الفهرس 
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Abstract
The present work was carried out for isolation, purification and selection of efficient endophytic bacterial isolates associated with some medicinal plants due to their PGP activities and production of bioactive secondary metabolites. As well as, verify their effects, to induce the systemic resistance in basil plants (Ocimum basilicum) in presence of Fusarium oxysporium (biotic stress). Addtionally, evaluate the effect of the most potent strains as a biofertilizer and bicontrol agent on growth and disease severity of Ocimum basilicum (basil) and Coriandrum sativum (coriander). The results could be summarized as follows:
1. Isolation of endophytic microorganisms
Eighty nine endophytic bacterial isolates were isolated by two techniques from surface sterilized roots of seven medicinal plants. The percentage of bacterial isolates were 21.34%, 19.10%, 17.97%, 15.73%, 10.11%, 8.98% and 6.74% from Rosmarinus officinalis, Pelargonium graveolens, Ocimum basilicum, Saliva officinalis, Anethum graveolens, Coriandrum sativum and Origanum majorana respectively.
2. Primary screening of PGP traits
Primary screening includes five experiments; the ability of endophytic isolates to secrete exoenzymes to hydrolyzed three main component of plant cell wall (celluloses, pectinases and amylases). As well as, production of two phytohormones (indol acetic acid and gibberellic acid). Results showed that out of 89 isolates; 44 isolates can hydrolyse the carboxy-methyl cellulose, 36 isolates can produce pectinase enzyme, and 28 isolates gave positive result for starch solubilization. For phytohormones production in broth medium, out of 89 endophytic isolates; twenty two isolates can produced IAA and 82 isolates produced GA3 that was determined as ppm.
3. Secondary screening
Secondary screening experiments depend on test the ability of twenty selected endophytic bacterial isolates, which were selected from primary screening to produce different bioactive compounds. Besides,
SUMMARY
Eman Adel Sayed Ahmed (2020), Ph.D., Fac. of Agric., Ain Shams Univ.
96
their abilities to antagonize some phytopathogenic fungi, represented as % of fungal growth reduction. Isolate S14, D2 showed the greatest inhibition on Fusarium solani B99 growth being 46.94 % and 43.9% fungal growth reduction, respectively. All the 20 selected isolates gave negative results of inhibition against Rhizoctonia solani Z7 except isolate S14 with 28.04% growth reduction. Isolate B3 and S14 recorded the highest values of % growth reduction against Fusarium oxysporium B27 and Sclerotium rolfsii B20. Among the twenty selected isolates, B3 isolate showed maximum antioxidant activity (64.50%), while isolate R3 isolated from Rose geranium plant gave the minimum antioxidant activity (18.53%). Additionally, the superior amount of total phenols was secreted by isolate D6, whereas the high amount of IAA was produced by isolate S14 giving 8.84 ppm.
Using Nessler’s reagent, all the 20 selected isolates gave positive result for NH3 production except isolates RO3 and S2. While thirteen isolates produced different amount of siderophores ranged from 38 by isolate S7 to 67.5 mMDFOM by isolate S14, four isolates; RO14, C8, R15 and M3 had a nitrogenase activity being 12, 6, 4 and 4 nmole C2H4/100 ml/h, respectively. Six bacterial isolates were selected from previous testes as the most efficient isolates. The six isolates can produce flavonoids ranged from 1.43 to 31.14 ppm. Also, all the six most efficient isolates can produce alkaloids except isolate S14. Four isolates (RO10, RO14, S14 and C8) were able to solubilize un-soluble phosphate. Data showed that the six isolates gave negative result for HCN production.
4. Colonization of endophytic inside the plant tissue
In this study, the most efficient isolates (6 isolates) were evaluated to colonize the plant tissue of two medicinal plants (Ocimum basilicum (basil) and Coriandrum sativum (coriander) by two methods as follow:
a- TTC stain technique
This technique depend on enzymatic action, where all the six isolates gave positive result which appeared in change of inoculated roots
SUMMARY
Eman Adel Sayed Ahmed (2020), Ph.D., Fac. of Agric., Ain Shams Univ.
97
color to red after treated with 2,3,5-triphenyl tetrazolium chloride (TTC) stain.
b- Transmission electron microscope examination (TEM)
Images by TEM showed the endophytic bacteria on surface of roots and colonize intercellular and intracellular space for cortex tissue of two medicinal plants basil and coriander.
5. Phenotypic characteristics
Based on some morphological and biochemical characteristics the most efficient isolates (six isolates) were belong to three genera; Enterobacter sp., Aeromonas sp. and Bacillus sp.
6. Hypersensitivity test
Hypersensitivity test proved that all the selected endophytic bacterial isolates (RO10, RO14, S14, D6, C8 and B3) are nonpathogenic bacteria.
7. Induce systemic resistance in basil plants under greenhouse conditions
Six bacterial isolates with multiple plant beneficial traits were chosen to evaluate their abilities to induce systemic resistance in infested basil plant. The results showed that the highest percentage of plant survival was 100% for all treatments of individual endophytic isolates compared with control treatment (fungal inoculation alone) which gave 40% for survival rate.
