae (Davis) (Hemiptera: Aphididae). Their results showed that nano Al2O3 and amorphous nano SiO2 were highly effective and nano TiO2 was moderately effective against L. pseudobrassicae. Goswami et al. (2010) studied the application of different kinds of nanoparticles, viz. silver (AgNP), aluminum oxide (AlNP) nanoparticles in the control of rice weevil; S. oryzae. They reported that hydrophilic AgNP was most effective on the first day. On day 2, AgNP and AlNP caused more than 90 % mortality. After 7 days of exposure, 95 and 86% mortality were reported with hydrophilic and hydrophobic AgNP and nearly 70 % of the insects were killed when the rice was treated with lipophilic SiNP. However, 100 % mortality was observed in case of AlNP. Stadler et al. (2010) studied the insecticidal activity of nanostructured alumina against two insect pests; S. oryzae and Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). They reported significant mortality after 3 days of continuous exposure to nanostructured alumina-treated wheat. Debnath et al. (2011) found that SiNPs were much more effective on the second instar larvae of Spodoptera litura F. (Lepidoptera: Noctuidae) especially, hydrophilic SiNPs which killed all the larvae within 24 hours after treatment. Jayaseelan et al. (2011) revealed that synthesized AgNPs possess excellent anti-lice and mosquito larvicidal activity, when exposed larvae to varying concentrations of synthesized AgNPs for 24 h. Their results suggested that the optimal times for measuring mortality effects of synthesized AgNPs were 33% at 5 min, 67% at 15 min and 100% after one hour. Kirthi et al. (2011) assessed the anti-parasitic activity of the synthesized zinc oxide nanoparticles (ZnO NPs) against the larvae of cattle tick Rhipicephalus (Boophilus) microplus, Canestrini (Acari: Ixodidae); head louse Pediculus humanus
capitis, De Geer (Phthiraptera: Pediculidae); larvae of malaria vector, Anopheles subpictus, Grassi and filariasis vector, C. quinquefasciatus, Say (Diptera: Culicidae). Authors exposed parasite larvae to varying concentrations of synthesized ZnO NPs for 24 h. The results suggested that the mortality effects of synthesized ZnO NPs were 43 % at 1h, 64 % at 3 h, 78 % at 6 h and 100 % after 12 h against R. microplus activity. In pediculocidal activity, the results showed that the optimal times for measuring mortality effects of synthesized ZnO NPs were 38 % at 10 min, 71% at 30 min, 83 % at 1 h and 100 % after 6 h against P. humanus capitis. Also, they observed one hundred percent lice mortality at 10 mg/L treated for 6 h. The mortality was confirmed after 24 h of observation period. The larval mortality effects of synthesized ZnO NPs were (37 %, 72 % and 100 %) and (43 %, 78 % and 100 %) at 6, 12, and 24 h against A. subpictus and C. quinquefasciatus, respectively. Ramyodevi et al. (2011) tested the efficacy of synthesized copper nanoparticles (Cu NPs); size range of 35-80 nm, against the larvae of blood-sucking parasites exposed to varying concentrations. They observed the larval mortality for 24 h. Results of their research demonstrated that the larval percent mortality observed in synthesized Cu NPs were 36, 49, 75, 93 and 100 % against A. subpictus; 32, 53, 63, 73 and 100 % against C. quinquefasciatus and 36, 47, 96, 88 and 100 % against R. microplus at concentration of 0.5, 1.0, 2, 4 and 8 mg/L, respectively. Barik et al. (2012) tested three types of SiNPs (lipophilic, hydrophilic and hydrophobic) to assess their larvicidal, pupicidal and growth inhibitor properties and also their influence on oviposition behaviour (attraction/deterrence) of mosquito species; Anopheles, Aedess and Culex. Authors found that application of hydrophobic SiNPs at 112.5 ppm was effective against mosquito species tested. On the other hand, the larvicidal effect of hydrophobic SiNPs on mosquito species tested was in the order of Anopheles stephensi > Aedes aegypti > Culex quinquefasciatus, and the pupicidal effect was in the order of A. stephensi > C. quinquefasciatus > Ae. aegypti. Their results of combined treatment of hydrophobic SiNPs with temephos in larvicidal test indicated independent toxic action without any additive effect.
Chakravarthy et al. (2012a) demonstrated that DNA tagged gold nanoparticles were effective against S. litura and would therefore be a useful component of an integrated pest management strategy. Chakravarthy et al. (2012b) examined the potential adverse effects of Ag and TiO2 nanoparticles on S. litura in laboratory. Their data of second instar S. litura larvae indicated that TiO2NPs showed maximum of 73.79 % larval mortality at 2400 ppm and the least was 18.50 percent at 150 ppm. The AgNPs caused maximum 56.89 percent mortality at 2400 ppm followed by 46.89 and 33.44 percent mortality at 1200 and 600 ppm, respectively. The authors concluded that the two tested nanoparticles proved effective against S. litura larvae and hence can be selectively used for suppression of the pest. Debnath (2012) tested the entomotoxicity of three types of SiNPs (hydrophilic, hydrophobic and lipophilic) on Sitophilius oryzae L. (Coleoptera: Curculionidae) and S. litura. The author reported high toxic effect of hydrophobic and lipophilic SiNPs reached 100 % after 7 and 14 days, respectively against S. oryzae, whereas mean mortality reached 100 % after 24 hours for hydrophilic SiNPs against S. litura. Debnath et al. (2012) applied two types of SiNPs (spherical, monosidperse) against second instar larvae of S. litura. Their results indicated that both types of SiNPs could effectively kill the insect larvae. The authors suggested that this silica based nanocide can be an alternative to the commercial insecticides which have numerous health hazards. Derbalah et al. (2012) evaluated some insecticides (indoxacarb, imidacloprid) and non-traditional methods (culture filtrate of Bacillus thuringiensis, Artemisia cina extract, clove oil and SiNPs) against Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) on tomato plants under greenhouse conditions. They found that SiNPs was the most effective treatment against this insect pest.
El-Samahy and Galal (2012) used five concentrations of SiNPs; 100, 200, 300, 400 and 500 ppm, to control A. craccivora and L. trifolii. Their results showed that the reduction percentages in the number of both insects were significantly increased as the concentration increased. Rouhani et al. (2012) conducted laboratory trials to determine the effectiveness of different concentrations (1, 1.5, 2 and 2.5 g/kg) of SiNPs and AgNPs on larval stage and adults of Callosobruchus maculatus F. (Coleoptera: Chrysomelidae) on cowpea seed. Results showed that both nanoparticles (silica and silver) were highly effective on adults and larvae with 100% and 83% mortality, respectively. Sabbour (2012) tested the two nanomaterials; alaminium oxide (Al2O3) and titanium dioxide (TiO2), against rice weevil S. oryzae under laboratory and store conditions. Results showed that nano Al2O3 was highly effective, while nano TiO2 had lower moderately effective against S. oryzae. Under laboratory conditions, the number of mortality of S. oryzae was significantly increased to 41.4±4.4, 47.8±5.8 and 50.6±3.6 individuals after treatment for 7, 21 and 45 days with 3 % TiO2, as compared to 1.0±2.8, 2.0±5.1 and 3.0±3.4, respectively, in the control. Under store conditions, their results showed that the nanoparticles significantly increased the number of mortality to 67.3±1.4 after 45 days of storage as compared to 3.8±3.8 individuals in the control. The author explained that accumulative mortality (%) of S. oryzae beetles increased gradually by increasing the period of exposure, and Al 2O3 had the highest cumulative mortality (73.3%) compared to TiO2 (59.7 %) after seven days. Zahir et al. (2012) assessed the efficacies of AgNPs; size of 25-80 nm with an average size of 52.4 nm, against the adult of S. oryzae. They conducted pesticidal bioassay tests at varying concentrations for 14 days. Their results suggested that the synthesized AgNPs have the potential to be used as an ideal eco-friendly approach for controlling this insect pest. El-Bendary and El-Helaly (2013) evaluated the effects of hydrophobic SiNPs (100, 150, 200, 250, 300 and 350 ppm) on the resistance of tomato plants to S.
littoralis. Their results indicated high toxic action at all concentrations. Moreover, they found high resistance in tomato plants against this insect pest especially at 300 and 350 ppm. Vani and Brindhaa (2013) revealed that amorphous SiNPs were highly effective against stored grain pest Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) causing 100% mortality. Abd El-Wahab and Anwar (2014) reported that copper (CuO) and zinc (ZnO) nanoparticles showed significant effect on the 2nd instar larvae of S. littoralis, with mortality percentage of 100 and 73.33%, respectively. Moreover, this nanoparticle caused some malformation and morphological changes by adsorption through the integument of the 2nd larval stage of S. littoralis. Abo-Arab et al. (2014) reported the insecticidal effect of nanostructured titanium dioxide (TiO2) and aluminum oxide (Al2O3) against S. oryzae and Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae), which are major insect pests in stored food. Data indicated that both insect species experienced significant mortality after 3 days of continuous exposure to treated maize. After 21 days of treatment, the mortality percentage was 61.66% for TiO2 and 95.33% for Al2O3 nanoparticles at the concentration of 1 gm/kg against S. oryzae, While mortality percentage was 60.66% for TiO2 and 91.66% for Al2O3 nanoparticles at the same mentioned concentration against S. zeamais. Results obtained clearly showed that the TiO2 nanoparticls had the lowest effect on the all tested parameters of S. oryzae or S. zeamais. Babu et al. (2014) used AgNPs to control the 3rd instar larvae of sugarcane white grub Lepidiota mansueta (Burmeister). Their results revealed that mortality of the larvae was significantly increased at all concentrations. Thus; they concluded that the synthesized AgNPs is economical, efficient and ecofriendly and it is strongly recommended as a hopeful challenger for agricultural and marine pests. El-Samahy et al. (2014) compared efficacy of SiNPs, neem oil (plant extract) and imidacloprid (chemical pesticide) against tomato leaf miner T. absoluta. Results
revealed the high efficacy of SiNPs, which did not differ significantly than imidacloprid. Salem et al. (2015) used aluminum oxide (Al2O3) and zinc oxide (ZnO) nanoparticles (2, 1, 0.5, 0.25 and 0.125 g/Kg crushed wheat) as stored product insect protectants against the stored product beetles; T. castaneum. Data obtained showed that the two nanoparticles significantly inhibited the number of progeny and weight loss (%) and the concentration of 2 g/kg wheat grain had the highest effect based on the LC50 values. Results revealed that ZnO had the most effect compared to Al2O3 nanoparticles.
2. Toxicity studies: 2.1. Effects of nanoparticles on germination and plant growth: Lu et al. (2002) showed that nanoscale SiO2 and TiO2 enhanced nitrate reductase activity in soybean, and apparently hastened its germination and growth. Bao-shan et al. (2004) applied exogenous application of SiNPs on Changbai larch (Larix olgensis) seedlings and found that SiNPs improved seedling growth and quality, including mean height, root collar diameter, main root length and the lateral roots number of seedlings. Shah and Belozerova (2009) tested silica, palladium, gold and copper NPs and found that all these NPs had a significant influence on lettuce seeds. Debnath et al. (2010) reported that nano Al2O3 has deleterious effects on plant growth, whereas non crystalline SiNPs has no such adverse effect on plants. Nair et al. (2011) observed better seed germination of rice in the presence of SiNPs.
Suriyaprabha et al. (2012) reported that SiNPs increased seed germination by providing better nutrients availability to maize seeds as well as pH and conductivity to the growing medium. Hassan et al. (2013) reported that the interaction of plant cell with the engineered nanomaterials leads to the modification of plant gene expression and associated biological pathways, which eventually affect plant growth and developments. Karunakaran et al. (2013) evaluated effect of SiNPs (50 nm) and different sources of silicon on maize seed germination. They found that SiNPs promoted seed germination percentage (100 %) in maize than conventional Si sources. Nadiminti et al. (2013) suggested that engineered nanomaterials give different effects on plant growth, depending on nanomaterials surface structure, size, shape, chemical composition, concentration, solubility and aggregation. Abou-Zeid and Moustafa (2014) studied the effect of AgNPs on germination percentage, seedling growth and mitotic cell division. The results showed significantly increased the germination percentage. Mean comparison showed that the highest germination percentages (98, 92 and 96%) were observed in the pretreated wheat (Beni Sweif 1 and Gemmieza 9) and barley (Giza 130) seeds, respectively. The results clearly revealed that shoot length, fresh and dry weights were slightly promoted by AgNPs, while root parameters were reduced compared with those of control. Sabaghnia and Janmohammdi (2014) evaluate the effects of SiNPs on lentil (Lens culinaris Medik.) under salt stress. Their results showed a significantly increase with SiNPs application in germination percentage and seedling growth. Authors concluded that adding of SiNPs could improve germination and seedling early growth under salinity stress.
Siddiqui and Al-Whaibi (2014) revealed that the application of SiNPs (12 nm) on tomato (Lycopersicum esculentum Mill.) significantly enhanced seed germination potential. SiNPs improved seed germination percentage, mean germination time, seed germination index, seed vigour index, seedling fresh weight and dry weight. Abdul Qados and Moftah (2015) evaluated the effects of silicon (Si) and SiNPs for ameliorating negative effects of salinity on germination, growth and yield of faba bean. Results of their study showed that both Si and SiNPs (2 mM) improved germination percentage, germination rate and mean germination time. Thus, the harmful effect of salt stress on vegetative growth was also alleviated by the addition of Si and SiNPs.
2.2. Genotoxicity effects: Due to the unique physicochemical properties of SiNPs, their interactions with different biological systems have been studied extensively, but the effect on plant cell has received little attention.
