الفهرس 
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Abstract
In the present study, pure colony of Azotobacter sp. (FWT) was molecularly characterized and treated different times with various doses of chemical mutagen N-methyl-N-nitro-Nnitrosoguanidine (MNNG) to induce mutations. The mutant cells were identified and tested for its genetic variability and their plant growth promoting potentiality on sweet melon seedling.
1. Molecular characterization of Azotobacter sp. strain.
PCR using genomic DNA of Azotobacter sp. (FWT) strain as template and universal bacterial primers for 16s rDNA, produced single fragment of 1500 bp. The amplified fragments of Azotobacter sp. (FWT) were put through nucleotide sequencing with the same primers, and the obtained sequences were compared with the NCBI Genbank database. The nucleotide sequence data can be found in the NCBI GenBank with accession number OR742885. The consensus sequence of 16S rRNA gene was obtained from the alignment of the two sequences of forward and reverse 16S rRNA primers. Comparison of amplified 16S rDNA sequence of the FWT strain of Azotobacter sp. with the possible sequences of NCBI GenBank showed an identity of 100% to the two Azotobacter chroococcum strains with GeneBank accession Nos. NR_114167 and NR_041035) and exhibited 99.89% similarity to Azotobacter chroococcum strain with accession no. NR_116305.
2. Induction of mutations in Azotobacter chroococcum FWT strain by using N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) as a chemical mutagen
2.1. The effect of different MNNG mutagen concentrations and exposure times on number and percentages of Azotobacter chroococcum FWT survivals:
The obtained results showed that treatment of Azotobacter chroococcum FWT with different concentration (50, 100 and 200µg/ml) of MNNG mutagen at three times (30, 60 and 120 min) was accompanied with reducing the numbers and percentages of Azotobacter chroococcum FWT survivals as compared to the untreated control. It was observed that the numbers and percentages of treated colonies were decreased with increasing the concentration and time exposing of mutagen. The highest number of survivals was obtained by untreated control (13150 cells with 100%). While, the lowest value (590 cells) were found after 120min in plates treated with 100mg/ml MNNG mutagen with 4.5% survivals. There was no survivals colony after treatment with 200 mg/ml of MNNG for 120 min.
2.2. The mutagenic effect of different MNNG mutagen concentrations on Azotobacter chroococcum FWT strain
The total number of Azotobacter chroococcum mutants which obtained after treatments with MNNG mutagen was 15 auxotrophs with frequency 0.65%. These mutants were symbolized from FM1 to FM15. The highest number of mutant cells (5cells) with frequency 1.92% was found in cells treated with 200mg/ml for 30min while the lowest value was one cell that found after treated with 200mg/ml for 60min with frequency 0.35%. Cells exposing to the two doses 50 and 100mg/ml for 30min and100mg/ml for 120min of MNNG mutagen didn’t gave mutants.
Among of them, ten mutants were identified while the other five gave unclear results so it was excluded from the identification but it used in the other experiments. It was found that 6 mutants had a single mutation: Isoleucineless (Isoleu-), Aspartic acid less (Asp-), Histidineless (His-), Prolineless (Pro-), Glutamic acid less (Glu-) while the other mutants were carried a double mutation: Vit.B1-/Thr-, Thr-/Thi-, Orn-/ Asp- and Vit.B9-/Vit.B6- .
3. Antibiotic resistance test of Azotobacter chroococcum FWT and their mutants on the presence of different antibiotics.
The actual antibiotic resistance of the wild type and 15 auxotrophs isolates was verified on Ashby-Sucrose agar medium supplemented with following antibiotics (μg/ml): Colistin(CT)10, Enrofloxacin (ENR) 5, Oxacillin (OX)5, Ceftriaxone (CRT)30, Azitromycin (AZM) 15; Ampicillin (AMP)10, Oxytetracycline (O) 30 and Amikcin (AK)30.
4. pH tolerance of Azotobacter chroococcum FWT and their mutants.
Data indicated that, all mutants were tolerant to pH levels less than 10 similar to the wild type, except the FM2 mutant which weakly grew at pH 5. All tested strains survived well at pH 11 whereas, FM2 mutant grew weakly at the same level. All the mutants grew well at pH12 similar to wild type strain, but the two mutants (FM2 and FM13) exhibited weakly growth at the same level and only the mutant FM6 couldn’t survived at pH12 level.
5. Salinity tolerance of Azotobacter chroococcum FWT and their mutant strains.
Salinity test of different Azotobacter chroococcum FWT and their mutants were done at different salt concentration (5 to 10%) of NaCl add to Ashby-Sucrose agar medium. It was found that all of wild type and their mutants grown well at concentrations of NaCl ranged from 5 to 7%, whereas, after increasing the concentration it was observed that wild type strain didn’t grow well. On contrast all tested mutants were grown and survived well at 10%NaCl concentration.
6. Effect of Azotobacter chroococcum FWT and their mutants on growth promotion of sweet melon (cv. Kahera 6) seedling under greenhouse condition.
6.1. Effect of Azotobacter chroococcum FWT and their mutants on some vegetative traits.
Different growth traits i.e., shoot and root length; leaves number, shoots / roots fresh and dry weight were recorded after 30 days of sowing. Data indicated that almost of the mutant strains of Azotobacter chroococcum showed considerable increase in all vegetative parameters (root length, shoot length, root fresh and dry weight and shoot fresh and dry weight) as compared to wild type.
6.2. Effect of Azotobacter chroococcum FWT and their mutants on germination% and growth indicators of sweet melon (cv. Kahera 6) seedling.
After 30 days of planting seeds in pots under greenhouse conditions germination percentage, seedling length and vigor index were recorded. Seed covered with the nine mutants FM7 to FM15 showed the highest percentage of seed germination (100%). While, the four mutants FM3 to FM6 gave similar results to wild type and untreated control (83.33%). The lowest values of germination percentages were found in seeds treated with the two mutants FM1 and FM2 with the same value (66.67%). The vigor index increased significantly in sweet melon seedling treated with almost of tested mutants (FM6 to FM15) as compared with both of wild type and untreated control treatments.
6. 3. Effect of Azotobacter chroococcum FWT and their mutants on N, P and K content in leaves of sweet melon seedling
The obtained results revealed that inoculation of sweet melon plants with almost of tested Azotobacter chroococcum strains particularly the three mutants (FM12, FM13 and FM8) increased the uptake of leaves N, P and K contents considerably higher than wild type and untreated control
6-4. Effect of Azotobacter chroococcum FWT and their mutants inoculation on photosynthetic pigments in leaves of sweet melon seedling.
Data revealed that inoculation with almost of all tested Azotobacter chroococcum strains considerably increased all photosynthetic pigments (chlorophyll a, b and carotenoids) in the sweet melon shoots as compared to untreated control treatment. Inoculation with the three mutants FM12, FM13 and FM8 gave higher percentages of photosynthetic pigments with a significant increase rather than both of wild type and untreated control treatments.
Finally, it could be concluded that treatment of Azotobacter chroococcum wild type strain with different MNNG concentrations may produce a new genetic parameter (Genotype) which could be used as a desirable bio-inoculates to improve growth and quality of sweet melon seedlings.