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Abstract This study has been carried out to isolate N2- fixing Azotobacter strains from leafy vegetables plants rhizosphere i.e. spinach (Spinacia oleracea) and corchorus (Corchorus olitorius). Isolates were characterized and identified biochemically and genetically. They were evaluated to recognize the impacts of most effective Azotobacter strains to be used as bio-fertilizers in sand soil, for improving spinach and corchorus vegetables production and nutritional values. The obtained results could be summarized as following: Part І: Microbiology results 1- Two rhizosphere soil samples collected from Mashtol region, Belbeas, Sharqiya Governorate, Egypt, with spinach and corchorus standing crops used for nitrogen fixers Azotobacter spp. isolation. 2- The Azotobacter like colony morphology was selected, exanimated, and purified. 3- The most effective four strains in atmospheric N2-fixation, phytohormones like substances production i.e. auxin, gibberellin and cytokinin, and the siderophores production ability were selected as bio-fertilizers. 4- Based on the 16s rRNA gene sequences identification, the isolates nomenclature as A. chroococcum strain S11 (Az. Msp7), Az. chroococcum AtP (Az. Msp8), Azotobacter sp. ART001 (Az. Msp13), and Az. chroococcum AcN1 (Az. Msp18).Part ІІ: Pot experiment Most effective four strains used as biofertilizers in a pot experiment at Soil and Water Research Department, Nuclear Research Center, Atomic Energy Authority, Abou Zaabal, Egypt. Spinach and corchorus plants were grown in the net house for about two months. Different parameters of spinach and corchorus plants were recorded, as well as evaluation of nitrogen mineral fertilizers utilization by using 15N tracer technique. All of the nitrogen derived from fertilizers percentage (%Ndff), nitrogen derived from air percentage (%Ndfa), and nitrogen derived from compost percentage (%Ndfc) for both leafy vegetable crops were determined using the emission spectrometer (NOI-6PC). The obtained results could be summarized as following: Experiment І: spinach (Spinacia oleracea) 1) The spinach leafs reflected the highest total chlorophyll concentration 2.77 mg/ml achieved by application of 25% mineral fertilizer + 75% compost + strain Azoto AtP. 2) The superior spinach whole plant fresh weight 53.02 g pot-1, whole dry weight 6.7 g pot-1 and leaf dry weight 5.93 g pot-1 were gained with 75% mineral fertilizer + 25% compost application in the presence of strain S11. 3) The spinach plants moisture percentage wasn’t significantly different between all treatments and strains. 4) Optimum nitrogen uptake by spinach shoot was 77.17 mg N pot-1 resulted from application of 75% mineral fertilizer + 25% compost treatment + strain S11. While the root-N uptake was 7.37 mg N pot-1 with strain ART001 and treatment 50% mineral fertilizer and 50% compost. 5) The 15N tracer technique results clarified that nitrogen derived from air was the dominant N source in spinach plants when inoculated with strain S11 and strain ART001 (33.9, and 33.5 mg pot-1, respectively), followed by nitrogen derived from fertilizer (Ndff) (15.4, and 12.5 mg pot-1, respectively), then the N derived from compost (Ndfc) (11.2, and 10.7 mg pot-1, respectively) came to the next. In somehow, both strains Azoto AtP and Azoto AcN1 showed little difference where the second source of nitrogen was the compost. The main N source was Ndfa (29.8, and 27.5 mg pot-1, respectively), followed by Ndfc (14.6, and 8.7 mg pot-1, respectively), then Ndff (12.2, and 8.4 mg pot-1 respectively). 6) Nitrate uptake by shoot was high 293.871 mg NO3 pot-1 as affected by combined treatment 75% mineral fertilizer + 25% compost in the presence of strain S11. It is known that the decrease of nitrate uptake into spinach shoot is more important than increasing. So, the minimum value of 46.75 mg NO3 pot-1 was obtained when plant treated with 100% compost in presence of strain Azoto AtP. 7) The highest phosphorus uptake of spinach’s shoot was 129.992 mg P pot-1 with fertilization treatment 75% mineral fertilizer + 25% compost plus strain S11. Furthermore, the spinach’s root phosphorus uptake showed the highest value 12.853 mg P pot-1 with application of 50% mineral fertilizer + 50% compost combined with strain ART001. 