الفهرس 
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Abstract
The present experiments were conducted in order to study some pathogens that infect the mulberry silkworm Bombyx mori, by isolating and defining them phenotypic and genotypic, and studying the influence of some natural and chemical compounds to control these pathogens to reduce the damage caused by them.
The obtained results could be summarized as follows:
5.1. Fungal diseases
5.1.1. Isolation, characterization and identification of the fungal pathogens
Two different fungal isolates (SW1 and SW2) belonging to genus Aspergillus were obtained. The classification was adopted according to the cultural and morphological properties of the fungal isolates. Both isolates showed granular colonies on PDA, both isolates were identified as Aspergillus fumigatus SW1 and Aspergillus flavus SW2.
5.1.2. Pathogenicity test for fungus strains
When the different stages of the silkworm were inoculated with A. fumigatus and A. flavus, a great similarity was found in the pathological symptoms that appear on the insect in all its stages. The pathological symptoms that appeared on the infected larvae for both species were recorded as follows:
5.1.2.1. First instar larvae
Newly hatched silkworm larvae inoculated with A. fumigatus and A. flavus separately died within 48 hrs. after inoculation without any external disease symptoms, as the larvae became dry and stuck to mulberry leaves. On dead larvae mycelial cluster developed quickly and formed conidia after few hours from death.
5.1.2.2. Second instar larvae
The second instar larvae did not show any external disease symptoms at the beginning of the infection, but larvae became brown during the fasting period and failed to molt, and most of the larvae died after three days of inoculation.
5.1.2.3. Third instar larvae
After 72 hrs. of inoculation, third instar larvae became slow moving and lost their appetite and the end-abdominal area became fossilized and black in color and the larvae died within few hours.
5.1.2.4. Fourth instar larvae
Newly molted fourth instar larvae showed small black spots at the end of the abdomen, these spots gradually increased in size and this area of the body became fossified, and larvae died within 24-48 hrs. In some larvae, the rectum was released out of the end of the abdomen during the fasting period, these larvae were unable to complete the molting process and died. Some of the larvae succeeded in completing the molting but these larvae died within 2-3 days from the beginning of the fifth larval instar.
5.1.2.5. Fifth instar larvae
Newly molted fifth instar silkworms had black spots on head, end of the abdomen, abdominal legs, and the respiratory stomata. These larvae died within few days. Some infected larvae succeeded in spinning their cocoons, but these cocoons were fragile, and the larvae died in the pre-pupal and pupal stage, some infected pupae reached the stages of adults, but these adults were deformed.
5.1.3. Effect of inoculating silkworm eggs with different concentrations of Aspergillus fumigatus and A. flavus
1.3.1. External pathological symptoms
When the surface of the egg was contaminated with Aspergillus fungi (A. fumigatus and A. flavus) during the incubation period, the fungus hyphae penetrated the egg shell and then reached the embryo where it penetrated the cuticle and multiplied inside the embryo, then a deformation of the embryo occurred inside the egg and therefore most eggs were not hatched. Some larvae were separated from the egg shell but could not get out and died inside the egg. Some larvae were able to bite the eggshell contaminated with the fungus and came out, but these larvae were infected with the fungus and died immediately after hatching or during the first or second instar.
5.1.3.2. Eggs hatchability
5.1.3.2.1. Aspergillus fumigatus
Inoculating silkworm eggs with different concentrations of A. fumigatus at different incubation periods significantly decreased hatching percentage, reaching 18, 6, 1 and 1% when treating eggs with concentrations 1×105, 1×106, 1×107 and 1×108 conidia/ml after one day of incubation, respectively, while it was in control 90%. When eggs were treated with the fungus three days after incubation, these percentages ranged between 19 and 54%. On the other hand, the percentage of hatching was not affected when eggs were treated after seven days of incubation.