2.2.1. Genotoxicity of nanoparticles on plant: Kumari et al. (2009) investigated cytotoxic and genotoxic impacts of four different concentrations; 25, 20, 75 and 100 ppm, of AgNPs (below 100 nm size) using root tip cells of Allium cepa. They observed no chromosomal aberration in the control treatment and the mitotic index value was 60.3 %. With increasing concentration of the nanoparticles, the authors noticed decrease in the mitotic index to 27.62 %. Also, authors studied the different cytological effects including the chromosomal aberrations and inferred from their study that AgNPs could penetrate plant system and may impair stages of cell division causing chromatid bridge, stickiness, disturbed metaphase, multiple chromosomal breaks and cell disintegration.
Panda et al. (2011) revealed the genotoxic effects of AgNPs using Allium test. They hypothesized that these nanoparticles induced DNA damage by triggering oxidative stress. El-Samahy and Galal (2012) examined the genotoxic effect of 10-20 nm SiNPs (100, 200, 300, 400 and 500 ppm) on mitotic divisions and chromosomal aberrations as well as changes in the seed storage proteins of M2 faba bean plants. The cytological analysis revealed that all SiNPs concentrations induced significant increase in the mitotic index, except 500 ppm, which did not differ significantly from the control. All concentrations of SiNPs induced significant increase in the percentage of chromosomal abnormalities which included stickiness, laggards, bridges, disturbed, micronuclei and binucleate cells. At the biochemical level, marked changes were observed in the M2 seed storage protein banding patterns. These changes included alterations in number of bands, band intensity, disappearance or appearance of certain bands. Patlolla et al. (2012) used of faba bean root-tip meristem to investigate the genotoxicity of four different concentrations; 12.5, 25, 50 and 100 mg/L, of AgNPs (60 nm). Their results demonstrated that AgNPs exposure significantly increased the number of chromosomal aberrations, micronuclei, and decreased the mitotic index in exposed groups compared to control. Results of this study demonstrated that AgNPs are genotoxic to plant cells. Gopinath et al. (2013) tested the efficacy of the synthesized gold noparticles (AuNPs); particle sizes in the range of 20-50 nm with the spherical nature, for the mitotic cell division and pollen germination. They suggested that AuNPs induces the mitotic cell division and pollen germination. Moreover, they found no cytotoxic effect of A. cepa root tip cells and Gloriosa superba pollen grains. Prokhorova et al. (2013) estimated genetic activity of AgNPs by Allium test and observed that no mutagenic effects were detected after treatment root meristem cells of A. cepa with 1.0, 2.5, 5.0 and 50 mg/liter AgNPs. Also, they found that AgNPs
in a concentration of 50 mg/liter significantly increased the mitotic index. In addition, nanoparticles in a dose of 5 mg/liter induced slight, but significant increase in mitotic index, but did not affect the ratio of phase indexes. On the other hand, exposure to AgNPs in concentrations of 1.0 and 2.5 mg/liter was not followed by modification of mitosis. Abou-Zeid and Moustafa (2014) studied the effect of AgNPs on mitotic cell division. Data obtained revealed that cytological changes in root tips of 72 h and 120 h germinated seeds were observed by disturbed chromosomes at metaphase and anaphase. They observed several types of chromosomal aberrations; chromosomes aneuploidy, binucleate cells, deletion chromosomes, deform nuclei, micronuclei, chromosomes fragment, and stickiness chromosomes. Authors found that the mitotic index significantly increased in the pretreated tested plants compared to the control. Their study inferred that AgNPs could penetrate plant system and might impair stages of cell division causing chromosomal aberrations. Rajeshwari et al. (2015) evaluated the cytogenetic potential of Al2O3 NPs in A. cepa root tip cells at a range of exposure concentrations (0.01, 0.1, 1, 10 and 100 μg/mL) and the oxidative stress generated. They noted a dose-dependent decrease in the mitotic index (42 to 28 %) and an increase in the number of chromosomal aberrations. Authors observed various chromosomal aberrations, e.g. sticky, multipolar and laggard chromosomes, chromosomal breaks and the formation of binucleate cells. Their finding suggested that the bio-uptake of Al2O3 led to reactive oxygen species generation, which in turn probably contributed to the induction of chromosomal aberrations.
2.2.2. Genotoxicity of SiNPs on other biological systems: 2.2.2.a. Effect of SiNPs on chromosomal aberrations: Kim et al. (2006) reported that SiO2-coated magnetic NPs of 50 nm did not induce chromosomal aberrations in Chinese hamster lung fibroblasts.
Wang et al. (2007) incubated WIL2-NS human cells for 6, 24 and 48 h with 0, 30, 60 and 120 microg/ml ultrafine crystalline SiO2 (UF-SiO2). They reported that treatment with 120 microg/ml UF-SiO2 for 24 h produced a fourfold increase in the frequency of micronucleated and binucleated cells in dose-dependent manner. The lowest dose of 30 microg/ml (24 h treatment) gave a statistically significant increase with cytotoxicity of less than 10 %. Park et al. (2011) investigated the potential of four well-characterized amorphous SiNPs to induce chromosomal aberrations and gene mutations using two in vitro genotoxicity assays. They used transmission electron microscopy (TEM) to verify the manufacturer's nominal size of 10, 30, 80 and 400 nm which showed actual sizes of 11, 34, 34 and 248 nm, respectively. The authors reported that the 80 (34) nm SiNPs induced chromosomal aberrations in the micronucleus assay using 3T3-L1 mouse fibroblasts. In addition, both the 30 (34) and 80 (34) nm SiNPs induced a dose related increase in gene mutations in mouse embryonic fibroblasts carrying the lacZ reporter gene, while those of 11 nm and 248 nm did not. Galal and El-Samahy (2012) employed the Drosophila melanogaster model to investigate the effects of exposure to 100, 250, 500 and 1000 ppm of SiNPs (10-20 nm) on chromosomal rearrangements. They found that SiNPs at the concentrations of 500 and 1000 ppm appeared to be more affective on salivary gland chromosomes than the other two concentrations. Uboldi et al. (2012) demonstrated that SiNPs with diameters between 15 nm and 300 nm and exposure times up to 72 h did not show any cytotoxic effect on Balb/3T3 mouse cells as measured by the MTT test; a colorimetric assay for assessing cell metabolic activity, and the Colony Forming Efficiency (CFE) assay. Furthermore, SiNPs did not induce morphological transformation in Balb/3T3 cells and did not result in genotoxicity, as shown by Cell Transformation Assay (CTA) and Micronucleus (MN) assay, respectively.
Lankoff et al. (2013) treated human peripheral blood lymphocytes with 10, 25, 50 and 100 mg/ml of vinyl-modified, aminopropyl/vinyl modified SiNPs and unmodified SiNPs for 50 h. They indicated that all three types of nanoparticles had no significant effect on the frequencies of chromosomal aberrations in lymphocytes. They also found markedly decreased mitotic indices in lymphocytes treated with 25, 50 and 100 mg/ml of unmodified and modified SiNPs.
2.2.2.b. Effect of SiNPs on genomic stability and integrity: Valko et al. (2006) observed that SiNPs induced the reactive oxygen species (ROS), especially the hydroxyl radical, a highly reactive molecule that may induce DNA strand breaks and oxidized bases. Jin et al. (2007) reported negative outcome in the Comet assay after exposure of A549 human lung epithelial cells to luminescent SiNPs (50 nm) for up to 72 hours. Wang et al. (2007) reported that there was no significant difference in DNA strand breakage as measured by the Comet assay when incubated WIL2-NS human cells for 6, 24 and 48 h with 0, 30, 60 and 120 microg/ml ultrafine crystalline SiO2 (UF-SiO2). On the other hand, they found a significant increase in induced mutant frequency at 120 microg/ml as detected by the hypoxanthine guanine phosphoribosyltransferase mutation assay. Barnes et al. (2008) tested the genotoxicity of commercial colloidal and laboratory-synthesized SiNPs on 3T3-L1 fibroblasts using Comet assay that assesses breaks in both single- and double-stranded DNA. They found that no significant genotoxicity was observed for SiNPs (4 or 40 μg/ml) under the conditions of 3, 6 and 24 h incubations. Landsiedel et al. (2009) observed positive results for SiNPs in two types of genotoxicity assays; i.e. the micronucleus assay and the gene mutation assay.
Yang et al. (2009) reported very mild positive results on DNA damage in Comet assay after exposure of mouse embryo fibrolasts (3T3) to different concentrations of SiNPs (20 – 400 nm). Nabeshi et al. (2011a) suggested that the well-dispersed amorphous SiNPs of particle size 70 nm (nSP70) penetrated the skin barrier and caused systemic exposure in mouse and induced mutagenic activity in vitro. Nabeshi et al. (2011b) investigated the relationship between the surface properties of SiNPs and their cytotoxicity against a murine macrophage cell line (RAW264.7). The authors exposed RAW264.7 cells to SiNPs either unmodified (nSP70) or modified with amine (nSP70-N) or carboxyl groups (nSP70-C). Their results showed that nSP70-N and nSP70-C have a smaller effect on DNA synthesis activity by comparison to unmodified nSP70. They concluded that the surface properties of SiNPs play an important role in determining their safety. Chen et al. (2012) evaluated in vitro genotoxicity of a non-copper nano-silica polymer modified composite. They applied Comet assay to the determination of DNA damage and used means of Ames test using Salmonella typhimurium TA98 and TA 100 tester strains with and without metabolic activation to test mutagenic activity. The authors concluded that the non-copper nano silica modified composite did not exhibit in vitro genotoxicity. Downs et al. (2012) investigated the effects of amorphous SiNPs (15 and 55 nm) in an in vivo Comet/micronucleus (MN) combination assay and in an in vitro MN assay performed with human blood. They also measured DNA damage in liver, lung and blood cells and micronuclei in circulating reticulocytes, after 3 consecutive intravenous injections to male Wistar rats at 48, 24 and 4 h before sacrifice. Their results revealed that SiNPs caused a small but reproducible increase in DNA damage and micronucleated reticulocytes when tested at their maximum tolerated dose. On the other hand, they did not observe genotoxic effects at lower doses, and the in vitro MN assay was also negative.
Jennifer and Maciej (2013) showed that nanoparticles can cause epigenetic and genomic changes which may stimulate cancer progression. Lankoff et al. (2013) determined the effect of different doses (10, 25, 50 and 100 ug/ml) of unmodified and vinyl-modified, aminopropyl/vinyl modified SiNPs on DNA damage in human peripheral blood lymphocytes after 2 and 24 h. They indicated no significant increase of basal DNA strand breaks in cells treated with all three types of silica as revealed by Comet assay.
2.2.2.c. Effect of SiNPs on gene expression: Chen and von Mikecz (2005) reported that SiNPs had an impact on nuclear integrity by forming intranuclear protein aggregates and resulting in inhibition of replication, transcription and cell proliferation. Huang et al. (2008) indicate that uptake of mesoporous silica nanoparticles (MSNs) into human mesenchymal stem cells (hMSCs) did not affect the cell viability, proliferation and regular osteogenic differentiation of the cells. However, the internalization of MSNs had a significant effect on the transient protein response and osteogenic signal in hMSCs. Gong et al. (2010) observed that with increasing SiNPs dose, the global level of mRNA expression of MBDs (methyl-CpG-binding domain protein) gradually decreased which resulted in a decrease of genomic DNA methylation status. Yang et al. (2010) studied gene expression profiles after exposure to amorphous SiNPs in human epidermal keratinocytes (HaCaT cells). They found that, at 10 mg/L (the only reported slightly cytotoxic concentration), a down regulation of oxidative-stress associated proteins (Prx1, Prx6, Trx, GSTP1) may indicate a reduced antioxidant capacity following the induction of cytotoxicity by particle exposure. Similarly, changes in molecular chaperones and energy metabolism-associated proteins were indications for silica-induced cytotoxicity.
Lu et al. (2011) reported an increase in p53 level and a decrease in Bcl-2 level in hepatoma cells after treatment with SiNPs (20 nm). On the other hand, in hepatic cells, cytotoxic effect of SiNPs was slightly. Galal and El-Samahy (2012) explained in vivo biological effects of SiNPs (100, 250, 500 and 1000 ppm) using D. melanogaster fruit fly. They found that SiNPs induced specific changes in the number and intensity of total protein as well as the activity of esterase and peroxidase isozymes. These toxic effects were closely related to the concentration used.
MATERIALS AND METHODS The present investigation was carried out at the Experimental Farm and Field Crop Pests Research Laboratory of Plant Protection Research Institute (PPRI), Sakha Agricultural Research Station (SARS), Kafr El-Sheikh, Egypt, as well as the laboratories of Economic Entomology and Genetics Departments, Faculty of Agriculture, Kafrelsheikh University, Egypt. Field experiments were carried out during the two growing seasons; 2012/13 and 2013/14 for faba bean as well as 2013 and 2014 for soybean.
1. Seed material: Seeds of faba bean (Sakha 1 variety) and soybean (Crawford variety) were obtained from Food Legumes Research Section, SARS, Kafr El-Sheikh, Egypt. Sakha 1 variety was found to be relatively susceptible to infestation with cowpea aphid; A. craccivora, and faba bean leafminer; L. trifolii. Also, Crawford variety found to be susceptible to infestation with cotton leafworm; S. littoralis.
2. Silica nanoparticles: Silica nanoparticles (SiNPs); 10-20 nm in diameters, were purchased from NanoTech Egypt Co., Dreamland, Wahat Road, 6th October, Egypt, in the spherical form (99.99 % purity). Six different concentrations of SiNPs; 75, 150, 225, 300, 375 and 425 ppm, were used in this study.
