الفهرس 
MATERIALS AND METHODSOnly 14 pages are availabe for public view
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Abstract
Actinobacteria represent the most prominent group of microorganisms, which produce a vast number of bioactive compounds especially the Streptomyces genus which is the richest source of natural products especially antibiotics, enzymes, vitamins, hormones and other bioactive compounds. Streptomyces strains are potent producers of a wide range of secondary metabolites with diverse biological activities, which include medicinally and agriculturally important compounds.
The research aims to study the biological activity of some local strains of actinobacteria, with aims to produce antimicrobial substances and using them in the biological control of some phytopathogenic microorganisms. To achieve this goal, the following steps were performed:
1. Isolation of some local isolates of actinobacteria from different Egyptian soils.
2. Purification and identification of the local isolates up to genus.
3. Screening the purified actinomycete isolates according to their biological activities (antimicrobial activity).
4. Selection and identification of isolate showing high biological activities up to species.
5. Optimization of growth and productivity of the most active strain.
6. Application experiments for the use of antimicrobial substances produced by the selected strain to biocontrol of phytopathogenic microorganisms.
The most important results obtained can be summarized as follows:
1- Sixty actinobacterial isolates were isolated from 27 different soil samples collected from 8 governorates of Egypt. All isolates were purified using starch nitrate agar medium and identified up to genus according to their cultural, morphological and physiological characteristics. They were found to belong to genus Streptomyces, as they form well developed branching, non-septate, non-fragmented aerial mycelium bearing along spore chains and non-motile spores which not borne in verticillate sporophores.
2- Sixty Streptomyces isolates were screening according to their antimicrobial activity against some pathogenic fungi, bacteria and virus, where 3 separate experiments were performed as follows:
a. Sixty Streptomyces isolates were screening according to their ability to secrete the antifungal activity against the following pathogenic fungi Fusarium oxysporum f. sp. Lycopersici, Alternaria alternata MTCC 1362 and Rhizoctonia solani ATCC 76144. It was found that 33 isolates have an inhibitory effect on the former fungi. Accordingly, a statistical analysis was conducted for 33 isolates that had an inhibitory effect on fungi causing previous diseases, and the results showed significant differences between isolates and the best isolate that gave the highest inhibition zone was MSQ21, which was gray in its color. The diameter of the halo inhibition on the fungus is as follows: F.oxysporum 32mm, Alt.alternata32mm and R.solani 18mm.
b. Sixty Streptomyces isolates were screening according to their ability to secrete the antibacterial activity of the following pathogenic Ralstonia solanacearum type3 biovar2. It was found that 9 isolates have an inhibitory effect on the former bacteria. Accordingly, a statistical analysis was conducted for 9 isolates that had an inhibitory effect on bacteria causing previous diseases, and the results showed significant differences between isolates and the best isolate that gave the highest inhibition zone was MSQ21, which was gray in its color. The diameter of the halo inhibition on the bacteria was as follows: Ralstonia solanacearum 18 mm.
c. Sixty Streptomyces isolates were screening according to their ability to secrete the antiviral activity of the following pathogenic ToMV on Datura metal and the antiviral inhibition were divided to three categories high > 75 %, moderate 50-75 % and low < 50%). Streptomyces isolates B1, B3, MCA10, C2, QDE10, D4, SFG10, F2, F5, F7, MSQ21, FJB70, K3, M1, M2, M3, QMH51, M6, M8, S2 and N1 showed high inhibition. Accordingly, it was found that the best 5 isolates that have an inhibitory effect on the previous virus are MCA10, F5, MSQ21, FJB70 and QMH51, with a percentage of 88%.
from the previous results, it was found that the best isolate that gives antimicrobial activities was Streptomyces MSQ21, where the percentage of inhibition zone against the different phytopathogenic microorganisms was F.oxysporum (77.8%), Alt.alternata (75.5%) and R.solani (66.7%), Ralstonia solanacearum (80%) and ToMV (88%). Accordingly, the Streptomyces isolate MSQ21 was defined according to their cultural, morphological, physiological and biochemical characteristics. It’s of interesting to mention that in all standard references examined (Shirling and Gottlieb 1968a, b and 1972; Kuster, 1972; Pridham and Tresner, 1974 and Bergey’s Manual, 2012), no similar species was found to Streptomyces isolate MSQ21. Therefore, the phylogenetic analysis based on 16SrRNA gene sequencing of the selected Streptomyces isolate MSQ21 was studied, and it was found to belong to S. maritimus (strain nov) (925 nucleotides), and documented in GenBank under the accession number of LC638499.1.
