Nigella Sativa) by Using Azotobacter As N2 Biofertilizer

Annals of Agricultural Science (2014) 59(1), 95–108

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ORIGINAL ARTICLE Improving the productivity and quality of black cumin (Nigella sativa) by using Azotobacter as N2 biofertilizer

Samah M. Abdel-Aziez a,*, Wedad E. Eweda b, M.G.Z. Girgis b, Bouthaina F. Abdel Ghany a a Soil Fertility and Microbiology Dept. of Microbiological Unit, Desert Research Center (DRC), Cairo, Egypt b Agricultural Microbiological Dept., Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, Cairo, Egypt

Received 18 November 2013; accepted 13 March 2014 Available online 10 August 2014

KEYWORDS Abstract Thirty one rhizosphere soil samples were collected from different gavernorates and local- Azotobacter chroococcum; ities cultivated with different standing crops. Samples were used for the isolation of N2-fixer Azo- Nigella sativa; tobacter sp. isolates. The purified isolates were identified as Azotobacter chroococcum. The purified Antibacterial effect isolates were tested for their N2 fixation activity, phosphates dissolving ability, production of plant growth promoting substances, exopolymer secretion, siderophores production, salicylic acid forma- tion, and some enzymatic production. Out of these purified isolates namely Azo.4, Azo.5, Azo.9 and Azo.23 found to be more significant in the production of the aforementioned activities as compared with the other purified isolates. The four purified isolates were tested for some biochemical activities (hormonal activity and enzymes production) and used to prepare the effective microbial inoculants for black cumin (Nigella sativa) seeds. Results show that mixed inoculation with the four biofertil-

izer strains and using half dose of recommended N2-fertilizer enhanced the densities of the total microbial microflora, phosphate dissolving bacteria, azotobacters colonization, CO2 evolution in the rhizosphere of the inoculated plants and plant growth features in comparison with uninoculated plants (control). The effect of the crude oils of the produced black cumin seeds on some human pathogenic bacteria was studied. It was found that the crud fixed oil extracted from the seeds of plants has powerful antibacterial properties against this diverse genus of bacteria. However, the influence was different and depending on the tested bacterial strain. ª 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.

Introduction

* Corresponding author. Medicinal plants are a unique type of natural product requiring E-mail address: [email protected] (S.M. Abdel-Aziez). special consideration due to their potential impact of people’s Peer review under responsibility of Faculty of Agriculture, Ain-Shams health. Therefore, improving the productivity and quality of University. http://dx.doi.org/10.1016/j.aoas.2014.06.014 0570-1783 ª 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. 96 S.M. Abdel-Aziez et al. various medicinal and aromatic plants in Egypt was an ultimate Microscopical examination was carried out to make sure the goal, in the latter decade, to meet the increase of population and purity of cultures by Gram’s staining, then kept on slants of to avoid chemical therapy side effects on human health through specific medium for the microorganism subculturing for the utilization of the medicinal herbs. Aromatic plants containing purified isolates that were usually done every month and main- volatile and fixed oils are belonging to this family and they rep- tained on the same medium mentioned before at 4–5 C until resent an important source of the national income of Egypt for used. local consumption and export. One of the most important plants containing volatile and fixed oil is black cumin family Ranuncul- Screening of bacterial isolates aceae is an herbaceous indigenous plant in the Mediterranean region. Seeds of this plant have been used for centuries as a spice Determination of N2-fixation and food preservative, as well as a traditional medicine for the The thirty one purified isolates were tested for their N2 fixation treatment of various diseases, including skin infections activity according to the microKjeldahl method described by Schleicher and Saleh (2000) and Goreja (2003). The antimicro- (Jackson, 1958). Nitrogenase activity was determined for the bial effects of black cumin seeds against different pathogenic most active four isolate in N2-fixation by acetylene reduction microbes were investigated (Hosseinzadeh et al., 2007; Salman activity technique (ARA) according to (Schollhorn and et al., 2008; Abu-Al-Basal, 2009 and Abou-Zied, 2011). Burris, 1967). Biofertilizers have the ability to access a major part of nutrients for growing Plant along with growth-promoting factors. These Assessment of phosphate solubilizing ability benefits play an effective role in reduction of chemical fertilization and also result in higher crop yield (Cordovilla et al., 1999). The purified thirty one bacterial isolates were tested for phos- Plant growth-promoting rhizobacteria (PGPR) are bacteria of phate dissolving capability qualitatively by inoculating each promoting plant growth by colonizing the plant root. For a long isolates on modified Bunt and Rovira medium. Estimating period PGPR were mainly used for assisting plants to uptake the clear zone around the developed colonies after an incuba- nutrients from the environment or preventing plant diseases. tion period of four days at 28 C. Their phosphate dissolving Recently, the application of PGPR has been extended to reme- potency was also determined quantitatively by inoculating diate contaminated soils in association with plants Zhang et al. Bunt and Rovira medium, which was previously prepared with (2007). A technology combining plant and microorganisms, the addition of 0.9 g rock phosphate per 100 ml medium, with mainly PGPR inoculated into the plant roots, may promote a 1 ml of bacterial suspension. The inoculated media were synergistic action leading to improved plant growth. This may incubated for ten days at 28 C. After incubation period, each overcome many of the limitations of single organisms and pro- sample was centrifuged at 6000 rpm for 15 min, then superna- vide a useful and more powerful approach for enhancing reme- tant was used in determining the quantity of dissolved phos- diation of contaminated environments (Khoramdel et al., 2008). phorus according to method adopted by Watanabe and The biofertilizers include mainly the nitrogen fixing, phosphate Olsen (1965). solubilizing and PGPR microorganisms (Goel et al., 1999). The beneficial effect of bio-N-fertilizers application is the improve- Production of exopolysaccharides (EPS) ment of nitrogen contents, as well as the improvement of the The exopolysaccharides’ Production of the purified isolates of physical and chemical properties of the yield (Sayed and azotobacters was determined by the method described by Giti- Hossein, 2011). The present study aimed to evaluate the efficacy Emtizi and Habibi, 2004. of microbial inoculants (Azotobacter chroococcum) and their influence in conjugation with different levels of inorganic Production of salicylic acid(SA) and siderophores N-fertilizer on the rhizosphere biological activities, yield and The activity of Azotobacter isolates to produce salicylic acid yield components of black cumin grown in sandy soil. and siderophores was determined in Standard succinate medium according to (Meyer et al., 1992) and Low-Fe synthetic Materials and methods medium according to (Reeves et al., 1983) respectively.

