USE OF MICROORGANISMS AS BIOFERTILIZERS FOR SOME PLANTS ALI SALAMA ALI SALAMA

USE OF MICROORGANISMS AS BIOFERTILIZERS FOR SOME PLANTS BY ALI SALAMA ALI SALAMA A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Agriculture (Agricultural Microbiology) Department of Agricultural Microbiology Faculty of Agriculture Zagazig University 2006

USE OF MICROORGANISMS AS BIOFERTILIZERS FOR SOME PLANTS BY ALI SALAMA ALI SALAMA B.Sc. Agriculture (Agricultural Genetics and Genetic Engineering), Faculty of Agriculture, Zagazig University, (2001) Under the Supervision of: Prof. Dr. S. H. Salem... Prof. of Agric. Microbiology, Agric. Microbiology Dept. Faculty of Agriculture, Zagazig University Prof. Dr. Fatma I. El-Zamik... Prof. and Head of Agric. Microbiology Dept. Faculty of Agriculture, Zagazig University Dr. Howaida M. L. Abd El-Basit... Assistant Prof. of Agric. Microbiology, Agric. Microbiology Dept. Faculty of Agriculture, Zagazig University

Approval Sheet USE OF MICROORGANISMS AS BIOFERTILIZERS FOR SOME PLANTS BY ALI SALAMA ALI SALAMA B.Sc. Agriculture (Agricultural Genetics and Genetic Engineering), Faculty of Agriculture, Zagazig University, (2001) approved by: This thesis for M. Sc. Degree has been Prof. Dr. S. H. Salem... Prof. of Agric. Microbiology, Agric. Microbiology Dept. Faculty of Agriculture, Zagazig University Prof. Dr. E. M. Gewaily... Prof. of Agric. Microbiology, Agric. Microbiology Dept. Faculty of Agriculture, Zagazig University Prof. Dr. Wedad T. Ewada... Prof. of Agric. Microbiology, Agric. Microbiology Dept. Faculty of Agriculture, Ain Shams University Prof. Dr. Fatma I. El-Zamik... Prof. and Head of Agric. Microbiology, Agric. Microbiology Dept. Faculty of Agriculture, Zagazig University Date of examination: / / 2006

ABSTRACT This work was carried out in the laboratory and greenhouse of Agric. Microbiology Dept. at the Faculty of Agriculture, Zagazig University, Egypt, in order to isolate and select efficient indigenous strains of Rhizobium leguminosarum bv. vicieae, Azospirillium spp. and Azotobacter spp. which isolated from different locations in Sharkia governorate to use them as biofertilizers for broad bean (Vicia faba L.) and wheat (Triticum aestivum L.) plants. Thirty two Rhizobium isolates were selected as effective strains for nodulation on broad bean, 18 isolates of Azospirillum, and 14 isolates of Azotobacter were efficient in nitrogenase activity in liquid cultures. Five isolates showing high values in nitrogen fixation of R. leguminosarum bv. vicieae and the highest five effective isolates of each of Azotobacter spp. and Azospirillum spp. were used for GA 3, IAA and IBA production in liquid cultures. The selected bio-fertilizers were examined on the effect on growth and productivity of broad bean and wheat plants which cultivated in both sandy and clay soils under different levels of inorganic and organic N fertilizers in six greenhouse experiments. The results showed that the isolate RZ11 was the highest efficient isolate of R. leguminosarum bv. vicieae in plant growth, nodulation, nitrogenase activity and production of growth promoting substances. In addition the isolate ZH21 of Azotobacter spp. and isolate ASH21 of Azospirillum spp. reflected the highest activity of nitrogenase enzyme and production of growth promoting substances in liquid cultures. The inoculation of broad bean and wheat plants grown on both sandy and clay soils with the selected bio-fertilizers (R. leguminosarum bv. vicieae, Azotobacter spp and zospirillum spp) is importance to obtain the best performance in growth, yield and total nitrogen and mineral contents.

ACKNOWLEDGMENT This work has been carried out under the supervision and direction of Prof. Dr. Samir H. Salem Prof. of Microbiology, Agric. Microbiology. Dept. Fac. Agric., Zagazig University. To him I express my deepest gratitude for suggesting this problem, valuable assistance, progressive criticism, keeping interest and guidance. I also wish to express my thanks and gratitude to Prof. Dr. Fatma I. El-Zamik Prof. and head of Agric. Microbiology Department, Fac. Agric., Zagazig University for her supervision, unlimited help and effort during the preparation of this manuscript. Thanks are also due to Dr. Howaida M. L. Abd El-Basit Assistant Prof. of Microbiology,in the same Department for supervising this work and here valuable advise and guidance Gratitude are also due to all the staff members and colleagues in Agric. Microbiology. Dept. Fac. Agric., Zagazig University for their encouragement and providing facilities throughout this work.

CONTENTS Title of contents Page I-INTRODUCTION... 1 II-REVIEW OF LITERATURE... 4 III- MATERIALS AND METHODS... 43 IV-RESULTS AND DISCUSSION... 71 4.1. Testing the efficiency of different isolates of Rhizobium, Azotobacter and Azospirillum... 71 4.1.1. Testing the efficiency of Rhizobium isolates... 71 4.1.1.1. Nodulation... 72 4.1.1.2. Nitrogenase activity... 73 4.1.1.3. Plant dry weight... 74 4.1.1.4. Nitrogen content... 75 4.1.1.5. The phage reaction of Rhizobium isolates... 78 4.1.2. Testing the efficiency of N 2 -fixation by Azotobacter and Azospirillum isolates in liquid culture... 79 4.1.3. Determination of growth promoting substances in liquid cultures of Rhizobium, Azotobacter and Azospirillum isolates... 82 4.1.3.1. GA 3, IAA and IBA in Rhizobium isolates... 82 4.1.3.2. GA 3, IAA and IBA in Azotobacter isolates... 83 4.1.3.3. GA 3, IAA and IBA in Azaspirillium isolates... 83 4.2. Response of broad bean plants to bio-fertilizers and inorganic N-fertilizers under cultivation in sandy and clay soils... 86 4.3. Response of broad bean plants to bio and organic fertilizers under cultivation in sandy and clay soil... 109