3- The effect of different factors on the production of the antibacterial activity against R.solanacearum was studied by using the new strain of S. maritimus MSQ21. The effect of different carbon sources such as glucose, glycerol, dextrin, galactose and sucrose, in addition to some plant residues such as corn cobs, rice straw and wheat straw by substitution instead of starch in an environment of starch and nitrate, was also studied. By statistical analysis, significant differences were found between carbon sources and accordingly show that the best suitable carbon source for increasing the antibacterial activity of Streptomyces maritimus strain MSQ21 against Ralstonia solanacearum was glycerol because it gave the highest inhibition zone (20mm) and the lowest mycelial dry weight (90 mg/50ml. The effect of different nitrogen sources such as calcium nitrate, sodium nitrate, ammonium nitrate, ammonium sulfate, urea, peptone, beef extract and yeast extract by substitution instead of potassium nitrate in the starch nitrate medium was also studied, and by statistical analysis, significant differences were found between nitrogen sources and accordingly it was found that the best nitrogen source for antibacterial activity was ammonium sulfate because it gave the highest antibacterial activity (27mm of inhibition zone) and lowest mycelial dry weight (90mg/50ml). Based on the previous results, glycerol and ammonium sulfate were the best sources of carbon and nitrogen.
4- The production of the antibacterial activity of R.solanacearum was maximized by studying the previous nutritional factors with environmental factors by the new strain S.maritimus strain MSQ21 through the response surface methodology, which was carried out in two steps of the Plackett-Burman design and the central compound design.
5- Selection of the most significant variables using Plackett-Burman design by entering 11 factors, it gave 12 runs, and the results obtained revealed that the inhibition zone diameters formation by S.maritimus strain MSQ21 against R.solanacearum was ranged from 4 to 28 mm. The largest inhibition zone diameter (28 mm) was measured in run number 1 with the medium consisting of glycerol (22.5 g/l); (NH4)2SO4 (1.3 g/l) and adjusted pH to 8.5, inoculated with (1ml/50ml medium( and then incubated at 30±2°C and on rotary shaker 225 rpm for 4 days. Statistical analysis was done using ANOVA the analyzed results of S. maritimus strain MSQ21 suggests that out of 7 different independent variables, only three (glycerol, (NH4)2SO4 and agitation speed) were most significantly variables affected the antibacterial activity.
6- Improvement of antibacterial activity production by central composite design where the 3 variables which most affecting the antibacterial activity were entered at 3 different levels, and the results obtained revealed that the inhibition zone diameters formation (actual and predicted) by S. maritimus strain MSQ21 against R. solanacearum were ranged from 5 - 30 and 3.35 – 30.4 mm, respectively. The largest inhibition zone diameter in actual form (30 mm) and predicated form (30.4 mm) was appeared in run number 7 with the medium consisting of glycerol (25 g/l); (NH4)2SO4 (1.6 g/l) and incubated at 250 rpm, respectively. By statistical analysis using ANOVA, it was found that the coefficient of determination (R2) 0.931. This indicates a high correlation between the experimental and predicted values.
It could be concluded that the maximum antibacterial activity (30mm) produced by S. maritimus strain MSQ21 against R. solanacearum could be obtained by using glycerol- ammonium sulphate medium containing (g L-1): glycerol, 25; ammonium sulphate, 1.6; dipotassium hydrogen phosphate, 1; magnesium sulphate, 0.5; sodium chloride, 0.5; calcium carbonate, 3; ferrous sulphate, 0.01 and distilled water up to 1 liter and under the following enviromental conditions: temperature 30˚, agitation speed 250 rpm, inoculum size 1ml/50ml medium, incubation period 4 days and initial pH 8.5.
7- Accordingly, three pot experiments were designed to evaluate the efficiency of the antimicrobial activity produced by S. maritimus strain MSQ21 against some phytopathogenic microorganisms (F.oxysporum, R.solanacearum and ToMV) with the aim of biological control of infected tomato plants. The most important results can be summarized in the following.
a. The first experiment: It was designed to evaluate the efficiency of antimicrobial agent produced by S. maritimus strain MSQ21 on tomato plants infected with F.oxysporum fungus. The most important results can be summarized in the following.
• Growth parameters: Impact of inoculation with antimicrobial inoculum (MSQ21) on tomato plants infected with F.oxysporum fungus was enhanced the growth plants where significantly increased the plant height, stem diameter, number of leaves, fresh and dry weights as compared with only infected tomato plants (Control).
