الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 131 |  |  |
| | | from | 131 | | |
Abstract
This research was done in genetic department Faculty of Agriculture Minufiya University to characterize some Rhizobium leguminosarum isolated from Viciae faba nodules. That were planted at different heavy metal contaminated soils throughout Egyptian governorates including Minufiya, Giza, Asute, Kafer El-Shekh, Behera, Sharkia, Qalubia, Gharbia, El-Wady El-Geded. Soils were chemically analyzed to determine their contents of heavy metals. We choose four heavy metals Pb, Zn, Cu, Mn to detect their effects on Rhizobium leguminosarum isolates phenotypically and molecularly. Phenotypic characterization was done on their response to some antibiotics Streptomycin, Meropenem, Cefuroxime, Vancomycin, Oxacillin, Oxytetracyline, Neomycin, Bacitracin, Ampicillin and Kanamycin using cross fusion and their ability to grow at several pH levels ranging from (4 to 11). Furthermore their ability to grow at higher degrees of temperatures ranging from 30 – 45°C. Molecular characterization was done by plasmid isolated and detect their restriction pattern using EcoRI and MspI. In addition to isolate total soluble protein to detect the variations between the ten tested isolates. We can summarize the obtained results in the following: Chemical analysis to the soil from which Rhizobium isolates were obtained showed that, isolate number 8 was present in Pb (20.2 ppm) followed by the isolate number 6 was present in soil containing (18.66 ppm) of Pb in contrast to the isolate number 4 that was obtained from soil containing the least Pb concentration. Regarding Zn heavy metal in the soil ; it appeared that the highest Zn concentration was (54 ppm) where we got isolate number 8, followed by the isolate number 6 (32.6 ppm). This was followed by isolates number 7 and 9 that obtained from soil having 23 ppm. Concerning Cu heavy metal it appeared that only isolates 8 and 6 came from soil containing (16.9 ppm) and (13.83 ppm) respectively. All isolates showed ability to grow at 30 and 35°C, whereas isolates 1, 3, 5, 7, 8 and 9 showed ability to grow at 40°C. There was none of the ten tested isolates is able to grow at 45°C. Testing the ten isolates to their response to ten antibiotics, It appeared that all of them are sensitive to k30 and resistant to antibiotic B. To study the relatedness and similarities between them, It appeared that concerning degrees of temperature, ten isolate came to be in two clusters. Whereas tolerance to acid and alkaline put them in two clusters, one of them having seven isolates (1, 3, 5, 6, 7, 9 and 10) and the other one three isolates (2, 4 and 8). Response to ten tested antibiotics, put Rhizobium leguminosarum isolates in three clusters reflecting the range of similarities. The first cluster included isolate number one, another cluster included six isolates (1, 3, 5, 7, 8 and 9). Detecting the effect of four chosen heavy metals (Pb, Zn, Mn and Cu) on Rhizobial growth. Four bacterial isolates number (2, 3, 5 and 6) are able to grow in presence of 1 mM of Cu. Whereas isolates number (7, 8, 9 and 10) are able to grow in presence of lead at 1.5 mM. Rhizobial isolates number 5, 6 and 8 showed ability of growth at 1mM of Zn. Meanwhile, Rhizobial isolates number 1, 3, 5, 6, 7 and 8 are able to grow in presence of 6mM of Mn. Presence of one plasmid in all tested ten isolates with molecular weight 7000 bp except isolates 6 and 8 contain two plasmids with molecular weight7000 bp and another one at 9000 bp. We used two restriction endonucleases EcoRӀ and MspӀ to characterize plasmid DNA. Digestion pattern using MspΙ enzyme in plasmids that present in isolates six and eight reflect the homogeneity between them on number of fragments and their sizes. Plasmid in isolate number four appeared to be unique and contains only one recognition site for restriction enzyme MspΙ with molecular weight (7000 bp). Using MspΙ enzyme showed presence of two kind of plasmid isolates No. 1,2,3 and 5 gave two bands with molecular weights (5000 and 2000bp). Isolates No. 6 and 8 gave five bands with various molecular weights (6000,4000,3000,2000 and 1000bp) respectively. Isolates No. 9 and 10 gave two bands with the same molecular weight (5000 and another one with molecular weight 2000bp). • In the case of EcoRI restriction pattern, several isolates 1,2,3,5,7,9 and 10 appeared to be similar as they showed two fragments with molecular weights (5000 and 2000 bp). Whereas isolate number 4 appeared to have no recognition site for EcoRΙ restriction enzyme and showed less plasmid size. On the other hand isolates number six and eight appeared to have similar plasmids. They have four EcoRΙ recognition sites, giving rise to four fragments with molecular weights (6000,5000,3000 and 2000 bp). In fact we could mention presence of more than one plasmid in these two isolates, one plasmid that present in the rest of the isolates that gave two fragments (5000 and 2000 bp), and other one showed more two fragments that not present in them. Total soluble protein on SDS – PAGE three isolates 5, 8 and 9 that showed ability to grow in 1mM of Zn. There were some variations in sizes of bands in isolate 8 and 9. Four isolates number 2, 3, 5 and 6 that showed ability to grow in presence Cu. There are similar protein band except isolate number 2 showed two extra bands. Isolates number 7, 8 and 9 that were able to grow in presence of Pb showed less number of bands. Isolate number 5 showed the ability to grow in presence of Zn, Cu and Mn. whereas isolate 8 is able to grow in presence of Zn, Pb and Mn. Isolate number 9 is able to grow in presence of Zn and Pb. Our characterization to those isolates could be used to enhance symbiotic effectiveness in heavy metals (Pb, Zn, Cu and Mn) polluted soil.