ii 4.4. Response of broad bean plants to bio-fertilizers under cultivation in sandy and clay soils... 130 4.5. Effect of bio-fertilizers only or combined with inorganic or organic N-fertilizers on GA 3, IAA and IBA content in roots of broad bean plants... 152 4.6. Comparison the response of broad bean plants to application of biofertilizers only or combined with organic or inorganic N-fertilizers with control treatment in both sandy and clay soil... 155 4.7. Response of wheat plants to bio-fertilizers and inorganic N-fertilizers under cultivation in sandy and clay soils... 159 4.8. Response of wheat plants to bio-and organic fertilizers under cultivation in sandy and clay soils... 181 4.9. Response of wheat plants to bio-fertilizers under cultivation in sandy and clay soils... 202 4.10. Effect of bio-fertilizers only or combined with inorganic or organic N-fertilizers on GA 3, IAA and IBA content in roots of wheat plants... 221 4.11. Comparison between the different responses of wheat plants to application of bio-fertilizers only or combined with organic or inorganic N-fertilizers with control treatment in both sandy and clay soil... 224 V- SUMMARY AND CONCLUSION... 228 VI- LITIRATURE CITED... 233 VII- ARABIC SUMMARY...

iii LIST OF TABLES Table No. Title of Tables page 1 The locations of soil samples selected for isolation of rhizobia, azotobacter and azospirilla... 47 2 physical and chemical analyses of the soil under investigation... 57 3 Shoot and root dry weight (g/plant), total nitrogen content, nitrogenase activity and phage interaction in nodulation test of broad bean plants, as affected by inoculation with different isolates of rhizobia... 73 4 physical and chemical analyses of soil in deffernt locations of Sharkia governorate... 77 5 Nitrogenase activity by Azotobacter and Azospirillum isolates in liquid cultures... 81 6 GA 3, IAA and IBA in liquid culture of Rhizobium leguminosarum bv. vicieae, Azotobacter and Azospirillum isolates... 84 7 Effect of bio-fertilizers and inorganic N-fertilizers on number of nodules, dry weight of nodules (mg/plant) and nitrogenase activity of broad bean plants... 87 8 Effect of bio-fertilizers and inorganic N-fertilizers on shoot, root and whole plant dry weight (g/plant) of broad bean plants... 91 9 Effect of bio-fertilizers and inorganic N-fertilizers on total nitrogen content in shoot, root and whole plant (mg N/plant) of broad bean plants... 96 10 Effect of bio-fertilizers and inorganic N-fertilizers on total phosphorus content in shoot, root and whole plant (mg P/plant) of broad bean plants... 99

iv 11 Effect of bio-fertilizers and inorganic N-fertilizers on total potassium content in shoot, root and whole plant (mg K/plant) of broad bean plants... 103 12 Effect of bio-fertilizers and inorganic N-fertilizers on weight of 100 seeds (g), nitrogen % and protein % of broad bean plants... 106 13 Effect of bio and organic fertilizers on number of nodules, dry weight of nodules (mg/plant) and nitrogenase activity of broad bean plants... 110 14 Effect of bio and organic fertilizers on shoot, root and whole plant dry weight (g/plant) of broad bean plants... 114 15 Effect of bio and organic fertilizers on total nitrogen content in shoot, root and whole plant (mg N/plant) of broad bean plants... 118 16 Effect of bio and organic fertilizers on total phosphorus content in shoot, root and whole plant (mg P/plant) of broad bean plants... 121 17 Effect of bio and organic fertilizers on total potassium content in shoot, root and whole plant (mg K/plant) of broad bean plants... 125 18 Effect of bio and organic fertilizers on weight of 100 seeds (g), nitrogen % and protein % of broad bean plants... 128 19 Effect of bio-fertilizers on number of nodules, dry weight of nodules (mg/plant) and nitrogenase activity of broad bean plants... 132 20 Effect of bio-fertilizers on shoot, root and whole plant dry weight (g/plant) of broad bean plants... 136 21 Effect of bio-fertilizers on total nitrogen content in shoot, root and whole plant (mg N/plant) of broad bean plants... 139

v 22 Effect of bio-fertilizers on total phosphorus content in shoot, root and whole plant (mg P/plant) of broad bean plants... 142 23 Effect of bio-fertilizers on total potassium content in shoot, root and whole plant (mg K/plant) of broad bean plants... 146 24 Effect of bio-fertilizers on weight of 100 seeds (g), nitrogen % and protein % of broad bean plants... 149 25 Effect of bio-fertilizers only or combined with inorganic or organic N-fertilizers on GA 3, IAA and IBA in root of broad bean plants... 153 26 Comparing the response of broad bean plants to application of bio and inorganic, bio and organic and bio-fertilizers only in sandy soil... 156 27 Comparing the response of broad bean plants to application of bio and inorganic, bio and organic and bio-fertilizers only in clay soil... 158 28 Effect of bio-fertilizers and inorganic N-fertilizers on shoot, root and whole plant dry weight (g/plant) in wheat plants... 160 29 Effect of bio-fertilizers and inorganic N-fertilizers on nitrogenase and dehydrogenase activities inrhizosphere of wheat plants... 165 30 Effect of bio-fertilizers and inorganic N-fertilizers on total nitrogen content in shoot, root and whole plant (mg/plant) in wheat plants... 167 31 Effect of bio-fertilizers and inorganic N-fertilizers on total phosphorus content in shoot, root and whole plant (mg/plant) in wheat plants... 171 32 Effect of bio-fertilizers and inorganic N-fertilizers on total potassium content in shoot, root and whole plant (mg/plant) in wheat plants... 174