3. SiNPs efficiency assays: Efficiency of SiNPs against insect pests was determined under field conditions for both faba bean main insects, while it was evaluated under two different experimental conditions (field and laboratory) for soybean insect.
3.1. Efficiency of SiNPs against faba bean main insect pests:
3.1.1. Field procedures: The experimental area was divided into two parts; the first part was used for studying the efficacy of SiNPs concentrations against A. craccivora, while the second one was used for studying the effect of the same SiNPs concentrations against L. trifolii. One meter wide stripe were leaved free without cultivation between the two parts. Seven treatments have been applied in each part. Faba bean seeds were planted in three replicates (each of 6×7 m) per treatment in Randomized Complete Block Design (RCBD). The first treatment was the control which sprayed with water. The others were treated with the different concentrations of SiNPs. The normal cultural practices of growing were applied as usual without using any insecticides.
3.1.2. Treatment against A. craccivora infestation: Seeds of faba bean were planted in mid-November during the two seasons; 2012/13 and 2013/14. Usually, the application of insecticides against aphid infestation should be used only if necessary, but in this experiment, the time of application was in mid-March during the two seasons. SiNPs were applied using low volume spray ordinary knapsack sprayer with one atomizer ensuring full coverage to the plants (200 liters were used per feddan). Numbers of A. craccivora; nymphs and adults, were directly recorded in the field by a suitable lens before treatments and after 1, 3, 5, 7, 10 and 15 days of spraying. Fifteen plants were used per replicate. The percentages of reduction were calculated using Henderson Tilton's formula (Henderson and Tilton, 1955) as follow:
Reduction % = [1 −
treatment after × control before ] × 100 treatment before × control after
3.1.3. Treatment against L. trifolii infestation:
Faba bean seeds were planted in mid-November. The same concentrations of SiNPs were applied against L. trifolii after two months from sowing. Randomized samples (each of 25 leaflets per replicate) were collected before treatments as well as after 1, 3, 5, 7, 10 and 15 days of spraying. Samples were examined under stereoscopic binocular microscope for counting L. trifolii larvae inside the tunnel between the upper and lower leaf surface. The percentage of L. trifolii reduction for each treatment was calculated using Henderson Tilton's formula as previously (Henderson and Tilton, 1955).
3.2. Efficiency of SiNPs against soybean main insect pest; S. littoralis: 3.2.1. Experimental design and treatments under field conditions: Seeds of soybean were planted in three replicates (each of 6×7 m) per treatment in Randomized Complete Block Design (RCBD). Seven treatments were applied. The first treatment was the control which sprayed with water. The others were treated with the different concentrations of SiNPs. The normal cultural practices were applied as usual without using any insecticides. Soybean seeds were planted in the first week of May during the two seasons; 2013 and 2014. Treatments of SiNPs were applied when about 25% of plant leaves were consumed by S. littoralis larvae. Randomized sample of ten plants per replicate were examined before treatment and after 1, 3, 5, 7, 10 and 15 days of spraying. Samples were examined by shake the plant in white bucket for counting live and dead larvae. The percentage of S. littoralis reduction for each treatment was calculated using Henderson Tilton's formula (Henderson and Tilton, 1955).
3.2.2. Biological studies on S. littoralis under laboratory conditions:
To examine the effect of SiNPs on toxicological and biological aspects of S. littoralis, a laboratory experiment was applied. Larvae of S. littoralis reared on castor-bean leaves and maintained at constant laboratory conditions of 25±2 °C and 65±5 % R.H. (El-Defrawi et al., 1964) were used for this study. Silica NPs was prepared in 50 ml total volume for each concentration. Treatments were carried out in three replicates (each of four plants) for each concentration. Soybean plants were completely sprayed with SiNPs and left to dry at room temperature. Fifty neonate larvae were released and allowed to feed on the treated plants; covered with double muslin cloth, for 15 days. Other three replicates of untreated plants were provided for the same period as a control. Larvae of S. littoralis were examined for live and dead ones to determine the mortality percentages. Survival larvae were transferred under laboratory conditions to feed on untreated soybean leaves till pupation. Larvae were examined daily to determine the different biological aspects such as: larval duration, pupal period, adult longevity, number of eggs laid per female and hatchability percentage.
4. Collecting of predators associated with insect pests: In order to collect predators in faba bean and soybean fields, sample of fifteen infested plants was examined per replicate. Samples were taken before as well as after 1, 3, 5, 7, 10 and 15 days of treatment. Plants were kept into box (30 × 20 × 10 cm) provided with four glass tubes in each side (Fig. 1) to help the predators to go outside the box under effect of natural light. Number of predators was recorded and reduction percentage was calculated using Henderson Tilton's formula (Henderson and Tilton, 1955).
Carton box
Glass tube
Fig. (1): The carton box used for predator collecting. 5. SiNPs toxicity assays: Toxic effect and genotoxicity of SiNPs on faba bean plants were tested by several biological assays.
5.1. Pollen vitality: Pollen samples were collected and dispersed on a drop of 2% aceto-carmine stain placed on separate slides. Slides were warmed gently until the coloration was intense enough for distinction to be made between stained and unstained pollen grains. A total of 1000 pollen grains of each treatment were counted for vital pollen grains. Pollen vitality was expressed as the percentage of number of stained pollen grains to total number of pollen grains.
5.2. Growth experiment:
Seed samples of M2 generation; which were collected after maturity and fruiting of faba bean treated with SiNPs, were used for this experiment. Germination and seedling growth bioassays were calculated in a Complete Randomized Design with three replications. Ten seeds of each replicate were allowed to germinate and grow in a 15 cm diameter Petri dish lined with Whatman No. 1 filter paper moistened with distilled water. After seven days, germination percentage in any Petri dishes was calculated as follow:
Germination percentage =
Number of germinated seeds × 100 Total number of seeds
Seedling growth in terms of shoot length was measured after one month from germination.
5.3. Cytological analysis: Five seeds of faba bean of each replicate were germinated as previously. The root tips of germinated seeds (1.5-2 cm) were cut and fixed in a fresh solution of glacial acetic acid and absolute ethanol in the ratio of 1:3 for 24 h and then stored in 70 % ethanol until used for cytological analysis. The fixed roots were boiled in glacial acetic acid to break connections among cells to facility mashing root tip. Then, root tips were stained with 2% aceto-carmine (Darlington and La Cour, 1976) and squashed on a slide to be examined. At least 3000 cells/treatment (1000 cell per replicate) were examined for each treatment and control. Cells were screened under a light microscope for mitotic index, numbers and types of abnormalities. The mitotic index and percentage of abnormal cells were calculated using the following formulas:
Mitotic Index (MI) =
Total dividing cells × 100 Total dividing and non dividing cells
Percentage of abnormal cells =
Total abnormal cells × 100 Total dividing cells
5.4. Molecular analysis: 5.4.1. Genomic DNA extraction: Total genomic DNA was isolated from young healthy leaves of the M2 faba bean plants by using the DNeasy Plant Mini Kit (QIAGEN GmbH, Cat. No. 69104).
5.4.2. Polymerase Chain Reaction (PCR): a. Template DNA: Polymerase Chain Reactions were conducted using extracted genomic DNAs (1µl) as templates. b. Primers: Six arbitrary random 10-mer primers with the following sequences (Table 1) were used. All primers were introduced from Bio Basic Inc., Canada.
Table (1): The used primers and their nucleotide sequences Primer
Nucleotide sequence (5´→3´)
OPA20 OPB1 OPB6 OPB7 OPB12 OPB14
GTT GCG ATC C GTT TCG CTC C TGC TCT GCC C GGT GAC GCAG CCT TGA CGCA TCC GCT CTG G
c. Amplification reaction mixture: Polymerase Chain Reaction (PCR) was performed using DreamTaq Green PCR master Mix, 2X (Thermo Scientific, #K1081) in a volume of 20 μl reaction mixture containing the following components: Components
Amount in μl
Template DNA
1.0
Primer (20 µM)
1.0
Master mix (2X)
10
Sterile distilled water
8
PCR was done using the PCR thermocycler machine from Perkin-Elmer, Gene Amp. 2400.
d. PCR amplification: The PCR amplification was performed according to Williams et al. (1990) with some modifications of cycling programme. The amplification conditions were as follows: Step
Temperature (°C)
Duration
Cycles
Denaturation
95
3 min
1
Denaturation
95
30 sec
Annealing
32
30 sec
40
Extension
72
3 min
Final extension
72
5 min
1
Hold
4
Until removed
1
e. PCR products analysis: The amplified DNA products from RAPD analysis were separated on 1.5 % agarose gel, stained with ethidium bromide and visualized under ultraviolet light. A known DNA Ladder (50 bp DNA Ladder ready-to-use, Cat-no: 300003, GeneON) was run against the PCR products.
5.4.3. Genomic template stability test: Genomic template stability (GTS) was calculated by the following equation: GTS (%) = (1 - a/n) × 100; since a is the number of polymorphic bands detected in each treatment, and n is the number of total bands detected in the control. Polymorphism observed in RAPD profile included disappearance of a normal band and appearance of a new band in comparison to control RAPD profile (Luceri et al., 2000; Atienzar et al., 2002; Qari, 2010).
5.5. Biochemical analysis: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) of seed storage proteins was carried out by using 12.5 % polyacrylamide gel. The electrophoresis was done in a vertical slab gel of 16cm × 14cm × 1mm dimension.
5.5.1. Preparation of reagents:
The stock solution for seed storage protein electrophoresis was prepared according to Laemmli (1970). i) Extraction buffer (0.125 M Tris) Only 1.625 g Tris-base was dissolved in 80 ml of distilled water, pH was adjusted to 6.8 with concentrated HCl and then made up the volume to 100 ml with distilled water. The solution was stored at 4oC for further use. ii) Acrylamide solution (30 %) Thirty grams of acrylamide and 0.8 g of methylene bis-acrylamide were dissolved in 50 ml of distilled water. When acrylamide was completely dissolved, volume was made up to 100 ml and stored under refrigerated condition.
iii) Separating gel buffer (1.5 M Tris) Only 18.17 g of Tris-base were adjusted to pH 8.8 with concentrated HCl and the final volume was made up to 100 ml with distilled water. It was stored under refrigerated condition. iv) Stacking gel buffer (0.5 M Tris) Only 6.06 g of Tris-base were dissolved in 80 ml of distilled water. The pH was adjusted to 6.8 with concentrated HCl and the final volume was made
up to 100 ml with distilled water. The solution was stored under refrigerated condition. v) Sodium dodecyl sulphate (SDS) 10% (w/v) One gram of SDS was dissolved in 8 ml of distilled water and the final volume was made up to 10 ml with distilled water. vii) Electrode buffer [0.05 M Tris, 0.375 M glycine, 0.1% (w/v) SDS] Six grams of Tris-base, 28.2 g glycine, 1g SDS were dissolved in 500 ml of distilled water and the volume was made up to 1000 ml. The pH was not adjusted, but confirmed that it was near 8.8±0.2. This buffer was stored at 4°C. viii) Sample buffer (0.06M Tris-HCl; pH 6.8, 2% SDS, 10% glycerol, few crystals of Bromophenol blue) Only 1.2 ml of 0.5 M Tris-HCI (pH 6.8), 0.1 g (w/v) SDS, 1 ml glycerol and 0.25 ml mercaptoethanol were mixed and made up a volume of 6.25 ml with distilled water. Few crystals of bromophenol blue were added to the solution, mixed thoroughly and stored at room temperature.
5.5.2. Preparation of samples: The dry M2 seeds of faba bean; whose parents were treated with SiNPs, were decoated and milled to fine powder. Two hundred fifty mg of powder were taken in 1.5 ml Eppendorf tube, and 1 ml of extraction buffer was added. Then, the samples were centrifuged at 12000 rpm for 15 min. The clear supernatants were transferred into new tubes and stored at -20°C until using.
For sample loading, 100 μl of the extracted protein from each sample was taken in clean Eppendorf tube and dissolved in 400 μl of sample buffer.
Then, this solution was kept in boiling water for 10 min. These samples were used for loading.
5.5.3. Preparation of gel: i) Separating gel (12.5%) It was prepared by mixing 20 ml of 30 % acrylamide solution, 16.4 ml of distilled water, 12.5 ml of separating gel buffer, 0.5 ml of 10 % SDS, 0.5 ml of 10 % APS and 0.04 ml of TEMED and quickly poured into the gel plates leaving a margin of 3.0 cm on the upper side and the gel was allowed to polymerize. ii) Stacking gel (4%) It was prepared by mixing 4.0 ml of 30% acrylamide, 3.0 ml stacking gel buffer, 12.0 ml distilled water, 0.2 ml of 10% SDS, 0.4 ml of 10% APS and 0.04 ml of TEMED. The stacking gel solution was poured on the top of the separating gel. The gel was allowed to polymerize for 30 min.
5.5.4. Electrophoresis: Fifty μl of protein extract were loaded into the wells of stacking gel. A current of 20 mA was applied until the tracking dye crossed the stacking gel. Later the current was increased to 25 mA. The electrophoresis was stopped when the tracking dye reached the bottom of the stacking gel.
5.5.5. Staining: Coomassie brilliant blue R-250 was used for staining. Coomassie blue (0.5 %) was prepared by dissolving 1 g of coomassie brilliant blue R-250 in 100 ml methanol, 20 ml acetic acid and 80 ml distilled water. After dissolving
of coomassie blue, it was filtered through Wattman No. 1 filter paper and used for staining. The gel was immersed in coomassie blue solution for overnight under room temperature. Then, gel was destained, using destaining solution which was prepared by mixing 227 ml of methanol (45 %), 46 ml acetic acid (10 %) and 227 ml of distilled water, until the bands were clearly visible. After destaining, photographs of the gel were taken. Different molecular weights (MW) of bands were determined against pre-stained high molecular weight standard marker (PiNK Prestained Protein Marker, Cat. No. MWP02) ranged from 15 to 175 kDa.