5.1.3.2.2. Aspergillus flavus
Inoculating silkworm eggs with different concentrations of A. flavus at different incubation periods decreased significantly hatching percentage, ranged from 48 to 9%, 63 to 9% and 80 to 58% when treating eggs with concentrations from 1×105 to 1×108 conidia/ml after one, three and seven days of incubation, respectively, while it was in control 90%.
5.1.4. Effect of inoculating silkworm larvae with different concentrations of Aspergillus fumigatus and A. flavus in spring and summer seasons
5.1.4.1. Aspergillus fumigatus
5.1.4.1.1. In spring season
When inoculating first instar larvae with different concentrations (1x108, 1x107, 1x106 and 1x105 conidia/ml) of A. fumigatus, no external symptoms were observed at beginning, but as the disease progressed some larvae lost their appetite and became inactive, the larvae had difficulty molting and died. Mortality was recorded from the eighth day of infection. The mortality percentage continued to increase until it reached 85.7% at the higher concentration and 64.3% at the lower concentration after 26 days of infection.
Silkworm larvae inoculated at the first day of the second instar larvae, recorded low percentage of mortality (4.2%) at concentration 1x108 on the tenth day of infection. Mortality increased gradually until the end of the fifth instar, reaching 100% in 1×108 and 72.7% for 1×105.
When larvae were inoculated at the first day of the third instar larvae, no mortality were recorded during the first 5 days of infection. On the sixth day of infection, low mortality percentage was recorded (14.3%) at the higher concentration of the fungus. Mortality increased gradually until it reached 76.9 and 53.8% for 1×108 and 1×105, respectively after 22 days of infection.
When larvae were inoculated at the first day of the fourth instar larvae, mortality percentages ranged between 5 and 20% after 14 days of infection, most of the larvae died before and during cocoon spinning, where the percentage of mortality reached 88.9% at higher concentration.
When larvae were inoculated at the first day of the fifth instar larvae, on the tenth day of infection, 5% mortality was recorded at the higher concentration only. At the end of the fifth larval instar (12 days after infection) mortality percentage ranged between 5.3 and 21.1% for concentrations from 1×105 to 1×108, while the other larvae were able to spin cocoons, where the percentage of cocooning ranged between 75 and 90%. The percentage of pupation ranged between 75 to 80% in and the percentage of adult emergence ranged between 60 to 75% in concentrations from 1×108 to 1×105. The percentages of cocooning, pupation and adult emergence in the control were 95%.
Although the percentage of cocooning was high when the fifth instar larvae were inoculated with different concentrations of A. fumigatus, but these cocoons were weak and fragile, the statistical analysis showed that there were significant differences in the weight of fresh cocoons and cocoon shells and cocoon shell ratio between the control and different concentrations of the fungus.
5.1.4.1.2. In summer season
When larvae were inoculated with different concentrations of A. fumigatus at the first day of the first instar larvae in summer season, 100% mortality were recorded in all concentrations within 2 to 10 days of inoculation. Also, all larvae died within 6 to 16 days of infection when larvae were inoculated at the first day of the second instar.
When larvae were inoculated at the first day of the third instar larvae, the percentage of mortality reached 100% in all concentrations during 10 to 18 days of infection. When larvae were inoculated at the first day of the fourth instar larvae, the mortality percentage ranged between 94.1 and 100% at the end of the fifth instar larvae (14 days after infection).
When larvae were inoculated at the first day of the fifth instar larvae, the mortality percentage ranged between 33.3 and 66.7% after 8 days of infection (the end of the fifth larval instar). The percentages of cocooning and pupation ranged between 26.7 and 53.3%.The percentage of adult emergence ranged between 20.0 and 53.3%. In the control, the percentages of cocooning and pupation were 80.0% and adult emergence was 73.3%. Statistical analysis showed significant differences in the weight of fresh cocoons and cocoon shells and cocoon shell ratio between the control and different concentrations.
5.1.4.2. Aspergillus flavus
5.1.4.2.1. In spring season
When silkworm larvae were inoculated with different concentrations of A. flavus at the first day of the first instar larvae in spring season, mortality was recorded from the sixth day of infection (the end of the second larval instar), where the percentage of mortality ranged from 13.3 to 46.0%. The mortality rate continued to increase until it reached 100% after 22 days of infection (at the end of the fifth larval instar).