vi 33 Effect of bio-fertilizers and inorganic N-fertilizers on weight of 1000 grains (g), nitrogen % and protein % in wheat plants... 178 34 Effect of bio and organic fertilizers on shoot, root and whole plant dry weight (g/plant) in wheat plants... 182 35 Effect of bio and organic fertilizers on nitrogenase and dehydrogenase activities in rhizosphere of wheat plants... 186 36 Effect of bio and organic fertilizers on total nitrogen content in shoot, root and whole plant (mg/plant) in wheat plants... 189 37 Effect of bio and organic fertilizers on total phosphorus content in shoot, root and whole plant (mg/plant) in wheat plants... 193 38 Effect of bio and organic fertilizers on total potassium content in shoot, root and whole plant (mg/plant) in wheat plants... 197 39 Effect of bio and organic fertilizers on weight of 1000 grains (g), nitrogen % and protein % in wheat plants... 200 40 Effect of bio-fertilizers on shoot, root and whole plant dry weight (g/plant) in wheat plants... 204 41 Effect of bio-fertilizers nitrogenase and dehydrogenase activities in rhizosphere of wheat plants... 207 42 Effect of bio-fertilizers on total nitrogen content in shoot, root and whole plant (mg/plant) in wheat plants... 209 43 Effect of bio-fertilizers on total phosphorus content in shoot, root and whole plant (mg/plant) in wheat plants... 212

vii 44 Effect of bio-fertilizers on total potassium content in shoot, root and whole plant (mg/plant) in wheat plants... 216 45 Effect of bio-fertilizers on weight of 1000 grains (g), nitrogen % and protein % in wheat plants... 218 46 Effect of bio-fertilizers only or combined with inorganic or organic N-fertilizers on GA 3, IAA and IBA in root of wheat plants... 222 47 Comparing the response of wheat plants to application of bio and inorganic, bio and organic and bio-fertilizers only in sandy soil... 225 48 Comparing the response of wheat plants to application of bio and inorganic, bio and organic and bio-fertilizers only in clay soil... 227

viii LIST OF FIGURES Fig. No. Title of Figure page 1 Map of Sharkia governorate showing localities of isolation... 44 2 Effect of bio-fertilizers and inorganic N-fertilizers on number and dry weight of nodules in broad bean plants grown in sandy and clay soils... 88 3 Effect of bio-fertilizers and inorganic N-fertilizers on dry weight of broad bean plants grown in both sandy and clay soils... 92 4 Effect of bio-fertilizers and inorganic N-fertilizers on total nitrogen content (mg/plant) of broad bean cultivated in both sandy and clay soils... 97 5 Effect of bio-fertilizers and inorganic N-fertilizers on total phosphorus content (mg/plant) of broad bean cultivated in both sandy and clay soils... 100 6 Effect of bio-fertilizers and inorganic N-fertilizers on total potassium content (mg/plant) of broad bean cultivated in both sandy and clay soils... 104 7 Effect of bio-fertilizers and inorganic N-fertilizers on grain yield, nitrogen (%) and protein (%) in seeds of broad bean cultivated in both sandy and clay soils... 107 8 Effect of bio and organic fertilizers on number and dry weight of nodules in broad bean plants grown in sandy and clay soils... 111 9 Effect of bio and organic fertilizers on dry weight of broad bean plants grown in both sandy and clay soils.. 115 10 Effect of bio and organic fertilizers on total nitrogen content (mg/plant) of broad bean cultivated in both sandy and clay soils... 119

ix 11 Effect of bio and organic fertilizers on total phosphorus content (mg/plant) of broad bean cultivated in both sandy and clay soils... 122 12 Effect of bio and organic fertilizers on total potassium content (mg/plant) of broad bean cultivated in both sandy and clay soils... 126 13 Effect of bio and organic fertilizers on grain yield, nitrogen (%) and protein (%) in seeds of broad bean cultivated in both sandy and clay soils... 129 14 Effect of bio-fertilizers on number and dry weight of nodules in broad bean plants grown in sandy and clay soils... 133 15 Effect of bio-fertilizers on dry weight of broad bean plants grown in both sandy and clay soils... 137 16 Effect of bio-fertilizers on total nitrogen content (mg/plant) of broad bean cultivated in both sandy and clay soils... 140 17 Effect of bio-fertilizers on total phosphorus content (mg/plant) of broad bean cultivated in both sandy and clay soils... 143 18 Effect of bio-fertilizers on total potassium content (mg/plant) of broad bean cultivated in both sandy and clay soils... 147 19 Effect of bio-fertilizers on grain yield, nitrogen (%) and protein (%) in seeds of broad bean cultivated in both sandy and clay soils... 150 20 Effect of bio-fertilizers and inorganic N-fertilizers on dry weight of wheat plants grown in both sandy and clay soils... 167 21 Effect of bio-fertilizers and inorganic N-fertilizers on total nitrogen content (mg/plant) of wheat cultivated in both sandy and clay soils... 168 22 Effect of bio-fertilizers and inorganic N-fertilizers on total phosphorus content (mg/plant) of wheat cultivated in both sandy and clay soils... 172

x 23 Effect of bio-fertilizers and inorganic N-fertilizers on total potassium content (mg/plant) of wheat cultivated in both sandy and clay soils... 175 24 Effect of bio-fertilizers and Inorganic N-fertilizers on grain yield, nitrogen (%) and protein (%) in grains of wheat cultivated in both sandy and clay soils... 179 25 Effect of bio and organic fertilizers on dry weight of wheat plants grown in both sandy and clay soils... 183 26 Effect of bio and organic fertilizers on total nitrogen content (mg/plant) of wheat cultivated in both sandy and clay soils... 190 27 Effect of bio and organic fertilizers on total phosphorus content (mg/plant) of wheat cultivated in both sandy and clay soils... 194 28 Effect of bio and organic fertilizers on total potassium content (mg/plant) of wheat cultivated in both sandy and clay soils... 198 29 Effect of bio and organic fertilizers on grain yield, nitrogen (%) and protein (%) in seeds of wheat cultivated in both sandy and clay soils... 201 30 Effect of bio-fertilizers on dry weight of wheat plants grown in both sandy and clay soils... 205 31 Effect of bio-fertilizers on total nitrogen content (mg/plant) of wheat cultivated in both sandy and clay soils... 210 32 Effect of bio-fertilizers on total phosphorus content (mg/plant) of wheat cultivated in both sandy and clay soils... 213 33 Effect of bio-fertilizers on total potassium content (mg/plant) of wheat cultivated in both sandy and clay soils... 217 34 Effect of bio-fertilizers on grain yield, nitrogen (%) and protein (%) in seeds of wheat cultivated in both sandy and clay soils... 219

