الفهرس 
Biological studiesOnly 14 pages are availabe for public view
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Abstract
Callosobruchus phaseoli (Gyllenhal) (Coleoptera: Bruchidae) was found and recorded for the 1st time in Israel in 1987, where it was infesting seeds of Dolichos lablab. This insect can infest many legume seeds like cowpea, broad bean, chickpea, lentils and mungbean. Also, C. phaseoli was found in Al-Hadiqa Al-Dawlia, Alexandria, Egypt and causes damage to lablab seeds.
C. phaseoli individuals were collected from lablab seeds, although this species was not believed to be present like an economic pest in Egypt and is not considered as a pest of legume seeds, but it may reach the level of a key insect-pest in the future if it was neglected. The early C. phaseoli infestations of some legumes could probably be due to cross- infestation from lablab that seeds may be stored near or in the same warehouse with other legumes.
The objectives of the present study were to
1. Study the biology of C. phaseoli on different legume seeds.
2. Evaluate the insecticidal efficacy of certain botanical oils and pure compounds admixed with different legume seeds against C. phaseoli.
3. Estimate the joint action of malathion combined with certain botanical oils and pure compounds.
4. Evaluate the residual effect of botanical oils and pure compounds after storing legumes for different periods (1 and 2 months).
5. Assess the residual effect of botanical oils and pure compounds on physical and viable properties of legume seeds such as swelling and germination.
6. Study the efficacy of certain botanical oils and pure compounds (peppermint oil, eucalyptus oil and eugenol) on some biochemical systems (enzymes) of C. phaseoli adults with primary propose of explaining the mode of action of natural compounds.
The obtained results could be summarized as follows:
1-Biological studies
1.1-Susceptibility of some leguminous seeds to the infestation by Callosobruchus phaseoli adults (choice and no-choice tests)
Results of the choice test indicated that the mean number of deposited eggs /female was greatest with lablab and cowpea, successively. The lower mean number of deposited eggs was 2.60 and 22.73 eggs /female in case of lentils and broad bean seeds. No hatched eggs were observed with lentil and kidney bean seeds. Moreover, the tested legume seeds could be arranged discendingly according to the percentages of the weight loss as follows: lablab> cowpea> broad bean> chickpea> lentil and kidney bean, respectively.
Results of the no-choice test indicated also that maximum mortality of 48.01% was obtained after one week from placing the insects on Kidney bean seeds, but lower mortality of 1.08% was observed on lablab seeds. The highest number of eggs was recorded on lablab followed by cowpea. The results also showed that the weight loss increased as the infestation increased.
2.1.2-Effect of the maternal and paternal age of C. phaseoli on offspring life-span and the daily number of deposited eggs in relation to the type of legume seeds
The offspring of younger parents (0-1day old) was found to take significantly longer time than others resulting from older parents (1-2 and 2-3 days old) in different hosts, e.g. lablab, cowpea, broad bean, and chickpea. Generally, the generation of C. phaseoli decreased as the maternal and paternal age increased. The life-span was shorter (38.15 days) in cowpea followed by chickpea, lablab and broad bean, (39.40, 39.88 and 41.44 days, in respect).
Concerning the maternal and paternal age effect on the number of daily offspring eggs of C. phaseoli, the greatest mean number of eggs was shown with younger parents. Also, it was found that the greatest mean number of eggs was noticed in the first day of oviposition period (17.77 and 12.22 eggs/female) on lablab and cowpea. The mean number of eggs decreased as the ovipostion period increased. The highest ovipostion period (10 days) was registered on lablab, while it was decreased to 6 days cowpea and to 4 days for either broad bean or chickpea. Female fecundity has been affected significantly by the type of the host (legumes).
1.3-Relationship between C. phaseoli eggs density on the tested seeds and hatchability percentage
The number of deposited eggs increased as the seed size increased. Lablab seeds harboured less number of eggs compared with the comparatively big sized seeds of broad been and chickpea that bear more eggs. Hatchability percentage was found to be decreased as the egg density on seeds increased for all the tested hosts.
