Potential role of Nostoc muscorum and Nostoc rivulare as biofertilizers for the enhancement of maize growth under different doses of n-fertilizer

Strains of the genera Nostoc (7 species) and Phormidium (one species) were isolated from soil. Cyanobacterial isolates were tested for their ability to form associations with the roots of wheat seedlings grown in liquid culture as well as fixing of atomospheric N2. The present study revealed that Nostoc colonization of wheat was tight association while phormidium colonization was loose association. In case of Nostoc association, its growth was in the form of aseriate packages on root surface. Moreover, our study reported that Nostoc muscorum isolate No. (12) had the ability to penetrate epidermal cells. In microcosms experiment, cyanobacterial isolates positively affected wheat growth as compared to the non-heterocystous isolate, Phormidium. Nostoc rivulare and Nostoc muscorum isolate No. (12) were more efficient in nitrogen fixing activity as compared to the rest of isolates. In case of 2,4dichlorophenoxyacetic acid induced maize roots, nitrogenase activity of Nostoc significantly enhanced as compared to the untreated maize roots. Nostoc muscorum or Nostoc rivulare colonized externally at the junction of the para-nodules, and also abundance of Nostoc on the induced maize roots increased as compared to the untreated maize roots. Nitrogenase activity and abundance of Nostoc muscorum or Nostoc rivulare co-cultivated with maize roots was increased up to 1.7 times in the absence of combined nitrogen (nitrates) as compared to the nitrate treated plants. In pot experiments, biofertilization by Nostoc muscorum or Nostoc rivulare significantly increased shoot length and leaf area of maize either alone or in combination with N-fertilizer at 50 and 100 kg N/ha. Combination of biofertilization and Nfertilization, especially at 100 kg N/ha had more effect on the growth of maize compared to the biofertilization alone, as well as nitrogenase activity.


INTRODUCTION
The use of chemical fertilizer is considered inevitable for obtaining optimum yield of crops.But it has been observed that continuous use of chemical fertilizers may *Corresponding author.E-mail: essam_92003@yahoo.com.affect soil health and may lead to a negative impact on soil productivity.Bio-fertilization technique is used beside mineral fertilizers for the plant nutritional requirements in agriculture attempts for minimizing the use of mineral fertilizers due to their high cost as well as to avoid the environmental pollution problem (Bloom, 1998).
N 2 fixing cyanobacteria improve crop production by acting as natural fertilizers as they increase both carbon and nitrogen status of soils (Lange et al., 1994).Acea et al. (2003) had reported that the addition of Cyanobacteria to soils help in increasing of soil fertility.Several studies had reported that cyanobacteria can produce extracellular polymeric substances which help them to overcome conditions of water stress and also bind soil particles to be increased from structure and fertility of soil (Hill et al., 1994).Flaibani et al. (1989) had reported that exopolysaccarides producing cyanobacteria play a significant role in reclaiming and fertilization of desert soils.
El-Shahed and Abdel-Wahab (2006) showed that cocultivation of N. rivulare and the wheat exhibited nitrogenase activities both in the presence or absence of nitrate but results also indicated that addition of nitrates significantly reduced nitrogenase activity.Several studies reported that colonization of cyanobacterial strains on the plant cultivars depends on cyanobacterial isolate, plant, and content of nitrogen and pesticides in liquid and soil cultures (Gantar et al., 1991a, b;Spiller et al., 1993;Patnaik et al., 1994).One strategy had been used the auxin (2,4-D) to induce the formation of tumor-like growth on roots called para-nodules.Previous studies reported that Azospirillum, Herbazospirillum and cyanobacterium Nostoc sp strain 2S9 B colonized paranodules of wheat roots and had ability to fix the nitrogen (Yu and Kennedy, 1995;El-Komy and Abdel-Wahab, 1998;Gantar and Elhai, 1999).El-Shahed and Abdel-Wahab (2006) showed nitrogenase activity of N. rivulare with 2,4-D (1 ppm) significantly enhanced as compared to those nontreated with 2,4-D.
It is well known that nitrogen fertilization plays a significant role in improving rice yield.Surendra et al. (1984) had reported that nitrogen addition in the soil increased its fertility, development of leaf area and productivity of plant crops.Fadl-Allah et al. (2010) had reported that biofertilization with 50% N-fertilization enhanced growth of wheat and also increased N-yield of wheat.Hussein and Radwan (2001) had found that increasing nitrogen application rates increased number of grains per spike, grain weight and wheat productivity.El-Kalla et al. (1988) had concluded that increasing nitrogen application rates up to 75 kg N/ha increased plant height, leaf area, spike weight and wheat productivity.Abd El-Rahman et al. (1992) had indicated that the application of 144 kg N/ha to the wheat plant enhanced spike length, 1000-grain weight, number of grains/spike and grain yield compared to the different weights of nitrogen fertilizer.Several research groups had studied effect combination between biological and chemical fertilization in the flooded rice system (Jeyaraman and Purushothaman, 1988) and reported that bio-fertilization was a better alternative for the extensive use of nitrogen fertilizers in rice production.
As an effort in this respect, the present study aims at isolation and selection of the most efficient nitrogen fixing cyanobacterial isolates in order to be used as inoculants.Colonization of nitrogen fixing cyanobacteria to cereals roots and their effects on the plant growth were investigated through hydroponic solution (BG11 medium).Our study also aim to induce roots of maize by 2,4-D for formation of paranodules which are colonized with N. revulare and N. muscorum and its effect on nitrogenase activity.Field experiments were conducted to evaluate the effect of inoculation with Nostoc isolates selected as inoculants on the growth and yield of maize plant grown in sandy soil in absence or presence of different levels of nitrogen fertilizer in order to determine the extent to which the nitrogen-fixers could replace or decrease the application of nitrogen fertilizer.

