الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Biocontrol depended on the use of microorganisms to inhabit plant diseases caused by pathogenic fungi present a reliable alternative to common strategies especially chemical fungicides, which have raised serious concerns of food contamination and environmental pollution. Microorganisms, which secrete a complex of mycolytic enzymes, are possible biological control agents of plant diseases; chitinase plays an important role in the biocontrol of plant pathogenic fungi. Most of the known rhizobacteria that serve as biocontrol agents produce and excrete chitinase. The chitinase enzymes produced by these organisms hydrolyze the chitin polymer present in the cell wall and degrade the fungal mycelia. Genes encoding for these chitinases have been cloned from various bacterial and fungal species. The isolated gene coding for the chitinase can be transferred into another biocontrol agent producing secondary metabolite or introduced into different plant species in order to enhance resistance against a broad range of fungal pathogens. The major aims of this study were isolation, identification, molecular characterization and study the antagonistic activity of the potent broad spectrum local chitinolytic bacterial isolates against soil borne pathogenic fungi, evaluation of their antagonistic activity against Rhizoctonia solani and Fusarium oxysoprum. Detection, Isolation, and nucleotides sequence of ChiA gene from the most potent isolate.
The results of this study can be summarized as follow:
1. Seventeen bacterial isolates have been successfully isolated and purified from a total of three rhizosphere soil samples, collected from rhizosphere of different commercial plants located in three different agriculture areas in Egypt.
2. Two of these isolates exhibited different sizes of clear zones on solid chitinase-inducing medium, evidencing differences in their chitinolytic activities.
3. Isolates S3-C and S1-C gave the widest clear zone and subsequently were selected for further studies.
4. The selected bacterial isolates S3-C and S1-C were identified based on morphological, physiological, biochemical characteristics presented in Bergey’s Manual of Systematic Bacteriology and 16s rRNA. The results revealed that Isolates S3-C and S1-C were identified as Bacillus cereus.
5. SDS-PAGE analysis of cells of isolates S3-C and S1-C revealed that the protein banding patterns of the two isolates and reference strains different from each other. Furthermore, Bacillus cereus they gave slightly different protein profile of total protein cells. Since the latter exhibited one clear band at (≈ 40 KDa) in S1-C while the former exhibited one clear band at ≈ 35 KDa lacked in the latter. Also, the pattern showed that the two isolates S1-C and S3-C were slightly different from each other.
6. The ability of the selected chitinolytic isolates to inhabit the growth of pathogenic fungi including Rhizoctonia solani and Fusarium oxysoprum have been tested in in vitro antifungal assay. Our isolates antagonized all the tested phytopathogens in different manner. Isolates S3-C and S1-C were the most effective isolates, they had the largest inhibition zones of all isolates, in addition; both isolates suppressed the growth of all tested fungi.
7. SDS-PAGE analysis of crude supernatants proteins secreted by the most potent bacterial isolates S3-C in unconcentrated M9 media amended by colloidal chitin as a chitinase inducer have been carried out. Proteins in the cell-free culture supernatant of the most effective isolates Bacillus cereus S3-C were concentrated by ammonium sulfate precipitation. The results revealed that the chitinase activities induction by colloidal chitin as the substrate has been recorded in the proteins fractions 80% of isolate S3-C. The fraction 80% was given the highest activity also the band corresponding to ChiA gene was detected in SDS-PAGE at ≈ 95 KDa.
8. PCR was used to detect the presence of chiA gene in local isolate B.cereus S3-C using chiA gene-specific primers (Wen at el., 2002).The gene of interest was evident in all isolates and reference strain, full length of chiA gene from the Bacillus Cereus S3-C which exhibited the High chitinase activity and antifungal properties. Amplification of 1080 bp fragment was performed using pair of primers chiA CHIS-F and CHIS-R on complete genomic DNA of isolate B.cereus S3-C. The size of amplified product was identical to the expected amplified fragment 1080 bp.
9. Two pairs of specific PCR primers were designed based on the published Bacillus Cereus chiA sequences to amplify the full-length chiA gene of S3-C local isolate. The full-length of chitinase gene (chiA) from the local isolate of Bacillus cereus S3-C was detected by pair of primers CHIS-F and CHIS-R and it was 1080 bp.
10. Two experiments were made to use the most active antagonistic isolate of B.cereus S3-C for suppression the damping off disease one in Potato under greenhouse and other under growth room conditions. We can observe the source of variance (SOV) was returned to B.cereus S3-C factor in reduction of R.Solani and F.oxysoprum factors which are soil borne potato fungal pathogens under greenhouse and growth room conditions.