Rhizosphere soil samples Production of phytohormones All purified bacterial isolates were tested for the promoting During January 2009 and June 2009, thirty rhizosphere soil activity on plant seedlings by measuring the elongation in samples of different crops were collected from some govern- shoots and roots as indicated by El-Shazly (2003). Phytohor- orates and locations in U.A.R. (Table 1). mones were also determined quantitatively for selected effective microorganisms using by high performance liquid Isolation, purification and characterization of the bacterial chromatography (HPLC) according to the modified method isolates of Rizzolo et al. (1991).

Indole production The isolation of N2-fixing Azotobacter was carried out as pro- posed by Thompson and Skerman (1979). Thirty one N2-fixing Indole acetic acid (IAA) production potential of these isolates Azotobacter isolates were subjected to purification trials by was tested in Ashby’s nitrogen free broth supplemented with successive streaking on nitrogen deficient of modified Ashby’s 0.005 M concentration tryptophan at 28 C. The concentration medium (Abdel-Malek and Ishac, 1968). Nutrient agar of IAA in the culture broth after 3 days of incubation was medium was used for growth, maintenance and checks the determined by spectrophotometric method using Salkowski purity of the isolates (Jacobs and Gerstein, 1960). reagent as described by Mali and Bodhankar (2009). Improvement of productivity and quality of black cumin 97

Table 1 Mechanical and chemical characteristics of experimental soil used for pot experiment of black cumin. Soil sample Mechanical analysis Chemical analysis Sand Silt Clay % Soil texture pH EC OM% OC% TN% C/N Cations meq lÀ1 Anions meq lÀ1 % % dS mÀ1 Ratio ++ ++ + + À À À À Ca Mg Na K CO3 HCO3 Cl SO4 El sadat area 95.37 0.69 0.51 Sandy soil 8.3 0.958 0.48 0.28 0.01 28 1.745 1.648 5.672 0.513 - 2.618 5.400 1.270

Bioassay of cytokinin cumin plant. These strains were Azo.4, Azo.5, Azo.9 and In order to study the ability of Azotobacter isolates to release Azo.23. cytokinin in the growing medium, puriﬁed bacterial isolates were grown in Ashby’s medium for 7 days at 28 C and the Preparation of plants and soil materials microbial growing cultures were taken to study the qualitative Sandy soil was collected from the top layer (20 cm depth) from bioassay test of cytokinin using Radish (Raphanus sativus) desert in El sadat Area-West of Nile Delta, Monuﬁa. Egypt, cotyledons according to Lethman (1968). air dried, sieved thorough a 2 mm sieve and thoroughly mixed. Organic manure (compost) was thoroughly mixed with the Bioassay of Gibberellins experimental soil and left for a month before cultivation at À1 The method used for bioassay of gibberellins was that adopted the rate of 1% w/w (75 g pot ). Pot diameter is 20 cm that by Bentley-Mowat (1966) and Jones and Varner (1967). contained 10 kg/sandy soil.

Some enzymes production Inorganic fertilizers and organic manure Azotobacter chroococcum isolates were cultured in Boucher’s The mineral fertilization used for the plant adopted in this minimal medium (BMM) supplemented with 0.1% citric acid investigation was applied at the rates of 50, 100, 150 and À1 which was used as deﬁned medium at 28 C. Growth on vari- 200 kg fed respectively. Seeds of black cumin were succes- ous carbon sources was assayed in BMM supplemented with sively washed and immersed for 30 min in heavy cell suspen- 8 À1 0.2% (w/v) carbon source. Polygalacturonic acid, pectin and sions of Azotobacter chroococcum strain (10 cells ml ) and carboxyme-thylcellulose were used as a sole carbon sources a mixture of strains at the ratio of 1:1:1:1. Half percent of car- for the determination of polygalacturonase, pectin lyase and boxy methyl cellulose (CMC) was used as an adhesive agent. cellulase production, respectively. Optical density of the Seeds of control treatments were treated in the same manner Cultures was measured at 600 nm and incubated in a but using N-deﬁcient medium instead of bacterial culture. 300-rpm on shaker at 28 C for 4 days (Allen et al., 1997 and The inoculated seeds were air dried at room temperature for Tans-Kersten et al., 1998). 2 h before sowing. Ten seeds of each treatment were planted in each pot. Pots were directly irrigated after sowing to provide Polygalacturonase production suitable moisture for inocula. The seedlings were thinned one month after cultivation into 3 plants per pot. The polygalacturonase production was determined by measur- Compost was used at rate of 15 m3/feddan, and it has an ing the release of reducing groups using the dinitrosalicylic organic carbon of 20.84%, total N of 1.24%, organic matter acid reagent DNS assay according to Odeniyi et al. (2009). of 36.56%, C/N ratio of 16.8, soluble NH4+ of 330 ppm, soluble NOÀ of 270 ppm, total P of 0.58%, total K of 1.15%, Pectin lyase assay 3 total Fe of 1.26%, total Mn of 578 ppm, total Zn of Pectin lyase in the supernatant of cultures was determined 130 ppm, total Cu of 136 ppm, E.C (1:10) of 6.10 and pH spectrophotometrically at wavelength of 235 according to the (1:10) 8.8. method described by Silva et al. (2005).