Cyanobacterium and growth condition
Soil samples collected from Minia Governorate, Egypt, is illustrated in Figure 1.The roots of selected plants were removed and gently shaken to remove free soil on surface.Five grams of free soil were placed in flask that contained 100 ml of sterile water after thoroughly shaking suitable dilutions were prepared.One ml of soil suspension was transferred to each sterile Petri-dish, which was then poured with melted but cooled nitrogen free BG11 medium (Rippka and Herdman, 1992) which is supplemented with agar for solidification (1%), at light intensity 3000 lux.Three plates were prepared for each sample; the plate's cultures were incubated at 24°C ±1 for 15 days according to the method described by Abdel-Hafez et al. (2000).Developing cultures were identified according to Desikachary (1959).Pure isolates were maintained on N2 containing BG11 medium for further studies.

Nitrogenase activity of free living cyanobacterial isolates culture (in vitro)
Nitrogenase activity was evaluated in 15 days old culture using the acetylene reduction technique which was described by Hardy et al. (1973), using Gas Chromatograph, ATIUNICAM 610-GLC (UK) equipped with a glass column filled with activated alumina.A 10 ml aliquot of each culture was transferred to a flask with a 50 ml total volume, which was sealed with tight stopper.A 10% of free volume of air was replaced with pure acetylene using a gas tight syringe.Flasks were incubated for 2 h at 30°C.Results were expressed as nmole of C2H4 per µg Chlorophyll a.

Host plant
Seeds of host plant were first rinsed five times in sterile distilled water prior to surface sterilization.After two minutes, seeds of selected plants were sterilized with a mixture of 1:1 solution of hydrogen peroxide (30%) and ethyl alcohol (70%), then they were rewashed with the distilled water and germinated on a sterile filter paper in sterile 9 cm Petri dishes for 2-3 days.Uncontaminated seedlings were transferred and grown in glass beakers or pots for experiments.

Hydroponic growth conditions
The method described by Gantar and Elhai (1999) was used for growing plant host in hydroponic solution (Rippka and Herdman, 1992).Pre-germinated host seeds were grown in sterile 250 ml glass beakers instead of tested tubes.250 ml of sterile hydroponic solution was added to each beaker containing sterile foam rubber plate previously perforated with a cork borer to support the wheat seedling.This medium was used for testing the effect of combined nitrogen after the addition of NaNO3 at a rate of 1.5 g/L..