6. Statistical analysis: All data were expressed as means±standard error (SE). Statistical analysis was conducted using Analysis of Variance (ANOVA) to compare the significance of differences between SiNPs concentrations. The results were considered significant at P < 0.05. Data for reduction percentage were subjected to Analysis of Variance for Randomized Complete Block Design followed by Duncan's Multiple Range Test; DMRT (Duncan, 1955). Pollen vitality, germination and shoot length as well as cytological analyses were calculated in a Complete Randomized Design with three replicates. The data were subjected to One-Way Analysis of Variance followed by LSD test to compare the significance of differences between means.
RESULTS AND DISCUSSION The obtained results are presented under three parts as given below: 1. Efficiency of SiNPs against faba bean and soybean main insect pests. 2. Effect of SiNPs on predators associated with studied insect pests.
3. Toxicity of SiNPs on faba bean plants.
1. Efficiency of SiNPs against insect pests:
1.1. Efficiency of SiNPs against faba bean main insect pests: Efficiency of SiNPs was evaluated against two faba bean main insect pests; A. craccivora and L. trifolii.
1.1.1. Effect of SiNPs on A. craccivora: Data of 2012/13 season presented in Table (2) showed that the highest reduction percentage in A. craccivora numbers (94.36 %) was observed after one day of SiNPs application at 425 ppm. After 3 and 5 days, the two and the four highest concentrations, respectively, gave completely protection against A. craccivora. Moreover, application of SiNPs for 7, 10 and 15 days gave completely protection against A. craccivora at all concentrations. In general, the highest reduction percentages were recorded at 375 and 425 ppm with mean values of 98.31 and 99.06 %, respectively.
Table (2): Reduction percentages of Aphis craccivora populations after SiNPs application during 2012/13 and 2013/14 seasons Day
Conc.
Mean±SE (ppm)
1
3
5
7
10
15
2012/13 season 75
29.67
52.75
96.76
100.00 100.00 100.00 79.86±17.81a
150
26.61
70.09
98.79
100.00 100.00 100.00 82.58±17.25a
225
70.93
83.02
100.00 100.00 100.00 100.00
92.33±7.21b
300
79.12
89.01
100.00 100.00 100.00 100.00
94.69±5.08b
375
89.86 100.00 100.00 100.00 100.00 100.00
98.31±2.39c
425
94.36 100.00 100.00 100.00 100.00 100.00
99.06±1.33c
2013/14 season 75
60.18
84.67
96.54
100.00 100.00 100.00
90.23±9.17a
150
66.99
88.18
98.50
100.00 100.00 100.00
92.28±7.63a
225
80.94
91.08
100.00 100.00 100.00 100.00
95.34±4.56b
300
86.14
94.81
100.00 100.00 100.00 100.00
96.83±3.25b
375
94.49
98.94
100.00 100.00 100.00 100.00
98.91±1.27c
425
94.37 100.00 100.00 100.00 100.00 100.00
99.06±1.33c
Means followed by the same letter are not significantly differ at 0.05 probability level.
Data obtained in 2013/14 season (Table 2) revealed that the highest concentrations of SiNPs were the most effective against A. craccivora infestation. A complete protection was shown after 3 days of treatment at 425 ppm, while the concentrations of 225, 300, 375 and 425 ppm induced completely protection after 5 days. When the percentages of reduction were recorded after 7, 10 and 15 days, they were 100 % at all SiNPs concentrations. In general, the same trend was observed as in 2012/13 season, since the two highest concentrations revealed the highest percentages of reduction (98.91 and 99.06 %) with no significant differences.
1.1.2. Effect of SiNPs on L. trifolii: Data of 2012/13 season showed that the reduction percentages in L. trifolii larvae were generally increased by concentrations increase (Table 3). Application of the four highest concentrations of SiNPs; 225, 300, 375 and 425 ppm gave the highest reduction in L. trifolii larvae with values of 88.10, 86.63, 89.17 and 88.99 %, respectively, which did not differ significantly from each other. After one day, the percentages of reduction for these concentrations were 43.13, 36.44, 42.43 and 38.62 %, respectively. These percentages reached 100 % after 7, 10 and 15 days. During the second season (2013/14), data presented in Table (3) revealed that application of 425 ppm occurred the highest reduction in the larvae number (86.81 %) followed by 225, 300 and 375 ppm which did not differ significantly from each other, while differ significantly from the concentration of 425 ppm. The highest reductions of 86.90 and 96.07 % were obtained at 425 ppm after the examination periods of 3 and 5 days, respectively, and reached 100 % after 7, 10 and 15 days.
Table (3): Reduction percentages of Liriomiza trifolii larvae after SiNPs application during 2012/13 and 2013/14 seasons Conc.
Day
Mean ± SE
(ppm)
1
3
5
7
10
15
2012/13 season 75
15.73 64.01 78.07
85.97
87.94
82.61
69.06±15.87a
150
30.48 99.34 77.48
89.13
89.33
92.32
79.68±14.50b
225
43.13 99.46 85.99 100.00 100.00 100.00 88.10±13.12c
300
36.44 99.48 83.84 100.00 100.00 100.00 86.63±14.67c
375
42.43 99.60 92.99 100.00 100.00 100.00 89.17±13.32c
425
38.26 99.60 96.07 100.00 100.00 100.00 88.99±14.38c 2013/14 season
75
20.45 72.16 75.80
92.31
87.09
83.70
71.92±15.16a
150
14.17 63.96 73.85
89.65
90.97
88.71
70.22±17.02a
225
37.75 72.56 81.85
98.32
99.13
100.00 81.60±13.98b
300
40.07 80.68 88.72 100.00 100.00 100.00 84.91±13.48b
375
25.74 81.13 92.37 100.00 100.00 100.00 83.20±16.81b
425
37.91 86.90 96.07 100.00 100.00 100.00 86.81±14.14c
Means followed by the same letter are not significantly differ at 0.05 probability level.
These results are in agreement with Debnath et al. (2010), they reported that SiNPs can be effectively used to control Mustard aphid (L. pseudobrassicae). Also, ElSamahy and Galal (2012) found that the reduction percentages in the numbers of A.
craccivora and L. trifolii were significantly increased as the concentration of SiNPs increased. Ulrichs et al. (2005) reported that a wide range of agricultural insect pests can be controlled by the use of SiNPs. Barik et al. (2008) reported that SiNPs has been utilized as a nano-insecticide and its insecticidal property was suggested due to its absorption ability into the cuticular layer of insect pests. Use of surface modified hydrophobic nanosilica to control a range of agricultural insect pests was reported by Rahman et al. (2009). Derbalah et al. (2012) reported that SiNPs played a serious role in controlling the leaf miner T. absoluta insects under greenhouse conditions. Likewise, Elsamahy et al. (2014) suggested the promising role of SiNPs in reducing T. absoluta larvae number. On the other hand, Liu et al. (2006) revealed that porous hollow silica nanoparticles (PHSNs) loaded with validamycin (pesticide) have been successfully employed as an efficient and controlled release formulation for water soluble pesticides.
1.2. Efficiency of SiNPs against soybean main insect pest; S. littoralis: Efficiency of SiNPs against S. littoralis was evaluated under both field and laboratory conditions.
1.2.1. Effect of SiNPs on S. littoralis under field conditions: The reduction percentages in the number of S. littoralis larvae under field conditions during 2013 and 2014 seasons were presented in Table (4). Table (4): Reduction percentages of Spodoptera littoralis larvae after SiNPs application under field conditions during 2013 and 2014 seasons Conc. (ppm)
Day Mean±SE 1
3
5
7 2013 season
10
15
75
24.58
46.16
57.28
85.10
92.26
95.20
66.76±16.51a
150
31.61
50.03
63.28
86.88
93.17
95.60
70.09±15.04ab
225
18.50
50.83
64.97
88.31
97.71
98.35
69.78±18.17a
300
51.51
71.01
72.69
100.00
100.00
100.00
82.54±11.85b
375
57.33
74.49
86.96
100.00
100.00
100.00
86.46±10.14c
425
54.01
79.44
89.92
100.00
100.00
100.00
87.23±10.52c
2014 season 75
29.77
48.36
60.36
86.74
93.18
96.18
69.10±15.66a
150
29.50
51.63
63.65
86.39
94.12
96.07
70.23±15.38a
225
31.23
53.52
70.79
91.90
97.33
99.40
74.03±15.85b
300
50.25
64.48
77.96
100.00
100.00
100.00
82.12±12.39c
375
55.59
69.27
89.97
100.00
100.00
100.00
85.81±10.98c
425
53.33
68.25
90.29
100.00
100.00
100.00
85.31±1.51c
Means followed by the same letter are not significantly differ at 0.05 probability level.
Data showed that the highest reduction percentages; for the two seasons, were observed at all SiNPs concentrations after 7, 10 and 15 days. These reduction percentages reached to 100% after application of 300, 375 and 425 ppm for the same examination periods.
In general, data presented in Table (4) for 2013 season revealed that applications of 375 and 425 ppm of SiNPs; which did not differ significantly, occurred the highest reduction in the larvae number (86.46 and 87.23 %, respectively) followed by 300 ppm (82.54 %) which differed significantly. During 2014 season, the three highest concentrations revealed the highest percentages of reduction with no significant differences. The percentages of reduction were 82.12, 85.81 and 85.31 % at 300, 375 and 425 ppm, respectively. These results are in agreement with Debnath (2012), who revealed that SiNPs were effective against the leaf worm S. litura especially in its hydrophobic and lipophilic cases. Moreover, Debnath et al. (2012) found that silica based nanocide can be alternative to the commercial insecticides against this leaf worm. El-Bendary and El-Helaly (2013) reported that nano-silica application on the tomato plants can minimize the issues caused by S. littoralis providing resistance to the moderate level. In general, results of this study demonstrated that the concentrations of SiNPs were more effective on A. craccivora than the other two insect pests; L. trifolii and S. littoralis. A complete protection was obtained after 7 days of treatment with the two lowest concentrations; 75 and 150 ppm. The reduction percentages in the number of A. craccivora were increased as the concentration of SiNPs increased. SiNPs decreased almost A. craccivora insects with the highest concentration; 425 ppm, after just one day of treatment and reached 100% at concentrations of 225, 300 and 375 ppm after 5 days of treatment. On the other hand, efficiency of SiNPs concentrations against the other two insect pests reached 100 % after 7 days of treatment with the three highest concentrations during 2013/14 season (for L. trifolii) and the two studied seasons (for S. littoralis). In addition, completely protection was observed after application of the four highest concentrations of SiNPs against L. trifolii during 2012/13 season.
1.2.2. Effect of SiNPs on S. littoralis under laboratory conditions:
a. Mortality percentage: Efficacy of SiNPs as an insecticidal agent was tested on S. littoralis larvae under the laboratory conditions. Mortality percentage was checked after 15 days of feeding larvae on soybean plants treated with SiNPs. As shown in Fig. (2), highly significant differences in mortality percentage means were observed among SiNPs concentrations. At the two lowest concentrations; 75 and 150 ppm, there was no significant difference in mortality percentage, while it differed significantly compared to that recorded in control treatment (4.67 %). The mortality percentage of 225, 300, 375 and 425 ppm treated larvae increased significantly as the concentration increased. However, insignificant difference was recorded between the two highest concentrations; 375 and 425 ppm. This result indicates that SiNPs were toxic against the larvae of S. littoralis.
b. Biological aspects: Corresponding to the biological aspects of S. littoralis larvae reared on soybean plants treated with SiNPs, larval duration, pupal period, adult longevity, number of eggs laid per female and hatchability percentage were determined (Table 5). The larval duration were not affected with SiNPs at the lowest concentrations; 75 and 150 ppm, compared to control, while it was increased at the other four concentrations with no significant differences.
100
e
d
80 Mortality %
e
c
60
40 20 0
b
b a control
75 ppm
150 ppm
225 ppm
300 ppm
375 ppm
425 ppm
4.67
16.00
20.67
60.00
73.67
94.67
95.33
Fig. (2): Mortality percentages of Spodoptera littoralis larvae after 15 days of feeding on soybean plants treated with SiNPs. Different letters indicate significant differences at 0.05 probability level.
Table (5): Biological aspects of Spodoptera littoralis reared on soybean leaves treated with SiNPs under laboratory conditions Conc. (ppm) Contro l 75
150
225
300
Larval duration (day) 14.00±0.71 a
13.80±0.84
Pupal period (day) 9.20±1.30a
Adult longevity (day)
Egg/female
Hatchability
(No.)
(%)
a
258.75±8.54d
79.50±3.32c
13.20±0.84
255.00±12.91
a
d
66.50±6.21b
13.60±0.55
a
9.80±0.84 a
14.60±1.14
13.00±1.22
14.40±0.89
a
b
a
250.00±9.13d
62.50±6.45b
19.00±0.71
13.80±1.48
13.80±1.48
196.25±14.93
61.25±8.54a
b
b
a
c
b
19.80±1.10
16.80±0.84
13.00±1.00
160.00±10.80
b
c
a
b
59.25±9.88a
375
425
20.40±1.52
17.00±1.58
14.00±1.58
121.25±13.15
b
c
a
a
53.00±2.45a
21.00±0.71
16.20±1.79
13.80±1.48
b
c
a
115.00±5.77a
48.75±4.79a
Different superscript letters within each column indicate significant differences at 0.05 probability level.