1.4-Oviposition preferences of C. phaseoli females offered seeds bearing different numbers of emergence holes
Female preferred clean seeds for oviposition than those already have different bearing emergence holes. The greatest number of eggs was laid on clean cowpea seeds were 7.66-11.00 eggs and 7.66 eggs on average were laid on cowpea seeds with zero-one bearing hole. There was a reduction in total laid eggs as the number of holes increased and the mean number of laid egg was greatest on lablab followed by cowpea, broad bean and chickpea in descending order.
2-Toxicological studies
2.1- Residual film technique
The insecticidal efficacy of the tested oils (peppermint oil, eucalyptus oil, clove oil) and bioassayed after 72hrs showed that peppermint oil was highly toxic (LC50= 0.161%) followed by eucalyptus oil (LC50= 0.212%), while clove oil showed low toxicity (LC50= 0.452%) against C. phaseoli.
The results of LC50 values of pure compounds (eugenol, menthol) indicated that eugenol was more effective (LC50= 0.196%) than menthol, which gave LC50 value of 0.448%.
2.2- Admixture of certain tested materials with legume seeds
2.2.1 -Admixing with cinnamon dust
Cinnamon powder was more effective on treated cowpea seeds when it was tested against C. phaseoli. The highest concentration of 30 mg/kg gave higher reduction percentage of emerged adults of F1 calculated by 88.80%. The loss of cowpea seeds was also high at that higher cinnamon concentration and lower percentage of 11.28% in the second generation.
2.2.2-Effect of peppermint oil on C. phaseoli adults on different legume seeds
Peppermint oil achieved the higher efficacy to control legume seeds (lablab, cowpea, broad bean and chickpea) infested by C. phaseoli and therefore, it can be used for controlling this insect-pest at a higher dose that ranging between 0.3 - 0.5%. The insect showed higher sensitivity when broad bean seeds treated with peppermint oil and no emerged adults were observed at concentration of 0.3% followed by lablab and chickpea 0.4%, in respect. On the other hand, the insect showed lower sensitivity to peppermint oil when it was admixed with cowpea seeds. No emerged adults were observed when the adult parents exposed to a concentration of 0.5% peppermint oil.
2.2.3-Effect of Eucalyptus oil on C. phaseoli adults on different legume seeds
Maximum mortality of 100% of eucalyptus oil admixed with chickpea and broad bean seeds were gained at the concentrations of 0.4% and 0.5%, respictively. Eucalyptus oil gave moderate effect of 80.33% and 78.33% mortality at the concentration of 0.5% with lablab and cowpea seeds, respectively. Complete protection of lablab, cowpea, chickpea and broad bean was achieved after treating the seeds with eucalyptus oil at concentrations of 0.6, 0.5, .0.4 and 0.4%, respectively.
2.2.4-Effect of clove oil on C. phaseoli adults on different legume seeds
Clove oil treatment was found to have a weak effect when it was used at the concentration of 0.5% that has been admixed with all legume seeds, except for lablab seeds. At dose of 4.00% clove oil, the observed mortality after one week of treating cowpea seeds reached 96.66%. Complete mortality of C. phaseoli individuals was found after exposing them to broad bean and chickpea admixed with 1% and 1.5% of clove oil, in respect.
Reduction of adults emergence reached100% at concentration of 3% of clove oil admixed with cowpea seeds. Complete reduction was also observed at the concentration of 0.8% with broad bean and chickpea. It was found that the second emerged generation consumed more seeds in high seeds loss due to the slight effect of the tested concentration.
2.2.5-Effect of eugenol on C. phaseoli adults on different legume seeds
Data showed that eugenol gave 100% mortality after one week exposure period, when it was admixed with all tested legumes seeds at concentration of 0.6%. Again, the highest tested concentration (0.6%) gave good protection against the tested insect and showed the insecticidal potency of eugenol when it was admixed with all tested legumes seeds.