I. INTRODUCTION The intensive agricultural farming system as practical in Egypt, where the crop rotation consists of 2 or 3 crops per year, are main reasons for the high consumption of chemical fertilizers. Intensive methods of farming and food production are having unfavorable consequences on the quality of food, environment and on animal and human health. In these systems, agriculture is treated like other industries, with an emphasis on efficiency and maximum productivity regardless of their impacts on human health and ecology. The amount of nitrogen applied to the soil in intensive agricultural systems varies considerably, depending upon the crop being grown, the soil type, and the previous cropping history of the soil. Nowadays, the harmful effects on the environment of heavy use of N fertilizers are becoming more evident. Furthermore, the fossil fuels which are used in the production of N fertilizers are becoming scarcer and more expensive. Therefore, there is a great need to search for all possible avenues to improve biological nitrogen fixation and its use by farmers through bio-fertilization process (Hussien et al., 1997). Nitrogen fixation by the legume-rhizobium symbiotic partnership represents an inexpensive alternative to the use of chemical nitrogen fertilizers in the production of food protein and oil. The process requires that the host crop be adequately nodulated by Rhizobium bacteria effective in nitrogen fixation. Inoculation of legumes with suitable rhizobial strains is carrying out in many countries to ensure nodulation (Brock et al. 2003).

2 A wide range of bacteria in rhizosphere can promote plant growth, orchestrated by rhizosphere bacteria that communicate with the plant using complex chemical signals, gibberellins, glycolipids and cytokinins are now beginning to be fully appreciated in terms of their biotechnological potential. A critical process that occurs on the surface of the plant and particularly in the root zone, is associative nitrogen fixation in which the nitrogen fixing microorganisms is on the surface of the plant root, the rhizoplane, as well as in the rhizosphere. This process is carried out by representatives of the genera Azotobacter and Azospirillum. Recent evidence sugests that their majour contribution may not be in nitrogen fixation but in production of growthpromoting hormones that increase root hair development and thus greater ability of the plant to take up nutrients. This is an area of research that is particularly important in tropical agricultural areas (Prescott et al. 2005). Inoculation with indigenous Azospirillum strains is important procedure when studying their inherent capacity to benefit crops. In some cases, indigenous can perform better than introduced strains in promoting the growth of crops due to their superior adaptability to the environment (Gunarto et al. 1999). On the other hand, application of organic fertilizers was shown to enhance the incidence and activities of promoting plant rhizobacteria and stimulating plant growth (Khamis and Metwally, 1998). For all reasons, there is a widespread interest in the use of combination of mineral fertilizers and biofertilizers as an

3 alternative and cheap source for chemical fertilizers. From the economic point of view the cost of inoculantes is not usually a constraint to their use by farmers who outlay capital for seed. Inoculant cost will seldom exceed 1 % of the seed cost (Bohlool et al., 1992). This study was conducted with the following objectives: 1. To isolate and select of efficient isolate of endigenous Rhizobium leguminosarum bv. vicieae, Azospirillum spp and Azotobacter spp from Sharkia governorate soils and use them as bio-fertilizers in the next study. 2. To study the effect of selected bio-fertilizers with or without different levels of inorganic N-fertilizers in two soil textures on the growth of broad bean and wheat plants. 3. To study the response of broad bean and wheat plants to the selected bio-fertilizers with or without different levels of organic amendment under cultivation in sandy and clay soils. 4. To study the effect of selected bio-fertilizers only in two soil textures on the growth of broad bean and wheat plants. 5. To study the effect of bio-fertilizers only or combined with organic or inorganic N-fertilizers on GA 3, IAA and IBA content in roots of broad bean and wheat plants.

4 II. REVIEW OF LITERATURE The term "Biofertilizers" or more appropriately "microbial inoculant" can be generally defined as preparations containing live or latent cells of efficient strains of nitrogen-fixing, phosphate solubilizing or celluloytic microorganisms used for application to seed, soil or composting areas with the objective of increasing the numbers of such microorganisms and accelerating certain microbial processes to augment the extent of the availability of nutrients in a form which can be easily assimilated by plants. Subba Rao, 1993 added also that, in a larger sense, the term may be used to include all organic resources (manures) for plant growth which are rendered in an available form for plant abstraction through microorganisms or microorganisms plant associations or interactions. Recently, Subba Rao (1999) mentioned that biofertilizers are carried based preparations containing beneficial microorganisms in available state intended for seed or soil application and designed to improve soil fertility and help plant growth by increasing the number and biological activity of desired microorganisms in the root environment. Biofertilizers are ecofriendly and cannot at any rate replace chemical fertilizers that are indispensable for getting maximum yield of crops. In general, biofertilizers are environment friendly, low cost agricultural input with maximum output. These biofertilizers are to play an important role in enhancing crop productivity through nitrogen fixation, phosphate solubilization, plant hormone productivity, ammonia excretion, siderophore formation and to control various plant disease (Pankhurst and Lynch, 1995;

