الفهرس 
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Abstract
Bacteriophages specific to Bacillus subtilis were enriched from a soil sample collected from rhizosphere soil of wheat plants, growing in the Experimental Farm of the Faculty of Agriculture, Minia University, Minia, Egypt,. The spot test was used for detection of phages in the collected rhizosphere soil sample.
Bacteriophages of B. subtilis were found to be of widespread occurrence in soil from where the sample had been taken. The single plaque isolation technique was used to purify phages. Ten single plaques of phages specific to B. subtilis, each having different morphology were picked and kept as single phage isolates.
One hundred ml of high titre phage suspension was prepared for each phage isolate, to be used in this study. The titres ranged from 2.7x1010 to 4.5x1012 pfu/ml.
The different characteristics (Optimum pH, host range, Thermal inactivation point, sensitivity to ultraviolet irradiation, DNA restriction endonuclease pattern and particle size and morphology) of the 10 phage isolates were studied to find out if these phages are belonging to one phage type or different types.
Each of the ten phage isolates of B. subtilis was tested against four different bacillus species (B. subtilis, B. cereus, B. megaterium and B. polymyxa). All phage isolates were infectious to B. subtilis (the main host). Whereas, none of the ten phages was infectious to the other tested bacillus species.
The optimum pH for phage infection of all phage isolates was found to be pH 9
According to the similarity in the thermal inactivation point, bacteriophages were grouped. The ten bacteriophage isolates were divided into five groups (group A, B, C, D and E). Each group comprised the phage isolates which exhibited the same thermal inactivation point.
Sensitivity of the isolated phages to UV radiation (at wave length 254 nm) was studied. Exposure to UV at wave length of 254 nm inactivated the isolated phages at different exposure times. Accordingly, the isolated phages were divided to five groups (group A, B, C, D and E). Each group comprised number of phage isolates which inactivated after the same exposure time.
Interestingly, the five groups of phages which divided on the basis of the thermal inactivation point were found to be the same as those classified on the basis of the sensitivity to UV radiation. Such results may indicate that the phages of each group are belonging to a single phage type.
The DNA of each phage isolate was extracted. To compare the DNA restriction patterns of these phage isolates, DNAs were separately digested with the restriction enzyme EcoRI. The DNA fragments were screened by electrophoresis on 1% agarose gel. The phages of each group exhibited identical DNA restriction pattern. Such results may indicate that the phage isolates which involved in one group represent one phage type and it can be concluded that, the ten phage isolates of B. subtilis were found to be belonging to five phage types. These five phage types were designated Bs1, Bs2, Bs3, Bs4 and Bs5, as they were divided previously in groups A, B, C, D and E.
The ten phage isolates were negatively stained with uranyl acetate and examined by electron microscope. The electron micrographs indicated that all phages were found to be of head and tail types. phages of each group exhibited similar particle size and morphology. i.e. no doubt, the ten phage isolates are belonging to five types which were previously designated Bs1, Bs2, Bs3, Bs4 and Bs5.
Specific antiserum was obtained for each phage type by immunizing white New Zealand rabbits with the phage types using subcutaneous and intramuscular injections.
Slide agglutination test indicated that all phage types showed heavy agglutination with their specific antisera. i.e. the isolated phage types have the immunogenicity and antigencity properties.
The double immunodiffusion test (Ouchterlony test) was carried out to determine the serological relationships between the five phage types under study. The obtained results indicated that the five phage types (Bs 1, Bs 2, Bs 3, Bs 4 and Bs 5) are serologically related to each other.
The latent and rise periods as well as burst size of each phage type were determined using one-step growth curves. The latent periods for phages Bs1, Bs2, Bs3, Bs4 and Bs5 were found to be 25, 30, 25, 25 and 25 minutes, respectively. The rise periods were estimated for Bs1, Bs2, Bs3, Bs4 and Bs5 and found to be 15, 15, 10, 15 and 15 minutes, respectively. Moreover, the calculated burst sizes for Bs1, Bs2, Bs3, Bs 4 and Bs 5 were 229, 253, 429, 246 and 400 pfu/cell, respectively.
In an attempt to protect B. subtilis against phage infection B. subtilis was prepared in form of alginate immobilized cells. Moreover, a phage resistant mutant was successfully isolated.
In soil cultivated with wheat plants, densities of sporeforming bacteria in rhizosphere soils of the plants inoculated with the different forms of inocula were tested.
The obtained results indicated that at any sampling time, rhizosphere of wheat plants inoculated with the immobilized cells contained much higher numbers of sporeforming bacteria than in those inoculated with the free cells.
Presence of phages, markedly reduced numbers of sporeforming bacteria in rhizosphere soils of wheat plants inoculated with B. subtilis in form of free cells, as compared to those inoculated with free cells in absence of phages. Furthermore, in plants inoculated with immobilized cells of B. subtilis plus their specific phages, slight reduction in numbers of these microorganisms in rhizosphere soils was observed, as compared to those inoculated with immobilized cells in absence of phages.
No pronounced differences in numbers of the sporeforming bacteria were detected in rhizosphere soil of wheat plants inoculated with the phage resistant mutants plus phages as compared to those inoculated with these mutant in absence of phages.
A marked increase in numbers of sporeforming bacteria in rhizosphere soils of wheat which inoculated with the immobilized cells of B. subtilis was observed as compared to those inoculated with the phage resistant mutant either in presence or in absence of phages.
At any sampling time, the rhizosphere soil of wheat plants which inoculated with the immobilized cells of B. subtilis contained much higher numbers of sporeforming bacteria than those inoculated with the free cells.
Moreover, in absence of phages inoculation of wheat plants with the wild types (free cells) or phage resistant mutant of B. subtilis led to significant increase in hight, fresh and dry weight of the plants as compared to the uninoculated ones.
In addition, at any sampling time the plant hight, fresh and dry weight of wheat plants inoculated with the wild type plus phages was lower than in the other treatments.
On the basis of the obtained results it can be concluded that, the depressive effect of the bacteriophages can be avoided by application of the bacterial inocula in form of alginate immobilized cells. Moreover, isolation of phage resistant mutants of such desired bacteria can be used as well to avoid the phage attack.
Therefore, application of immobilized cells or phage resistant mutant of such desired bacteria as biofertilizers is highly recommended to avoid the phage attack and to promote the efficiency and maintenance of these microorganisms in the soil.