الفهرس 
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Abstract
In developing countries, adoption of new agricultural technologies is often hampered by a lake of chemical fertilizers. The high cost of chemical fertilizers, particulary nitrogen ones, is often prohibitive to small and marginal farmers. In developed countries, where very high rates of nitrogen fertilizers are used, pollution is often a concern. It is therfore, important to explore alternative sources of nitrogen to partially meet the nitrogen requirment. Biological nitrogen fixation offers this alternative, and also offers an economically attractive and ecologically sound means of reducing external inputs and improving internal resources. In this context, the aim of the present thesis was to evaluate whether non-symbiotic nitrogen-fixing Azotobacter and/or Azospirillum (as nitrogen biofertilizers) have the potential of increasing yield or decreasing nitrogen fertilizer requirements. As an effort in this respect, the present study was planned to study the following points:
1-Isolation and selection of the most efficient nitrogen-fixing bacterial isolates (Azotobacter and Azospirillum) in order to be used as inoculants.
2-Sensitivity of these selected isolates towards different concentrations of certain pesticides, salts and antibiotics.
3-Antagonism between sporeformers and actinomycetes isolated from the rhizosphere of either tomato or onion plants and the most efficient nitrogen-fixing bacterial isolates.
4-Antagonism between the most efficient nitrogen-fixing bacterial isolates and certain pathogenic fungi.
5-Colonization and persistence of Azotobacter and Azospirillum which selected to be used as inoculants in the rhizosphere and rhizoplane of either tomato or onion plants and their effects on the plant growth, through series of greenhouse experiments, using two soil types.
6-Evaluation of seedling-roots inoculation of either tomato or onion plants with Azotobacter and/or Azospirillum selected as inoculants on the growth and yield of tomato or onion grown in two soil types in absence or presence of different levels of nitrogen fertilizer in order to determine the extent to which nitrogen-fixers could replace the application of nitrogen fertilizer.
The obtained results could be summarised as follows:
Part I :
1-Fifty-one and thirty-five representative isolates of Azotobacter and Azospirillum respectively were isolated at random from the rhizosphere and rhizoplane of tomato and onion plants grown in different locations of Minia goveronrate. After purification, the isolates were tested for their efficiency for nitrogen-fixation using the ambient assay of N-ase activity and the most efficient five isolates of either Azotobacter (Z6, Z36, Z43, Z46, Z51) or Azospirillum (S1, S2, S5, S7, S8) .
2-Variations have been observed between the five isolates of either Azotobacter or Azospirillum regarding their tolerance to actelic concentrations.
3-Two Azotobacter isolates, (Z6 and Z43) and one Azospirillum isolate (S7) were found to be tolerant to actelic concentrations up to 5ppm compared with the other tested isolates. Gradual decrease in the growth for all investigated Azotobacter and Azospirillum isolates were observed at concentrations higher than 5 ppm of actelic.
4-The presence of marshal in the growth medium at low concentration (1.0) ppm had no effect on the Azotobacter and Azospirillum isolates except the isolate number (Z36) of Azotobacter and isolates number S1, S5 of Azospirillum. Marshal at concentrations of 5 ppm or more significantly affected the growth of all the tested Azotobacter and Azospirillum isolates.
5-Generally, it could be concluded that, the rate of inhibitory effect of the tested pesticides on growth of N2-fixing bacterial isolates depended on the type and concentrations of pesticide used as well as the type of the microorganism under study.
6- All the tested Azotobacter isolates gave a maximum increase in their growth in the medium contained 1.0% NaCl with the exception of the isolate Z43, which grew well only in the medium with no added NaCl. Two Azotobacter isolates, (Z6 and Z46) were found to be more tolerant to sodium chloride concentrations up to 2% compared with the other tested ones. Gradual reduction in the growth for all investigated isolates was observed at concentrations higher than 2% NaCl except the isolates Z6 and Z46 which were tolerant to concentrations higher than 2%.
7-Out of the five tested Azospirillum isolates, two (S2 and S5) continued their growth in media contained 2% NaCl. With increasing sodium chloride concentrations more than 2%, the isolate S5 was not able to grow, however the isolate S2 was tolerant to salt concentrations up to 4%.
8-The results showed that the presence of penicillin in the growth medium at low concentrations 10.0 and 20.0 ppm had no effect on all the tested Azotobacter and Azospirillum isolates, except the isolate Z51 of Azotobacter and isolates S1 and S7 of Azospirillum which were not very active at these concentrations.
9-Increasing penicillin concentration to 30 ppm did not affect the growth of the studied Azotobacter or Azospirillum isolates. The isolates, Z6 and Z43 of Azotobacter were found to be more tolerant to Penicillin at concentrations up to 40 ppm compared with the other tested isolates. On the other hand, the isolate (S2) followed by the isolates S5 and S8 of Azospirillum were more tolerant to penicillin at concentrations up to 40 ppm compared with the other tested ones.