Data recorded for pupal period revealed that the three highest concentrations of SiNPs (300, 375 and 425 ppm); which did not differ significantly, were the most effective in increasing pupal period followed by 150 and 225 ppm; which did not differ significantly. On the other hand, pupal period did not affect with 75 ppm concentration in comparison with control (9.20 days). The adult longevity was not affected with all SiNPs concentrations compared to control (13.60 days). With respect to number of eggs laid per female, data presented in Table (5) revealed that applications of 375 and 425 ppm of SiNPs; which did not differ significantly, occurred the highest reduction in eggs number (121.25 and 115.00 eggs, respectively) followed by 300 ppm (160.00 eggs) and 225 ppm (196.25 eggs) which differed significantly. There were no significant differences between the two lowest concentrations; 75 and 150 ppm, and control treatment (258.75 eggs). With this respect, El-Bendary and El-Helaly (2013) reveled that spray of SiNPs on tomato plants influence the feeding preference of S. littoralis ultimately increasing the resistance in plants. It affects the reproductive potential of insect females causing a reduction in population density of insect. The percentage of hatchability was reduced significantly at all SiNPs concentrations compared to control (Table 5). The highest reductions in hatchability percentage were recorded at the three highest concentrations which did not differ significantly (59.25, 53.00 and 48.75 for 300, 375 and 425 ppm, respectively).
2. Effect of SiNPs on predators associated with insect pests: To our best knowledge, this is the first study to illustrate the toxicity of SiNPs on predators associated with faba bean and soybean main insect pests.
2.1. Effect of SiNPs on predators in faba bean field: Six species of predators were collected from faba bean plants treated with SiNPs; Coccinella undecimpunctata, Cydonia vicina isis, Cydonia vicina nilotica, Scymnus interruptus, Peaderus alfierii and Chrysoperla carnea. These were the most abundant species of predators in faba bean plants. Obtained results in 2012/13 season (Table 6) showed that the examined predators were affected negatively by different SiNPs treatments. The predators number was significantly reduced in dose-dependent manner till 300 ppm concentration. The lowest reduction percentage was observed at 75 ppm, it ranged from 9.94 % (after one day) to 100 % (after 10 and 15 days of SiNPs application) with an average of 58.89 %. On the other hand, the highest reduction percentages were recorded at 300, 375 and 425 ppm; which did not differ significantly, with average values of 97.42, 96.61 and 96.61 %, respectively. The same trend was observed in 2013/14 season (Table 6), since applications of all SiNPs concentrations showed negative effect on the examined predators. Application of 75 ppm gave the lowest reduction in pedators number with mean value of 67.39 %. Likewise, the three highest concentrations revealed the highest reduction percentages of 100%, followed by 225 and 150 ppm (90.53 and 83.22 %, respectively) which differed significantly. Effect of SiNPs on predators reached 100 % after application of the three highest concentrations at all examination periods.
Table (6): Reduction percentages of predators in faba bean field after SiNPs application during 2012/13 and 2013/14 seasons Conc. (ppm)
Day Mean±SE 1
3
5
7
10
15
2012/13 season 75
9.94
21.84
46.58
74.96
100.00
100.00
58.89±22.45a
150
56.82
69.58
78.32
100.00
78.32
82.67
77.62±8.26b
225
78.08
56.94
100.00
84.53
100.00
100.00
86.59±9.98c
300
100.00
100.00
84.52
100.00
100.00
100.00
97.42±3.65d
375
79.68
100.00
100.00
100.00
100.00
100.00
96.61±4.79d
425
79.66
100.00
100.00
100.00
100.00
100.00
96.61± 4.79d
2013/14 season 75
31.22
15.29
81.25
100.00
100.00
100.00
67.39±22.08a
150
87.64
71.57
100.00
66.46
66.46
73.64
83.22±7.76b
225
85.01
76.91
81.24
100.00
100.00
100.00
90.53±6.17c
300
100.00
100.00
100.00
100.00
100.00
100.00
100.00±0.00d
375
100.00
100.00
100.00
100.00
100.00
100.00
100.00±0.00d
425
100.00
100.00
100.00
100.00
100.00
100.00
100.00±0.00d
Means followed by the same letter are not significantly differ at 0.05 probability level.
2.2. Effect of SiNPs on predators in soybean field: All predator species detected on soybean plants after application of SiNPs were the same as those found on faba bean plants, in addition to species of Orius spp. Data of 2013 season (Table 7) showed that the reduction percentages in predator numbers were generally increased by concentration increased till 300 ppm concentration and then it decreased again at 375 ppm (74.45 %). The lowest reduction percentage was observed at 75 ppm with an average of 57.24 %. Applications of SiNPs at 300 and 425 ppm; which did not differ significantly, gave the highest reduction percentage in predator numbers with mean values of 79.87 and 79.81 %, respectively. During 2014 season, data revealed that application of 300 and 425 ppm of SiNPs; which did not differ significantly, reduced the number of predators to 78.96 and 76.54 %, respectively. The percentages of reduction were 55.65% at the lowest SiNPs concentration; 75 ppm. Interestingly, predators number was increased after one day of treatment with 75 ppm SiNPs to 11.44 and 10.21 % during 2013 and 2014 seasons, respectively. Moreover, it increased to 3.52, 2.41 and 1.01 % after one day of treatment with concentrations of 150, 375 and 425 ppm, respectively, during 2014 season.
Table (7): Reduction percentages of predators in soybean field after SiNPs application during 2013 and 2014 seasons Day
Conc.
Mean±SE (ppm)
1
3
5
7
10
15
2013 season 75
-11.44
35.70
70.15
77.15
88.44
83.46
57.24±22.22a
150
4.50
53.12
84.92
75.77
89.77
100.00
68.01±20.16b
225
19.84
53.12
88.66
87.89
100.00
100.00
74.92±18.48c
300
36.48
55.23
100.00
87.52
100.00
100.00
79.87±15.84d
375
3.26
43.43
100.00
100.00
100.00
100.00
74.45±24.00c
425
7.06
71.78
100.00
100.00
100.00
100.00
79.81±21.58d
2014 season 75
-10.21
28.87
74.94
74.16
82.03
84.10
55.65±22.02a
150
-3.52
43.52
84.89
76.58
94.62
100.00
66.02±22.78b
225
18.44
37.34
89.62
89.29
100.00
100.00
72.45±20.41c
300
22.67
58.24
96.47
96.36
100.00
100.00
78.96±18.43d
375
-2.41
42.22
100.00
100.00
100.00
100.00
73.30±25.23c
425
-1.01
60.26
100.00
100.00
100.00
100.00
76.54±23.78d
Means followed by the same letter are not significantly differ at 0.05 probability level.
Results of this study previously demonstrated that the concentrations of SiNPs were more effective on A. craccivora than the other two insect pests; L. trifolii and S. littoralis. Moreover, SiNPs had indirect effect on the predators associated with A. craccivora and S. littoralis. These results and those mentioned above on the effects of SiNPs on insect pests may be due to that application of SiNPs led to kill the insects which these predators attack. As mentioned preciously, this is the first study that provides information on the effect of SiNPs on associated predators. A few studies have focused on the positive impact of silica in increasing predator numbers. Moraes et al. (2004) revealed that silicon application increased the degree of the predator Chrysoperla externa attractiveness in wheat plants decreasing preference to greenbug aphid S. graminum. This action of silica may be due to the deleterious effect on the predator feeding source.
3. Toxicity of SiNPs on faba bean plants: Plants as an important component of the ecosystems need to be included when evaluating the overall toxicological impact of the SiNPs in the environment.
3.1. Effect of SiNPs on pollen vitality: As presented in Fig (3), all SiNPs applications significantly increased faba bean pollen vitality, except 300 ppm which recorded 97.30 % with no significant difference compared to control treatment (96.89 %). The other concentrations of 75, 150, 225, 375 and 425 did not differ significantly from each other. Few studies have been conducted on the potential effects of silica on pollen vitality. Miyake and Takahashi (1986) showed that silica increases pollen fertility on soybean plants. On the other hand, Hou et al. (2006) revealed that silicon result in reduced pollen fertility, affecting fruit yield.
3.2. Effect of SiNPs on seed germination and shoot length: All concentrations of SiNPs; except the highest concentration 425 ppm, significantly increased seed germination of faba bean compared to control (Table 8). The germination value in the control was 76.67% and the maximum value of 96.67% was observed at 75 ppm. It was decreased by increasing SiNPs concentration at 150, 225, 300 and 375 ppm which did not differ significantly than each other. The highest SiNPs concentration (425 ppm) significantly inhibited the seed germination compared to the control. Thus, the highest concentration of SiNPs exerted a toxic effect on germination of the seeds of faba bean.
b
100
a
a
a
b
a
a
Pollen vitality %
80 60 40 20 0
Control 96.89
75 ppm 150 ppm 225 ppm 300 ppm 375 ppm 425 ppm 98.29
98.59
98.89
97.3
98.7
98.6
Fig (3): Effect of SiNPs on faba bean pollen vitality. Different letters indicate significant differences at 0.05 probability level.
Table (8): Effect of SiNPs on seed germination and shoot length of faba bean Concentration
Germination
Shoot length
(ppm)
(%)
(cm)
Control
76.67 ± 8.82ab
5.86 ± 0.91ab
75
96.67 ± 3.33c
6.04 ± 0.47ab
150
86.67± 6.67bc
6.01 ± 0.20ab
225
86.67 ± 3.33bc
7.35 ± 0.90bc
300
90.00 ± 5.77bc
8.09 ± 0.58c
375
86.67 ± 3.33bc
6.91 ± 0.27bc
425
60.00 ± 5.77a
4.52 ± 0.45a
Different superscript letters within each column indicate significant differences at 0.05 probability level.
Siddiqui
and
Al-Whaibi
(2014)
found
that
application
of
8 g/L of SiNPs significantly enhanced the characteristics of seed germination in tomato. These findings were in agreement with Nair et al. (2011), they observed better germination of rice seeds in the presence of FITC-labelled SiNPs. Data generated by Yuvakkumar et al. (2011) in laboratory and field studies showed that SiNPs increased seed germination and water use efficiency. Exogenous application of SiNPs improves seed germination of soybean by increasing nitrate reductase (Lu et al., 2002) and also by enhancing seeds ability to absorb and utilize water and nutrients (Zheng et al., 2005). The improvement in germination characteristics of seed as a result of SiNPs may be due to the absorption and utilization of SiNPs by seeds (Suriyaprabha et al., 2012). An increase in germination parameters by the application of SiNPs may be effective for the growth and yield of crops. With respect to shoot length, results in Table (8) indicated that faba bean shoot length did not differ significantly than the control at the concentrations of 75 and 150 ppm. The shoot length values were significantly increased as SiNPs increased at 225 and 300 ppm. The highest value (8.09 cm) was observed at 300 ppm concentration. The shoot length was decreased again as concentration increased, since its value reached 4.52 cm at 425 ppm. This result was in constant with that of seed germination, where the highest concentration of SiNPs exerted a toxic effect on shoot length. The effect of SiNPs on seed germination and shoot length may be due to penetrate the thick seed coat and support water uptake inside seeds (Khodakovskaya et al., 2009).
3.3. Cytological effects of SiNPs on mitosis: Cytological analysis of mitotic activity (a measurement of actively dividing cells known as the mitotic index), alterations in the mitotic phase, and individual cell aberrations are key parameters by which plant growth may be evaluated.
3.3.1. Mitotic index and frequency of mitotic phases: The mitotic frequencies of faba bean meristemic cells were scored in M 2 root tips treated with different concentrations of SiNPs. The total number of proliferating cells and the number of cells at various mitotic stages were calculated. Mitotic stages; prophase, metaphase, anaphase and telophase, under normal conditions were presented in Fig. (4). Cytological results showed that application of all SiNPs concentrations caused significantly increase in faba bean mitotic indices compared to control (10.49 %) as reported in Table (9). The highest value of mitotic index (30.05 %) was recorded at 75 ppm. This means that SiNPs concentrations prolonged cell division time and shortened interphase in cell division, thus division cycle shortened. On the other hand, mitotic index values did not differ significantly at 150, 225, 300 and 375 ppm, which decreased significantly compared to 75 ppm. This reduction in mitotic index could be explained as a result of inhibition of DNA synthesis at Sphase (Sudhakar et al., 2001) or blocking in G2 phase of the cell cycle, preventing the cell from entering mitosis (El-Ghamery et al., 2000). Again, markedly increased mitotic index was found in cell treated with the highest concentration; 425 ppm, of SiNPs which is indicative of the induction of molecular changes in the genetic material, suggesting either DNA lesion or interference with cell cycle (Baeshin and Qari, 2003).
Fig. (4): Mitotic phases in faba bean root tip cells under normal conditions: a) prophase; b) metaphase; c) anaphase; d) telophase. Magnification is 1000X.
Table (9): Mitotic index and percentage of mitotic phases (relative to dividing cells) of faba bean root tip cells treated with SiNPs Mitotic phase
No. of Concentration examined (ppm) cells
No. of dividing cells
Prophase
Metaphase
Anaphase
Telophase
N
N
%
N
N
35
7.54
75
%
%
Mitotic index (%)
%
Control
5066
464
243 52.37
16.16 111 23.92
10.49±3.59a
75
3872
1169
782 66.66 127 10.82 129 10.99 131 11.16
30.05±7.32c
150
3776
582
286 49.14 124 21.30
15.64
15.42±0.76ab
3397
601
223 37.10 160 26.62 101 16.81 116 19.30
16.04±1.81ab
3230
459
118 25.76 146 31.88
91
19.87 103 22.49
14.21±1.29ab
3202
507
198 39.05 113 22.29
79
15.58 117 23.08
15.81±1.04ab
3464
808
370 45.74 135 16.69 102 12.61 201 24.85
23.25±2.31bc
225 300 375 425
Values followed by the same letter are not significantly differ at 0.05 probability level.
