الفهرس 
Rep -PCR analysisOnly 14 pages are availabe for public view
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Abstract
This research was conducted in the genetics laboratory –Faculty of Agriculture, Menoufia University. This was carried out to detect the genetic diversity and symbiotic efficiency in 25 rhizobial isolates from legume plants namely Faba bean. They were isolated from different sites in menofia Governorate (Shebin Elkom –Tala - Berkit Elsaba –Ashmoun – Menouf – Elshohada – Quisna – Elbagour - Elsadat City).
This study was performed with six objectives:
1- Isolation and morphological identification of rhizobial isolates that reside in faba bean (Vicia faba from different geographical areas throughout menofia governorate.
2- Biochemical characterization of rhizobial isolates.
3- To study the phylogenetic relationship among chosen isolates by 16SrRNA gene.
4- To study the diversity of genomic DNA among isolates by Repetitive element sequence-based PCR (rep-PCR).
5- To amplify the nifH gene among chosen isolates by using Pcr technique.
6- To measure the efficiency of nitrogen fixation among the selected isolates by doing a field experiment.
Obtained data summarized in the followings:
All morphological tests proved that all tested isolates were rhizobia. They were gram negative bacteria, motile and rod shaped.
Biochemical tests showed some variations in their response and produced different chemicals. The biochemical tests included various tests, carbon sources, antibiotics, different rangs of pH, different concentration of NaCl and other biochemical tests.
• Carbon sources included: Glucose, Fructose, Sucrose, Lactose, Sorbitol, Mannose, and Maltose.
• Antibiotics discs included: Ampicillin (AM-10), Ofloxacin (OFX-5), Clindamycin (DA-2), Trimethoprim / sulphamethoxazole (SXT-25), Cloxacillin (CX-1), Tobramycin (TOB-10), Meropenem (MEM-10), Amoxycillin / Clavulanic acid (AMC-30), Rifamycin (RF-30), Cephalexin (CL-30), Oxacillin (OX-1), Gentamicin (CN-10), Streptomycin (S-10), Imipenem (IPM-10) and Chloramphenicol (C-30).
• pH ranges included: 4,5,6 and 8.
• NaCl concentration ranges included: 0.5%,1%,2% and 4%.
For phylogeny study 16SrRNA gene was isolated from all rhizobial isolates by using PCR amplified and DNA sequencing. Nucleotide sequences of every isolate were compared to known nucleotide sequences in gene bank using nucleotide BLAST and we obtained accession numbers for all isolates understudy on gene bank.
The 16S rRNA gene fragments of isolates was 1500 base pairs. The DNA sequences of 16SrRNA genes comparison lead to detection of twenty five species of Rhizobium leguminosarum. Also, nifH gene region was amplified for all isolates. Phylogenetic tree based on partial 16SrRNA sequence demonstrated two clusters; first cluster contained three groups with similarity from 98 to 100%. the second subcluster contained two groups.
The rep-PCR results were summarized. The polymorphic and monomorphic bands were produced from the PCR amplification. About 100 bands resulted from the six rep-PCR primers. Out of them, 21 bands were monomorphic with a monomorphism average of 21%, while 79 bands were polymorphic bands with a polymorphism average of 79 %. The highest polymorphism among R. leguminosarum isolates was revealed by BOX-A1 primer (88.3%), followed by that revealed by ScoT-12 primer (87.5%). However, the lowest polymorphism was 68.75% resulted from application of rep-16 primer.
We used reflict primers for amplification of nif H gene as presented. All amplified nif H showed the same molecular weight which came to be 336bps.
Least symbiotic efficiency appeared in isolates numbers MNF-9(87.95), MNF-15(84.77), MNF-13(73.98) and MNF-12(78.46), and the highest symbiotic efficiency appeared in isolates MNF-21(169.3) and MNF-23(138.72).