Preparation of N2-fixing Azotobacter inoculant Cellulase assay technique In order to prepare a suitable inoculant of the four selected Cellulase enzyme was determined by the technique described most effective strains that were used as a biofertilizer, azoto- by Odeniyi et al. (2009). bacter mixture, heavy cell suspension of the selected Azotobac- ter chroococcum was obtained by growing separately on Identification of selected azotobacter isolates modified Ashby’s medium, mixed v/v from each strain to Azotobacters isolates were identified according to Krieg and obtain the desired concentration (1.8 · 108 cells/ml) and was Holt (1984) and Holt et al. (1994). kept at 4 C until applied to seeds.

Evaluation of the effectiveness of azotobacters inoculants on Treatments growth and productivity of black cumin plant The following treatments have been carried out A pot experiment was conducted under a greenhouse conditions at Desert Research Center (DRC), Cairo, Egypt to study 1. Without inorganic nitrogen fertilizer. the role of the most four active strains of the aforementioned 2. 1/4 the recommended dose (50 kg N feddanÀ1)+ tests Azotobacter chroococcum on the productivity of black *inoculation. 98 S.M. Abdel-Aziez et al.

3. 1/2 the recommended dose (100 kg N feddanÀ1)+ microbial activities in the rhizosphere soil as influenced by inoculation. inoculation of N2-fixer bacteria according to the method 4. 3/4 the recommended dose (150 kg N feddanÀ1)+ described by Anderson (1982). inoculation. 5. 5-Full recommended dose of N-fertilizer (200 kg N Determination of oil and fixed oil contents of black cumin seeds À1 feddan ) + inoculation. The oil percentage of black cumin seeds was determined 6. 6-Uninoculated treatment full dose of recommended according to the method described by British Pharmacopoeia nitrogen fertilizer (control). (1968). Soxhlet method was used for the estimation of fixed oil as stated by the A.O.A.C. (1990). * Inoculation: Inoculation was done using individual strain and/or mixed strain. Determination of antimicrobial activity of the extracted oil The antibacterial activity of black cumin oil against some Plant sampling and determinations pathogenic bacteria such as, (Staphylococcus aureus, Esche- The plants were kept for seven months. Samples were taken at richia coli, Salmonella sp., Shigella sp. and Listeria monocyto- three growth stages (vegetative, flowering and fruiting for gens) was evaluated by agar-diffusion technique using filter black cumin for pot experiment after 60, 150 and 210 days of paper disks according to Jacobs and Gerstein (1960). growth. Statistical analysis Soil physical and chemical analysis Data were subjected to an analysis of variance (ANOVA), Soil samples were mechanically analyzed according to the using a log transformation when necessary. When ANOVA methods described by Piper (1950). The electrical conductivity generated a significant F-value (P < 0.05), treatment means (EC) of the soil was measured in saturated soil according to were compared by Tukey’s LSD-test. Experiment was method described by Jackson (1958). Soluble anions, cations carried out with three replicates per treatment Snedecor and soil pH were determined in saturated soil according to and Cochran (1982). The least significant difference the method described by (Richards, 1954). Organic carbon (L.S.D) was used to differentiate means (Waller and was determined using the rapid titration method. Duncan, 1969).

Plant measurements Results and discussion NPK in plant samples and seeds determinations The chemical analysis was carried out on the dry samples Thirty one rhizosphere soil samples that were collected from obtained from the different treatments according to Hach different governorates with different crops were chosen for iso- et al. (1985). Nitrogen content in plant samples was deter- lation of N2-fixing Azotobacters (Table 2). Microscopic exam- mined by modified microKjeldahl method as described by ination and cultural characteristics of the bacterial isolates A.O.A.C. (1990). Phosphorus and potassium were determined revealed that they could be placed in two groups according using the method described by Murphy and Riley (1962) and to their morphological futures. Sixteen bacterial isolates were Cottenie et al. (1982) respectively. placed in group (1) spherical, gram negative, motile, produced Nitrogen content in the seeds was determined by modified diffusible dye light visible yellow-green pigments (i. e Azo1, 3, microKjeldahl method as described by A.O.A.C. (1990). Total 7, 8, 12, 16, 17, 18, 20, 21, 22, 24, 26, 28, 30 and 31). Fifteen bacterial isolates in group (2) large ovoid cell occur in pairs proteins (%) = N (%) · 6.25 (James, 1995). and some form cyst, produced non-diffusible brown pigment, Fresh and dry weight, plant height and number of branches) active motile and gram negative (i.e. Azo2, 4, 5, 6, 9, 10, 11, 13, 14, 15, 19, 23, 25, 27 and 29). From data recorded in Tables The total Fresh and dry weight of whale plant was recorded in 2 and 3 a significant differences in the potential of nitrogen fix- g/plant. Plants were dried by oven at 70 C until reached to a ation of the isolates were detected. Isolates Azo.9 and Azo.23 constant weight (Black et al., 1965). Measuring of Plant height obtained from the rhizosphere of Alfa alfa and Wheat respec- by cm/plant and the number of branches per plant. tively were higher in fixed nitrogen being 200 ppm, 18.462 lmol C2H2/100 ml/24 h and 102 ppm, 6.511 lmol The microbiological determinations C2H2/100 ml/24 h, respectively followed by the moderate iso- Samples were subjected to the determination of densities of lates i.e., Azo.4 and Azo.5 which recorded 44.00 ppm, total microbial flora on Bunt and Rovira (Buntt and Rovira 5.940 lmol C2H2/100 ml/24 h and 41 ppm, 4.257 lmol C2H2/ (1955), with decimal plate count technique, Azotobacters 100 ml/24 h, respectively. Those isolates showed variable spp., on modified Ashby’s liquid N-deficient medium (Abdel- degrees of metabolic activities in phosphate solubilization. Iso- Malek and Ishac, 1968). Using Most Probable number lates Azo.4 and Azo.5 were recorded the higher activity being (MPN) technique. 55 and 38 ppm of phosphorus, plant growth promoting substances and salicylic acid activity, were detected in isolate Estimation of carbon dioxide evolution rates (CO2 evolution) in Azo.4 which recorded 16.4 and 18.81 cm in shoot and root the rhizosphere soil length respectively, comparing to the control which gave (7, 5 cm) respectively. The maximum siderophores’ production The rates of CO2 evolution were periodically determined at the black cumin growth stages in rhizosphere soils to evaluate was recorded in Azo.23, Azo.4 being 59, 35 lg/ml and the max- Improvement of productivity and quality of black cumin 99

