الفهرس 
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Abstract
5.1 Azospirillum isolate:
In the present study Azospirillum isolate was randomly isolated from rhizosphere of wheat plants (Triticum aestivum) growing in the Experimental Farm at Faculty of Agriculture Minia University, Minia, Egypt. This bacterium was found to be common in the cultivated soil.
5.2. Bacteriophages:
Bacteriophages specific to Azospirillum were enriched from the collected wheat rhizosphere soil sample. The spot test was used for detection of phages. Bacteriophages of Azospirillum 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. Nine single plaques of Azospirillum phages, having different morphologies were picked and kept as single phage isolates.
5.3 Titre of the prepared phage suspensions:
One hundred ml of high titre phage suspension were prepared for each phage isolate of Azospirillum, to be used in this study. The titers ranged from 4.8x1011 pfu/ml to 6.2x1012 pfu/ml.
5.4 characterization of the isolated phages:
The different characteristics (Optimum pH, Thermal inactivation point, sensitivity to ultraviolet irradiation, longevity invitro and DNA restriction pattern) of the nine phage isolates of Azospirillum were studied to find out if these phages are different types or similar.
5.4.1 Determination of the optimum pH:
The optimum pH for infection of all phage isolates (9 phage isolates for Azospirillum) was found to be pH 8.
5.4.2 Sensitivity to ultraviolet irradiation:
Sensitivity of the isolated phages of Azospirillum to U.V. radiation (at wave length of 260 and 360 nm) was studied. No effect for U.V. radiation at wave length of 360 nm on the phage isolates was observed, even after exposure for 90 min. Whereas, U.V. at wave length of 260 nm inactivated the isolated phages at different exposure time. Accordingly, the isolated phages of Azospirillum were divided to three groups. Each group comprised number of phage isolates which inactivated after the same exposure time.
5.4.3 Thermal inactivation point (TIP):
According to the similarity in the thermal inactivation point, bacteriophages of Azospirillum were grouped. The nine bacteriophage isolates of Azospirillum were divided into three groups (group A, B and C). Each group contained the phage isolates which exhibited the same thermal inactivation point. Interestingly, the three groups of Azospirillum 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 U.V. radiation. Such results may indicate that the phages of each group of Azospirillum are belonging to a single phage type.
5.4.4 longevity in vitro:
The longevity in vitro of each phage isolate was determined using the spot test technique. Each of the nine phage isolates of Azospirillum was tested under room temperature. According to longevity in vitro, the isolated phages of Azospirillum were divided to three groups (A, B, and C). Each group comprised number of phage isolates which exhibited the same longevity in vitro. These groups were the same as those classified previously on the basis of sensitivity to U.V. radiation and thermal inactivation point.
5.4.5 DNA restriction endonuclease pattern of the isolated phages:
The DNA of each phage isolate of Azospirillum was extracted. DNAs of Azospirillum phages were digested by EcoRI and separated by 1% agarose gel electrophoresis. Phages of Azospirillum in each group classified on the basis of previously mentioned characteristics exhibited the same DNA restriction pattern with EcoRI and the same genome size.
On the basis of the obtained results, it can be concluded that. The nine phage isolates of Azospirillum were found to be belonging to three phage types. These three phage types were designated ØAz1, ØAz 2 and ØAz3.
5.4.6 Size and morphology of phage particles:
The three phage types of Azospirillum (ØAz1, ØAz 2 and ØAz3) were negatively stained with uranyl acetate and examined by electron microscopy. The three phage types of Azospirillum sp. were found to be of non-contractile tails. Phage type ØAz1 had isometric head of 76 Ø 3 nm in diameter and the shortest tail (38 Ø 2 nm) among the three phage types. The other two phage types (ØAz 2 and ØAz 3) were of long tails and isometric heads. According to the particle morphology of the three phage types, the phage type ØAz1 is seems to be belonging to family Podoviridae. Whereas, ØAz2 and ØAz3 are seems to be belonging to family Siphoviridae.
5.4.7 One step growth curve:
One step growth curve experiment was carried out for each phage type. The latent periods for ØAz1, ØAz2 and ØAz3 were found to be 10 min, 20 min and 15 min, respectively. Moreover, the burst sizes for ØAz1, ØAz2 and ØAz3 were estimated to be 75 pfu/cell, 93 pfu/cell and 82 pfu/cell, respectively
Generally, on the basis of the obtained results it can be concluded that, the plaque morphology is not an accurate technique to identify and classify the bacteriophages. Since in this study, the nine phage isolates of Azospirillum were found to be belonging to three phage types, although each of the nine phage isolates of Azospirillum was isolated with different plaque morphology. i.e. plaques of different morphologies can be formed by a single phage type. Therefore, the plaque morphology should not be used for phage identification and classification, whereas, it can be used as a rapid technique to purify the mixed phages.
Moreover, the optimum pH for phage infection which was used in this study to differentiate between the isolated phages, was found to be not an accurate technique as well to identify and classify the bacteriophages. Since, the optimum pH for all phage isolates of Azospirillum was found to be pH 8, in spite of the phage isolates were not a single type.