5 Pathak et al., 1997; Dadarwal et al., 1997 and Hedge et al., 1999). In Egypt, for instance, many types of peat based inoculants of Rhizobium, Azospirillum and phosphate dissolving bacteria are now produced and distributed to farmers under the supervision of General Organization for Agriculture Equalization Funds (GOAEF) which is belonged to Egyptian Ministry of Agriculture and Land Reclamation (Abou El-Naga, 1994). 2.1. Biological nitrogen fixation 2.1.1. Symbiotic nitrogen fixers (Rhizobium) The interaction of leguminous plant and bacteria of the genera Rhizobium, Bradyrhizobium and Azorhizobium result in the formation of root nodules, new organs in which the bacteria are able to fix and reduce atmospheric nitrogen into ammonia available for plant biosyntheses. Legumes are commonly inoculated with efficient nitrogenfixing strains of rhizobia for maximizing crop productivity. Nodule induction at high frequency by introduced inoculum strains has been readily demonstrated in instances where legumes have been grown on soil deficient in indigenous rhizobia (Salem 1962, 1969; Bell and Nutman, 1971; Roughley et al., 1976; Bromfield and Ayanaba, 1980; Brockwell et al., 1987; Somasegaran et al., 1988). Gewaily et al. (1981) carried out a pot experiment using sterilized sand to test the growth and N content of soybean inoculated with 6 different strains of Bradyrhizobium and broad bean inoculated with 8 different strains of R. Leguminosarum bv. vicieae only. They found that, strain No. 3409 belonging to

6 Bradyrhizobium out of six tested organisms, and four strains belonging to R. Leguminosarum bv. vicieae out of 8 used were categorized as efficient strains. Respiration rates of all these strains of rhizobia in pentoses or hexoses sugars were highly variable. When glycine was added, it stimulated the respiratory rates of some of the strains only, which were in general found to be quite efficient in N 2 -fixation in pot culture trials. In addition Wagner and Zapata (1982) used various reference crops and N 15 A-value to estimate symbiotic N 2 -fixation by inoculated broad bean. They found that N 15 A-value for broad bean were higher than those for any of the non N 2 -fixing crops, and the nitrogen fixed-estimated from A-value was approximately 140 kg ha -1. However, Abdallah et al. (1989) studied the biologically fixed nitrogen by three cultivars of broad bean i.e., Giza 2, Giza 3 and Giza 402, grown on alluvial clay loam soil in the presence of 20 kg N ha -1 and ph 8.1 using N 15 dilution technique with barley and wheat as reference crops. They found that amount of N 2 - fixed varied according to the broad bean cultivar, the highest fixed quantity was recorded by Giza 3 followed by Giza 2 and Giza 402 being 113.9, 99.8 and 91.4 kg N ha -1, respectively. Also Hassan et al. (1990) estimated the N 2 -fixed by broad bean in three soil types namely clay, calcareous and sandy soils by using A-value technique and wheat as reference crop. They found that the amount of N 2 -fixed markedly differed depending upon inoculation treatment, dose of N fertilizers applied and soil type. They reported also that, the percentages of nitrogen derived from atmosphere in uninoculated plants were 32.95-69.40 %

7 compared with 40.51-75.56 % in inoculated ones. On the other hand, increasing dose of nitrogen fertilizer led to marked decrease in nodulation of broad bean and amount of N 2 -fixed. Also clay soil showed relatively lower percentage of nitrogen derived from atmosphere (40.5-63.91%) as compared with both calcareous (45.56-75-56%) and sandy (47.03-67.95%) soils. Moreover, Hussein et al. (1997) reported that Rhizobium inoculation significantly increased the number and dry weight of nodules of broad bean cultivated in newly reclaimed soil of Egypt. They showed that Rhizobium inoculation, combined with the highest rate of P and K, produced the highest number (51.7/plant) and dry weight (353.1 mg/plant) of nodules, dry weight of shoot and N-content as well as protein yield. The most important microorganisms used as inoculants, today are the rhizobia that added to legume seeds to ensure successful inoculation (Coyne, 1999). In early times, inoculation methods involved the transfer of soil from the roots of well-nodulated plants to the seeds at planting. However, during the past few decades, different types of inoculants have been developing and significant advances in formation technology and delivery of Rhizobium inoculants are presented. In addition, there is interest in developed co-inoculants containing other microorganisms which are able to improve legume growth (Mishra et al., 1999 and Rodelas et al., 1999). These include rhizobacteria which promote nodulation, nitrogen fixation, plant vigour and yield via such mechanisms as phytohormones, antibiotic or metal-binding compound production, bacteria or fungi which protect against specific root phathogens and other which aid in

8 nutrient supply via phosphate solubilization. For instance, Rodelas et al., (1999) pointed out that co-inoculants of broad bean with R. Leguminosarum bv. vicieae plus plant growth promoting Azotobacter and Azospirillum led to changes in total content concentration and/or distribution of the macro and micronutrients, K, P, Ca, Mg, Fe, B, Mn, Zn and Cu when compared with plants inoculated with Rhizobium only. Schulze et al., (2000) studied the efficiency of N 2 -fixation in Vicia faba L. in combination with different R. leguminosarum strains. They used three Rhizobium leguminosarum strains inoculated in Vicia faba in a pot experiment during vegetative and reproductive growth. Dry matter formation, N 2 fixation and the carbon (C) costs of N 2 fixation were determined in comparison with nodule free plants grown with urea. Nodule number and the capacity of different respiratory chains in the nodules were also measured. The C costs for N 2 fixation were in all cases significantly lower during reproductive growth compared to vegetative growth. Neither the latter nor the differences in C expenditure for N 2 fixation between the Rhizobium strains could be explained in terms of differences or shifts in the capacity of different respiratory chains in the nodules. In addition, Hamaoui et al. (2001) studied the effects of inoculation with Azospirillum brasilense on chick pea (Cicer arietinum) and broad beans (Vicia faba) under different conditions in greenhouse experiments with both legumes. Inoculation with Azospirillum brasilense significantly enhanced