When silkworm larvae were inoculated at the first day of the second instar larvae, low mortality percentages were recorded (4.2 to 16.7%) on the twelfth day of infection, the mortality percentage increased gradually until it reached the highest mortality percentage (77.3 to 90.9%) after 24 days of infection (end of the fifth larval instar).
When silkworm larvae were inoculated at the first day of the third instar larvae, ten days after infection, low mortality percentage (7.7%) was recorded at the higher concentration (1×108 conidia/ml), and death was not recorded in other concentrations. Mortality increased gradually until it reached 66.9 and 83.4% for 1×105 and 1×108, respectively, at the end of the fifth larval instar (24 days after infection).
When silkworm larvae were inoculated at the first day of the fourth instar larvae, the percentage of mortality was recorded after 12 days of infection, where it ranged between 5.0 and 10.0%. Most of the larvae died before and during spinning their cocoons, where the percentage of mortality reached 94.4% in higher concentrations.
When silkworm larvae were inoculated at the first day of the fifth instar larvae, 12 days after infection, the mortality percentages ranged between 5.3 and 31.6%, while the other larvae were able to spin the cocoons. where the percentage of cocooning ranged from 65 to 90%. Some of the larvae died inside the cocoons before turning into pupae, where the percentage of pupation ranged between 65 and 85%. The percentage of adult emergence ranged between 55 and 75%, while the percentages of cocooning, pupation and adult emergence in the control were 95%.
Although the percentage of cocooning was high when the fifth instar larvae were inoculated with A. flavus, these cocoons were weak and fragile. The results of the statistical analysis showed that there were significant differences in the weight of the cocoons and cocoon shells and cocoon shell ratio between the control and all concentrations.
5.1.4.2.2. In summer season
When larvae were inoculated with different concentrations of A. flavus at the first day of the first instar larvae in summer season, 100% mortality were recorded in all concentrations within 4 to 16 days of inoculation. Also, all larvae died within 18 days of infection when larvae were inoculated at the first day of the second instar.
When silkworm larvae were inoculated at the first day of the third and fourth instar larvae separately, the percentage of mortality increased gradually during the fourth and fifth instar until it reached 100% in all concentrations after 14 to 16 days of infection.
When silkworm larvae were inoculated at the first day of the fifth instar larvae, the mortality percentage ranged from 25.0 to 66.3% after 8 days of infection (the end of the fifth larval instar).
The percentages of cocooning and pupation ranged between 26.7 and 60.0%. The percentage of adult emergence ranged between 20.0 and 40.0%. In the control, the percentages of cocooning and pupation were 80.0% and adult emergence was 73.3%. Statistical analysis showed significant differences in the weight of fresh cocoons and cocoon shells between the control and different concentrations. No significant difference in cocoon shell ratio between all concentrations and the control.
5.1.5. Effect of antifungal agents on silkworm larvae infected with Aspergillus fumigatus and A. flavus
5.1.5.1. Aspergillus fumigatus
Dusting silkworm larvae with different concentrations of the fungicide Actamyl, one and 12 hrs. after inoculation with A. fumigatus (1x106 conidia/ml), reduced the mortality and increased the survival percentages. The highest mortality recorded in inoculated control larvae (larvae treated with A. fumigatus without the Actamyl treatment) reached 100% on the fourth day of inoculation. The lowest percentage of larval mortality (21.4 and 33.3%) were recorded when larvae were dusted with 6% of Actamyl after 12 hrs. and one hr. after inoculation.
Dusting silkworm larvae with different concentrations of the salicylic acid, one hr. and 12 hrs. after inoculation with A. fumigatus, reduced mortality and increased the survival percentages. The percentage of mortality in inoculated control larvae was 100% on the fourth day of treatment, while the mortality percentages decreased to 53.3% when the larvae were treated with salicylic acid at 15%, after one hr. of inoculation. Also, the larval mortality decreased to 20.0% after 12 hrs. of inoculation with the fungus.