Screening of N2 fixing cyanobacteria associated with wheat roots in sterile soil (Microcosms experiment)
Four germinated wheat grains were transplanted in pots (500 ml volume) containing 500 g of sterilized sandy soil (2 sand: 1clay).Pots containing germinated wheat grains divided into nine groups with three replicates for each treatment as illustrated in Plate (2) followed with inoculation of different cyanobacteria (10 6 heterocysts per seedling).Seedlings of the first group represent the control.While those of the other groups were inoculated with tested cyanobacterial isolates.Pots were irrigated with distilled water according to field capacity.Pots incubated at continuous illumination, at room temperature.After 25 days from cultivation, growth parameters and nitrogenase activity were measured.

Induction and colonization of para-nodules
Ganter and Elhai (1999) and El shahed and Abdel-Wahab (2006) described the used method.When the roots of maize seedlings were about 5 cm length, aliquots of N. muscorum and N. revulare culture were added to the beakers containing 250 ml hydroponic solution to maintain a cell density of 10 6 heterocysts per seedling.Aliquots of sterile 2,4-D solution were also added to give a final concentration of 0.5, 1.0 and 3.0 ppm and hence beakers were divided into five groups with three replicates.Non-inoculated and inoculated beakers without 2,4-D were used as controls at continuous light intensity of 300 lux at room temperature for 15 days.After five days from treatment with 2,4-D, para-nodules were well formed on the root.Roots were examined and photographed using Phase Contrast Microscope.When wheat seedlings were 15 days old, seedlings were harvested, growth parameters (represented in length of both root and shoot and weights of both root and shoot), nitrogenase activity and cyanobacterial abundance on root (Chl.a content) were determined.

Tetrazolium salt staining and microscopic examination
Para-nodules seedlings were incubated overnight with a solution of 0.025% triphenyltetrazolium chloride (TTC) to locate the site of strong reduction, such sites were detected and examined using a Kyowa, Japan, dissecting stereomicroscope.

Determination of cyanobacterial abundance on wheat root
The total cyanobacterial biomass in the root was determined as chlorophyll a content.The colonized roots were extracted in acetone 85% and chlorophyll a was estimated spectrophotometrically at 663 nm (Mackinney, 1941) and expressed as a root weight basis.

Greenhouse pot experiments
Six germinating grains were transplanted into a pot containing 3 kg soil (2:1 sand and clay) respectively.This was followed by inoculation with N. muscorum isolate No. (12) or N. rivulare (10 6 heterocysts per seedling).After one week from transplantation of

Nitrogenase assay in situ
Nitrogenase activity was assayed by acetylene reduction assay (ARA) using a Gas Chromatograph, ATIUNI CAM 610-GLC (UK) equipped with a glass column filled with activated alumina.The remaining seed and its detached root were aspectically washed in sterile nitrogen free mineral solution and were transferred to a 15 ml serum bottle containing 2 ml of that mineral solution.The serum bottles were stopped with sterile rubber stoppers and 10% of the gas reaction mixture was replaced with acetylene and injected at 30°C for 2 h.Results were expressed as nmole of C2H4 per gm of root (Turner and Gibson, 1980).

Determination of plant growth
The lengths of tested plants (cm) were measured and weighed for obtaining fresh weights (mg) and then dried in an oven at 105°C to constant mass for further analysis.
a) Determination of leaf area: Leaf area was determined according to Norman and Campbell (1994).b) Determination of the photosynthetic pigments: Chlorophyll a, Chlorophyll b and carotenoids were determined using the spectrophotometeric method recommended by Metzner et al. (1965).c) Determination of calcium and magnesium: The versene (disodium dihydrogen ethylenediamine tetraacetic acid) titration method (Schwarzenbeck and Biederman, 1948) was employed for the determination of Ca +2 and Mg +2 concentrations.d) Determination of Sodium and Potassium: Sodium and potassium were determined in the samples photometrically by flame photometry according to the method of Golterman et al. (1978).

