الفهرس 
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Abstract
Cabbage aphid Brevicoryne brassica cause significant damage to many crops of the Brassicaceae. It has agricultural importance because it is a vector of at least 20 viral pathogens that can cause diseases in crucifers. Pesticides are the most methods used in controlling different species of aphids and other pests, in Egypt and other world but pesticide residues have adverse impact on the environment and human health and on non - target species. The parasite Diaeretiella rapae is a very common parasite on aphids and plays an important role in regulating aphid populations. Thus, it is needed to develop new and modern strategies for the management of aphid and conserve the biological agents with the use of modern insecticide formulations based on nano- emulsion essential oils.
Thus, this study was carried out with the objective to provide information on the effects of the insecticides, micro and nano-emulsion oils formulation on D. rapae, and their host B. brassica and their potential use in IPM programs. We hope that the obtained results might exhibit more information that could help to development of an efficient, eco-friendlier and less hazardous products can used in controlling B. brassica, minimize the adverse effect on non-target species, and can use as a good element in brassica pest management. The results of this study can summarize as follows:
5.1. Laboratory studies
5.1.1. Toxicity of three pesticides against adult of B. brassica and parasite D. rapae using a thin film technique.
Results showed insecticides (indoxacarb, imidachloprid, and emamectin benzoate) were rapid and highly toxic, in which compounds gave effects to calculate their LC50 values after 24 hours and LC50 values were (49.28 ± 1.2, 52.02±4.2, and 176.0±63 Ug/cm2, respectively, without a large variation among LC50 values among indoxacarb and imidacloprid insecticide. Emamectin benzoate was the least toxic insecticide with significant differences when comparing its fiducial limits of LC50 with indoxacarb and imidacloprid. The same trend was observed when these insecticides were bioassayed with the same method of application against the endoparasite D. rapae mummies. Indoxacarb appeared to be more effective than imidacloprid and emamectin benzoate LC50 were 62.8±3.6, 73.44±2.46 and 163.50±5.7 Ug/cm2 for indoxacarb, imidacloprid and emamectin benzoate respectively with no significant differences between the values of indoxacarb and imidacloprid and significant differences between them and emamectin benzoate. The calculated selective ratio of these insecticides showed that imidacloprid was the most selective one gave selective ratio 141.7 % followed by indoxacarb (127.4 %), while emamectin benzoate was not selective (92.58%). The application of three tested insecticides, caused satisfactory toxicity in aphid numbers and less toxic on D. rapae mummies when treated with thin layer technique.
5.1.1.2. Toxicity of common insecticides against adult of B. brassica and parasite and selective ratio D. rapae using dipping technique.
When the tested insecticides were bio-assayed with immersion technique against D. rapae the results showed that insecticides were rapid and highly toxic, after 24 hours and the values of LC50 were (1.65± 9.94, 3.16±9.29 and 5.01±9.22 PPM respectively. Emamectin benzoate was the least toxic insecticide. In addition, the toxicity was highly effect when treated with this method against the parasitoids but more less that the toxicity on aphids LC50 values were (27.13± 0.34, 24.17±0.36 and 25.4±0.36) PPM for indoxacarb, imidacloprid and emamectin benzoate respectively. The selective ratios were high when compared with the method of thin layer technique (1644.2%, 764.87, and 506.4%) for indoxacarb, imidacloprid and emamectin benzoate respectively. The slopes of the LDP line for D. rapae were higher than the slopes of LDP line for B. brassicae.
5.1.1.3. Residual toxicity of insecticides to adult of D. rapae (three day-old) after different post-treatment intervals
The residual experiments showed that significant differences between insecticide treatments the corrected mortality percentage were 68.66, 67.66 and 50.00 after one days of exposure and after three days were 28.1, 18.6 and 77.00 for indoxacarb, imidacloprid, and emamectin benzoate respectively. The reproductive capacity of the parasite reduction % were the least one in indoxacarb treatment (30.37%) followed by imidacloprid (47.04%) and the most effective one was emamectin benzoate (52.66%). After three days exposure the least reduction percentage in reproductive capacity of the parasite was in imidacloprid treatment (18.6%) followed by indoxacarb and emamectin benzoate (28.1 and 77.00% respectively. After 7 days residual imidacloprid gave 8.8% followed by emamectin benzoate (22.4%) and the highest one was indoxacarb (52.5%). These results are due to irrespective of the residue age and their oxidative photolysis.
