الفهرس 
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Abstract
1- Chalkbrood disease, which is caused by Ascosphaera apis led to a tangible damage to honeybee colonies, as it infests the brood it leads to a decrease in honeybee population.
Survey of chalkbrood disease in different districts of Assiut governorate during 2009/2010 seasons revealed that the honeybee chalkbrood disease in honey bee colonies clearly has a widespread incidence and causing economic damage to apiaries .
Survey data showed that Al Fath and Al Badarie districts have the highest percentages of infestation during 2009/2010 seasons with chalkbrood disease. They were 15.8, 14.7% respectively, followed by Al Qusia, Manflout, Sahel Selim and Sedfa 7.8, 6.8, 6.8 and 5.6% respectively. However, low infestation percentages were noticed in Dirout 4.7%, Assiut 3.1%, El Ghanaiem, 2.3%, Abnoub 0.8% and Abou Tig district 1.1% .
Data showed that the natural infestation of chalk brood disease was recorded in the early of spring from February and extended to April, the disease disappeared during summer months, from June to August. But it started again in the beginning of autumn, during September and October months. However, infestation of honeybee with chalkbrood disease might take place during winter months
Reasons that might lead to the appearance of chalkbrood infestation on honeybee colonies at different districts of Assiut governorate in 2009/2010 seasons were suspected mainly due to the queen’s weakness, the increase in relative humidity due to poor ventilation and the transition of old combs.
So using an easy method for colonies treatment of such disease can consider of highly practical importance.
2) Six fungal isolates belonging to Ascosphaera apis were isolated from infested honeybee brood collected from different districts (Assiut, Dirout, Dashloot, Kom boha, Shotp districts of Assiut governorate. Isolated fungi which form sporocysts on cultivated medium were identified. Ascosphaera apis is a heterothallic organism that sporulates only when mycelia of opposite sex (designated + and -) come together.
3) Six biotype of isolated fungi were identified as Ascosphaera apis, two of them (A7 and A15) were able to form sporocysts on cultivated medium, however isolates (A3, A4, A8 and A9) did not form sporocysts on cultivated mMS medium. When (A8 and A9) grow together on the previous cultivated medium. They showed compatibility and formed Sporocysts in the line of isolates mating. Meanwhile, the white isolates (A3 and A4) showed no compatibility with each other and with (A8 and A9).
4) Honeybee colonies artificially inoculated with Ascosphaera apis ascospores by spraying or feeding at the 9th of November 2009. In the experiment in vivo, data indicated that no sign of infestation on honeybee brood was noticed by using artificially inoculation of the causal pathogen after one week. However, the first sign of chalkbrood disease was recorded at the 23rd of November 2009. Data revealed that there is a significant effect between the two methods of inoculation of honeybee colonies brood with ascospores of the causal pathogen of chalkbrood disease. Spraying honeybee brood was significantly superior in its disease incidence mean (2.05) rather than feeing method, which gave (1.3) mean of the disease incidence.
5) Sporocysts of Ascosphaera apis were found on black mummies or formed in vitro on cultivated medium, which examined under immersion lens (x 1000). Sporecysts diameter of Ascosphaera apis were measured the causal pathogen of chalk brood of honeybee was measured 47 to 140 μm in diameter, while individual spores are 3.0 to 4.0 μm by 1.4 to 2.0 μm.
6) Three different media were used for isolating Ascosphaera apis, the causal pathogen of honeybee chalkbrood disease, potato dextrose agar (PDA), Murashieg Skoog medium (mMS) with some modification and rice medium.
The Ascosphaera apis was grown well on the three media, but there were some differences in its growth rate. The best mean of Ascosphaera apis growth rate was recorded in the case of rice medium followed by mMS medium and lastly on PDA medium.
Data showed that Ascosphaera apis Pale buff strain (A7) had intensive growth (5.8 cm) on rice medium followed by white strain (A9), which gave nearly the same growth (5.8 cm) on the same medium. No significant differences between the strains previously mentioned.
Most strains of Ascosphaera apis showed moderate growth on rice medium. However, less growth was pronounced on PDA medium in case of white strain (A4, A7, A9 and A15) which gave 3.4, 3.5, 3.5 and 3.6 cm of growth zone respectively.
Meanwhile, good growth was induced on mMS medium in case of most strains of Ascosphaera apis.
7) Data revealed that adding Oyster mushroom mycelium powder at different concentrations to cultivation medium reduced the growth of Ascosphaera apis, in vitro. The highest reduction growth was pronounced at concentration of 1gm/l of the mycelium powder. No significant differences were noticed between the different concentrations. However, highly significant differences at 1% probability were noticed between the different Ascosphaera strains.
8) Data showed a simple correlation between disease incidence of chalkbrood disease and temperature or relative humidity inside the honeybee colony.
Temperature was found to be negatively correlated (-0.53). However, the relationship between relative humidity and disease incidence was found to be positive (0.51). It means that infestation of honey bee colonies with the chalkbrood is relative humidity dependent.
The multi-regression analysis of data indicated that (35.5%) of variability percentage of chalkbrood infestation mainly due to both of the two factors, temperature and relative humidity altogether. It was found that temperature was the most effective variable (20.32%) than the relative humidity (15.10%).