Physico-chemical properties of tested soil
Soil temperature was determined in situ using a Hg thermometer.
pH value was immediately measured after transportation to the laboratory using pH meter.Electrical conductivity of collected soils was measured using conductivity meter.Soil texture was determined by mechanical analysis through soil texture triangular.Cl -, Ca +2 and Mg +2 were determined by volumetric methods.Phosphate and ammonia were determined by spectrophotometric methods using a Perkin Elmer Spectrophotometer (Table 4).
a) Determination of chloride: Estimation of chloride in soil extract was performed according to the method described by Jackson (1960).b) Determination of carbonate: The carbonate is determined in soil samples by the use of phenolphethalein as indicator and standard solution of the hydrochloric acid (0.05 N) as recommended by Jackson (1960).c) Determination of orthophosphate: In general, all the glassware used in phosphorus determinations (bottles, pipettes, measuring cylinders, etc.) were washed with 10% H2SO4 to remove phosphates that may adhere to glassware and then rinsed with distilled water before use.The orthophosphate determination was performed according to the procedure reported in the American Public Health Association Publications (1995).d) Determination of ammonia: Ammonia was determined colorimetrically by the method adapted by Naguib (1964) e) Determination of nitrate: Nitrates were determined by sodium salicyate method (Deutsche Einheitsverfahren Zur Wasser-Abwasser Und Schlamm-untersuchung, 1960).

Statistical analysis
The triplicate sets of data for the various parameters evaluated were subjected to ANOVA (Analysis of variance) in accordance with the experimental design (Completely Randomized Design) using SPSS11 statistical packages to quantify and evaluate L.S.D values which were calculated at P level ≤ 0.05 (Steel and Torrie, 1960).

Nitrogenase activity of cyanobacterial isolates in vitro
All cyanobacterial isolates were tested for their ability to fix atmospheric nitrogen in vitro.Results in Table 2 showed that the eight isolates have the ability to fix nitrogen.

Colonization of wheat roots by different cyanobacterial isolates in hydroponic solution and their capacity on nitrogen fixing (in situ)
The isolated cyanobacteria were tested for their ability to colonize the roots of wheat plant in hydroponic solution.Results in Table 3 and Plate 1 showed that all cyanobacterial isolates have the ability to colonize the roots of wheat, and colonization of roots by N. passeriniamum, N. muscorum isolate No. (12), Phormidium molle was higher than other isolates.Results in Table 3 also showed that all cyanobacterial isolates have the ability to fix nitrogen in situ.The nitrogenase activity of N. muscorum isolate No. (12) and N. rivulare was the highest compared to other cyanobacterial isolates.Two different kinds of association between roots and cyanobacteria were observed, (a) loose attachement in case of Phormidium, (b) Tight association was observed in case of Nostoc isolates which showed tightly packed filaments of the Nostoc forming aseriate packages on a root surface.The first stage of colonization of wheat roots by these isolates was probably the migration of hormogonia, then hormogonia developed into long filamentous and the long filaments  developed of the aseriate stage which consisted of filaments tightly packed in a mucilaginous sheath.In addition to form a tight association, one strain (N.muscorum isolate No. ( 12)) also appeared to penetrate some root cells as cyanobacterial mass.

Effect of different cyanobacterial isolates on wheat growth
Different cyanobacterial isolates were tested for their ability to affect the growth of wheat growing in sterilized loamy sand soil.Results in Table 5 and Plate (2) showed that most cyanobacterial isolates increased wheat root length.Data also showed that cyanobacterial inoculation increased lengths of shoot, while Phormidium molle and N. muscorum isolate No. (11) showed non-significant effect in length of shoot.The tested isolates except Phormidium molle and N. passeriniamum caused a significant increase in fresh weights of root and shoot of wheat plant.Cyanobacterial inoculation positively affected pigment content, Phormidium molle treatment showed that the highest effect.Most cyanobacterial isolates showed a significant increase in plant leaf area, but N. punctiforme was not affected significantly in plant leaf area.Data also showed cyanobacterial inoculation clearly increased plant shoot and root dry weight, while Phormidium molle did not exhibited such effects.

