الفهرس 
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Abstract
Three isolates of root nodule bacteria of Vicia faba, one isolate of Cicer arietinum, one isolate of Glycine max, two isolates of Phaseolus vulgaris and one isolate of root nodule bacteria of Trifolium alexandrinum were isolated from root nodules of the host plants collected from the experimental farm of faculty of agric. and faculty of science, Minia university, Minia, Egypt. In addition, one strain of Rhizobium leguminosarum bv. viceae (RCR1044) and one strain of Rhizobium sp. (RCR3824) were kindly supplied by Cairo MIRCEN (Microbial Resource Center), faculty of agriculture, Ain Shams university, Cairo Egypt. These isolates were used in this study.
Bacteriophages specific to R. leguminosarum bv. viceae (RCR1044) and Rhizobium sp. (RCR3824), were enriched from soil samples collected from rhizosphere soil of Pisum sativum and Vigna unguiculata plants, respectively, growing in the experimental farm of faculty of agric. and faculty of science, Minia University, Minia, Governorate. Egypt. Five phage isolates specific to R. leguminosarum bv. viceae (RCR1044) and four phage isolates specific to Rhizobium sp. (RCR3824) were isolated from the collected soil samples. Different characteristics (the thermal inactivation point, sensitivity to U.V. radiation, host range as well as size and morphology of the isolated phages) were studied. On the basis of the studied characteristics the five phage isolates of R. leguminosarum bv. viceae (RCR1044) were found to be belonging to two phage types and designated RL1 and RL2. Whereas, the four phage isolates of Rhizobium sp. (RCR3824) were found to be belonging to three phage types and designated RS1, RS2 and RS3.
Protection of root nodule bacteria against bacteriophage attack:
A phage resistant mutant of R. leguminosarum bv. viceae (RCR1044) was successfully isolated via exposing of R. leguminosarum bv. viceae (RCR1044) to the virulent phages. The isolated mutant exhibited high resistant to the virulent bacteriophages. In addition, R. leguminosarum bv. viceae (RCR1044) was prepared in form of alginate immobilized cells to be used as inoculum for the host plant (Pisum sativum).
In a pots experiment presence of phages had no significant effect on nodulation, N% and growth of Pisum sativum plants inoculated with phage resistant mutant as compared to those inoculated with the wild type in absence of phages.
Moreover, presence of phages had no significant effect on number of root nodules/plant, N% and growth of Pisum sativum plants inoculated with the immobilized cells of R. leguminosarum bv. viceae (RCR1044) as compared to those inoculated with the free cells in absence of phages.
Accordingly, the depressive effect of the bacteriophages can be avoided by application of the bacterial inocula in forms of alginate immobilized cells or as a phage resistant mutant.
Effect of salinity on the root nodule bacteria
Effect of salinity on one isolate of root nodule bacteria of faba bean (Vicia faba) was examined in vitro in liquid cultures. Eight concentrations of NaCl (i.e. 1%, 2%, 3% upto 8%) were used and the optical density was taken as a measure for bacterial growth. The highest growth of the tested root nodule bacterial isolate was recorded in the normal medium with no added salt (control). Whereas, in the salinized media, the growth was gradually decreased with increasing the salt concentration. Growth of root nodule bacteria of Vicia faba was detected in salinized media upto 6% of NaCl /L. Whereas, no growth was detected in salinized media which contained 7% and 8% gm of NaCl /L.
An isolate of halophilic bacteria was picked from foreshore saline soil sample collected from Alexandria Governorate. This halophilic bacterial isolate was identified as Bacillus cereus by Cairo MIRCEN, faculty of Agriculture, Ain Shams university. The halophilic bacterial isolate was grown in midia containing different concentrations of NaCl (i.e. 1%, 2%, 3% upto 8%). The growth was measured as optical density at different incubation periods. the lowest growth values of the halophilic bacterial isolate was recorded in the normal medium with no added salt. The growth values increased with increasing NaCl concentration. The highest growth was achieved in media containing 8% NaCl/ L. Transformation was carried out using halophilic bacterial isolate (Bacillus cereus) as a donor and root nodule bacteria of Vicia faba as a recipient. The transformant exhibited remarkable growth in presence of the different NaCl concentrations (i.e. 1%, 2%, 3% upto 8%). The highest growth of the transformant was recorded in medium supplemented with 4% NaCl /L.