• Photosynthetic pigments: The chlorophyll a, b and carotenoid contents were significantly decreased in F.oxysporum infested tomato plants. On the otherhand, the plants inoculated with antimicrobial inoculum (MSQ21) showed significant increased in chlorophyll a, b, and carotenoid contents.
• Total protein contents: Total protein content was significantly increase in F.oxysporum infested tomato plants. On the otherhand, the plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in total protein contents
• Peroxidase activity (POD): peroxidase activity was significantly increase in F.oxysporum infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in peroxidase activity.
b. The second experiment: It was designed to evaluate the efficiency of antimicrobial agent produced by S. maritimus strain MSQ21 on tomato plants infected with R. solanacearum. The most important results can be summarized in the following.
• Growth parameters: Impact of inoculation with antimicrobial inoculum (MSQ21) on tomato plants infected with R. solanacearum was enhanced the growth plants where significantly increased the plant height, stem diameter, number of leaves, fresh and dry weights as compared with only infected tomato plants (Control).
• Photosynthetic pigments: The chlorophyll a, b and carotenoid contents were significantly decreased in R. solanacearum infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant increased in chlorophyll a, b, and carotenoid contents.
• Total protein contents: Total protein content was significantly increase in R. solanacearum infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in total protein contents
• Peroxidase activity (POD): peroxidase activity was significantly increase in R. solanacearum infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in peroxidase activity.
c. The third experiment: It was designed to evaluate the efficiency of antimicrobial agent produced by S. maritimus strain MSQ21 on tomato plants infected with ToMV virus. The most important results can be summarized in the following.
• Growth parameters: Impact of inoculation with antimicrobial inoculum (MSQ21) on tomato plants infected with ToMV virus was enhanced the growth plants where significantly increased the plant height, stem diameter, number of leaves, fresh and dry weights as compared with infected tomato plants (Control).
• Photosynthetic pigments: The chlorophyll a, b and carotenoid contents were significantly decreased in ToMV infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant increased in chlorophyll a, b, and carotenoid contents.
• Total protein contents: Total protein content was significantly increased in ToMV infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in total protein contents
• Peroxidase activity (POD): peroxidase activity was significantly increased in ToMV infested tomato plants. OActinobacteria represent the most prominent group of microorganisms, which produce a vast number of bioactive compounds especially the Streptomyces genus which is the richest source of natural products especially antibiotics, enzymes, vitamins, hormones and other bioactive compounds. Streptomyces strains are potent producers of a wide range of secondary metabolites with diverse biological activities, which include medicinally and agriculturally important compounds.
The research aims to study the biological activity of some local strains of actinobacteria, with aims to produce antimicrobial substances and using them in the biological control of some phytopathogenic microorganisms. To achieve this goal, the following steps were performed:
1. Isolation of some local isolates of actinobacteria from different Egyptian soils.
2. Purification and identification of the local isolates up to genus.
3. Screening the purified actinomycete isolates according to their biological activities (antimicrobial activity).
4. Selection and identification of isolate showing high biological activities up to species.
5. Optimization of growth and productivity of the most active strain.
6. Application experiments for the use of antimicrobial substances produced by the selected strain to biocontrol of phytopathogenic microorganisms.
The most important results obtained can be summarized as follows:
1- Sixty actinobacterial isolates were isolated from 27 different soil samples collected from 8 governorates of Egypt. All isolates were purified using starch nitrate agar medium and identified up to genus according to their cultural, morphological and physiological characteristics. They were found to belong to genus Streptomyces, as they form well developed branching, non-septate, non-fragmented aerial mycelium bearing along spore chains and non-motile spores which not borne in verticillate sporophores.
2- Sixty Streptomyces isolates were screening according to their antimicrobial activity against some pathogenic fungi, bacteria and virus, where 3 separate experiments were performed as follows:
a. Sixty Streptomyces isolates were screening according to their ability to secrete the antifungal activity against the following pathogenic fungi Fusarium oxysporum f. sp. Lycopersici, Alternaria alternata MTCC 1362 and Rhizoctonia solani ATCC 76144. It was found that 33 isolates have an inhibitory effect on the former fungi. Accordingly, a statistical analysis was conducted for 33 isolates that had an inhibitory effect on fungi causing previous diseases, and the results showed significant differences between isolates and the best isolate that gave the highest inhibition zone was MSQ21, which was gray in its color. The diameter of the halo inhibition on the fungus is as follows: F.oxysporum 32mm, Alt.alternata32mm and R.solani 18mm.