10-The presence of choloramphenicol in the growth medium at low concentration 10.0 and 20.0 had no effect on the Azotobacter and Azospirillum isolates except the isolates Z36 and Z43 for Azotobacter and isolates S1, S7 for Azospirillum.
11-Out of the five Azotobacter isolates, three (Z6, Z46 and Z51) were found to be more tolerant to choloramphenicol at concentrations up to 40 ppm compared with the other tested isolates. On the other hand, the isolates S2, S5 and S8 were more tolerant to choloramphenicol at concentrations up to 40 ppm compared with the other tested ones.
12-Generally, it could be said that the rate of both stimulatory and inhibitory effects of the investigated antibiotics on the growth of the tested nitrogen fixing bacterial isolates depended on the kind and concentration of antibiotic used as well as the type of microorganism under investigation.
13-Results showed that 10 isolates of sporeformers (20%) and 4 isolates of actinomycetes (8%), showed antagonistic relations towards the studied Azotobacter and Azospirillum isolates, indicating their capability to produce inhibitory metabolites but with variable degrees.
14- Azotobacter isolates Z43 and Z46 showed good antagonistic properties towards Fusarium oxysporum (inhibition zone between 5 to 7 mm.) indicating their capability to produce inhibitory metabolites to such fungus. Azospirillum isolate S2 showed good inhibitory effect (inhibition zone 9 mm.) towards the same fungus.
15-Azotobacter isolates did not show any effect aganist Rhizoctonia solani, however Azospirillum isolates S5 and S7 showed marked antigonistic effect towards such fungus, while the rest of the isolates did not show any effect. Furthermore, Azotobacter and Azospirillum isolates did not show any effect towards Sclerotium rolfesii.
Part (II):
1- Total bacterial counts as well as the non-symbiotic nitrogen-fixing bacteria (Azotobacter and Azospirillum) in root-free soil as well as in rhizosphere soils and rhizoplane samples of both plants uninoculated or inoculated were higher in the clay loam soil than in sandy one and remained higher throughout the experimental period.They were also found to be much higher in the rhizoplane (washed reptured roots) of either tomato or onion plants than in the rhizosphere ones.
2-In the two soil types tomato plants supported highest numbers of the studied microorganism in both rhizosphere and rhizoplane as compared with those determined in onion plants during the different growth phases.
3-The obtained results generally also indicate that numbers of total bacterial as well as Azotobacter and Azospirillum were found to be higher in the rhizosphere soils of both studied plants than in non-rhizosphere one, resulting in positive rhizosphere effect. The calculated R/S ratios obtained for either tomato or onion grown in clay loam or sandy soil, show that there were increases in the rhizosphere effect with plant age. However, these values differed according to type of plant and growth stage of either plant or soil type. The calculated R/S ratio showed that inoculation of either tomato or onion plants supported the growth of bacteria in their rhizosphere compared to the uninoculated rhizosphere in both clay loam and sandy soils.
4- Inoculation of tomato or onion plants with any of the most efficient isolates of Azotobacter or Azospirillum resulted in similar trends concerning R/S ratios and total count of bacteria in rhizosphere and rhizoplane samples.
5- Counts of Azotobacter were relatively higher in the rhizosphere and rhizoplane of both tested plants inoculated with any of the five selected Azotobacter isolates grown in both investigated soils compared with those calculated in the compatible uninoculated ones resulting in a strong positive rhizosphere effect (R/S ratio), and this is more obviously pronounced in sandy soil indicating the successful inoculation of either tomato or onion seedling-roots with the selected Azotobacter isolates.Generally, however, the effect of such inoculation on Azotobacter population differed according to type and the stage of plant growth, type of soil, and type of isolate used as inoculant.
6-In the rhizosphere and rhizoplane of both investigated plants grown in both soil types, also a considerable increase in the population of Azospirillum is observed throughout the experiment perid as a result of seedling-root inoculation with any of the used Azospirillum isolates compared with those in the uninoculated ones. Such increase resulted in a strong positive rhizosphere effect (R/S ratio) which was more pronounced in sandy soil than in clay loam ones, indicating the successful inoculation of both tested plants with Azospirillum.
7-The density of Azospirillum in the rhizoplane of both tested plants was found to be higher than those in the compatible rhizosphere ones and this was more observed in the inoculation treatment indicating the establishment of the inoculated Azospirillum within the root.
8-The growth of either tomato or onion plants as indicated by the average length, fresh and dry weights of plant as well as the nitrogen uptake was generally enhanced as a result of seedling-roots inoculation with any of the selected Azotobacter or Azospirillum isolates, which clearly indicates the benificial effect of such inoculation. The degree of enhancement varied from isolate to another.
9-The enhancing effect of seedling-roots inoculation with Azotobacter or Azospirillum isolates on the growth of tomato or onion plants differed according to the type of inoculant, type of soil and the type of plant.