Table 2 Biochemical activities of bacterial isolates. Zonal distribution Biochemical activities Isolate code Locality Standing N. ppm P. ppm Hormonal activity Production of plant Sh.L R.L Siderophores Salicylic acid Exopolymer (lg/ml, 410 nm) (lg/ml, 527 nm) (mg/ml) Sohag Wheat 16.00 18 10.06 7.34 À 2.2 1.66 Azo.1 Sohag Wheat 33.00 21 8.4 5.13 17.6 À 1.87 Azo.2 Sohag Bean 32.00 13 12.8 9.26 30 À 3.42 Azo.3 Sahl El-Ttina Wheat 44.00 55 16.4 18.81 35.0 7.4 1.79 Azo.4 Wadi El-Natrun Wheat 41.00 38 15.5 18 29.3 À 1.65 Azo.5 Sohag Alfalfa 14.00 15 11.8 8.61 13.0 2 1.62 Azo.6 Sidi-Barani Onion 20.00 17 10.3 7.89 11.3 4.7 1.42 Azo.7 Sidi-Barani Wheat 36.00 17 11.5 9.41 ÀÀ1.30 Azo.8 Maruit Alfalfa 102.00 22 13.5 11.56 ÀÀ7.49 Azo.9 Kom aumbo Dates 24.00 18 11.13 9.21 22.2 À 1.22 Azo.10 El-Maghara Garger 21.60 12 9.84 6.14 ÀÀ1.12 Azo.11 El-Maghara Onion 20.00 11 12.71 10.1 18.0 À 1.10 Azo.12 El-Maghara Wheat 15.90 14 13. 4 11.83 ÀÀ1.40 Azo.13 Ras Sudr Wheat 20.03 6 10.31 8.61 ÀÀ1.03 Azo.14 Ras Sudr Tomato 20.14 8 8.9 7.12 12.5 À 1.14 Azo.15 Ras Sudr Onion 11.11 11 10.64 8.38 20.5 À 1.11 Azo.16 Sohag Wheat 19.50 15 11.2 9.3 ÀÀ1.50 Azo.17 Sohag Bean 30.30 13 7.84 5.54 20.0 À 1.30 Azo.18 Sohag Grasses 35.00 8 11.83 8.17 ÀÀ1.80 Azo.19 Sohag Grasses 33.00 7 12.4 8.13 12.0 À 1.70 Azo.20 Sohag Alfalfa 32.00 12 9.78 7.96 ÀÀ1.20 Azo.21 Toshka Onion 11.30 13 11.4 8.54 ÀÀ1.30 Azo.22 El-quntra south Wheat 200.00 24 12.18 9.45 59.0 À 8.15 Azo.23 El-Maghara Alfalfa 16.00 11 13.4 11.81 15.6 À 1.10 Azo.24 Kom aumbo Dates 33.00 11 7.5 5 ÀÀ1.11 Azo.25 Kom aumbo Garger 32.00 14 8 6 19.0 À 1.44 Azo.26 Siwa Onion 44.00 17 11.2 8 ÀÀ1.70 Azo.27 Siwa Wheat 41.00 9 9 10.6 18.9 À 1.90 Azo.28 Ras Sudr Wheat 14.00 12 7.5 9.5 22.0 À 1.20 Azo.29 Ras Sudr Tomato 20.00 13 6.4 8 ÀÀ1.30 Azo.30 Ras Sudr Onion 36.00 16 8.1 10 12.0 À 1.66 Azo.31 Terms in bold mean isolates were selected.

imum production of salicylic acid recorded in Azo.4 being Azo.5, Azo.9 and Azo.23 were identified as Azotobacter 7.4 lg/ml. chroococcum. Exhibiting high in the biochemical activities, these isolates were characterized and identified. The four isolates were iden- Characterization and identification of N2-fixing Azotobacters isolates tified according to Krieg and Holt (1984) and Holt et al. (1994). Table 3 shows the of Azotobacter isolates. Based on the above characters, this isolates could be classified as Azoto- The following characteristics were determined: bacter chroococcum and could be differentiated from the other Azotobacter spp. The most active Azotobacter isolates in EPS Morphological characteristics production were recorded in the isolates Azo.23, Azo.9, Azo.4 and Azo.5 being 8.2, 7.5, 1.8 and 1.7 mg/ml. The maxi- According to the morphological characteristics and fixed nitro- mum siderophores’ production was recorded in isolates gen fixing ability 31 bacterial isolates were most likely belong- Azo.23, Azo.4 being 59 and 35 lg/ml and the maximum pro- ing to the genus Azotobacter chroococcum according to duction of salicylic acid recorded in isolate(4) being 7.4 lg/ Bergey’s manual of systematic bacteriology (Yao and New, ml, Table 2. Our results were in line with some investigators, 1984). and they recorded that siderophores are low-molecular-weight compounds that are secreted by microorganisms to chelate Physiological characteristics iron from the environment (Hofte, 1993) who stated that their modes of action in suppression of disease were thought to be