b. Sixty Streptomyces isolates were screening according to their ability to secrete the antibacterial activity of the following pathogenic Ralstonia solanacearum type3 biovar2. It was found that 9 isolates have an inhibitory effect on the former bacteria. Accordingly, a statistical analysis was conducted for 9 isolates that had an inhibitory effect on bacteria causing previous diseases, and the results showed significant differences between isolates and the best isolate that gave the highest inhibition zone was MSQ21, which was gray in its color. The diameter of the halo inhibition on the bacteria was as follows: Ralstonia solanacearum 18 mm.
c. Sixty Streptomyces isolates were screening according to their ability to secrete the antiviral activity of the following pathogenic ToMV on Datura metal and the antiviral inhibition were divided to three categories high > 75 %, moderate 50-75 % and low < 50%). Streptomyces isolates B1, B3, MCA10, C2, QDE10, D4, SFG10, F2, F5, F7, MSQ21, FJB70, K3, M1, M2, M3, QMH51, M6, M8, S2 and N1 showed high inhibition. Accordingly, it was found that the best 5 isolates that have an inhibitory effect on the previous virus are MCA10, F5, MSQ21, FJB70 and QMH51, with a percentage of 88%.
from the previous results, it was found that the best isolate that gives antimicrobial activities was Streptomyces MSQ21, where the percentage of inhibition zone against the different phytopathogenic microorganisms was F.oxysporum (77.8%), Alt.alternata (75.5%) and R.solani (66.7%), Ralstonia solanacearum (80%) and ToMV (88%). Accordingly, the Streptomyces isolate MSQ21 was defined according to their cultural, morphological, physiological and biochemical characteristics. It’s of interesting to mention that in all standard references examined (Shirling and Gottlieb 1968a, b and 1972; Kuster, 1972; Pridham and Tresner, 1974 and Bergey’s Manual, 2012), no similar species was found to Streptomyces isolate MSQ21. Therefore, the phylogenetic analysis based on 16SrRNA gene sequencing of the selected Streptomyces isolate MSQ21 was studied, and it was found to belong to S. maritimus (strain nov) (925 nucleotides), and documented in GenBank under the accession number of LC638499.1.
3- The effect of different factors on the production of the antibacterial activity against R.solanacearum was studied by using the new strain of S. maritimus MSQ21. The effect of different carbon sources such as glucose, glycerol, dextrin, galactose and sucrose, in addition to some plant residues such as corn cobs, rice straw and wheat straw by substitution instead of starch in an environment of starch and nitrate, was also studied. By statistical analysis, significant differences were found between carbon sources and accordingly show that the best suitable carbon source for increasing the antibacterial activity of Streptomyces maritimus strain MSQ21 against Ralstonia solanacearum was glycerol because it gave the highest inhibition zone (20mm) and the lowest mycelial dry weight (90 mg/50ml. The effect of different nitrogen sources such as calcium nitrate, sodium nitrate, ammonium nitrate, ammonium sulfate, urea, peptone, beef extract and yeast extract by substitution instead of potassium nitrate in the starch nitrate medium was also studied, and by statistical analysis, significant differences were found between nitrogen sources and accordingly it was found that the best nitrogen source for antibacterial activity was ammonium sulfate because it gave the highest antibacterial activity (27mm of inhibition zone) and lowest mycelial dry weight (90mg/50ml). Based on the previous results, glycerol and ammonium sulfate were the best sources of carbon and nitrogen.
4- The production of the antibacterial activity of R.solanacearum was maximized by studying the previous nutritional factors with environmental factors by the new strain S.maritimus strain MSQ21 through the response surface methodology, which was carried out in two steps of the Plackett-Burman design and the central compound design.
5- Selection of the most significant variables using Plackett-Burman design by entering 11 factors, it gave 12 runs, and the results obtained revealed that the inhibition zone diameters formation by S.maritimus strain MSQ21 against R.solanacearum was ranged from 4 to 28 mm. The largest inhibition zone diameter (28 mm) was measured in run number 1 with the medium consisting of glycerol (22.5 g/l); (NH4)2SO4 (1.3 g/l) and adjusted pH to 8.5, inoculated with (1ml/50ml medium( and then incubated at 30±2°C and on rotary shaker 225 rpm for 4 days. Statistical analysis was done using ANOVA the analyzed results of S. maritimus strain MSQ21 suggests that out of 7 different independent variables, only three (glycerol, (NH4)2SO4 and agitation speed) were most significantly variables affected the antibacterial activity.