5.1.1.4. IOBC category of the three tested insecticides, to D. rapae
Results concluded that all tested insecticides were classified as IOBC category 2 slightly harmful) except emamectin benzoate after one day post treatment showed moderately harmful category to D. rapae.
5.1. 2. Biochemical studies (Esterase’s activity)
5.1.2.1. esterase’s isozyme analysis:
The patterns of cabbage aphid (B. brassica) treated with different pesticides doses for esterase’s enzyme system showed 2 enzymatic bands in control treatments. Results showed that esterase’s enzyme systems depend on that type of insecticide and the dose of insecticides use the degradation type of the insecticides. In general, it appears three clear dark esterase’s bands in cabbage aphid insects treated with emamectin (0.1ppm) while the other doses gave two bands. Regarding insects treated with imidacloprid, the treatment three esterase’s bands while the other treatment gave 2 bands. Finally, treatment with 0.1ppm of indoxacarb gave two esterase’s bands while the other treatment gave only one clear band.
The esterase’s patterns of D. rapae treated with different pesticides (emamactin, imidacloprid and indoxacarb) Results showed the untreated control insects exhibited only 1 clear dark esterase isozyme band. While as D. rapae treated with emamactin gave generally 6 bands except treatment with 12.5ppm which gave only 5 faint bands. Treatment with 25 ppm gave all fainted bands. While treatments with indoxacarb gave all fainted bands except 25 ppm treatment which gave 1 dark band. Results explained the highly selective effect of the three tested insecticides on the parasite D. rapae results showed that many types of esterase’s enzymes were found in the parasite toxicated with insecticides (6 bands) while it was in the toxicated aphids from two to three bands. Other explanation to the selectivity is bio activation of indoxacarb when ingested at mide gut to N-decarbomethoxyllated S-metabolites, which are potent voltage-dependent sodium channel blockers, is the toxic mechanism of action in insects include sucking insect pests, the higher toxicity relative to indoxacarb, and certainly much more rapidly than in parasite which no ingest the insecticide.
5.1.3. Physical and chemical tests of Nano-emulsions prepared formulations from essential oil.
5.1.3.1. Particle size
Results showed that not all oils had Nano size in the presence of its milky color and without sonication exposure. The particle size of Nano-E was measured by 10.4±0.59, 66.5±0.4, 13.4± 0.63m 64.1± 0.39 and 90.7±0.75 nm for Camphor, Peppermint, Jojoba, Chamomile, and Garlic, respectively. Nano –emulsion formulations respectively. The particle size of Macro-E was 1466± 58, 12840±1.4, 1648± 0.84, 3234±1.1 and 13264± 320 in macro. - E of Camphor, Peppermint, Jojoba, Chamomile, and Garlic, respectively.
5.1.3.2. pH values
Macro-E had higher pH than 7, while Garlic and camphor had maximum pH values (it ranged from 7.4 to 7.8 in Macro-E while it ranged from 6.71 to 7.4 in Nano-emulsions.
5.1.3.3. Electrical conductivity
The EC value ranged from 0.164 to 0.169 m mols /cm (Macro-E) and 0.101 to 0.132 m moles /cm (Nano-E). There was less variation in EC value within Nano-E and Macro-E.
Nano-E was characterized with excellent transparency and higher (UV/VIS) transmission percentage as compared with Mac-E and had milky color and lower transmission percentage ranging from 22.71 to and 5.5 % for Mac. -E while it ranged from 91.32 to 35.0% in Nano- E.
5.1.3.4. Mechanical stability of the formulation:
The results showed the stability of Nano-E toward precipitation after 20, 40, 60 minutes centrifugation. However, higher trace precipitation was recorded after 120 min. Thermal stability in Macro-E and Nano-E was stable at 4±1, 25±1 and 40±1 C° for thirty days.