Part (III):
1-Generally, the studied vegetative growth characters of either tomato or onion plants significantly increased by inoculation and/or nitrogen fertilization in the two seasons for both soil types. Furthermore, the growth measurements of either tomato or onion plants increased significantly by increasing nitrogen fertilization levels.
2-Tomato growth characters i.e plant hieght, number of branches per plant, fresh and dry weights per plant as well as N-uptake increased significantly by inoculation compared with uninoculated ones in the two seasons for both soil types.
3-Comparing the three inoculation treatments {i.e Azotobacter, Azospirillum and Azotobacter+Azospirillum (mixed) } it is worthy to note that, inoculation with Azotobacter increased the aformentioned tomato growth characters more than the inoculation with Azospirillum alone or in conjunction with Azotobacter in the two seasons for sandy soil. While in clay loam soil it was obvious that, inoculation with the mixed culture (Azotobacter + Azospirillum) was found to be the best treatment in the two seasons.
4-The interaction effect (A×B) between inoculation (A) and nitrogen fertilization levels (B) was highly significant on the studied growth characters, for clay loam and sandy soil in the two seasons.
5-Increasing nitrogen fertilization levels tended to increase the early, total and marketable yields and decreased the unmarketable one in the two seasons for both soil.The highest means of yield and yield quality were recorded from the fertilization by 100% of the RDNF while the lowest values were obtained from the unfertilized one.
6-Inoculation with Azotobacter and/or Azospirillum increased the early, total and marketable yield and decreased the unmarketable one as compared with uninoculated treatments in the two seasons for both investigated soils. The highest values were obtained from the inoculation with the mixed culture (Azotobacter + Azospirillum) compared to inoculation with each solely. The interaction effect (A×B) on the early, total, marketable and unmarketable yields was found to be highly significant in both soil types in the two seasons.
7-It is clearly obvious that inoculation with Azotobacter and/or Azospirillum in absence of external nitrogen fertilization resulted in an increase in tomato yield production accompanied by improvment in its quality in both soil types during the two seasons. The yields obtained from such treatments were obviously found to be similar to those obtained by 25% of the RDNF alone, indicating that any inoculation treatment could contribute to an amount equivalent to 25% of the RDNF or even more. Such contributions of the nitrogen fixing inoculants were also noticed in the presence of any nitrogen fertilization level; e.g. inoculation in conjunction with one fourth the RDNF brought about the same yield production or even more as fertilization with one half the RDNF only. Furthermore, inoculation coupled with 50% or 75% of the RDNF brought about the same yield production or even more as the fertilization with 75% or 100% of the RDNF solely. The effect of inoculation on crop yield and yield quality was more pronounced in case of the mixed culture (Azotobacter + Azospirillum) than each of them solely. Hence, one could conclude that the inoculation could save at least 25% of the RDNF. So, on adopting the present inoculation technique, there is no need to increase the nitrogen fertilization level above 75% of the RDNF.
8-Results clearly indicate that inoculation the seedling-roots of onion as well as the nitrogen fertilization had a highly significant effect on the all tested growth characters in both seasons for the two soils. Increasing nitrogen fertilization levels up to the full N-dose (100% of the RDNF) tended to increase all checked growth characters with highly significant differences.
9-High values of growth characters have been significantly produced as a result of inoculation with the mixed culture (Azotobacter+Azospirillum) compared to inoculation with each separetly.
10-Inoculation with Azotobacter and/or Azospirillum in the presence of different nitrogen fertilization levels significantly increased the abovementioned plant growth characters. The highest values were obtained from onion seedling-root inoculated with the mixed culture (Azotobacter+Azospirillum) in the two seasons for both soils. The over all picture of the obtained data clearly indicate that, inoculation with Azotobacter and/or Azospirillum coupled with 75% of the RDNF gave slightly higher values of the growth characters as compared with uninoculated onion fertilized by the full nitrogen dose (RDNF). The effect of inoculation on the studied growth characters was more pronounced in sandy soil.
11-Inoculation and/or nitrogen fertilization significantly increased almost all the yield parameters. The results showed that these parameters increased with increasing nitrogen fertilization levels in absence or presence of inoculation. It could be conducted that the hieghest values of the abovementioned parameters resulted from onion plants fertilized with the full nitrogen dose (RDNF) and inoculated with the mixed culture (Azotobacter+Azospirillum) in both soils in the two seasons. Also the mixed culture was found to be the best treatment for these studied parameters in absence of nitrogen fertilization. Herein again, results indicate clearly that inoculation of onion seedling roots with the mixed culture (Azotobacter+Azospirillum) in conjunction with 75% of the RDNF resulted in high total yield with best quality similar to or even better than those obtained from the uninoculated but fertilized onion plant with the full nitrogen dose (RDNF).