Signiﬁcant differences in the potential of N2-ase activity of solely based on competition for iron with the pathogen these isolates were also detected. Using the identiﬁcation crite- (Duijff et al., 1999). Interestingly, siderophores can induce sys- ria described by Thompson and Skerman (1979), according to temic resistance (ISR) (Leeman et al., 1996). Salicylic acid is the aforementioned references four isolates namely Azo.4, also a plant hormone and is implicated in the induction of sys- 100 S.M. Abdel-Aziez et al.

temic acquired resistance in plants (Enyedi et al., 1992). How- ever, the morphological, cultural and physiological characteristics indicated that those were most likely belonging to the g/100 ml) l

ABA ( genus Azotobacter. Among the tested bacterial isolates namely Azo.4 and Azo.5 obtained from the rhizosphere of wheat were proven to be the best isolates in phosphate solubilizing and hormonal productivity and the isolates namely Azo.23 and g/100 ml) l

IAA ( Azo.9 were the best isolates in nitrogen ﬁxation activity. There- fore, the most effective Azo.4, Azo.5, Azo.9 and Azo.23 were selected for further study. 3 The biochemical characteristics of the selected Azotobacter sp. GA (mg/100 ml)

Production of plant growth promoting substances The ability of the selected effective isolates to produce growth g/100 ml) l

Cytokinin ( promoting substances was tested qualitative using the bioassay test in the culture media after 7 days at 28 C revealed that azotobacter isolates have the ability to produce and release cytokinins as measured by radish cotyledons bioassay, and the isolates gave the highest amount of releasing cytokinins as compared to other different tested isolates. Generally, all

Indole production (ppm) the obtained Azotobacter isolates can produce and release cytokinin by the following order: Azo.4 > Azo.5 > A- zo.9 > Azo.23. These results are in agreement with Holland (1997b). Azotobacter isolates have the ability to produce and release gibberellins as measured by radish sorghum bioassay,

% of Increases Gibberellins and Azotobacter isolates gave the highest amount of releasing gibberellins as compared to other different tested isolates. Generally, all obtained Azotobacter isolates can produce and release gibberellins by the following order Azo.9 > A- zo.4 > Azo.23 > Azo.5. The most active isolates were Azo.4

Qualitative% of Increases cytokinin Quantitative being 65.8 ppm followed by isolate Azo.5 being 39.8 ppm followed by Azo.23 being 30.2 ppm and Azo.9 being 18.2 ppm. isolates. On the other hand the selected effective Azotobacter isolates were tested for producing the phytohormones quantitatively Pectin lyase(PL) using high performance liquid chromatography (HPLC). Data in Table 3 revealed that Azo.4 produced the highest amount of Azotobacter cytokinin and indole acetic acid being 2296.7, 41.25 lg/100 ml.

The highest amount of gibberellic acid GA3 and abscisic acid -ﬁxers

2 was recorded by Azo.9 being 71.51 mg/100 ml and 6.19 lg/ 100 ml. Polygalacturonase (PG)

Enzymatic production Some enzymatic activities of isolate Azo.23 were high in nitro- Enzyme productionCellulase (Cx) Hormonal activity genase and cellulase production followed by isolates Azo.9, Azo.4 and Azo.5. Isolate Azo.5 was high in polygalacturonase followed in descending order by isolates Azo.4, Azo.9, Azo.23, and Azo.23, Azo.9 were the less active in pectin lyase. However the azotobacter isolate Azo.23 recorded the highest of all Azo- /100 ml/24 h) 2

H tobacter isolates in ﬁxing atmospheric nitrogen in selected 2 effective bacterial isolates (18.46 lmol/ml) siderophores’ production (59.0 lg/ml) cellulase production, exopolymer produc- mol C l

( tion (8.15 mg/ml). Isolate Azo.4 characterized by highest phosphate solubilization (55 ppm), indole acetic acid (65.8 ppm), gibberellins (69.41 mg/100 ml), cytokinin production (156.65 mg/100 ml), and Salicylic acid production Biochemical and microbial activities for selected N (6.19 lg/100 ml) pectin lyase compared with others. Isolate

5923 4.257 6.511 18.462 ++ + +++ +++ ++Azo ++.9 revealed +++ + 636 + the 200 highest 370 37.50 in 50.00 abscisic 39.8 75.00 acid 30.2 18.2 hormonal. 239.8 597.2 1.59Azo 634.2.5 6.95 1.36 71.51 3.60 0.809 0.583 3.12 6.19 .4 5.940 ++ ++ ++ 679 68.75 65.8 2296.7 69.41 41.25 1.35 recorded the highest isolate in polygalacturonase, pectin lyase Table 3 Azotobacter isolate Nitrogenase activity(ARA) Azo Azo. Azo. Azo. Table 3. mrvmn fpoutvt n ult fbakcmn101 cumin black of quality and productivity of Improvement

Table 4 Total microbial counts (T.MC), CO2 evolution and Densities of Azo. in the rhizosphere of black cumin (Nigella sativa) as affected by inoculation with Azotobacter chroococcum and/or inorganic N-fertilization.