2.2.6-Effect of menthol on C. phaseoli adults on different legume seeds
No emerged adults of both first and second generations were found at the concentrations of 0.8, 1.0, 0.6 and 1.0% of menthol admixed with lablab, cowpea, chickpea and broad bean, respectively. Weight loss percentage of treated legume seeds with menthol after the emergence of the second generation was approximately 2 fold as that of the first generation. So, menthol can be used at higher concentrations (0.8% and more) to achieve complete protection to legume seeds infested by C. phaseoli.
2.2.3- Joint action of malathion combined with certain botanical oils and pure compounds against C. phaseoli adults at different legume seeds
The mixtures of malathion (at LC25) and either peppermint or clove oils (at LC25) were the most effective mixtures tested in all varieties of the tested seeds giving observed mortality of 100% and co-toxicity factor of 100(=potentiation). Therefore, malathion can be admixed with botanical oils and pure compounds to be effective protectants against C. phaseoli.
2.4-Residual effect of the tested compounds on eggs deposition and emergence of C. phaseoli adults after seeds storage
The highest mortality percentages after a storage period of one month were 30.00, 75.00, 100.00 and 100.00% and they were recorded for those treated stored seeds of lablab, cowpea, broad bean and chickpea, respectively admixed with peppermint oil after one week exposure period followed by eucalyptus oil. Conversely, menthol showed the lowest mortality percentages of 7.03, 38.33, 68.33 and 10.00% for those treated seeds of lablab, cowpea, broad bean and chickpea, respectively. Complete adults reduction was observed with stored treated seeds with peppermint oil except lablab (91.02%), but lower reduction percentages were observed with those seeds treated with clove oil. Peppermint oil and eucalyptus oil caused long protection of legume seeds after a storage period of one month than the other tested compounds.
The highest mortality percentages were achieved with stored peppermint oil treated legume seeds after a storage period of two months. Also, maximum reduction percentages of emerged adults were observed with peppermint oil and eucalyptus oil (68.64, 68.51, 100.00 and 100.00%) and (63.55, 41.39, 87.16 and 96.44%) with lablab, cowpea, broad bean and chickpea, respectively. Lower reduction percentages were found with clove oil and eugenol.
Protective effect of the tested botanical oils and pure compounds against C. phaseoli was decreased as the time of storage increased. Clove oil achieved low protection effect after a storage period of one month and no protection effect after two months storage. On the other hand, peppermint oil and eucalyptus oil showed longer protection of legume seeds stored for one and two months.
The botanical oils and pure compounds caused high efficacy as insecticides against the adults of the tested insect, therefore, these compounds could be used before storing the seeds in warehouses.
2.5-Residual effect of the tested botanical oils and pure compounds on legume seeds swelling
Peppermint oil achieved highest swelling percentage of the selected and examined legume seeds (lablab, cowpea and broad bean) followed by eucalyptus oil, while menthol showed the lowest swelling percentage with all legume seeds, except for lablab seeds. Thus, the evaluated essential oils and pure compounds admixed with legume seeds showed positive effect on seeds swelling. These treated seeds be suitable for human consumption.
2.6-Residual effect of botanical oils and pure compounds on legumes seeds Germination
The tested compounds caused an adverse effect on lablab seeds germination, while these compounds had a positive effect on seed swelling. After period of storage (5 weeks), peppermint oil affected seed germination, but by the end of storage periods (8 weeks) cowpea seeds germination was increased to 100%. Peppermint and clove oils did not affect significantly, cowpea seeds germination if they were treated with either tested concentrations and they germinated after 8 weeks post-treatment. Peppermint oil and clove oil are suitable for the protection of cowpea seeds against insect infestation without adverse effect on seeds germination.