Nitrogen fixing efficiency of different cyanobacterial isolates associated with wheat in situ
Results in  12) were the greatest among other isolates.According to these results, both N. rivulare and N. muscorum isolate No. (12) were selected for further studies.

Effect of different 2,4-D concentrations and inoculation with N. rivulare or N. muscorum on growth of maize seedlings (15 days old)
Results in Table 7 showed that treatment of seedlings of

Nitrogenase activity (In situ) of N. rivulare and N. muscorum associated with maize at different 2,4-D concentrations (15 days old)
Data presented in Table 8 showed that nitrogenase activity assayed by acetylene reduction assay of N. rivulare or N. muscorum associated with maize plants treated with 2,4-D was higher than those nontreated with 2,4-D.Generally, nitrogenase activity increase in the presence of 2,4-D.

Abundance of N. rivulare and N. muscorum (chlorophyll a content) colonized maize roots at different concentrations of 2,4-D (15 days old)
Data presented in Table 8 showed that colonization of N rivulare or N. muscorum to maize roots enhanced by increasing 2,4-D concentration as compared with inoculated roots and not treated with 2,4-D.

Effect of different 2,4-D concentrations on numbers of para-nodules of maize seedlings inoculated with N. rivulare or N. muscorum
Results of our study indicate that para-nodules were developed mainly on the main roots as swollen projections after 5 days of 2,4-D treatments.The greatest number was found at the tip of the root (Plate 3a).Data presented in Table 9 also showed that high numbers of para-nodules (per plant) were obtained at the range of 0.5 -1.0 ppm 2,4-D.The application of 2,4-D at a rate of 1.0 ppm did not repress plant development.

Colonization of para-nodules of maize with N. rivulare or N. muscorum
When nodulated seedlings were inoculated with N. rivulare or N. muscorum and were inoculated overnight in a solution of 0.025% triphenyltetrazolium chloride (TTC), the whole para-nodule structure was stained red indicating that these structures had been colonized by N. muscorum which found a possible better site for N 2 fixation as indicated in (Plate 3b).Light microscopy examination revealed that N. rivulare or N. muscorum colonized the para-nodules externally at both the basal connection between the nodule and the root and at the top of the nodules as loosely arranged filaments as indicated in (Plate 3c).

Nitrogenase activity of N. muscorum and N. revulare colonized maize roots (treated 1 ppm 2,4-D) as affected by the presence or absence of nitrates
Results in Table 10 showed that co-cultivation of maize seedlings with N. rivulare and N. muscorum

Effect of N. rivulare or N. muscorum isolate No. (12) inoculation on the growth and nitrogen fixing of maize grown in pot experiment through the addition of different doses of combined nitrogen
Results in Tables 11 and 12 and Plate (4a, b, c) showed that plants fertilized with ammonium sulphate only showed improvement in measured parameters expressed as plant height, weights (dry and fresh) of both root and shoot and leaf area.Plants inoculated with N. muscorum isolate No. ( 12) and N. rivulare in t eh eshrh eh of combined nitrogen (half and full dose) showed a significant increase more than those of non-inoculated and fertilized plants (controls).
Results also indicated that the growth of maize inoculated with N. muscorum isolate No. ( 12) at half dose or full dose of combined nitrogen significantly increased compared with growth of maize inoculated with N. rivulare at half dose or full dose of combined nitrogen.Results in Table 12 showed that the addition of half or full dose of combined nitrogen caused an increase in pigment content and magnesium content in shoot.Nfertilization of maize caused a small increase in Ca +2 and Mg +2 content in shoot compared with the control plants.Results also indicated that inoculation of maize with N. muscorum isolate No. (12) and N. rivulare at addition of half or full dose of combined nitrogen improved from pigment content as well as potassium and sodium amount in shoot compared with maize treating alone with half dose or full dose of combined nitrogen.At the same time, it caused a small increase in calcium and magnesium content in shoot of maize.