81
13.92
91
Data in Table (9) also showed that SiNPs exhibited an effect on the percentage of mitotic stages, where the percentage of prophase increased with a corresponding decreased in the percentages of the other stages. This prophase accumulation could also occur as a result of disturbance or breakdown of spindle apparatus (Abu Ngozi and Duru, 2006). 3.3.2. Percentage and types of abnormalities:
3.3.2. Percentage and types of abnormalities: Data in Table (10) presented the cytological effect of SiNPs on faba bean root tip cells as the percentage of cells showing abnormalities. Cytological analysis of root tip cells revealed a universal increase in the percentage of abnormalities after all SiNPs treatments as compared to their respective control (1.22 %). The frequency of abnormalities was significantly increased as the concentration of SiNPs increased, except the concentrations of 375 and 425 ppm which significantly decreased the abnormality percentages again compared to the other concentrations. Although the two highest concentrations decreased the abnormalities, they increased in percentage of aberrations compared to control. The maximum percentages of aberrations (44.17 and 43.72 %) were recorded at concentrations of 225 and 300 ppm, respectively, which did not differ significantly. Results in Table (10) also showed that percentages of abnormalities were increased in metaphase stage than the other mitotic stages. This result was in constant with the above described results of the effect of SiNPs on increasing the prophase percentage than the other stages, which could occur as a result of disturbance or breakdown of spindle apparatus (Abu Ngozi and Duru, 2006).
Table (10): Percentage of abnormal mitotic cells (relative to dividing cells) of faba bean root tip cells treated with SiNPs Abnormal mitotic phase Concentration (ppm)
No. of dividing cells
No. of mitotic abnormalities Prophase
Metaphase
Anaphase Telophase
N
%
N
%
N
%
N
%
Abnormal cells (%)
Control
464
7
0
0
7
1.51
0
0
0
0
1.22±0.76a
75
1169
194
33
2.82
99
8.47
55
4.70
7
0.60
16.77±3.00b
150
582
145
33
5.67
85
14.60
26
4.47
1
0.17
24.73±2.26bc
225
601
266
53
8.82
146
24.29
59
9.82
8
1.33
44.17±2.57e
300
459
198
37
8.06
114
24.84
44
9.59
3
0.65
43. 72±3.25e
375
507
174
49
9.66
74
14.60
50
9.86
1
0.20
34.43±1.09d
425
808
223
71
8.79
91
11.26
54
6.68
7
0.87
28.03±2.19c
Values followed by the same letter are not significantly differ at 0.05 probability level.
As presented in Fig. (5) and summarized in Table (11), treatment of faba bean with SiNPs revealed various types of chromosomal abnormalities in M2 root tips. The different concentrations of SiNPs produced similar types of chromosomal abnormalities. The most frequent aberrations were C-metaphase, disturbance, stickiness, bridges, laggards and fragments.
The chromosomal aberrations might be induced by the following ways: Firstly, chemical compounds directly affect DNA and lead to chromosomal aberration. Secondly, chemical compounds could disturb the synthesis of DNA and protein, or the translation of RNA, so that no materials relating to the chromosomal movement could be formed, and the chromosomal aberration is occurred eventually. Thirdly, chemical compounds can prevent the reestablishment of the chromosome under normal conditions through interfering with the normal repairing of some damages to the new refusions, such as the rearrangement of chromosomal bridges, loops and fragments. Any such irreversible DNA damages will lead to the chromosomal aberrations. Types of abnormalities such as C-metaphase, disturbance, stickiness, bridges, laggards as observed in the present investigation point out to direct effect of SiNPs on spindle apparatus causing defective formation of the spindle fibers (Polit et al., 2000; Usciati et al., 2004). C-metaphase (Fig. 5a), the major chromosomal aberration in the cells treated with 75, 150, 225 and 300 ppm, is commonly associated with spindle poisoning as reported by Shahin and El-Amoodi (1991). Inhibition of spindle formation by SiNPs may result in such type of chromosomal aberrations. Disturbance (Fig. 5b, d, h); the other major chromosomal aberration at the same concentrations; 75, 150, 225 and 300 ppm, may be caused by enhanced disturbances of spindle function (Jain et al., 2000).
Fig. (5): Types of chromosomal aberrations induced in faba bean root tip cells treated with SiNPs. a) C-metaphase; b) disturbed metaphase; c) metaphase with fragments; d) disturbed anaphase; e) sticky prophase; f) sticky telophase; g) chromosomal bridge in anaphase; h) disturbed anaphase with multibridges; i) telophase with chromosomal bridge and fragment; j) telophase with lagging chromosome; k) anaphase with laggards; l) anaphase with forward lagging chromosome. Magnification is 1000X.
Table (11): Types and percentage of abnormalities (relative to dividing cells) of faba bean root tip cells treated with SiNPs C-metaphase
Concentration (ppm) Control 75 150 225 300 375 425
Disturbance
Stickiness
bridge
laggard
fragment
N
%
N
%
N
%
N
%
N
%
N
%
2
0.43
4
0.86
0
0.00
0
0.00
1
0.22
0
0.00
78
6.67
46
3.94
38
3.25
14
1.19
11
0.94
7
0.59
53
9.11
40
6.87
33
5.67
7
1.20
7
1.20
5
0.86
104
17.30
75
12.48
53
8.19
14
2.33
16
2.66
4
0.67
73
15.90
58
12.64
37
8.06
15
3.27
11
2.39
4
0.87
49
9.66
36
7.10
54
10.65
18
3.55
2.56
4
0.79
45
5.57
59
7.30
73
9.03
23
2.85
1.36
12
1.49
13 11
Chromosome stickiness was also recorded in considerable percentages, especially at SiNPs concentrations of 375 and 425 ppm (Fig. 5e, f). Stickiness of chromosomes might have resulted from increased chromosome contraction and condensation or possibly from the depolymerization of DNA. There is an agreement that stickiness reflects highly toxic and usually irreversible effect that probably leads to cell death (Akaneme and Amaefule, 2012). This may be the cause of decrease the percentage of abnormalities at the two highest concentrations; 375 and 425 ppm (34.43 and 28.03, respectively) due to cell death. The presence of chromosome stickiness is an indication of SiNPs affecting organization of chromatin, and suggests a possible role by which this cytotoxic agent may impact the physical and chemical properties of DNA, protein, or both, ultimately leading to improper folding of chromatin (El-Ghamery et al., 2003; Talukdar, 2013; Teerarak et al., 2010). Types of aberration such as chromosomal bridges were found in the anaphase or telophase cells (Fig. 5g, h, i). The bridges noticed in the cells are probably formed by breakage and fusion of chromosomes and chromatids (Haliem, 1990). Bridges may be due to the chromosomal stickiness and subsequent failure of free anaphase separation or may be attributed to an unequal translocation or inversion of chromosome segment (Gomurgen, 2000). Laggards (Fig. 5j, k, l) may be explained on the basis of abnormal spindle formation and failure of chromosome movement (Haiba et al., 2011). They are a potential source of aneuploidy because they lost the ability to attach by spindle fibers. Laggards do not participate to the normal division and cause genetic disequilibriums between daughter cells (Truta et al., 2011). Recep et al. (2012) stated that the occurrence of chromosomal laggards at anaphase was due to the failure of the chromosomes or acentric chromosome fragments to move to either of the pole. Acentric fragments (Fig. 5c, i) were low at all SiNPs concentrations, whoever the highest value (1.49 %) was found at the highest concentration; 425 ppm. Most
chromosomal aberrations lead to the formation of chromosomal fragments without centromeres which are crucial for proper chromosomal division. Therefore a chromosomal fragment is likely to be lost from one of the daughter cells formed after cell division. The chromosomal aberrations noticed in this study indicate that treatment of faba bean with SiNPs can lead to mutation or can act as clastogene. The same result reported by Njoku et al. (2011) who found that altered chromosomes may possible have altered DNA and gene sequences. The highest frequencies of abnormalities; which caused by the two SiNPs concentrations of 225 and 300 ppm, was accompanied by decreased in mitotic indices. Smaka-Kincl et al. (1996) suggested that the cytotoxicity level can be determined by the decreased rate of the mitotic index. The reason is possibly that SiNPs at these concentrations does more harm to cells and leads to increasing chromosomal aberrations. On the other hand, SiNPs at 75 ppm caused clear effect on mitotic index increasing, which caused the lowest percentage of mitotic aberrations compared to the other SiNPs concentrations Several studies evaluated the cytotoxic potential of SiNPs. These studies showed that the cytotoxicity of SiNPs was cell-type specific (NTP, 2009). One investigation based on the micronucleus assay found that these nanoparticles indeed induce chromosomal damage (Wang et al., 2007). Despite these abnormalities, there is limited evidence to suggest that SiNPs are genotoxic and some recent studies utilizing the Comet assay have demonstrated that SiNPs ranging in size from 20 to 400 nm do not exert significant genotoxicity (Jin et al., 2007; Barnes et al., 2008). Galal and El-Samahy (2012) revealed that SiNPs at 100 and 250 ppm were not able to cause mutagenicity on chromosomal levels in Drosophila salivary chromosomes. Finally, from the above mentioned results it was evident that SiNPs affected faba bean on cytological levels, which can even lead to DNA damage.
3.4. Effect of SiNPs on genomic DNA:
3.4.1. RAPD profile: Random Amplified Polymorphic DNA (RAPD) technique was used to detect variations between DNA extracted from faba bean treated with SiNPs and the control. RAPD profiles generated from faba bean DNA using six oligonucleotide primers were presented in Fig. (6). Profiles generated by these primers revealed differences between control treatment and different SiNPs concentrations, with visible changes in the number and the intensity of amplified DNA fragments. Data presented in Table (12) show the total number of bands, polymorphic bands and percentage of polymorphism for DNA-RAPD profiles of faba bean treated with SiNPs. The total number of bands scored for the six primers was 58 bands (9.67 bands in average per primer), out of them 36 bands were polymorphic and 22 bands were monomorphic. Percentage of polymorphic bands was 62.07 %. All of the six primers exhibited polymorphic patterns of the amplified DNA fragments. Changes in the RAPD profile (number and size of the amplification products) obtained from each treatment depending on the sequence of primer used.
Fig. (6): RAPD profiles of genomic DNA extracted from M2 faba bean seedlings treated with different concentrations of SiNPs using primers OPA20, OPB01, OPB06, OPB07, OPB12 and OPB14. Lane M: DNA marker, lane 1 for control, lanes 2, 3, 4, 5, 6 and 7 for 75, 150, 225, 300, 375 and 425 ppm SiNPs, respectively.
Table (12): Total number of bands, polymorphic bands and percentage of polymorphism in DNA-RAPD profiles of faba bean treated with SiNPs Concentration (ppm) Primers
Total bands
Polymorphic bands
Polymorphism (%)
control
75
150
225
300
375
425
OPA20
3
3
3
3
4
3
4
4
1
25.00
OPB01
9
11
9
4
10
10
3
12
10
83.33
OPB06
8
9
11
11
8
6
9
16
9
56.25
OPB07
11
8
7
10
12
6
10
15
9
60.00
OPB12
3
2
2
3
5
5
5
6
4
66.67
OPB14
4
5
5
5
3
3
2
5
3
60.00
Total
38
38
37
36
42
33
33
58
36
62.07
3.4.2. Genomic template stability: DNA variation which was induced in faba bean cells treated with different concentrations of SiNPs was shown in Table (13). As can be seen, six random primers generated a total of 38 RAPD bands in control treatment. Bands number ranged from three bands for primers OPA20 and OPB12 to eleven bands for primer OPB07. The six tested primers gave specific and stable results, with apparent changes in the number and the intensity of amplified DNA bands. The different concentrations of SiNPs gave variable bands; compared to control, as reflected by changes in band intensity (increase/decrease), disappearance of bands, and appearance of new bands. An increase in band intensity was the major event arising in the patterns generated from faba bean DNA treated with concentrations of 75, 150, 225 and 300 ppm. The concentration of 300 ppm showed the highest increase in band intensity. On the other hand, all concentrations of SiNPs increased the disappearence of normal band compared to the control. These changes in disappearent bands were increased as SiNPs concentrations increased, except the the concentration of 300 ppm which increased the disappearent bands to only one band. The highest changes in bands numbers (both appearance of new bands or disappearance of normal bands) was appeared in faba bean treated with the highest concentration of SiNPs; 425 ppm. Number of appeared and lost RAPD bands (six and ten bands, respectively) was much increased after application of this concentration.
Table (13): Changes in DNA-RAPD profile of faba bean treated with different concentrations of SiNPs
No. of bands in control
Concentration (ppm)
a
OPA20
3
0
0
0
0
0
0
0
3
0
0
0
0
1
0
2
0
0
0
2
1
1
0
2
0
OPB01
9
2
0
1
0
0
0
1
1
0
5
0
3
1
0
2
1
1
0
2
0
0
6
0
2
OPB06
8
1
0
3
1
3
0
7
0
3
0
5
0
0
0
4
2
1
0
1
1
3
0
4
0
OPB07
11
0
4
1
3
0
5
1
2
0
2
1
2
0
0
3
0
0
6
0
6
0
2
1
2
OPB12
3
0
1
1
0
0
1
2
0
0
0
0
0
2
0
2
0
2
0
2
0
2
0
2
0
OPB14
4
1
0
1
0
1
0
1
0
1
0
1
0
0
1
1
0
0
1
0
0
0
2
0
0
Total
38
4
5
7
4
4
6
12
6
4
7
7
5
4
1
14
3
4
7
5
8
6
10
9
4
Primer
75
150
225
300
375
425
b
c
d
a
b
c
d
a
b
c
d
a
b
c
d
a
b
c
d
a
b
c
d
a+b
9
10
11
5
11
16
a+b+c+d
20
28
23
22
24
29
GTS (%)
76.32
73.68
71.05
86.84
71.05
57.89
a: appearance of new band, b: disappearance of normal band, c: increase in band intensity, d: decrease in band intensity, a+b: polymorphic bands, GTS: genomic template stability.