8) High value of potassium uptake by spinach’s shoot 15.494 mg K pot-1 was achieved by fertilization treatment 75% mineral fertilizer + 25% compost combined with strain S11. Furthermore, the spinach’s root potassium uptake showed the highest value 1.574 mg K pot-1 with the application of 75% mineral fertilizer + 25% compost combined with strain S11. 9) Shoot total carbohydrates percentage of 3.42% was induced with combined treatment 25% mineral fertilizer + 75% compost plus strain Azoto AcN1. 10) The shoot oxalate contents showed the lowest value 0.55 mg/ml when plant treated with 100% compost plus strain S11, and strain Azoto AtP; application 75% mineral fertilizer + 25% Compost + strain Azoto AtP; application 50% mineral fertilizer + 50% Compost, and 25% mineral fertilizer + 75% compost, when both treatments combined with strain ART001. 11) Increase of Vitamin C content 7.02 g /100g in spinach’s shoot was detected with the treatment 100% mineral fertilization combined with strain Azoto AtP. 12) The maximum Vitamin B1 (Thiamine) content 0.465 g/100g of spinach’s shoot induced by the fertilization rate 75% mineral fertilizer + 25% compost, combined with strain Azoto AtP. 13) The superior vitamin B2 (riboflavin) content 2.396 g/100g plant in spinach’s shoot was achieved by strain Azoto AtP combined with 50% mineral fertilizer + 50% compost. Experiment ІІ: corchorus (Corchorus olitorius) 1) The corchorus leafs reflected the highest total chlorophyll concentration 35.67 SPAD when 25% mineral fertilizer + 75% compost in the presence of strain S11, was applied. 2) The highest corchorus whole plant fresh weight 75.1 g pot-1 was obtained by 75% mineral fertilizer + 25% compost treatment plus strain ART001. While whole dry weight 18.1 g pot-1 and leaf dry weight 7.3 g pot-1 both were with 100% mineral fertilizer treatment plus the same strain. 3) The corchorus plants moisture percentage showed no significant difference with both 100% compost, and 75% mineral fertilizer + 25% compost when both treatments combined with strain ART001. 4) The corchorus leaf optimum nitrogen uptake was 59.2 mg N pot-1 and the upper corchorus’s stem nitrogen uptake 24.4 mg N pot-1 were resulted with 100% mineral fertilizer plus strain S11. Came to the next, corchorus’s root nitrogen uptake was 42.3 mg N pot-1 induced by addition of 50% mineral fertilizer + 50% compost plus strain S11. 5) The 15N tracer technique gave the chance to distinguish the N uptake from the different nitrogen sources, which clarified that the highest N source in corchorus plants when they were treated with strain S11,strain Azoto AtP, strain ART001, and strain Azoto AcN1 was Ndfa (34.5, 31.1, 33.8 and 32.1 mg pot-1, respectively), followed by Ndfc (30.1, 28.3, 33.2 and 19.8 mg pot-1, respectively), then Ndff (24.6, 26.1, 15.1 and 10.3 mg pot-1, respectively). 6) The total carbohydrates percentage (4.08%) in corchorus leafs was obtained with the fertilization treatment of 50% mineral fertilizer + 50% compost + strain Azoto AtP. Also, the corchorus’s stem total carbohydrate percentage (1.79%) was detected with the application of 25% mineral fertilizer mixed with 75% compost combined in the presence of strain ART001. Came to the next, the value of corchorus’s root total carbohydrate percentage (2.25%) induced with 75% mineral fertilizer + 25% compost combined with strain Azoto AcN1. 7) It appears that the optimum phosphorus uptake by corchorus’s leaf (51.7 mg P pot-1). Furthermore, high corchorus’s stem phosphorus uptake 41.2 mg P pot-1 was detected with the application of 100% mineral fertilizer + strain ART001. Corchorus’s root phosphorus uptake 55.8 mg P pot-1 was resulted with Azoto AtP combined with 100% mineral fertilizer. 