When propolis powder was used on larvae inoculated with A. fumigatus, it was obvious that the 5% concentration had no significant effect in reducing the percentage of mortality neither 1 h. nor 12 hrs. after inoculation, but the use of the concentration 15% led to a reduction in the percentage of mortality to 33.3% when used 12 hrs. after inoculation.
Using henna powder on the larvae inoculated with A. fumigatus recorded the lowest percentages of mortality (46.7 and 40.0%) and the highest percentage of survival (53.3 and 60.0%) when henna powder was used at a concentration of 15%, after one hr. and 12 hrs. after inoculation with fungus, respectively.
5.1.5.2. Aspergillus flavus
Dusting silkworm larvae with different concentrations of the fungicide Actamyl, one and 12 hrs. after inoculation with A. flavus (1x106 conidia/ml), reduced the mortality percentages. The highest mortality recorded in inoculated control larvae reached 61.1% on the eighth day of inoculation, treatment with 6% Actamyl reduced the percentage of mortality (14.3 & 9.1%) after 1 and 12 hrs. of inoculation, respectively, the larval survival increased from 40.5 to 85.1% when using concentrations from 2 to 6% after 12 hours of inoculation.
The lowest percentage of larval mortality (16.7%) was recorded when larvae were treated with 15% of salicylic acid 12 hrs. after inoculation with A. flavus. The larval survival in treated larvae over inoculated control increased from 30.2 to 72.8% when using concentrations from 5 to 15% after 12 hrs. of inoculating the larvae with the fungus.
The percentage of mortality in silkworm larvae was 61.1% when larvae were inoculated with A. flavus, this percentage decreased to 20.0% and 25.0% after treating larvae 15% of propolis powder 1 and 12 hrs. after inoculation.
When henna powder was used on larvae inoculated with A. flavus, a decrease in mortality occurred. The lowest percentage of mortality was obtained at 15% after 1 and 12 hrs.
5.2. Bacterial diseases
5.2.1. Isolation of bacterial pathogens
Three different bacterial isolates were obtained from the silkworm larvae infected with the bacterial flacherie disease. The isolates were given the codes: SW3, SW4 and SW5.
5.2.2. Identification of bacterial pathogens
According to phenotypic identification (cultural, morphological, and biochemical characteristics), the isolate SW3 was identified as a genus of Escherichia, the isolate SW4 was identified as a genus of Staphylococcus while the isolate SW5 was identified as a genus of Serratia. Then the identification was confirmed with genotypic characteristics using 16s rRNA, where E. coli SW3 isolate had a sequence identity of 97% with E. coli strain RE10 16S ribosomal RNA gene, as well as that Staphylococcus sp. SW4 isolate had a sequence identity of 95.7% with Staph. Sciuri strain MF02 16S ribosomal RNA gene and Serratia sp. SW5 isolate had a sequence identity of 98.04% with S. rubidaea gene for 16S rRNA, partial sequence, strain NBRC 12973.
5.2.3. Pathogenicity tests for bacterial strains
When silkworm larvae were inoculated with E. coli, Staph. sciuri and S. rubidaea, Separately at the first day of the third instar larvae, during the initial stages of infection no external symptoms were observed on the larvae, but as the disease progressed (8-10 days after infection), some larvae were lost their appetite, had slow movement, then severe vomiting occurred, then these larvae died within few hours from the onset of symptoms. The internal tissues began to decompose and the dead larval body became soft and dark brown in color, and a foul smell was emitted from the dead larvae
5.2.4. Effect of inoculating silkworm larvae with different concentrations of bacterial strains
5.2.4.1. Escherichia coli SW3 strain
When silkworm larvae were inoculated with different concentrations (1×109, 1×107, 1×105 and 1×103 CFU/ml) of E. coli at the first day of the third instar larvae, the mortality percentage was 13.3% at all concentrations after 10 days of inoculation. At the end of the fifth larval instar (after 18 days of infection), the mortality percentages ranged between 42.8 and 93.3% for concentrations from 1×109 to 1×103.
