الفهرس 
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Abstract
Genitical and biochemical analysis of cell wall degrading enzyme genes in some plant pathogenic and non-pathogenic fungi Three fungus were used in recent study: Aspergillus flavus, Aspergillus niger and Trichoderma viride to investigate the changes in their analytical activity after treatment with different substrates. Biochemical and molecular responses of these fungus were determined. 1- Materials and methods • Three fungus were used in recent study: Aspergillu flavus, Aspergillus. niger and Trichoderma viride • Inhibition and inducing effects of nine different substrates were investigated Aspirin, clove oil, tea tree oil, Trichoderma extract, cinnamon oil, rosemary oil, chitosan, nano-silica and nano-selenium. 1.2. Measurements 1.2.1. Fungus characterization 1.2.2. Minimum inhibitory concentration 1.2.3. Methods of applying treatments with different studied materials Three plating methods were used to investigate the inhibition or inducing effects of used substrates: - Agar plate diffusion plate method - The agar disk diffusion method - Fungi-substrate contact method 1.2.4. Reducing sugar assay 1.2.5. Analysis of gene expression of some analytical enzyme encoding genes such as: cellulases, chitinases and pectinases. 2. Results Obtained results could be summeriezed in the following points 2.1. Fungus characterization According to the partial sequence analysis of PCR product using ITS rRNA gene primers and their blast result on NCBI database obtained results revealed that used organisms have high similarity percentage with the known strains on data base. 2.2. MIC analysis Determination of minimum inhibitory concentration revealed that: • MIC values of clove oil were: 1 ppm against A. flavus and A. niger and 0.8 ppm • againist T. viride. Summary 64 • MIC values of tea tree oil were: 0.4 ppm against A. flavus , A. niger and T. viride. • MIC values of aspirin were: 7275 ppm against A. flavus, 3825 ppm against A. niger and 7125 ppm againist T. viride. • MIC values of Trichoderma viride extract: the results showed that it does not complete inhibition against A. flavus and A. niger, but only reduced growth 2.3. Methods of applying treatments with different studied materials • Screening methods revealed that clove oil, tea tree oil and aspirin have inhibitory effects for all studied fungus.Trichoderma extract showed an inhibitory effect against A. niger and A. flavus. • Rosemary oil, cinnamon oil, chitosan, Nano silica and Nano selenium showed a stimulation effect for studied fungus. 2.4. Reducing sugar assay: The effect of the materials used on the analytical activity was determined by estimating the reduced sugar production. • Treatment with cloves, tea tree oil, aspirin and T. viride extract significantly reduced the production of reduced sugars. • For A. flavus, the highest reduction effect was recorded for clove oil treatment followed by tea tree oil, then aspirin then T. viride extract. • It was observed that the decrease in the values of reduced sugar is associated with the effect of inhibition of the materials used on the production of enzymes that degrade cellulose, and the production of enzymes is affected when the growth of fungi is inhibited. • The highest efficiency in producing reduced sugar in A. flavus was recorded under cinnamon oil treatment followed by chitosan, rosemary oil , ,Nano selenium then Nano silica with significant differences between treatments • Our results point to the role for cinnamon oil, rosemary oil, chitosan, Nano silica, and Nano selenium in enhancing cellulases enzyme activity and increasing glucose production by A. flavus. • For A. niger, treatment with clove oil, tea tree oil, aspirin and T. viride extract induced a significant reduction in the production of reduced sugars. • The lowest value of produced sugar was recorded in A. niger treated with aspirin followed by tea tree oil, clove oil, then T. viride extract. • It was observed that the decrease in the values of reduced sugar is associated with the effect of inhibition of the materials used on the production of enzymes that degrade cellulose, and the production of enzymes is affected when the growth of fungi is inhibited. • The highest sugar production efficiency was recorded under rosemary oil treatment followed by chitosan, cinnamon oil nano silica then nano selenium. Summary 65 • Obtained results indicate to the role of cinnamon oil, rosemary oil, chitosan, Nano- silica and Nano-selenium in enhancing cellulases enzyme activity and increasing glucose production in A. niger. • For T. viride treatment with clove oil, tea tree oil, aspirin induced significant reduction in the production of reduced sugars. The lowest production of reducing sugar was recorded with tea tree oil treatment followed by clove oil, then aspirin. The highest value of reduced sugar was recorded under rosemary oil treatment followed by cinnamon oil chitosan, nano silica then nano selenium. 2.5. Analysis of gene expression Expression patterns of genes relevant to cell wall analysis were detected as changes in transcript amount using q RT-PCR analysis. The genes studied included: 2.5.1. A. flavus Expression patterns of genes relevant to cell wall analysis were detected as changes in transcript amount using q RT-PCR analysis. The studied genes included: - Pectinases, polygalacerturonase, (pec-A and pec-B) - Cellulases, exoglucanase (cbh-B and exo) and endoclucanase (egl-A and egl-B). • Obtained results revealed that treatment with chitosan induced up-regulation for all studied genes. where the highest stimulation effect was recorded for egl-A encoding gene with 36.757 folds compared to the control. • All studied genes were up-regulated as a result of treatment with Nano silica. The highest stimulation effect was for egl-A encoded gene with 6.906 fold in the amount of its transcript amount comparing with the control. • Aspirin treatment induced an increase in the transcript amount for only exo encoding gene with 2.228 folds comparing with control treatment. Transcript amount of pec-A, pec-B, cbh-B, egl-A and egl-B encoding genes were reduced under aspirin treatment. • Treatment with clove oil and tea tree oil induced an inhibition for all studied genes. 2.5.2. T. viride - Chitinase enzymes: endochitinase 42 (chit-42) and (chit-33) - Pectinases, polygalacerturonase, (pec-A and pec-B) - Cellulases: exoglucanase (cbh1) and endoclucanase (Egl B). • Obtained results revealed that treatment with chitosan showed significant up- regulation effects on the expression of all studied genes. The highest Summary 66 stimulation was recorded for pec-A encoding gene. where its transcript amount was increased with 1714.83 fold compared to the control treatment. • Treatment with nano-silica induced up-regulation for 5 studied genes. The highest stimulation effect was recorded for pec-A encoding gene. where it’s transcript amount was increased up to 5 folds comparing with control. Only pec-B encoding gene was down regulated under nano silica treatment. • All the studied genes were up-regulated under treatment with Nano- selenium. The highest stimulation was for Egl-B gene with five folds comparing with control treatment • Treatment with aspirin showed a significant up-regulation effect for chit33 encoding gene. The transcript amount was increased by 6571.2 fold comparing with control. Slight stimulation of Egl B gene (2 folds) was recorded after asprin treatment. The transcript amount of pec-A, pec-B, ech42 and cbh1 were reduced after aspirin treatment. • Based on these results, we recommend the use of chitosan, Nano silica and Nano selenium in the treatment of trichoderma fungus to increase the production of all selected genes in large scale for biological control and industry. Based on these results, we recommend either using inhibitors such as clove oil, tea tree oil and aspirin to inhibit the growth of A. flavus fungus by limiting the production of enzymes required for analysis of host tissues. Thus control the hazardous effect of these pathogenic fungus to plants and agricultural products as well as to human health.