9 nodulation by native rhizobia and improved root and shoot development when compared with non inoculated controls. Moreover, Zaied et al. (2002) tested the response of broad bean to inoculation with fungicide auxotrophic mutants induced in Rhizobium leguminosarum bv. vicieae. They found that some auxotrophic mutants induced significantly increased nodule developments, dry matter yield and nodule dry weight, in contrast, some other results in fewer nodule development. On the other hand, Ma-Wenbo et al. (2002) showed the strategies used by rhizobia to lower plant ethylene levels and increase nodulation. They found that the phytohormone, ethylene, acts as a negative factor in the nodulation process. Recent discoveries suggest several strategies used by rhizobia to reduce the amount of ethylene synthesized by their legume symbionts decreasing the negative effect of ethylene on nodulation. Dakora, (2003) reported that defining new roles for plant and rhizobial molecules in sole and mixed plant cultures involving symbiotic legumes. Rhizobia (species of Rhizobium, Bradyrhizobium, Azorhizobium, Allorhizobium, Sinorhizobium and Mesorhizobium) produce chemical molecules that can influence plant development including phytohormones, lipochito-oligosaccharide Nod factors, lumichrome, riboflavin and H 2 evolved by nitrogenase. Very low concentrations of lumichrome and H 2 released by bacteroids promote plant growth and increase biomass in a number of plant species grown under field and glasshouse conditions.

10 Although, Humphry et al. (2003) studied the genotypic and phenotypic characterization of the symbiotic properties of Rhizobium leguminosarum strains isolated from Jordanian Vicia faba. This study was conducted to compare Jordanian Rhizobium leguminosarum bv. vicieae from Vicia faba to their British counterparts to determine the relatedness of their chromosomal and symbiotic genotypes. It was determined that Jordanian isolates are genotypically distinct from the UK isolates, but from anovel group with Rlv, on the basis of chromosomal (glutamine synthetase gene) and nodulating V. sativa and V. cacca in addition to V. faba also a gene (Nodx) responsiple for conferring the ability to nodulate Pisum sativum cv. Afghanistan by R1v is described. More recently, Shumin et al. (2004) studied the enhancing phosphorus and nitrogen uptake of broad bean inoculating arbuscular mycorrhizal fungus and R. leguminosarum. They found that the dual inoculation of broad bean with R. leguminosarum and G. mosseae increased the plant height, chlorophyll content and number and weight of nodules of broad bean. The biomass of broad bean increased by 21.5 and 20.7 % when inoculated with G. mosseae alone and combined with R. leguminosarum, respectively. Mycorrhizal colonization increased by approximately 12 % with the inoculation of R. leguminosarum, the acid phosphatase and alkaline phosphatase activities of broad bean increased compared with the control when inoculated with both G. mosseae and R. leguminosarum. Also, phosphorus and nitrogen uptake increased by more than 50.9 and 22.0 %, respectively.

11 2.1.2. Non symbiotic nitrogen fixers The association between N 2 -fixing bacteria and root of non-legume plants, the organisms are present on rhizosphere and rhizoplane of the root zones. Studying the bacterial inoculation conditions such as, size of bacterium inoculum, time of inoculation, amount and concentration of organic matter added, mineral in soil and interaction between bacterial strains and host plant an increases of 10-30 % were reported in grain and forage yields with wheat, corn and sorghum after inoculation with Azospirillum ( Subba Rao, 1993). Döbereiner (1974) reported that contribution of biological nitrogen fixation in Barzial may reach to 70 % for sugar cane and up to 50 % in cereals through the activity of endophytic diazotroph in non-legume. 2.1.2.1. Azospirillum Azospirillum, an associative microaerophillic nitrogen fixer commonly found in loose association with roots of cereals and grasses which is of great interest. High nitrogen fixation capacity, low energy requirement and abundant establishment in the roots of cereals and tolerance to high soil temperature (30-40 C) are responsible for its suitability under tropical conditions (Hedge et al., 1999). Azospirilla are metabolically versatile and can grow vigorously in presence of nitrogenous compounds present in soil but as soon as the external combined nitrogen supply is exhausted, the bacteria switch on to diazotrophy. The ability to fix nitrogen is unaffected by presence of combined nitrogen sources, and may account for the beneficial response of

12 Azospirillum inoculation in field receiving mineral fertilizers (Fages, 1994; Bashan and Holguin, 1997 and Hedge et al., 1999). Of late, attention has been shifted from Azotobecter to Azospirillum as an inoculant due to its widerspread distribution in soil, association of crops grown on acidic to alkaline ph range, easy to culture and identification because of its curved form and type of motility and is relatively efficient in utilization of carbon support N 2 -fixation. The application areas for Azospirillum and Azotobacter are overlapping. However, in general, Azospirillum is more appropriate for cereals and azotobacter for non-food grain crops such as sugarcane, cotton, potato and other vegetables (Chhonkar and Tilak 1997 and Hedge et al., 1999). The mechanism of the bacterization resulting in yield increase with decrease or increase in N-concentration may be attributed to enhanced N 2 -fixation or increased N assimilation by plant (Aggarwal and Chaudhary, 1995 and Bashan and Holguin, 1997) enhanced mineral uptake in the plant (Stancheve et al., 1995) improved root growth and functions (Sarig et al., 1992 and Fallik et al., 1994) nitrate production in nitrate respiration (Bothe et al., 1992). In vitro Azospirillum lipoferum produces siderophores may which improve ironnutrition of plant (Hedge et al., 1999) produces in vitro the phytohormones IAA, gibberllins, cytokinin and ethylene. These phytohormones, especially IAA play an essential role in plant growth stimulation in general and in stimulating symbiosis between legumes and rhizobia (Bashan and Holguin 1997) and

13 effects plant all metabolism from outside suggests that bacteria are capable of excreting and transmitting a signal(s) which crossed the plant cell wall and is recognized by plant membranes and promoted nitrogen fixation. The modes of action of Azospirillum proposed over the last three decades point to the possibility that perhaps there is no major single mechanism involved. The combined activities of all the involved mechanisms may be responsible for the large measured effect of Azospirillum spp. (Bashan and Holguin, 1997). Egorenkova et al. (2000) noted the investigation of the initial stages of interaction of the bacterium Azospirillum brasilense with wheat seedling root adsorption and root hair deformation. They showed that the adsorption of azospirilla on root hair of soft spring wheat rapidly increased in the first hours of incubation going then to a plateauphase. In addition, Deaker and Kennedy (2001) studied the the improved potential for nitrogen fixation in Azospirillum brasilense SP7.5 associated with wheat nifh expression as a function of oxygen pressure. They showed a strong correlation between nitrogenase activity and nifh expression was found in pure cultures. nifh expression was maximal at 0.5 % oxygen in pure cultures of both the wild type SP7 and spontaneous mutant SP7.5. Moreover, Kaushik et al., (2001) tested the selection and evaluation of Azospirillum barasilense strains growing at a sub-optimum temperature in rhizocoenosis with wheat. They showed that significant increase in plant growth parameters; the overall response to inoculation was better in cultivar HD2285.