Vegetating stage (60 days) Flowering stage (150 days) Fruting stage (210days) No ¼N ½N ¾N 1N No ¼N ½N ¾N 1N No ¼N ½N ¾N 1N Plant stage cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g cfu/g 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 .x10 .x10 .x10 .x10 .x10 .x10 .x10 .x10 .x10 .x10 .x10 .x10 .x10 .x10 .x10 Azo Azo Azo Azo Azo Azo Azo Azo Azo Azo Azo Azo Azo Azo Azo /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. /100g dry soil/24hr. T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 T.M.Cx10 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 measurements CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO

Uninoculated 20 1.0 8 30 1.3 9 35 1.4 9 40 1.5 20 45 1.9 24 35 2.6 30 80 3.1 35 70 3.5 37 40 3.7 40 70 4.7 29 33 1.5 20 35 3.5 28 40 2.2 24 45 3.7 22 35 2.2 21 (control)

Azo.4 40 2.1 23 45 2.3 36 48 2.3 30 50 2.5 30 64 2.6 35 102 4.2 51 148 4.2 55 121 5.1 48 50 4.9 58 130 5.5 59 57 2.3 43 43 3.8 49 49 3.9 39 44 3.5 41 50 3.5 40

Azo.5 45 2.3 30 50 2.4 32 40 2.5 33 55 3.1 35 80 3.5 40 105 4.3 54 120 4.4 65 120 4.9 59 55 4.6 69 160 4.7 72 49 2.4 42 58 3.6 55 56 4.1 46 45 4.2 35 44 3.7 44

Azo.9 30 2.4 39 45 2.4 35 70 2.6 32 80 2.9 33 75 3.2 35 100 3.7 45 164 4.7 59 150 5.5 67 80 5.7 66 150 4.9 69 49 2.6 42 51 3.6 47 55 4.2 41 56 4.3 36 68 3.3 45

Azo.23 49 2.4 29 50 2.5 30 45 2.6 37 60 3.2 33 54 3.1 35 110 3.9 49 110 5.7 57 130 5.3 69 60 4.9 68 140 4.7 66 50 2.5 39 62 4.1 48 63 3.9 50 70 4.1 40 74 4.1 40

Mixed 60 4.1 40 90 4.7 44 111 4.9 47 71 4.8 45 88 4.5 60 190 7.3 70 214 8.1 77 249 9.9 97 71 9.7 79 220 8.7 75 70 5.9 49 89 6.1 65 120 8.3 73 77 7.4 54 89 7.7 59 inoculation 5 4 Initial total microbial count : 28x10 cells/g dry soil Initial CO2 evolution:0.5mg Initial total azotobacters density : 9x10 CO2/100g dry soil/24hr. cells/g dry soil. Factors: Factors: Factors: stage of plant growth = 0.363 stage of plant growth = 0.029 stage of plant growth = 0.294

Biofertilizers = 0.513 Biofertilizers= 0.041 Biofertilizers= 0.416

N-Fertilizrs = 0.469 N-Fertilizrs = 0.038 N-Fertilizrs = 0.379

Interaction stage of plant growth x Biofertilizers Interaction stage of plant growth x Biofertilizers Interaction stage of plant growth x Biofertilizers x N-Fertilizers =0.316 x N-Fertilizers =0.157 x N-Fertilizers=1.600 102 S.M. Abdel-Aziez et al.

Table 5 Effect of inoculation with Azotobacter chroococcum and/or inorganic N-fertilization on the plant height and Dry weight of black cumin (Nigella sativa) plant at different growth stages.

Plant height(cm) and Dry weight(g/plant) Vegetating stage (60 days) Flowering stage (150 days) Fruting stage (210days)

Plant stage No ¼N ½N ¾N 1N No ¼N ½N ¾N 1N No ¼N ½N ¾N 1N (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (cm) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) (g/plant) Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Dry weight Plant height Plant height Plant height Plant height Plant height Plant height Plant height Plant height Plant height Plant height Plant height Plant height Plant height Plant height Plant height Measurements Measurements

Uninoculated (control) 6 2.38 8 2.99 9 3.41 10.5 4.39 11 5.0 17 7.53 18 9.61 20 15.81 22 17.50 23 19.11 23 11.75 25 18.00 28 23.0 32 25.22 34 26.17

9 6.71 12 9.60 12.5 12.66 13 13.68 16 14.70 26 20.68 27 30.69 27.5 42.67 31 42.91 32 45.86 28 27.75 32 38.72 35 49.75 38 48.91 39 52.94 Azo.4

8.5 5.70 11 9.71 13 11.70 14 13.73 15 14.79 26 17.71 26.5 30.76 29 42.89 29.5 42.79 31 44.77 30 38.10 34 40.89 36 43.74 37 49.75 40 53.30 Azo.5

9 5.69 10 8.76 12 11.68 13 12.79 14.5 13.69 25 16.70 26 29.70 27 39.81 27 42.82 31 43.89 34 27.32 36 30.61 37 43.30 38 48.15 40 52.07 Azo.9

8 6.76 10 9.74 12 12.69 13.5 12.74 14 14.76 26 17.87 26.5 32.83 27 41.74 29 43.89 30 46.85 29 30.35 35 33.48 36 41.49 38 43.55 41 53.44 Azo.23

Mixed 9 8.12 15 14.42 16 15.49 18 14.55 21 15.50 30 27.56 31 40.61 34 55.83 33 54.81 33 54.88 38 35.18 40 45.0 45 63.52 43 61.20 44 60.14 inoculation Factors: Plant height Factors: Dry weight stage of plant growth)= 0.294 stage of plant growth = 0.053 Biofertilizers= 0.415 Biofertilizers = 0.076 N-Fertilizrs = 0.379 N-Fertilizrs= 0.069 Interaction stage of plant growth x Biofertilizers x N-Fertilizers = 1.600 Interaction stage of plant growth x Biofertilizers x N-Fertilizers =0.293

Effect of Azo. chroococcum and or inorganic nitrogen inoculation with biofertilizers, plant growth stages and N-min- fertilization on the microbial colonization and CO2 evolution in eral fertilization. Inoculation with azotobacters markedly rhizosphere of black cumin (Nigella sativa) plants increased total microbial densities in black cumin rhizosphere. Supplementation with the half dose of inorganic N-fertilizers gave an additional increase in total microbial counts in the rhi- Microbial colonization zosphere of black cumin. Obtained data indicate that the high- Results recorded in Table 4 reveal that total microbial flora est numbers at the flowering stage of black cumin plants and azotobacters densities were significantly affected by inoculated with a mixture of Azo. chroococcum and received