Genomic template stability test; performed for the qualitative measurement of changes in RAPD profiles, showed considerable effect at the given concentration of SiNPs. All concentrations showed low GTS compared to control. The highest concentration; 425 ppm, exhibited the lowest value of GTS (57.89 %). Changes observed among RAPD profiles obtained from control and SiNPs treatments may be induced by directly and/or indirectly interact with genomic DNA. This damage includes loss of one or more nucleotides which can lead to alterations of DNA sequence. These results indicate that SiNPs may interact with DNA causing genotoxic effect. These results were in agreement with Lankoff et al. (2013) who determined the effect of different doses of unmodified and surface modified SiNPs (10, 25, 50 and 100 ug/ml( on DNA damage in human peripheral blood lymphocytes after 2 and 24 h. Their results revealed that unmodified SiNPs exhibited genotoxic properties at high doses.
3.5. Effect of SiNPs on seed storage proteins: SDS-PAGE analysis was carried out on M2 faba bean seed storage proteins whose parents were previously treated with different concentrations of SiNPs. The protein banding patterns were illustrated in Fig. (7) and Table (14). The obtained data revealed that changes in protein patterns were limited. Comparing with the control, the recorded changes were expressed as variations in the number of separated bands; disappearance or appearance of certain bands, and alterations in band intensity.
Fig. (7): Gel image (a) and its diagram (b) of faba bean seed storage proteins treated with SiNPs. Lane M: protein molecular marker ranged from 15 to 175 kDa. Lane 1 for control and lanes 2, 3, 4, 5, 6 and 7 for 75, 150, 225, 300, 375 and 425 ppm SiNPs, respectively.
Table (14): Seed storage protein banding patterns of faba bean treated with SiNPs Concentration (ppm)
No. of Bands
Control
75
150
225
300
375
425
1
++
++
++
++
++
++
++
2
++
+
+
+
-
+
+
3
+
+
+
+
+
+
+
4
+
+
+
+
+
+
+
5
+
+
+
+
+
+
+
6
+
+
+
+
+
-
-
7
+
+
+
+
+
+
+
8
+
+
+
+
+
+
+
9
+
+
+
++
++
+
++
10
+
+
+
+
+
+
+
11
-
+
+
+
+
-
+
12
-
+
+
+
+
+
+
13
++
++
++
++
++
++
++
14
++
+
+
+
+
+
+
15
+
+
+
+
+
-
-
16
+++
+++
+++
+++
+++
+
++
17
++
++
++
++
++
++
++
18
++++
++++
++++
++++
++++
++++
++++
19
++
++
++
++
++
++
++
20
++
++
++
++
++
++
++
21
++
++
++
++
++
++
++
22
++
++
++
++
++
++
++
23
+
+
+
+
+
+
+
24
+++
+++
+++
+++
+++
+++
+++
25
++
++
++
++
++
++
++
26
++
++
++
++
++
++
++
27
++
++
++
++
++
++
++
Total
25
27
27
27
26
24
25
- : Absent + : Very faint ++ : Faint +++ : Dark ++++ : Very dark
The total number of bands was 27 bands. Seed proteins profile of the control was found to have 25 bands, out of them 22 bands were found to be common in all treatments (monomorphic bands). The remaining bands No. 2, 6 and 15 (with MW of ~416, ~279 and ~63 kDa, respectively) were polymorphic, since it did not appear at 300 ppm for band No. 2 and at 375 and 425 ppm for bands No. 6 and 15, respectively. The disappearance of these bands could be explained on the basis of mutational event at the regulatory genes that prevent or attenuate transcription (Muller and Gottschelk, 1973). Induction of bridges and laggards by SiNPs may lead to the loss of genetic material. Therefore, some electrophoretic bands were disappeared due to the loss of their corresponding genes (Abdelsalam et al., 1993). On the other hand, the highest number of protein bands (27 bands) was recorded at 75, 150 and 225 ppm. Thus, two polymorphic protein bands were appeared by application of these three concentrations. The first band No. 11 (MW of ~112 kDa) was expressed as a result of treatment with all SiNPs concentrations,
except the concentration of 375 ppm. The second appeared polymorphic band; No. 12 (MW of ~105 kDa), was expressed as a result of treatment with all SiNPs concentrations and did not appear in the control. The overexpression of some proteins as a result of the three lowest concentrations could be due to the fact that the SiNPs interact with cellular proteins such as those involved in the cell division process (Brunner et al., 2006). Considering bands intensity, comparing with the control, there was an increase in the intensity of band No. 9 (MW of ~161 kDa) in protein profile produced by 225, 300 and 425 ppm treatments. Whereas, bands No. 2 and 14 (MW of ~416 and ~73 kDa, respectively) decreased in their intensity as a results of treatment with all concentration of SiNPs, although band No. 2 did not appear in the treatment of 300 ppm. Also, band No. 16; with MW of ~54 kDa, decreased in its intensity as a result of treatment with the concentrations of 375 and 425 ppm, compared to control. The increase in bands intensity could be attributed to gene(s) duplication that resulted from cytological abnormalities induced by application of SiNPs. The presence of bridges and laggards support this conclusion. This conclusion is in agreement with those of Gamal El-Din et al. (1988), they noticed that increasing the number of genes encoding for the different protein subunits through doubling of chromosome number from 12 to 24 in faba bean caused an increase in band intensity. It could be noticeable that the changes in the protein banding patterns did not show the dose-response relationship, but they were more pronounced in the two highest concentrations; 375 and 425 ppm. These alterations in protein expression in the presence of SiNPs provided further evidence of the toxicological effects of these nanoparticles. This result was in agreement with those of Geiser et al. (2005), Park and Park (2009) and Taylor et al. (2010), they reported that nanoparticles can enter into the cytoplasm and cell organelles due to their unique properties.
Based on the available evidence, it could be concluded that, despite the promising applications of SiNPs against faba bean and soybean main insect pests, there are still doubts regarding their safety. As determined by various methods (pollen vitality, germination and shoot length, chromosomal aberrations, genomic template stability and seed storage protein), result of the present study showed that SiNPs has considerable inhibitory effect on seed germination and shoot length at the highest concentration of 425 ppm. Moreover, it increased chromosomal aberrations in faba bean root tip cells to its maximum values at 225 and 300 ppm. Also, SiNPs affected the faba bean DNA as indicated by a decrease in genomic template stability values, especially at the highest concentration; 425 ppm. Alterations in protein expression in the presence of SiNPs provided further evidence of the toxicological effects of these nanoparticles. These changes in the protein banding patterns were more pronounced in the two highest concentrations (375 and 425 ppm). All these results showed that the highest concentrations of SiNPs may exhibit genotoxic effects on the treated plants. Thus, it could be recommended to use SiNPs with concentrations less than 225 ppm which were suitable concentrations in the control of faba bean and soybean main insect pests with less toxic effect on associated predators and plants. On the other hand, more detailed information on the molecular interaction and biochemical machinery that is altered in an organism upon contact with a SiNPs is needed.
SUMMARY This study was carried out at the Experimental Farm and Field Crop Pests Research Laboratory of Plant Protection Research Institute (PPRI), Sakha Agricultural Research Station (SARS), Kafr El-Sheikh, Egypt, as well as the laboratories of Economic Entomology and Genetics Departments, Faculty of Agriculture, Kafrelsheikh University, Egypt. Faba bean (Sakha 1 variety) as well as soybean (Crawford variety) was chosen to be relatively susceptible to infestation with insect pests. Six different concentrations of SiNPs (75, 150, 225, 300, 375 and 425 ppm) were tested against faba bean (A. craccivora and L. trifolii) and soybean (S. littoralis) main insect pests.
The aim of this study was to: 4. Evaluate the efficiency of SiNPs against faba bean and soybean main insect pests. 5. Study the effect of SiNPs on the associated predators. 6. Investigate the biosafety and genetoxicity of SiNPs on faba bean plants through cytological, molecular and biochemical analyses. Efficiency of SiNPs against insect pests was determined under field conditions for both faba bean main insects, while it was evaluated under two different experimental conditions (field and laboratory) for soybean insect. Filed experiments were carried out during the two growing seasons; 2012/13 and 2013/14 for faba bean as well as 2013 and 2014 for soybean.
The obtained results can be summarized as follows:
I. Efficiency of SiNPs against insect pests: 1- The highest reduction percentages in A. craccivora numbers were observed at 375 and 425 ppm SiNPs in both 2012/13 and 2013/14 seasons. The recorded mean values in 2012/13 season were 98.31 and 99.06 % at concentrations of 375 and 425 ppm, respectively. While in 2013/14 season, these values were 98.91 and 99.06 % for the same two concentrations, respectively. 2- For reduction percentages of L. trifolii larvae, the highest recorded mean values in 2012/13 season; which did not differ significantly, were 88.10, 86.63, 89.17 and 88.99 % for SiNPs at 225, 300, 375 and 425 ppm, respectively. While, the highest mean value in 2013/14 season was 86.81% at 425 ppm followed by the mean values of 81.60, 84.91 and 83.20 %; which did not differ significantly at 225, 300 and 375 ppm, respectively. 3- Concerning efficiency of SiNPs against S. littoralis under field conditions, the highest reduction percentages in the larvae number (86.46 and 87.23%, which did not differ significantly) were observed at 375 and 425 ppm, respectively; during 2013 season, followed by 300 ppm (82.54 %). The same three concentrations; 300, 375 and 400 ppm, revealed the highest reduction percentagse during 2014 season, with no significant differences. 4- With respect to effect of SiNPs on S. littoralis under laboratory conditions, the mortality percentage increased significantly as the concentration increased. However, insignificant difference was recorded between the two highest concentrations; 375 and 425 ppm (94.67 and 95.33%, respectively). Also, highly significant differences were observed among SiNPs concentrations for biological aspects of S. littoralis (larval duration, pupal period, number of eggs laid per female and hatchability percentage).
II. Effect of SiNPs on predators associated with insect pests: 1- Predators number in faba bean field was significantly reduced in dosedependent manner till 300 ppm concentration. The lowest reduction percentage was observed at 75 ppm with an average of 58.89 and 67.39 % for the two season; 2012/13 and 2013/14, respectively. On the other hand, the highest reduction percentages were recorded at 300, 375 and 425 ppm; which did not differ significantly, with mean values of 97.42, 96.61 and 96.61% in 2012/13 season, respectively. These values reached 100 % in 2013/14 season for the same three concentrations. 2- Results obtained for predators in soybean field were roughly the same. The lowest reduction percentage was observed at 75 ppm with mean values of 57.24 and 55.65 % for 2013 and 2014 seasons, respectively. In contrast, applications of SiNPs at 300 and 425 ppm gave the highest reduction percentage in predator numbers with no significant differences. These values were 79.87 and 79.81 % during 2013 season, and 78.96 and 76.54 % during 2014 season at the concentrations of 300 and 425 ppm, respectively.
III. Toxicity of SiNPs on faba bean plants: 1- All SiNPs applications significantly increased faba bean pollen vitality, except 300 ppm which did not differ significantly than control. On the other hand, all the other concentrations of 75, 150, 225, 375 and 425 did not differ significantly than each other. 2- For seed germination and shoot length, all concentrations of SiNPs; except the highest concentration 425 ppm, significantly increased seed germination compared to control. The maximum value of 96.67% was observed at 75 ppm. While, the concentration of 425 ppm significantly inhibited the seed
germination (60.00 %) compared to the control (76.67%). With regard to shoot length, the concentrations of 75 and 150 ppm did not differ significantly than control, while the highest value (8.09 cm) was observed at 300 ppm concentration. Then, shoot length was decreased again as concentration increased, since it reached 4.52 cm at 425 ppm. 3- Concerning cytological analysis, all SiNPs concentrations caused significantly increase in faba bean mitotic indices compared to control (10.49 %). The highest value of 30.05 % was recorded at 75 ppm. Values of mitotic index did not differ significantly at 150, 225, 300 and 375 ppm, which decreased significantly compared to 75 ppm. Also, SiNPs exhibited an effect on the percentage of the different mitotic stages, where the percentage of prophase increased with a corresponding decreased in the percentages of the other stages. 4- Cytological analysis of root tip cells revealed a universal increase in the percentage of abnormalities after all SiNPs applications as compared to their respective control (1.22 %). The frequency of abnormalities was significantly increased as the concentration of SiNPs increased, except the concentrations of 375 and 425 ppm which significantly decreased the abnormality percentages again compared to the other SiNPs concentrations. The maximum percentages of aberrations (44.17 and 43.72%) were recorded at 225 and 300 ppm SiNPs, respectively, which did not differ significantly. Treatment of faba bean with SiNPs revealed various types of chromosomal abnormalities; the most frequent aberrations were Cmetaphase, disturbance, stickiness, bridges, laggards and fragments. 5- Concerning the molecular studies, RAPD-PCR analysis for six oligonucleotide primers revealed differences between control treatment and
different SiNPs concentrations, with visible changes in the number and the intensity of amplified DNA fragments. The highest changes in bands numbers (both appearance of new bands or disappearance of normal bands) was appeared in faba bean treated with the highest concentration of SiNPs (425 ppm). On the other hand, an increase in band intensity was the major event arising in the DNA patterns treated with concentrations of 75, 150, 225 and 300 ppm. With respect to Genomic template stability (GTS) test; which performed for the qualitative measurement of changes in RAPD profiles, all SiNPs concentrations showed low GTS compared to control. The highest concentration; 425 ppm, exhibited the lowest value of GTS (57.89 %). 6- For seed storage protein, changes in protein patterns were limited comparing with the control. The recorded changes were expressed as variations in the number of separated bands; disappearance or appearance of certain bands, and alterations in band intensity. The total number of bands was 27 bands. Seed proteins profile of the control was found to have 25 bands, out of them 22 bands were monomorphic. Changes observed in the protein banding patterns did not show the dose-response relationship, but they were more pronounced in the two highest concentrations (375 and 425 ppm) than the other treatments. Finally, it could be concluded that; despite the promising applications of SiNPs against faba bean and soybean main insect pests, the highest concentrations may exhibit toxic or genotoxic effect on associated predators as well as the treated plants. Thus, it could be recommended to use SiNPs with concentrations less than 225 ppm which were suitable concentrations in the control of faba bean and soybean main insect pests with less toxic effect on associated predators and plants.