8) High potassium uptake of corchorus’s leaf was 1.66, and 1.65 mg K pot-1 was detected with the application of 100% mineral fertilizer + strain S11, and ART001, respectively. So, the corchorus’s stem potassium uptake value was 1.49 mg K pot-1 induced by the application of 100% mineral + strain ART001. The corchorus’s root potassium uptake recorded 1.51 mg K pot-1 was achieved with strain S11 combined with 100% compost. 9) The superior vitamin C content 7.06 g/100g plant corchorus was found with the 75% mineral fertilizer + 25% compost treatment plus strain ART001. Also, high vitamin C 5.46 g/100g stem was gained with strain Azoto AtP, and strain Azoto AcN1 when combined with 100% compost fertilization, and strain ART001 combined with 100% mineral fertilizer. While in the roots, the highest vitamin C value recorded 2.36 g/100g was gained when strain Azoto AcN1 plus 75% mineral + 25% compost was applied. 10) Vitamin B1 (Thiamine) showed the highest content 2.95 g/100g in corchorus’s leaf with application of 100% compost in the presence of strain Azoto AcN1. Otherwise, the corchorus’s stem vitamin B1 showed high concentration 3.5 g/100g with fertilizer applied at 100% mineral fertilizer combined with strain S11. The corchorus’s root vitamin B1 showed a value about 3.52 g/100g when Azoto AcN1 strain combined with 50% mineral fertilizer + 50% compost. 11) The superior value of vitamin B2 (riboflavin) 1.08 g/100g in corchorus’s leaf was obtained with the application of 100% compost plus strain Azoto AcN1. As well as, the corchorus’s stem vitamin B2 showed high concentration 1.84 g/100g with 100% compost combined with strain S11. The corchorus’s root vitamin B2 reflected a value of 1.917 g/100g with Azoto AcN1 combined with 25% mineral fertilizer + 75% compost.not clear such as the previous strains, upon the mineral and nutritional values contents in spinach’s shoot. Also, spinach results indicated that to enhance the NPK content and nutritional value of consumed shoot of spinach plants, only 75% of recommended mineral fertilizer level in agriculture report (250 kg Ammonium sulfate/ Fed.) combined with Strain S11, and Azoto AtP is enough since it has the same effect as 100% mineral fertilization rate. In the other turn, if the aim is to produce spinach plants with minimum levels of both nitrate and oxalate, only the organic fertilizers combined with same recommended strains could be used. from the corchorus experiment, results showed that application of Strain S11 as biofertilizer which had superior production for cytokinin and siderophore with complete mineral fertilizer rate (100 kg Ammonium sulfate/ Fed.) enhanced leafs NK uptake. While the 25% of recommended mineral fertilizer + 75% compost from recommended dose enhanced the total chlorophyll. As well as, Azoto AtP strain which had highest nitrogen fixation ability, and the highest production of siderophores enhanced the plant moisture content and total carbohydrate percentage when combined with 50% mineral fertilizer + 50% compost treatment. On another hand, the role of strain ART001 which had the highest indole acetic acid production, affected by the fertilization treatment. With complete mineral fertilizer rate (100 kg Ammonium sulfate/ Fed.) enhanced plant dry, leafs dry weight, and P uptake. While with the 75% of recommended mineral fertilizer + 25% compost from recommended dose enhanced the fresh weight, plant moisture, and vitamin C, and B1 content. Finally, used Azoto AcN1 strain as biofertilizer which had upper gibberellin production ability, enhanced the corchorus’s leafs vitamin content (Vit. B1, and B2) with complete compost dose (15 – 20 m3 fed.-1) as the only source of fertilizers.Therefore, corchorus results indicated that to enhance the NPK content and nutritional value of consumed leafs of corchorus plant, only 75% of recommended mineral fertilizer level in agriculture report (100 kg Ammonium sulfate/ Fed.) combined with ART001 is enough since it has the same effect as 100% mineral fertilization rate. |