Table 6 Effect of inoculation with Azotobacter chroococcum and/or inorganic N-fertilization on number of branches and weight of 100 seeds of black cumin (Nigella sativa) in the fruiting stag (after 210 days of growth). Treatment Number of branches/plant No 1/4N N1/2 N3/4 1N Uninoculated 7 14 14 21 25 Azo.4 14 28 35 35 42 Azo.5 21 23 28 28 35 Azo.9 21 28 28 35 42 Azo.23 19 28 35 36 40 Mixed inoculation 28 42 63 56 56 Weight of 100 seeds (g) No 1/4N 1/2N 3/4N 1N Uninoculated 0.16 0.20 0.22 0.24 0.25 Azo.4 0.25 0.27 0.27 0.29 0.30 Azo.5 0.23 0.26 0.27 0.25 0.27 Azo.9 0.28 0.28 0.31 0.25 0.26 Azo.23 0.22 0.24 0.24 0.27 0.31 Mixed inoculation 0.31 0.34 0.35 0.32 0.36 Number of branches/plant Weight of 100 seeds (g) Factors: Biofertilizers = 0.730 Factors: Biofertilizers = 0.007 N-Fertilizers = 0.667 N-Fertilizers = 0.007 Interaction biofertilizers · N-Fertilizers = 2.309 Interaction Biofertilizers · N-Fertilizers = 0.126 Improvement of productivity and quality of black cumin 103 a half dose of mineral N-fertilizers. Total microbial densities in has a stimulatory effect on the growth of a symbiotic N2-fixers rhizosphere of black cumin plants inoculated with Azotobacter in rhizosphere of the tested plants. This is in accordance with alone, received full dose of N-fertilizer at the flowering stage, several reports which concluded that the form and rate of N- were in the second order after plants inoculated with a mixture fertilizers may be limiting factors and exhibited a negative of biofertilizers. The CO2 evolution was significantly increased effect on the development of N2-fixers in various ecosystems in black cumin rhizosphere to reach its maximal rate at flower- Van Berkum and Neyra, 1979 while heavy doses of N-fertiliz- ing stage then, decreased thereafter at the fruiting stage. The ers inhibited N2-fixation (Pedersn et al., 1978). The effect of highest rates of CO2 evolution were obtained in rhizosphere inoculation with Azo. chroococcum on the total bacterial of black cumin plants inoculated with a mixture of Azo. chroo- counts and azotobacter counts could be attributed to the net coccum and received a half dose of inorganic N-fertilizer at the effect of plant – microorganism and soil treatments, interac- flowering stage (being 9.9 mg CO2/100 g dry rhizosphere soil/ tion on each of the three factors could be either positive, neu- 24 h). This was followed by the amount of CO2 emitted from tral or negative. However, that black cumin plant roots has the rhizosphere of plants inoculated with a mixture of Azoto- more positive effect than that of the plant roots. bacter chroococcum and received 3/4 dose of N-fertilizer at Among the several species, Azotobacter chroococcum hap- the same stages of growth (being 9.7 mg CO2 /100 g dry rhizo- pens to be the dominant inhabitant of the rhizosphere. There sphere soil/24 h). It was noticed that CO2 evoluted from the have been many reports the beneficial effects of Azot. chroo- rhizosphere of black cumin plants inoculated with the mixture coccum on growth and yield of various agriculturally impor- significantly overcame the amount emitted in rhizosphere of tant crops. It benefits plants in multiple ways, which includes those inoculated with, a single strain of Azo. chroococcum.It 1. ability to produce ammonia, vitamins and growth sub- was noticed that the vegetative stage gave the lowest levels of stances that enhance seed germination; 2. production of indole CO2 evolution. Soil CO2 evolution as an indication of micro- acetic acid and other auxins which enhance root growth and bial activity in plants rhizosphere soil, it was found that it aid in nutrient absorption; 3. inhibition of phytopathogenic always coincides with the densities of different microorganisms fungi through antifungal substances. 4. Production of sidero- + enumerated in rhizosphere, which simultaneously increased by phores which solubilize Fe3 and suppress plant pathogens inoculation with the mixture of biofertilizers. Evolution of through iron deprivation (Dubey et al., 2012). CO2 rates was mostly in line with total microbial densities determined in the rhizosphere. Inoculation with azotobacters Plant growth performance alone or in mixture influenced the growth and multiplication of azotobacters in rhizosphere giving the highest counts in Plant height and dry weight comparison with other treatments (Table 4). It was found that Results presented in Table 5 point out that plant height was azotobacters numbers were higher in the rhizosphere inocu- significantly affected by inoculation with the mixture of Azo. lated by the mixture followed by the inoculation with Azo. chroococcum and mineral N-fertilizer. The highest heights of chroococcum alone. Maximum numbers of azotobacters were plant, were recorded when black cumin plants were inoculated observed at the flowering stage in rhizosphere plants received with Azo. chroococcum alone or a mixture of bacterial strains a half dose of the recommended N-fertilizer. Low concentra- in ascending order in the presence of half dose of N-fertilizer. tion of inorganic N-fertilizer (half the recommended dose) The plant height measured in a mixture of bacteria plus half