Liquid cultures of both root nodule bacteria of Vicia faba and the transformant were prepared to be used as inocula for the host plant (Vicia faba). The inoculated plants were subjected to irrigation with salinized water (0.5% NaCl and 1% NaCl). Control plants inoculated with root nodule bacteria of Vicia faba and irrigated with tap water were also involved. Inoculation of Vicia faba plants with non-transformed root nodule bacteria (recipient) and irrigation with salinized water (0.5% NaCl or 1% NaCl) resulted in significant reduction in values of fresh and dry weight of plants, number of the formed nodules/plant and nitrogen percent as compared to those irrigated with tap water.
No significant differences in the values of the tested measurement were detected in plants inoculated with the salt tolerant transformant and irrigated with salinized water (0.5% NaCl or 1% NaCl) as compared to those inoculated with the non-transformed bacteria and irrigated with tap water.
Effect of high temperature on the growth of root nodule bacteria
In this study the thermal stability of root nodule bacterial isolate of Glycine max. was tested. This isolate was grown in liquid medium and incubated at different temperature degrees (30, 35, 40, 45, 50 upto 60°C). The bacterial growth was measured as optical density.
The maximum growth of root nodule bacterial isolate of G. max. was achieved when their cultures were incubated at 35°C. The growth tended to increase at 35°C with increasing the incubation period upto the end of the experiment (120 hours). Lower growth values were detected at incubation temperature from 40°C to 60°C.
An isolate of thermophilic bacteria was isolated from a mature compost sample. This thermophilic bacterial isolate was identified by Cairo MIRCEN, faculty of Agriculture, Ain Shams university and was found to be Bacillus stearothermophilus.
The thermophilic bacterial isolate (Bacillus stearothermophilus) was grown in nutrient broth medium and incubated at different temperature degrees (30, 35, 40, 45, 50 upto 60°C). The growth was measured as optical density. The highest growth values were recorded with incubation at 60°C, whereas, the lowest growth values were observed at 30°C.
As a result of the transformation process, a transformant was obtained for root nodule bacterial isolate of G. max. which was used as a recipient and the thermophilic bacterial isolate which was used as a donor. The optimum temperature for growth of the root nodule bacteria of soybean was found to be 35°C. Higher incubation temperature resulted in marked reduction in the growth of these bacteria. On the other hand, 60°C was found to be the most convenient temperature for the growth of thermophilic bacterial isolate (Bacillus stearothermophilus). As a result of transferring a plasmid of the thermophilic bacterial isolate into the root nodule bacteria of G. max., a transformant was obtained for the root nodule bacteria of (G. max.). The transformant of root nodule bacteria of G. max. exhibited remarkable growth at the high incubation temperatures even at 60°C. The highest growth values recorded for the transformant was achieved at 45°C.
In vivo study was carried out to ensure that the efficiency of the transformed root nodule bacteria in nodulating host plants and in fixing nitrogen did not negatively affect by transformation. Liquid cultures of both the root nodule bacteria of G. max. (recipient) and the transformant were used as inocula for the host plants (soybean). When soybean plants were 50 days old no significant differences were detected in values of fresh and dry weight of plants inoculated with the root nodule bacteria (recipient) and of those inoculated with the transformant. Moreover, no differences in the nodulation and nitrogen fixation efficiency of the recipient and transformant were detected, since, no significant differences were observed in number of nodules and plant N% in plants inoculated with root nodule bacteria (recipient) and those inoculated with the transformant isolate.