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الملخص العربي أجريت هذه الدراسة في المزرعة البحثية وكذلك معمل بحوث آفات المحاصيل الحقلية بمعهد بحوث وقاية النباتات -محطة البحوث الزراعية بسخا – كفر الشيخ ،باإلضافة إلى معامل قسمي الحشرات اإلقتصادية والوراثة بكلية الزراعة -جامعة كفرالشيخ -مصر. تم أختيار صنف الفول البلدي (سخا )1وصنف فول الصويا (كراوفورد) الحساسة لإلصابة باآلفات الحشرية الختبار ستة تركيزات مختلفة من جزيئات النانوسيليكا (،011 ، 227 ، 171 ، 57 057و 527جزء في المليون) ودراسة فاعليتها ضد اآلفات الحشرية الرئيسية لمحصولى الفول البلدي (من اللوبيا A. Craccivoraو صانعة أنفاق أوراق الفول البلدي )L. trifoliiو فول الصويا (دودة ورق القطن .)S. littoralis
تمت هذه الدراسة بهدف : -1تقييم فاعلية جزيئات النانوسيليكا ضد اآلفات الحشرية الرئيسية التي تهاجم محصولي الفول البلدي وفول الصويا. -2دراسة تأثير جزيئات النانوسيليكا على المفترسات الحشرية المرتبطة بتلك اآلفات. -0دراسة اآلمان الحيوي والسمية الوراثية لجزيئات النانوسيليكا على نباتات الفول البلدي من خالل التحليالت السيتولوجية والجزيئية والبيوكيميائية. تمت دراسة فاعلية جزيئات النانوسيليكا ضد آفتى الفول البلدي تحت الظروف الحقلية خالل موسمي 10/2112و ،15/2110بينما تم تقييم فاعليتها ضد دودوة ورق القطن التي تهاجم فول الصويا تحت كال من الظروف الحقلية (خالل الموسمين الزراعيين 2110و )2115والظروف المعملية.
وتم تلخيص النتائج المتحصل عليها فيما يلي:
أوال -فاعلية جزيئات النانوسيليكا ضد اآلفات الحشرية: -1لوحظت أعلى نسبة خفض في أعداد من اللوبيا A. craccivoraعند تركيزات النانوسيليكا 057 و 527جزء في المليون في كال موسمي الدراسة .حيث كانت متوسطات قيم الخفض خالل الموسم 10/2112هى 89،01و % 88،10عند التركيزات 057و 527جزء في المليون على الترتيب ،كما وصلت قيم الخفض خالل الموسم 15/2110عند نفس التركيزات الى 89،81و 88،10على الترتيب. -2فيما يخص نسب الخفض في تعداد يرقات صانعة أنفاق أوراق الفول البلدي ، L. trifoliiفقد كانت أعلى المتوسطات والتي لم تختلف معنويا هى 98،15 ، 90،00 ، 99،11و % 99،88عند تركيزات السيليكا 057 ، 011 ، 227و 527جزء في المليون على الترتيب وذلك خالل موسم .10/2112في حين وصلت أعلى قيمة لمتوسط الخفض خالل موسم 15/2110إلى % 90،91 عند التركيز 527جزء في المليون والتي تالها القيم 95،81 ، 91،01و % 90،21التي لم تظهر إختالفات معنوية عند التركيزات 011 ، 227و 057جزء في المليون ،على الترتيب. -0بالنسبة لفاعلية جزيئات النانوسيليكا ضد يرقات دودة ورق القطن S. littoralisتحت الظروف الحقلية على نباتات فول الصويا ،فقد لوحظت أعلى نسب الخفض في تعداد اليرقات ( 90،50و - )% 95،20والتي لم تختلف معنويا -عند التركيزات 057و 527جزء في المليون على الترتيب ،وتبعها القيمة % 92،75عند التركيز 011جزء في المليون ،وذلك خالل موسم .2110أيضا اظهرت نفس التركيزات الثالثة ( 011 ، 057و 527جزء في المليون) أعلى نسب الخفض في تعداد اليرقات -والتي لم تختلف معنويا -خالل موسم .2115 -5وفيما يتعلق بتأثيرات جزيئات النانوسيليكا ضد يرقات دودة ورق القطن S. littoralisتحت الظروف المعملية ،فقد زادات نسبة الموت معنويا بزيادة التركيز .بينما لم تسجل إختالقات معنوية بين التركيزين 057و 527جزء في المليون ( 85،05و % 87،00على الترتيب) .أيضا لوحظت إختالفات عالية المعنوية بين تركيزات النانوسيليكا المستخدمة عند دراسة بعض النواحي البيولوجية لآلفة مثل :مدة الطور اليرقي ،فترة طور العذراء ،عدد البيض التي تضعه كل أنثى باإلضافة الي نسبة فقس البيض.
ثانيا -تأثير جزيئات النانوسيليكا على المفترسات الحشرية المرتبطة باآلفات الحشرية:
-1لوحظ إنخفاض تدريجي معنوي في عدد المفترسات المرتبطة بمن اللوبيا A. craccivoraوذلك بزيادة تركيز جزيئات النانوسيليكا حتى التركيز 011جزء في المليون .وقد سجلت أقل نسبة خفض عند التركيز 57جزء في المليون بمتوسط 79،98و % 05،08خالل موسمي الدراسة 10/2112 و 15/2110على الترتيب .على الجانب األخر ،سجلت أعلى متوسطات لنسب الخفض (، 85،52 80،01و - )% 80،01والتي لم تختلف معنويا -عند التركيزات 057 ، 011و 527جزء في المليون على الترتيب خالل موسم .10/2112بينما وصلت هذه القيم إلى %111خالل موسم 15/2110لنفس التركيزات الثالثة السابقة. -2بالنسبة للمفترسات المرتبطة بدودة ورق القطن S. littoralisعلى نبات فول الصويا ،فقد سجلت أقل نسبة خفض عند التركيز 57جزء في المليون بقيم متوسطات بلغت 75،25
و% 77،07
خالل موسمي الدراسة 2110و 2115على الترتيب .على العكس أدت معامالت النانوسيليكا بالتركيزات 011و 527جزء في المليون إلى إرتفاع نسب الخفض في أعداد المفترسات ألقصاها بدون أية اختالفات معنوية ،حيث سجلت القيم 58،95و % 58،91خالل الموسم ،2110بينما سجلت القيم 59،80و % 50،75خالل الموسم ، 2115على الترتيب.
ثالثا -سمية جزيئات النانوسيليكا على نباتات الفول البلدي: -1أظهرت معامالت النانوسيليكا زيادة معنوية في حيوية حبوب اللقاح عند كل التركيزات المستخدمة، عدا التركيز 011جزء في المليون والذي لم يختلف معنويا عن المعاملة الضابطة .بينما لم تختلف باقي التركيزات معنويا عن بعضها البعض. -2فيما يتعلق بنسبة إنبات البذور وطول المجموع الخضري ،أظهرت النتائج أن جميع تركيزات النانوسيليكا -ماعدا التركيز األعلى 527جزء في المليون -سببت زيادة معنوية في نسبة إنبات بذور الفول البلدي مقارنة بالمعاملة الضابطة .وقد سجلت أعلى قيمة ( )% 80،05عند التركيز 57 جزء في المليون .في حين أن التركيز 527جزء في المليون خفض إنبات البذور معنويا إلى 01،11 %مقارنة بالمعاملة الضابطة ( .)% 50،05وبالنسبة لطول المجموع الخضري ،فإنه لم يختلف كال التركيزين 57و 171جزء في المليون عن المعاملة الضابطة ،في حين سجلت أعلى قيمة (9،18 سم) عند التركيز 011جزء في المليون لتنخفض مرة أخرى بزيادة التركيز إلى 5،72سم عند التركيز 527جزء في المليون.
-0على المستوى الخلوي لوحظ ان جميع تركيزات النانوسيليكا سببت زيادة معنوية في الدليل الميتوزي للفول البلدي مقارنة بالمعاملة الضابطة ( )%11،58حيث سجلت أعلى قيمه ( )%01،17عند التركيز 57جزء في المليون .ولم تختلف قيم الدليل الميتوزي معنويا عند التركيزات 227 ، 171 011 ،و 057جزء في المليون والتي أظهرت إنخفاضا معنويا مقارنة بالتركيز 57جزء في المليون .أيضا أظهرت جزيئات النانوسيليكا تأثيرا واضحا على المراحل الميتوزية المختلفة ،حيث أزدادت نسبة الطور التمهيدي مع حدوث إنخفاض واضح في نسب األدوار الميتوزية األخرى. -5أيضا أظهر التحليل السيتولوجي لخاليا القمة النامية أن جميع تركيزات النانوسيليكا سببت زيادة في نسب الخاليا الغير طبيعية مقارنة بالمعاملة الضابطة ( )%1،22وقد ازدادت نسبة تلك الخاليا معنويا بزيادة التركيز ،فيما عدا التركيزين 527 ، 057جزء في المليون والتي خفضت نسبة الخاليا الغير طبيعية معنويا مقارنة بتركيزات النانوسيليكا األخرى .وقد سجلت النسبة األكبر من الشذوذات الكروموسومية ( 55،15و )% 50،52عند التركيزين 227و 011جزء في المليون على الترتيب واللذان لم يختلفا معنويا عن بعضهما .كما أدت معاملة الفول البلدي بجزيئات النانوسيليكا إلي ظهور أنواع مختلفة من الشذوذات الكروموسوية ،حيث كانت األنواع التالية هي األكثر تكرارا :االستوائي الكولشيسيني ،االضطرابات ،االلتصاقات ،الجسور ،التلكئات والشظايا الكروموسومية. -7وبالنسبة للدراسات الجزيئية ،أظهرتحليل RAPD-PCRلستة من الدالئل الجزيئية وجود أختالفات بين المعامة الضابطة ومعامالت النانوسيليكا المختلفة؛ وظهرت تلك اإلختالفات في صورة تغيرات في عدد وكثافة حزم الـ DNAالمتضاعفة .وقد نتج التغير األكبر في عدد الحزم -سواء ظهور حزمة جديدة أو إختفاء حزمة من المعاملة الضابطة -في نباتات الفول البلدي المعاملة بالتركيز األعلى ( 527جزء في المليون) .على الجانب االخر ،كانت الزيادة في كثافة الحزم هي الحدث األكثر وضوحا في صورة الـ DNAالمعامل بالتركيزات 227 ، 171 ، 57و 011جزء في المليون .وبتقدير ثبات القالب الجينومي ) (GTSوالذي يعتبر مقياس كمي للتغيرات الحادثة في تحليل الـ ، RAPDاظهرت جميع تركيزات النانوسيليكا انخفاض GTSمقارنة بالمعاملة الضابطة .وقد سجلت أقل قيمة للـ )% 75،98( GTSعند أعلى تركيز مستخدم ( 527جزء في المليون). -0ومن خالل تحليل البروتينات المخزنة بالبذرة ،ظهرت تغيرات طفيفة في صورة البروتين لتركيزات النانوسيليكا المختلفة مقارنة بالمعاملة الضابطة .حيث سجلت تغيرات في عدد -ظهور او اختفاء حزمة -وكثافة الحزم المنفصلة .وكان العدد الكلي للحزم هو 25حزمة .أظهرت صورة بروتين
البذرة للمعاملة الضابطة عدد 27حزمة أشتركت منها 22حزمة في كل المعامالت .لم تظهر التغيرات المالحظة في صورة حزم البروتين عالقة تتفق مع التركيزات إال أنها كانت أكثر وضوحا مع أعلي تركيزين ( 057و 527جزء في المليون) مقارنة بالمعامالت األخرى. وعليه يمكن استخالص أنه بالرغم من التطبيقات الفعالة لجزيئات النانوسيليكا في مكافحة اآلفات الحشرية الرئيسية لمحصولى الفول البلدي وفول الصويا ،إال أن التركيزات العالية ربما تظهر تأثيرا ساما على المفترسات الحشرية المرتبطة بها فضال عن السمية الوراثية على النباتات المعاملة .لذا توصي الدراسة بإستخدام جزيئات النانوسيليكا بتركيزات أقل من 227جزء في المليون والتي تعتبر تركيزات مناسبة في مكافحة اآلفات الحشرية الرئيسية للمحصولين بأقل تأثير سام على المفترسات الحشرية والنباتات المعاملة.