14 Also, Swedrzynska and Sawicka (2001) studied the effect of inoculation with population numbers of Azospirillum bacteria on winter wheat, oat and maize. They showed that the inoculation of cereals with Azospirillum barsilense bacteria contributed to the increase of their number in soil. On the other hand, Pinheiro et al. (2002) studied the adsorption and anchoring of Azospirillum strains to roots of wheat seedlings. They showed that strains of A. brasilense, originally isolated from surface sterilized wheat roots (SP 245, SP 107) or with a proven ability to infect the interior of roots (SP 245), showed no greater ability to anchor to the roots than other Azospirillum strains isolated from wheat rhizosphere (SP 246) or from the rhizosphere or rhizosphere soil of other gramineae (SP 7, cd, S 82). Recently, Amoo et al. (2003) investigated the effect of Azospirillum inoculation on some growth parameters and yield of three wheat cultivars. They showed that inoculation increased growth parameters and yield of wheat cultivars, the effect being directly dependent on the strain-cultivar combination. Creus et al. (2004) studied the water relations and yield in Azospirillum-inoculated wheat exposed to drought in the field. They showed that the grains harvested from Azospirilluminoculated plants had significantly higher Mg, K and Ca than non-inoculated plants. Neither drought nor inoculation changed grain P, Cu, Fe and Zn contents. 2.1.2.2. Azotobacter Azotobacter, a free-living heterotrophic, nitrogen-fixing bacteria encountered in neutral to alkaline soils not provides the

15 nitrogen but produce a variety of growth promoting substances. Some of these growth promoting substances are indole acetic acid, gibberllins, B vitamins and antifungal substances. Another important characteristic of Azotobacter association with crop improvement is excretion of ammonia in the rhizosphere in the presence of root exudates which helps in modification of nutrient uptake by plant (Narula and Gupta, 1986). Solubilization of different inorganic phosphates by A. chroococcum was also reported (Tilak and Singh, 1994). The mechanisms by which the plants inoculated with Azotobacter derive positive benefits in terms of increased grain, plant bio mass and N uptake which are attributed to small increase in N input from biological nitrogen fixation, development and branching of roots, production of plant growth hormones, enhancement in uptake of No 3, NH + 4, H 2 Po 4, K, Rb and Fe, improved water status of plants, increased nitrate reductase activity and antifungal compounds (Wani et al., 1988 and Hedge et al., 1999). Several experiments conducted in temperate regions of the world showed that nitrogen fixation in Azotobacter inoculated soil is not more than 10 to 15 kg of N/ha/year, depending on the availability of carbon source (Subba Raw, 1999). On the other hand, Hedge et al. (1999), mentioned that in many cases, inoculants increase plant yield and such increase is statistically significant and also sometimes negative. Also, experiments with Azotobacter and crop plants cultivars at the Indian Agriculture Research Institute, New Delhi were carried out and the obtained results indicated that significant increases in

16 growth and yield of wheat, rice and vegetable crops could be obtained in pot trails. However, under field conditions, such uniform trends towards increases in yield are not always reproducible (Subba Rao, 1999). The benefits of Azotobacter inoculation in cereal production are well documented in wheat (Lakshminarayana et al., 1992). in barley (Tiwari et al., 1989); in sorghum (Lee et al., 1994); in maize (Gill et al., 1993), in rice (Wani, 1990) and in pearlmillet (Wani et al., 1988). Ishac and Mostafa (1998) evaluated the interaction of Azotobacter and vesicular arbuscular mycorrhizas and proposed several mechanisms to mediate Azotobacter. VAM interactions: hormonal effects, enhanced nutrients uptake, N 2 -fixation and increased resistance to soil borne pathogenic fungi. They reported that the synergistic interaction between Azotobacter and VAM may lead to substantial increase in growth, mineral uptake and yield of dually inoculated plants. Kumar et al. (2001) studied that the effect of phosphate solubilization strains of Azotobacter chroococcum on yield traits and their survival in the rhizosphere of wheat genotypes under field condition. They showed that seed inoculation of wheat varieties with phosphate-solubilizing and phytohormoneproducing A. chroococcum showed a better response over control. Sharma et al. (2001) etsted the survival of Azotobacter chroococcum in the rhizosphere of three different wheat crosses: effect of AM fungi. They found that effect of wheat genotypes

17 and AM inoculation on the proliferation and survival A. Chroococcum were prominent. Behl et al. (2003) investigated the interactions among mycorrhiza, Azotobacter chroococcum and root characteristics of wheat varieties. They found that a positive association between AMF infection in roots and Azotobacter survival in the rhizosphere was apparent. Vivek et al. (2004) studied the relative efficacy of Azotobacter chroococcum on tall and dwarf wheats (Triticum aestivum L.) in arid soils. They found that grain, straw yield and root biomass were higher with mutants, which maintained higher survival rate in rhizosphere during growth period of wheat crop. 2.2. Hormones and production of growth promoting substance by nitrogen fixing bacteria There is firm evidence that plant growth regulators like auxine, gibberellins, cytokinins and ethylene produced by plant are essential for their growth and development. There is also evidence that plant growth hormones produced bacteria can increase growth rates and improve yield of host plants (Barea and Brown, 1974). Azcon and Barea (1974) reported that culture supernatants of Azotobacter vinelandii and Azotobacter beijerinkii contain auxines, at least three of gibberellins-like substance and three of cytokinins-like substances. They added that the amount of hormones produced in these cultures are similar to those produced by Azotobacter paspali and Azotobacter chroococcum.