The percentages of cocooning pupation and adult emergence ranged from 6.7 to 53.3, 6.7 to 46.7 and 0.0 to 40.0% in concentrations from 1×109 to1×103, while in the control they were 93.3, 93.3 and 86.7%.
The average weight of the mature larvae, fresh cocoons and cocoon shells ranged from 1.02 to 1.65, 0.567 to 0.873 and 0.067 to 0.167 g, respectively, in concentrations from 1×109 to 1×103, while in the control they were 3.07, 1.560 and 0.303 g.
5.2.4.2. Staphylococcus sciuri SW4 strain
When silkworm larvae were inoculated with different concentrations (1×109, 1×107, 1×105 and 1×103 CFU/ml) of Staph. sciuri at the first day of the third instar larvae, the death of larvae started on the eighth day of inoculation. At the end of the fifth larval instar (after 18 days of infection). The mortality percentages reached 85.6, 64.2, 50.0 and 28.0% in concentrations from 1×109 to1×103.
The percentages of cocooning, pupation and adult emergence ranged from 13.3 to 73.3, 13.3 to 73.3 and 6.7 to 66.7% in concentrations from 1×109 to1×103, while in the control they were 93.3, 93.3 and 86.7%.
Statistical analysis showed that there were highly significant differences in the weight of mature larvae, fresh cocoons and cocoon shells between the control and the different concentrations
5.2.4.3. Serratia rubidaea SW5 strain
When silkworm larvae were inoculated with different concentrations of S. rubidaea at the first day of the third instar larvae, 10 days after infection the mortality percentages ranged between 20 and 53.3% for concentrations from 1×103 to 1×109, the mortality increased until it reached 93.3% at the end of the fifth larval instar (after 14-18 days of infection).
The percentages of cocooning, pupation and adult emergence ranged from 6.7 to 46.7, 6.7 to 40 and 6.7 to 33.3% in concentrations from 1×109 to1×103, while in the control they were 93.3, 93.3 and 86.7%.
Statistical analysis showed that there were highly significant differences in the weight of mature larvae, fresh cocoons and cocoon shells between the control and the different concentrations
5.2.5. Control of bacterial pathogenesis
5.2.5.1. Using antibiotic and antibacterial agents In vitro using agar well diffusion assay
Using Antibiotic (Ibiamox and Garamycin) and Kombucha were appeared inhibitor effect against tested pathogenic bacteria of E. coli, Staph. sciuri and S. rubidaea with inhibition zone diameter ranged from 10.0 to 18.3, 10.0 to 19.7, and 10.0 to 17.7 mm, respectively. Whereas, the tested plant extracts and essential oils did not appeared inhibitory effect against tested pathogenic bacteria, except olive oil which inhibited Staph. sciuri with diameter inhibition zone was 4.3 mm.
5.2.5.2. Applying Ibiamox and Kombucha to control bacterial diseases In vivo:
Third instar larvae of silkworm treated with Kombucha and Ibiamox 12 and 24 hrs. after inoculation with E. coli, Staph. sciuri and S. rubidaea (1x103 CFU/ml), reduced the mortality percentages. At the end of 5th instar the highest mortality recorded in inoculated control larvae reached 34.8, 34.8 and 30.4% for E. coli, Staph. sciuri and S. rubidaea respectively. The lowest percentage of mortality recorded when larvae were fed on antibiotic-treated mulberry leaves 24 hrs. after inoculation with S. rubidaea (4.3%), followed by E. coli and Staph. sciuri (8.7%). The percentage of mortality decreased to 8.7% when larvae were fed with mulberry leaves treated with Kombucha 24 hrs. after inoculation with E. coli, and reached 21% in larvae inoculated with Staph. sciuri and S. rubidaea.