5.1.3. Toxicity of Macro- emulsion oils (Macro-E) against adult of B. brassica and its parasite D. rapae (M’Intosh) using dipping technique.
5.1.3.1. Toxicity of Macro- emulsion oils (Macro-E) against adult B. brassica using dipping technique.
The results showed the LC50 values of Macro-E of Garlic, Camphor, Chamomile, Jojoba and Peppermint against the cabbage aphid, B. brasscae when applied with immersion technique. after 24 hours and were (1.58±0.21, 5.49±0.20, 3.01±0.19, 1.90±0.22, 7.76±0.65) ppm respectively. No significant differences were observed between LC50 values of Garlic, Camphor, Chamomile, and Jojoba while there were significant differences between them and Macro-E of Peppermint, which was the least toxic oils. The toxicity of Macro-E of all tested oils increased with the increased exposure period as shown in the decrease of LC50 values after 48 hours.
5.1.3.2. Toxicity of Macro-emulsion oils (Macro-E) against parasite D. rapae (M’Intosh) using dipping technique.
Results showed that LC50 values of tested Macro-E of Garlic, Camphor, Chamomile, Jojoba and Peppermint against parasite D. rapae when treated with immersion technique after 24 hours were (6.55±0.47, 5.18±0.49, 5.91±0.50, 7.31 ±0.55and 6.67±0.55), respectively. No significant differences observed between LC50 values of all tested oils. Macro-E of Camphor and Chamomile were the most highly toxic oils. Jojoba was the least toxic oils with no significant differences when comparing its LC50 with the other tested oils.
5.1.3.3. selective toxicity of Macro-emulsion oils (Macro-E) against parasite D. rapae (M’Intosh) using dipping technique.
Results showed that all macro emulsions of tested oils are good selective after 48 hours of exposure while after 24 h. camphor and peppermint oils showed no selective effect (94.35 and 85.95%). The maximum selective ratio was on garlic treatment followed by Jojoba (414.56 and 384.74).
5.1.4. Toxicity of Nano-emulsion oils (Nano-E) against adult of B. brassica and its parasite D. rapae using dipping technique.
5.1.4.1. Bioassay study of Nano-emulsion oils (Nano-E) prepared with the tested oils against adults of B. brassica using dipping technique.
Results showed that LC50 values were (1.09, 3.80, 1.82, 1.34 and 2.45 Garlic, Camphor, Chamomile, Jojoba and Peppermint against B. brasscae when applied with immersion technique respectively after 24 hours of exposure. Nano-E of Garlic, Chamomile and jojoba were the most highly toxic oils Camphor and Peppermint were the least toxic oils with significant differences when comparing its LC50 with the other tested oils. The toxicity of Nano- emulsion oils (Nano-E) of all tested oils increased with the increased the exposure period as shown in the decreasing of LC50 where they were (0.61±0.23, 1.02±0.22, 0.71±0.24, 0.64±0.24, 0.83±0.24). respectively. No significant differences were observed between LC50 values of the tested Nano- E oils after 48 hours of exposure. Also results indicated that Nano-E exhibited higher efficiency than that obtained with Macro-E. Nevertheless, both essential oils formulation exhibited lowest efficiency on aphids and parasites compared with insecticides.
5.1.4.2. Toxicity of Nano-emulsion oils (Nano-E) against parasite D. rapae (M’Intosh) using dipping technique.
Results showed that the toxicity of tested Nano-E of Garlic, Camphor, Chamomile, Jojoba and Peppermint against parasite D. rapae after 24 hours. No significant differences observed between LC50 values of all nano-emulsion of the tested oils. Nano-E of Garlic and Camphor followed by chamomile were the most highly toxic Nano- E oils. Peppermint and Jojoba gave the least toxic Nano -E tested oils.