Nitrogenase activity (in situ) of maize roots grown in pot experiment
Results in Table 11 showed that N. muscorum isolate No. (12) and N. rivulare inoculation showed nitrogen fixing capacity on maize roots.The addition of N-fertilizer enhanced the nitrogen fixing activity using both Nostoc spp.than those of non-fertilized plants.

DISCUSSION
In our study, we described the association between cyanobacterial isolates and wheat plant in liquid culture in which there were two types of associations: a) Loose association as in case of Phormidium, b) Tight association as in case of Nostoc.These types of association with wheat had been previously reported by Spiller et al. (1993) and Gantar (2000a).Tight association may be related to characteristic developmental life cycle of Nostoc: heterocyctous filaments, hormogonia and an aseriate stage that consists of heterocystous filaments packaged tightly together (Lazaroff, 1973).Polysaccharides by which cyanobacteria strains were produced may play an important role in the attachement of cyanobacteria to roots as similar as previous study had been reported by Robins et al. (1986) for the attachment In Microcosms experiment, results of the current study showed that inoculation of wheat plant with of cyanobacteria to plants cells.N. rivulare had been cyanobacterial isolates improved from wheat growth as well as positive effects of such association on plant growth which had been reported by Rai et al. (2000).The positive effects on wheat growth due to colonize cyanobacterial isolates the wheat roots, fix atmospheric N 2 and produce plant growth promoting compounds, as well as had been previously shown in Nostoc-wheat association (Gantar et al., 1995;Nanjappan et al., 2007;Sergeeva et al. 2002).
Preliminary studies showed that germinating maize seedling treated with high concentration of 2,4-D (5 ppm) alone exhibited fungal contamination compared with control plants in spite of sterilization of seeds with mixture of ethyl alcohol and hydrogen peroxide as well as had been previously shown by El-Shahed and Abdel-Wahab (2006).Thus, 2,4-D was applied up to the concentration of 3 ppm for the rest of the other plant in all our experiments.The present results showed that the auxin 2,4-D increased the colonization of roots of maize with N. muscorum or N. rivulare and hence, nitrogenase activity increased.These results are in accordance with previous results (Fadl-Allah et al., 2011;El-Shahed and Abdelwahab, 2006;Nilsson et al., 2002;Gantar and Elhai, 1999).The stimulatory effect of 2,4-D on colonization of cyanobacteria on root and nitrogenase activity specially in the absence of nitrates could be explained on the basis that either a) the auxin increases amounts of cyanobacteria bound to the root surfaces and thus the extent of N 2 fixation as well or b) 2,4-D had induced paranodules on plant roots and these could had provided suitable sites for cyanobacterial colonization (Fadl-Allah et al., 2011;El-Shahed, 2005;Gantar and Elhai, 1999).
In the Pot experiment, results of the present study showed that inoculation of maize plants with N. muscorum or N. rivulare caused a significantly increase in growth of maize plants, represented in plant height, leaf area, weight of plant as well as legume weight of wheat, pigment and minerals content.These results are in accordance with previous results (Fadl-Allah et al., 2010;Nanjappan et al., 2007;Al-Noim and Hamad, 2004;Rai et al., 2000).Growth stimulation by N. muscorum or N. rivulare could be attributed to production of the auxins/ bioactive molecules (Nisha et al., 2007;Biondi et al., 2004;Aziz andHashem, 2004, 2003;Sergeeva et al., 2002).The combination of biofertilization (N.muscorum or N. rivulare) and N-fertilization (half and full dose) significantly increased the growth of maize plants, compared to those of control non-inoculated and fertilized plants.These results are in accordance with previous observations (Fadl-Allah et al., 2010;Al-Noim et al., 2004;Nayak et al., 1986;Abou-Zeid et al., 1996;Hussein and Radwan, 2001;Bassal et al., 1996).The stimulatory effect of N-fertilizer addition on nitrogen fixing activity was due to stimulate root growth, as well as stimulated the growth of cyanobacterial population in soil cultivated with wheat, and thus colonization of cyanobacteria increased and also nitrogen fixing activity enhanced.Similar conclusions had been previously reported (Jha et al., 2001;Jha and Prasad;Singh et al., 1992;Wang, 1986).
Finally, our study shows that N. muscorum or N. rivulare is being a promising organism for achieving efficient association between cyanobacteria and nonlegume plants.However, further studies on these relationships will promote the practical application of para-nodules for improving the nitrogen nutrition of cereals.