6- Improvement of antibacterial activity production by central composite design where the 3 variables which most affecting the antibacterial activity were entered at 3 different levels, and the results obtained revealed that the inhibition zone diameters formation (actual and predicted) by S. maritimus strain MSQ21 against R. solanacearum were ranged from 5 - 30 and 3.35 – 30.4 mm, respectively. The largest inhibition zone diameter in actual form (30 mm) and predicated form (30.4 mm) was appeared in run number 7 with the medium consisting of glycerol (25 g/l); (NH4)2SO4 (1.6 g/l) and incubated at 250 rpm, respectively. By statistical analysis using ANOVA, it was found that the coefficient of determination (R2) 0.931. This indicates a high correlation between the experimental and predicted values.
It could be concluded that the maximum antibacterial activity (30mm) produced by S. maritimus strain MSQ21 against R. solanacearum could be obtained by using glycerol- ammonium sulphate medium containing (g L-1): glycerol, 25; ammonium sulphate, 1.6; dipotassium hydrogen phosphate, 1; magnesium sulphate, 0.5; sodium chloride, 0.5; calcium carbonate, 3; ferrous sulphate, 0.01 and distilled water up to 1 liter and under the following enviromental conditions: temperature 30˚, agitation speed 250 rpm, inoculum size 1ml/50ml medium, incubation period 4 days and initial pH 8.5.
7- Accordingly, three pot experiments were designed to evaluate the efficiency of the antimicrobial activity produced by S. maritimus strain MSQ21 against some phytopathogenic microorganisms (F.oxysporum, R.solanacearum and ToMV) with the aim of biological control of infected tomato plants. The most important results can be summarized in the following.
a. The first experiment: It was designed to evaluate the efficiency of antimicrobial agent produced by S. maritimus strain MSQ21 on tomato plants infected with F.oxysporum fungus. The most important results can be summarized in the following.
• Growth parameters: Impact of inoculation with antimicrobial inoculum (MSQ21) on tomato plants infected with F.oxysporum fungus was enhanced the growth plants where significantly increased the plant height, stem diameter, number of leaves, fresh and dry weights as compared with only infected tomato plants (Control).
• Photosynthetic pigments: The chlorophyll a, b and carotenoid contents were significantly decreased in F.oxysporum infested tomato plants. On the otherhand, the plants inoculated with antimicrobial inoculum (MSQ21) showed significant increased in chlorophyll a, b, and carotenoid contents.
• Total protein contents: Total protein content was significantly increase in F.oxysporum infested tomato plants. On the otherhand, the plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in total protein contents
• Peroxidase activity (POD): peroxidase activity was significantly increase in F.oxysporum infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in peroxidase activity.
b. The second experiment: It was designed to evaluate the efficiency of antimicrobial agent produced by S. maritimus strain MSQ21 on tomato plants infected with R. solanacearum. The most important results can be summarized in the following.
• Growth parameters: Impact of inoculation with antimicrobial inoculum (MSQ21) on tomato plants infected with R. solanacearum was enhanced the growth plants where significantly increased the plant height, stem diameter, number of leaves, fresh and dry weights as compared with only infected tomato plants (Control).
• Photosynthetic pigments: The chlorophyll a, b and carotenoid contents were significantly decreased in R. solanacearum infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant increased in chlorophyll a, b, and carotenoid contents.
• Total protein contents: Total protein content was significantly increase in R. solanacearum infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in total protein contents
• Peroxidase activity (POD): peroxidase activity was significantly increase in R. solanacearum infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in peroxidase activity.
c. The third experiment: It was designed to evaluate the efficiency of antimicrobial agent produced by S. maritimus strain MSQ21 on tomato plants infected with ToMV virus. The most important results can be summarized in the following.
• Growth parameters: Impact of inoculation with antimicrobial inoculum (MSQ21) on tomato plants infected with ToMV virus was enhanced the growth plants where significantly increased the plant height, stem diameter, number of leaves, fresh and dry weights as compared with infected tomato plants (Control).
• Photosynthetic pigments: The chlorophyll a, b and carotenoid contents were significantly decreased in ToMV infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant increased in chlorophyll a, b, and carotenoid contents.
• Total protein contents: Total protein content was significantly increased in ToMV infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in total protein contents
• Peroxidase activity (POD): peroxidase activity was significantly increased in ToMV infested tomato plants. On the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in peroxidase activity.
n the otherhand, the tomato plants inoculated with antimicrobial inoculum (MSQ21) showed significant decreased in peroxidase activity.