5.1.4.3. Potentiation effect of using Nano -E of different plant oils on other used by Macro. - E oils:
Data showed advantage percentages of Nano emulsion oils over another Macro. – E after 24 and 48 hours varied according to the plant oil used in the installation of the Nano - E. This advantage could be arranged in descending order as follows, peppermint, 316.7%, Chamomile, 165.4%, Garlic 145%, Camphor144.5% and Jojoba 141.8 after 24 hours of exposure. The highest potential values after 48 h. exposure was Nano- E Jojoba oil (151.6) followed by peppermint, (148.2) Nano E Comphor oil (135.3), Nano E Garlic oil (132.8%), Camommile oil (36.6%). While as against D. rapae the high potential percent was with Jojoba nano-E. oil (173.2%), Garlic oil (165.4). The Least potential percent was (138.1) with nano-E. Peppermint oil. Results indicated that the toxicity of Nano emulsion botanical oils depended on the source of oil, type of formulation (Macro. E or Nano - E and the time of exposure).
5.1.4.4. selective toxicity of Nano-emulsion oils (Nano-E.) against parasite D. rapae using dipping technique.
The data showed that all Nano-emulsion of tested oils are good selective after 24 hours of exposure, the maximum selective ratio was on garlic treatment followed by Jojoba (363.3 and 314.93). These selective percentages were higher after 48 hours (649.18, 331.3, 569.51, 659.38, 581.93% when the aphids were exposed for 48 hours.
4.2. Field studies
5.2.1. Survey of natural enemies associated with cabbage plants in field:
Results revealed the presence of the subsequent predators: - Paederus alfierii, Coccinella undecimpunctata, chrysoperla carnea, Amblyseius sp, Syrphus spp., and the parasites had been D. rapae, Aphidus sp, Trichogrammatoidea and lots of species of true spiders.
4.2.2. Efficiency of the tested insecticides against B. brassicae and beneficial arthropods in the cabbage field.
5.2.2.1. Efficiency of the tested insecticides against B. brassicae in cabbage field.
Results showed the reduction percent of various stages of B. brassicae after 24 confirmed significant variations among insecticides. The maximum potent insecticide was emamectin benzoate (83.8 reduction %) as compared with control followed by imidacloprid (81.6 R %) the least toxic towards cabbage aphids was indoxacarb (73.7%) after 24 hours.
5.2.2.2. Field evaluation of the tested insecticides against parasite D. rapaea and their residuals after 3, 5, and 7 days in a cabbage field.
Data showed that emamectin benzoate was the best at the parasite with an avg. reduction percentage of 88.6% followed by imidacloprid and the least toxic one was indoxacarb gave a discount percentage of 49.5%. Accordingly, indoxacarb had a degree of selectivity according to the Metcalf scheme (selective). Moreover, results of the greenhouse showed that it has IOBC class 2 (slightly harmful). Thus, indoxacarb can use in cabbage aphid integrated control in integration with parasite D. rapae.
5.2.2.3. Reduction in beneficial arthropods and selectivity in cabbage field ecosystem
5.2.2.4. Effect of application of insecticides on beneficial arthropods diversity in Cabbage
Results concluded that among the evaluated insecticides, the maximum efficacious insecticide identified to control B. brassica on cabbage is indoxacarb which may be utilized in cabbage aphid integrated control in integration with parasite D. rapae. It was selective in the field test and medium selective on other beneficial arthropods and had no effect on diversity of beneficial arthropods and equitability in the cabbage ecosystem.
5.2.3. Efficiency of the tested Macro-emulsion oils against B. brassicae and beneficial arthropods in the cabbage field.
5.2.3.1. Efficiency of the tested Macro-emulsion oils against B. brassicae in cabbage field.
Results showed that initial reduction of macro-E formulations after 24 hrs. of Garlic and Camphor (50.6 and 49 percentage) without any significant difference between them) followed by Chamomile 46.7 the least initial effect was observed in treatments of Jojoba, peppermint and chamomile. The average reduction percentages showed that Macro-E Garlic oil was the most toxic (62.7) followed by Chamomile oil (60.00). Another Macro. -E oils (Camphor, Peppermint, Jojoba) were similar in their effect with no significant differences between them.