9) Symbiotic bacteria associated with midgut of honeybee, have been isolated from healthy workers. The isolated bacteria gave the following morphological features and physiological reactions:
a- Four endospore-forming bacteria (B2, B4, and B10and B100) were isolated from midgut of honeybee (symbiotic bacteria). They had the following characters; rod shaped, motile, aerobic, Gram positive, Voges-proskaur positive, produced acid from glucose, arabinose, xylose and mannitol, and did not produce gas from glucose, hydrolyze urea, liquefied gelatin, tyrosinase negative, reduce nitrate, indole negative, grow well at pH 5.7 and 6.8, grow at concentrations 10% of NaCl, produce cream light brown pigment on nutrient agar medium and brown to black pigment on PDA medium.
b- Two non spore-forming bacteria (P1 and P5) were isolated from midgut of honeybee (symbiotic bacteria). They had the following characters; rod shaped, motile, aerobic, Gram negative, Voges-proskaur negative, produced acid from glucose, sucrose, D-Fructose, D-ribose, maltose, aesculin, Glycerol and did not produce acid from mannitol, gelatin liquefied negative, reduce nitrate, indole negative, H2S production negative, Starch hydrolysis negative, Citrate utilization positive, Growth at 40°C negative, Oxidation of glyconate negative, Yellow pigment with weak light under ultraviolet light.
c- The isolated endospore–forming bacteria, which was isolated from honeybee midgut (B2), could be identified as Bacillus subtilis, while non-spore forming bacteria (P1) could be identified as Pseudomonas fluorescence.
d- The antagonistic effectiveness of both Pseudomonas fluorescence and Bacillus subtilis bacteria was tested against Ascosphaera apis the causal pathogen of the chalkbrood disease in vitro.
10) Data demonstrated that Bacillus subtilis isolate (B2) gave the highest antagonistic effect against all Ascosphaera apis strains. This was pronounced in vitro on cultivated medium by inducing wide inhibition zone followed by Pseudomonas fluorescence (P1) isolate, however the least antagonistic effect against Ascosphaera apis was produced by Pseudomonas fluorescence (P5) Data also showed that there are highly significant differences at both 5 and 1% probability was noticed among Bacillus subtilis isolate (B2) and Pseudomonas fluorescence (P1) and the other bacterial strains tested.
11) Data demonstrated that Bacillus spp. have a higher count in healthy honeybees than those found in diseased one. It could be concluded that Bacillus subtilis, which is considered one of the acting bacteria in the (Bioassiut) formula, used in this experiment might play an active role with Pseudomonas fluorescence irrespective of that it was mainly missed in different periods.
12) Using the scanning electron microscope to examine mature sporocysts of Ascosphaera apis showed that it contained a large number of globules of varying sizes were scattering on the surface of diseased brood. However, sporocysts which formed in vitro, their walls of the immature stage were wrinkled and then became smooth as the mature sporocyst. Moreover, the interior and exterior surface of the sporocyst wall had numerous distinguishable papillae. However, some globules in the developing sporocysts began to form immature spores, which aggregated to form spore balls.
Ascosphaera apis hyphae was shown invaded by numerous bacterial cells of antagonistic bacteria Pseudomonas fluorescence in vitro. These bacteria have caused the disintegration and lysed the cell wall of the fungal hyphae. In this respect, mature Sporocyst of Ascosphaera apis also was invaded by a numerous bacterial cells of antagonistic bacteria Bacillus subtilis in vitro these bacteria lysed the cell wall of the mature sporocyst, forming a cavity and aggregated to go inside it.
Ascosphaera apis hyphae showed shrinking appearance when grown in vitro with antagonistic bacteria Bacillus subtilis on cultivated medium.
The over growth of oyster mushroom (Pleurotus ostreatus L.) on Ascosphaera apis in vitro was noticeably found when Ascosphaera apis and oyster mushroom the basidial fungus were grown face to face on the cultivated medium. The oyster mushroom has firstly antagonized the causal pathogen of honey bee chalkbrood and then the mycelium of oyster mushroom nearly covered all of the cystospores of Ascosphaera apis and its fungal mycelium has limited its additive growth due to the challenging by Pleurotus ostreatus fungus.
13) Data indicated that the percentage of chalkbrood infestation in the artificially infested colonies greatly decreased after feeding with bioagent (Bioassiut), after the first treatment.
All treated honeybee colonies by feeding them with bioagent (Bioassiut) completely overcame the disease, after the third treatment except colony No. 11, which the disease was abolished from it after the 4th treatment.
14) Data showed that the mean percentages of natural infestation of chalkbrood disease at the naturally infested colonies was 10.11%. Results indicated that the percentage of chalkbrood after feeding of infested colonies on bioagent (Bioassiut) significantly decreased from the second treatment (0.82 %). However, at the end of four feeding treatments the mean of percentage of chalkbrood infestation in all treated honeybee colonies with bioagent (Bioassiut) reached (0.0%) of infection.
Data also indicated that means of the percentage of chalkbrood infestation that have the same symbols from (a-e) had no significant differences, while there were highly significant differences at 5% between the mean of percentage of chalkbrood infestation at the date of 25/2/2010 and what follows it.
from the previous results, it could bee concluded that the use of such bio-formula (Bioassiut) in apiaries management and feeding infested honeybee colonies on it can be consider as a satisfying weapon for controlling such disease. Moreover, it’s considered beneficial and safe.