Inoculation basil plant with isolates S14, RO10 and D6 showed high significant increase in total dry weight being 34.01, 13.00 and 12.02 g/plant, respectively, compared with control treatments. Also, the photosynthesis pigments including chlorophyll a, chlorophyll b and carotenoids increased compared with control treatments. As well as, the isolate S14 showed the highest chl a, chl b and B- carotenoids compared with the other endophytic treatments. All bacterial treatments showed high content of nitrogen, phosphorus and potassium uptake for basil plants compared with the control (un-inoculated) treatments. Furthermore,
SUMMARY
Eman Adel Sayed Ahmed (2020), Ph.D., Fac. of Agric., Ain Shams Univ.
98
isolate Enterobacter sp. S14 showed high absorption of nitrogen, phosphorus and potassium in Ocimum basilicum plant.
In general, all the six bacterial treatments gave a high bioactive compound compared with controls treatments. Total phenols ranged from 0.07 g/100 g.f.w by isolate RO14 to 0.32 g/100 g.f.w by isolates S14. As well as, there is a significant effect between bacterial treatments and control treatments (without bacteria). In this respect, the total flavonoids were determined in the basil leaves to record high quantity. As well as, antioxidants activity ranged from 91.19 to 94.48 % in fresh leaves of basil plants treated with bacteria individually against 80 % without bacteria.
It was found that the highest activity of catalase was obtained by bacterial isolate RO10, RO 14 and B3. Bacterial isolates C8 induced significantly to produce the highest activity of peroxidase in plant whereas there is no significant difference between the other bacterial treatments. The polyphenol oxidase of plant increased significantly when inoculated by bacterial isolates RO10 and RO14 individually. Additionally, high value for polyphenol oxidase specific activity was recorded by Aeromonas sp. RO14 and Aeromonas sp. RO10.
8. Endophytic molecular identification
As a result to induced systemic resistance experiment, the three potent isolates (S14, D6 and RO10), which display a good effect on basil plant were chosen for molecular indentification. The bacterial isolates had the closest similarity to Enterobacter bugandensis, Aeromonas caviae and Bacillus tropicus with 99% similarty.
9. Performance of the enodphytic active strains as a mixed culture on basil and coriander plant under greenhouse conditions
A pot experiment was conducted at Horticulture Research Institute, using the bacterial inoculum as mixed culture of the most potent strains (Aeromonas caviae RO10 + Enterobacter bugandensis S14 + Bacillus tropicus D6) with Ocimum basilicum (basil) and Coriandrum sativum (coriander) grown in sandy loam soil infested with Fusarium oxysporium B27.
SUMMARY
Eman Adel Sayed Ahmed (2020), Ph.D., Fac. of Agric., Ain Shams Univ.
99
Diseases expression
a- Basil plant
The percentage of survival rate of plants inoculated with mixed culture in soil infested with phytopathogen Fusarium oxysporium B27 was 80% against 20% survival rate for infested plants without bacterial culture.
b- Coriander plant
The percentage of plant survival rate was 90% and 40% in infested soil with and without bacterial inoculum of endophytic strains, respectively.
Vegetative characteristics in plants
a- Basil plant
Significant increase of total dry weight and height of inoculated plants with endophytic bacteria and pathogenic fungus compared to un-inoculated plants with bacterial and fungal inocula.
b- Coriander plant
Significant increases in total dry weight, height and pigments content of Coriandrum sativum plants inoculated with mixed bacterial culture grown in soil infested with Fusarium oxysporium B27 over un-inoculated plants with fungal inoculum. The lowest data were recorded by untreated plants by endophytic inoculum with or without fungal inoculum.
Macronutrient content in plants
a- Basil plant
The values of NPK uptake of basil plant with mixed bacterial culture were 0.14, 0.15 and 0.07 g/plant against 0.07, 0.003 and 0.03 g/plant without bacterial culture in absence of fungal inoculum, respectively.
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Eman Adel Sayed Ahmed (2020), Ph.D., Fac. of Agric., Ain Shams Univ.
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b- Coriander plant
In infested soil, mixed bacterial inoculum recorded significant increases of N, P and K uptake being 5, 140 and 13 fold increases, respectively above the treatment without bacteria.
Redox compounds in leaves
a- Basil plant
The plants treated with mixed bacterial and fungal inocula gave the highest values of total phenols, flavonoids and antioxidants.
b- Coriander plant
Significant increases in bioactive compound of Coriandrum sativum plants, when inoculated with the mixed culture of the three endophytic bacterial strains in soil infested with or without Fusarium oxysporium B27. This observation was particularly obvious in total phenols, total flavonoids and antioxidant activity where about 240, 57 and 37% increases were recorded by treated plants with mixed bacterial inoculum
Enzyme activity in leaves
a- Basil plant
Catalase, peroxidase and polyphenol oxidase activities were significantly higher in plants inoculated with bacterial inoculum compared with the un-inoculated plants (control). The level of peroxidase achieved a maximum value in the bacterial treatments.
b- Coriander plant
The treated plants with endophytic inoculum showed significant increment in peroxidase and polyphenol oxidase activities than the other un-inoculated plants. In presence of fungal inoculum; application of endophytic bacteria exhibited 11.6 fold and 3.3 fold increases of peroxidase and polyphenol oxidase, respectively over un- inoculated
SUMMARY
Eman Adel Sayed Ahmed (2020), Ph.D., Fac. of Agric., Ain Shams Univ.
101
plants, whereas non-significant difference of catalase activity between all treatments.