18 Reynders and Vlassak (1979) found that all the tested strains of Azospirillum converted tryptophane to indole-3-acetic acid in pure culture. Gaskin et al. (1977) reported that auxines produced by Azospirillum stimulated the plant growth. Azospirillum produced plant growth substance, namly indole acetic acid, indole lactic acid, gibberellins and cytokinines like substance, such hormones induced the proliferation of lateral roots and root hairs which increase nutrient absorbing surfaces (Tien et al., 1979; Fouad, 1981 and Vose and Ruschel, 1981). Shank and Smith (1984) reported that the increase of grass crops after inoculation with Azospirillum spp is due to the stimulation effect of phytohormons produced by bacteria. Investigation about the mechanisms showed that the beneficial effects of most plant growth promoting rhizobacteria (PGPR) increased plant growth indirectly by changing the microbial balance in the rhizospheres (Klopper and Schroth, 1981), by producing iron chelating siderophores and by phytohormones or other plant growth enhancing compounds (Schippers, 1988). Kucey (1988) found that responses of wheat plants to inoculation with Azospirillum barsilense and Bacillus C-11-25 were similar to those caused by addition of gebberellic acid in growth pouches. Also, Taller and Wong (1989) reported that Azotobacter vinelandii can produce cytokinine like substance in culture medium. Grozier et al. (1988) and Fallik et al. (1989) stated that the isolate strains of Azospirillum are capable of producing some

19 hormones at very low levels namely indole lactic acid, indole-3- Butyric acid, indole-3-ethanole, abscisic acid, and several gibberellins and cytokinines In addition, to the beneficial effects of N 2 -fixing bacteria associated with roots of cereal crops, these bacteria are also reported to produce growth promoting substance which help increasing crop yield (De-Freitas and Germida, 1990). Baca et al. (1994) stated the indole-3-acetic acid (IAA) is excreted by different wild strains of Azospirillum spp. They added that microorganisms can produce IAA during the late stationary growth phase and significant increase in IAA production occurred when tryptophan is added. Strzelezyk et al. (1994) pointed out that some strains of Azospirillum produced cytokinine-like substance (CLS) and all tested strains of Azospirillum produced ethylene in presence of different carbon source like methionine, malate, succinate and pyruvate. Azospirillum can produce in-vitro the phytohormones IAA, cytokinine and gebberellines (Rademcher, 1994; Iosipenko and Iganov, 1995 and Patten and Glick, 1996). Response of cereals and vegetables to inoculation with some N 2 -fixing bacteria was attributed to the effect of plant growth regulators released by such microorganisms (Beylar et al., 1997 and Salamone et al., 1997). Amara and Dahodoh (1997) carried out a pot experiment to study the effect of inoculating wheat grains with plant growth promoting bacteria on yield and nutrients uptake. The inocula used were different species of Azotobacter and Azospirillum and Pseudomonas individually or in mixture and they improved grain

20 yield and total dry weight. Inoculation with mixture of these strains showed the highest value of straw yield and increased the uptake of N, P, K, Fe, Na, Zn, Mn and Cu. Dobbelaera et al. (1999) reported that auxine produced by Azospirillum is believed to play a major role in the plant growth promoting effect. In addition, El-Sawah (2000) found significant increase in N, P and K content of maize plant when seeds were inoculated with Azospirillum brasilense and Bacillus megatherium as well as low dose of mineral nitrogen fertilizers was applied. Zakharova et al., (2000) studied the effect of water-soluble vitamins on the production of indole-3-acetic acid by Azospirillum brasilense. They found that very low levels of vitamin B which added at 10-100 μg 1-1 affected production of indole-3-acetic acid in Azospirillum brasilense. The largest release of these phytohormone was observed after amendment with pyridoxine and nicotinic acid. Results of the study suggest a role of these vitamins which may fulfill in the regulation of indole-3-acetic acid synthesis in Azospirillum brasilense. Dobbelaera et al., (2001) found that Azospirillum are able to promote plant growth and increase yield in many crops, due to the production of plant growth promoting substances, which leads to an improvement in root development and increase in the rate of water and mineral uptake. Benizri et al., (2001) reported that certain rhizobacteria referred to as "plant growth promoting rhizobacteria (PGPR)" can contribute to the biological control of plant pathogens and improve plant growth. These bacteria enhance root development

21 either directly by producing phytohormones or indirectly by inhibiting pathogens through the synthesis of different compounds. Lapinskas (2001) studied the efficiency of combination of phytohormones and Rhizobium leguminosarum bv. trifolii and Sinorhizobium meliloti strains for Clover and Lucerne. They determenated that the phytohormones beta-indoleacetic acid (IAA, hetroauxin) and kinetin stimulated biomass growth (size of colony) of some Rhizobium leguminosarum bv trifolii and S. meliloti strains in field experiments, heteroauxine tended to increase the symbiotic efficiency of all the investigated Rhizobium leguminosarum bv trifolii strains. The dry matter yield increase made up 0.70-1.05 ton/ha. Yanni et al. (2001) evaluated the beneficial plant growthpromoting association of Rhizobium leguminosarum bv. trifolii with rice roots. They found that the inoculation increased total protein quantity per hectare in field grain thereby, increasing its nutritional value without altering the ratios of nutritionally important proteins. Studies using a selected rhizobial strain (E11) indicated that it produced auxine (indole acetic acid) and gibberellin (Tentatively identified as gibberellin; GA 3 ) phytohormones representing two major classes of plant growth regulators. Sangeeta et al. (2002) studied the potential of homologous and heterologous Azotobacter chroococcum strains as bioinoculant for cotton. These varied a lot in their ability to produce the phytohormone IAA and acetylene reduction activity. The effect of heterologous and homologous strains of