5.2.3.2. Efficiency of the tested Macro-emulsion oils against D. rapae in cabbage field.
Initial kill of all Macro-E oils is the same where the reduction percentage ranged from 58.8 to 57.5% without any significant difference while the Average reduction after 7 days are differed significantly. Jojoba and peppermint are medium selective while the others are degree selective.
5.2.3.2. Efficiency of the tested Macro-emulsion oils against beneficial arthropods in the cabbage field.
The average reduction percentages from initial effect and residual until 7 days after spray ranged from 48.8 t0 21.3 %. Camphor and Jojoba exhibited the least effect recording average reduction 21.2 and 22.2 Reduction percentage with degree of selectivity are good selective. The other Macro. -E oils showed degree of selectivity as selective.
5.2.3.4. Interspecific diversity and equitability of Entomophagous after application of Macro-emulsion oils in the Cabbage ecosystem.
The Diversity indexes after application of Macro-E were higher when all Macro-E oils applied except Camphor Macro- E. The value of equitability reached an environmental maximum; it did not differ from check treatment significantly. Results indicated the selectivity properties of these formulations from essential oils.
5.2.4. Efficiency of Nano-emulsion (Nano-E.) against B. brassicae and beneficial arthropods in the cabbage field.
5.2.4.1. Efficiency of the Nano-emulsion (Nano-E.) against B. brassicae cabbage field
Results showed that initial reduction of these formulations after 24 hrs. Garlic and Camphor showed (61.9 and 60.6 reduction percentage respectively without any significant difference between them followed by Chamomile 54.5 R percentage. The average reduction percentages showed that Nano-E of Garlic oil was the most toxic recording (76.9 R. %) followed by Camphor oil (73.8%). Another Nano. -E oils of Chamomile, Peppermint, and Jojoba were similar in their effect with no significant differences between them.
5.2.4.2. Efficiency of the Nano-emulsion (Nano-E.) against D. rapae in cabbage field
Initial kill of all Nano-E oils of Garlic, Chamomile and Peppermint gave the highest reduction percentage against D. rapae (76.00, 73.9 and 73.8 respectively) without any significant difference between them. The highest avg. reduction percentage observed in Nano– E. Jojoba oil treatment 72.5% followed by peppermint 69.5%. Their degree of selectivity was medium selective.
5.2.4.3. Efficiency of the Nano-emulsion (Nano-E.) against beneficial arthropods and non- target species in the cabbage field.
Results indicated that the tested Nano. E. oils did not completely reduce the beneficial arthropods numbers. The initial effect ranged from 59.1 to 65.00% reduction. The average reduction percentages of residual until 7 days post
spray ranged from 47.6 t0 59.9 %. Camphor exhibited the least effect recording average reduction 47.6 Reduction percentage with degree of selectivity selective degree. The other Nano-E oils showed degree of selectivity as medium selective according to scheme of (Metcalf 1973).
5.2.4.4. Interspecific diversity and equitability of Entomophagous after application of Nano-emulsion oils in the Cabbage ecosystem.
The Diversity indexes after application of Nano-E were exactly with maximum diversity in control treatment and not significantly differed than before treatment account. There are no significant differences between the values pre and post spray and did not differ than check treatment significantly. Results indicated the selectivity properties of these formulations from essential oils.
Our findings suggested that Nano-E of essential oils can successfully control B. brassicae and its application deserves to be considered as a potential tool for cabbage Integrated Pest Management.
“Plant essential oils (EOs) are eco- friendly control tools mainly due to their rapid biodegradability, low resistance phenomena and negligible toxicity towards non-target organisms”
The nano-formulations we have developed show an interesting potential to control B. brassicae, and their effectiveness can attribute both to the essential oils used and to the formulations themselves. Garlic Camphor and Jojoba essential oil may have contributed to the high efficacy of the insecticide formulations against B. brassicae emphasized the good insecticidal activity of the developed formulation in controlling B. brassica and selective or moderate selective activity against beneficial arthropods.