Eugenol showed the lowest germination percentage of those treated stored cowpea and broad bean seeds at the initial period of storage (5 weeks), nevertheless at the end of storage period (8weeks), germination reached 99.11% and 100%. It was also found that clove had no adverse effect if the seeds admixed with it at the tested concentrations of 0.5 or 0.75% and stored for 8 weeks.
All the evaluated materials caused no harmful effect and no adverse effect on chickpea seeds germination.
3- Biochemical Studies
3.1- In vivo effect of peppermint oil, eucalyptus oil and eugenol on the AST/GOT activity of C. phaseoli
The activity of ASpartate aminoTransferase (AST) was found to be increased as the concentration of peppermint oil, eucalyptus oil and eugenol increases. Also, the activity of AST was highest (89.40%) after exposing the adult to eucalyptus oil but it was (46.63) lowest with eugenol at (1/10) LC50. No significant differences were found between control and LC50 of peppermint oil. In case of eugenol, there were no significant differences in the activity of AST between (1/10) LC50 and (1/5) LC50 treatments.
3.2- In vivo effect of peppermint oil, eucalyptus oil and eugenol on the ALT/GPT activity of C. phaseoli
The activity of ALanine AminoTransferase (ALT) was increased as the concentration of the tested peppermint oil increased. Eugenol achieved the highest activity of 98.44% followed by eucalyptus oil (96.14%) and peppermint oil (74.54%) after exposing the adults to LC50of each.
3.3-In vivo effect of peppermint oil, eucalyptus oil and eugenol on the Alkaline Posphatase (ALP) activity of C. phaseoli
The inhibition of ALkaline Posphatase (ALP) was highest with the treatment of eucalyptus oil followed by peppermint oil and eugenol.
3.4-In vivo effect of peppermint oil, eucalyptus oil and eugenol on the Mixed Function Oxidases (MFO) activity of C. phaseoli
The exposure of C. phaseoli to different doses of peppermint oil and eucalyptus oil and Eugenol decreased the activity of Mixed Function Oxidases (MFO). Eugenol at the dose of LC50 increased the activity to be more than that of control giving an increase of 45.51% over the control (100%). Eugenol caused high MFO activity followed by eucalyptus and peppermint oil. MFO of C. phaseoli was still susceptible to both peppermint oil and eucalyptus oil, but, a little bit more resistant to eugenol.
from the previous results, the following points could be taken into consideration
1- C. phaseoli is an important insect because, it can infest legume seeds like cowpea, broad bean chickpea seeds, and therefore, the biologicak studies have been done in case of infestating legume seeds in storeds.
2- Admixing the botanical oils and pure compounds with leguminous can be adequate for protection of legume seeds from the infestation by the lablab beetle, since the tested oils and pure compounds prevent oviposition, egg hatching and adult emergence. Moreover, they are very safe for human and environment.
3- C. phaseoli are susceptible to botanical oils and pure compounds.
4- Admixing malathion with botanical oils and pure compounds can be an effective method for the application in warehouses before storage.
5- The tested compounds act as contact poisons, affecting certain enzymes (ALP and ALT) the respiration process.
6- If it is possible, the eradication of those lablab plants in Al-Hadika Al-Dawlia would be better after saving some seeds to be sterilized and grown later on, if necessary.
Future look
More attention must be taken in the future
• Mainting stock culture of the assigned insect to resume ecological, behaverioral and other studies.
• The obtained results of this thesis can be considered as a message to the caratine officers to check carefully the imported pulses.
• The contiuous survey of C. phaseoli in different rural places, of Eygpt, where cowpea crop in planted.
• Checking-up pulses in stores every three mounths as a protective procedure to prevent the insect spread.
• Many farmers plant lablab as on or namental shrub, these shurubs must be dislodeged.
• Another precaution should be arministered, that is not to raise cowpea adjacent to lablab farms, especially Upper-Egypt.
• Intiating IPM program for the insect, in case of its spreadimg as a key pest infestation important pulse in the stores.