Figure 1 .
Figure 1.General location map shows localities of soil samples.
Screening of different cyanobacterial isolates to improve the growth of wheat grown in microcosms experiment.Bar = 8 cm.
nitrogenase activity in the presence or absence of nitrate.Results also indicated that absence of nitrate caused a large increase in nitrogenase activity with N. muscorum.Nitrogenase activity of wheat co-cultivated with N. rivulare or N. muscorum was increased up to 1.7 times in the absence of nitrate compared with nitrate treated maize.Results in Table 10 also showed that colonization of N. rivulare or N. muscorum to maize increase in the absence of nitrate compared with the presence of nitrate.

Plate 4 .
Pot experiment of maize; a) Effect of different doses of combined nitrogen on growth of maize plant.Bar = 13.8 cm.b) Effect of inoculation with N. rivulare on maize growth at different doses of combined nitrogen.Bar = 13.8 cm.c).Effect of inoculation with N. muscorum isolate No. (12) on maize growth at different doses of combined nitrogen.Bar = 3.8 cm characterized by El-Shahed (2005) its ability to form tight association with wheat roots and fix the nitrogen.

Table 1 .
Isolation of cyanobacteria spp.from different localities in El Minia Governorate.
maize seedlings, Ammonium sulphate dose was added on three levels (100 kg N/ ha added on three levels, each stage 33.3 kg N/ ha).Pots were divided into 9 groups with three replicates for each treatment as illustrated in Plate (4a, b, c).At the beginning of the second week after sowing, plants were thinned down to four plants per pot.The experiment was performed in a wire proof greenhouse maintained at 30 ± 5°C under natural day light.Pots were irrigated with water as needed according to field capacity.After 45 days, Plants were harvested, growth parameters and nitrogenase activity were measured.

Table 2 .
Nitrogenase activity of cyanobacterial isolates in vitro.

isolate Nitrogenase activity (nmole C2H4 h-1 per µg of chl. a
Plate 1. Colonization of wheat roots with different Cyanobacterial spp.

Table 3 .
Colonization of wheat roots by different cyanobacterial isolates in hydroponic solution and their nitrogen fixation in situ.

Table 4 .
Physico-chemical properties of the used soil in the experiments.

Table 5 .
Effect of different cyanobacterial isolates on wheat growth and pigment contents.

Table 6 .
Nitrogen fixing efficiency (in situ) of different cyanobacterial isolates associated with wheat in microcosms experiment.

Table 6
Acetylene reduction assay of N. rivulare and N. muscorum isolate No. (

Table 7 .
Effect of different 2,4-D levels and inoculation with N. rivulare and N. muscorum isolate No. (12) on growth of maize seedlings (15 days old).

Table 9 .
Effect of different 2,4-D concentrations on numbers of paranodules of maize seedlings inoculated with N. rivulare and N. muscorum isolate No. (12).

Table 10 .
Abundance and Abundanceof N. rivulare and N. muscorum isolate No. (12) (chlorophyll a content) colonized maize roots (15 days old) as affected by presence or absence of nitrate.

Table 11 .
Effect of N. rivulare and N. muscorum isolate No. (12) inoculation on the growth and nitrogen fixation of maize grown in pot experiment through different doses of combined nitrogen.

Table 12 .
Effect of N. rivulare and N. muscorum isolate no.(12) inoculation on the pigment content and mineral content of maize grown in pot experiment through different doses of combined nitrogen.