Table 7 Effect of inoculation with Azotobacter chroococcum and/or inorganic N-fertilization on oil yield and oil constituents of black cumin (Nigella sativa) in the fruiting stage (after 210 days of growth). No 1/4N 1/2N 3/4N 1N Fixed oil % Uninoculated 11 18 18 19.7 20 Azo.4 24 26 26 30 32 Azo.5 25 28.5 27 25.5 20.7 Azo.9 22 23.3 26.3 30 33 Azo.23 25.7 28.5 30.8 31 31.5 Mixed inoculation 28 33.3 37.5 36 33.8 Oil constituents Lenolenic acid Palmitic acid 0 bio + 1/2N 65.14 15.35 Mixture + 1/2N 77.85 14.98 0 bio + 3/4N 70.69 18.85 Mixture + 3/4N 75.67 14.63 Full recommended dose 71.5 21.87 Mixture + 1N 73.02 21.24 Fixed oil% Factors: Biofertilizers = 0.598 N-Fertilizers = 0.546 Interaction Biofertilizers · N-Fertilizers = 1.337 104 Table 8 Effect of inoculation with Azotobacter chroococcum and/or inorganic N-fertilization on NPK in plants of black cumin (Nigella sativa). Treatment Total nitrogen% Vegetating Flowering stage Fruiting stage stage (60 days) (150 days) (210 days) No 1/4N N1/2 N3/4 1N No 1/4N N1/2 N3/4 1N No 1/4N N1/2 N3/4 1N Uninoculated 0.92 0.94 0.98 1.2 1.29 1.25 1.40 1.50 1.70 1.78 1.11 1.15 1.19 1.20 1.22 Azo.4 1.60 1.62 1.71 1.75 1.78 2.20 2.36 2.36 2.85 3.44 1.60 1.72 1.93 2.31 2.50 Azo.5 1.50 1.59 1.70 1.76 1.79 1.80 1.85 2.75 2.78 3.43 1.60 1.66 1.90 2.30 2.55 Azo.9 1.85 1.85 1.90 1.92 1.98 2.19 2.43 2.80 3.20 3.51 1.67 1.79 2.22 2.60 2.60 Azo.23 1.69 1.71 1.80 1.80 1.89 2.26 2.98 3.90 3.75 3.72 1.78 1.89 2.60 2.75 2.50 Mixed inoculation 1.90 2.33 2.58 2.60 2.68 2.30 3.57 4.25 3.90 3.99 1.90 2.40 3.20 3.00 2.90 Total phosphorus % Vegetating stage Flowering stage Fruiting stage (60 days) (150 days) (210 days) No 1/4N N1/2 N3/4 1N No 1/4N N1/2 N3/4 1N No 1/4N N1/2 N3/4 1N Uninoculated 0.28 0.33 0.40 0.42 0.45 0.36 0.38 0.43 0.46 0.50 0.33 0.37 0.41 0.44 0.48 Azo.4 0.33 0.40 0.44 0.48 0.52 0.40 0.42 0.50 0.54 0.55 0.35 0.40 0.48 0.49 0.52 Azo.5 0.32 0.38 0.41 0.45 0.50 0.39 0.41 0.48 0.53 0.53 0.37 0.39 0.47 0.51 0.52 Azo.9 0.30 0.37 0.40 0.43 0.47 0.39 0.40 0.45 0.52 0.53 0.36 0.38 0.48 0.50 0.53 Azo.23 0.30 0.37 0.41 0.44 0.49 0.40 0.42 0.49 0.51 0.52 0.38 0.39 0.47 0.49 0.50 Mixed inoculation 0.40 0.41 0.46 0.48 0.54 0.42 0.44 0.59 0.56 0.58 0.41 0.42 0.57 0.54 0.56 Total potassium % Vegetating Flowering Fruiting stage stage (60 days) stage (150 days) (210 days) No 1/4N N1/2 N3/4 1N No 1/4N N1/2 N3/4 1N No 1/4N N1/2 N3/4 1N Uninoculated 0.11 0.14 0.19 0.30 0.34 0.31 0.46 0.54 0.55 0.59 0.12 0.16 0.21 0.32 0.36 Azo.4 0.15 0.24 0.26 0.40 0.45 0.33 1.18 1.21 1.22 2.13 0.27 0.38 0.42 0.44 0.50 Azo.5 0.13 0.23 0.23 0.39 0.42 0.35 1.18 1.20 1.21 1.83 0.24 0.39 0.44 0.40 0.48 Azo.9 0.14 0.24 0.24 0.40 0.41 0.35 1.17 1.19 1.58 1.79 0.28 0.43 0.44 0.45 0.46 Azo.23 0.13 0.21 0.24 0.31 0.39 0.39 1.19 1.21 1.22 1.70 0.26 0.37 0.49 0.54 0.54 Mixed inoculation 0.17 0.39 0.47 0.48 0.50 0.52 1.29 2.79 2.67 2.18 0.29 0.47 0.59 0.68 0.68 Total nitrogen % Total phosphorus % Total potassium % Factors: stage of plant growth = 0.017 Factors: stage of plant growth = 0.002 Factors: stage of plant growth = 0.002 Biofertilizers = 0.024 Biofertilizers = 0.004 Biofertilizers = 0.004 ..AdlAize al. et Abdel-Aziez S.M. N-Fertilizers = 0.022 Factor for N-Fertilizers (LSD 0.05) = 0.003 N-Fertilizers = 0.003 Interaction stage of plant growth Interaction stage of plant growth Interaction stage of plant · Biofertilizers · N-Fertilizers = 0.022 · Biofertilizers · N-Fertilizers = 0.210 growth · Biofertilizers · N-Fertilizers = 0.210 Improvement of productivity and quality of black cumin 105 dose of mineral fertilizer was 45, respectively at the fruiting with findings mentioned by El-Sayed (2006) who found that stage in comparison with control (uninoculated), which gave number of branch/plant of black cumin plants was increased only 34 cm plant height. The minimal height of plants was in by application of biofertilizer in the combination of uninocu- the absence of N – fertilizer in treatments without inoculation. lated treatments. Moreover organic manure combined with No significant differences were recorded between plants biofertilizers significantly increased plant growth and fruit received full or half dose of inorganic N-fertilizer. The increase yield of Carum carvi plant compared with the control. in plant heights of plants inoculated with Azo. chroococcum The weight values of 100 seeds were significantly influenced may be due to the synthesizing of some plant growth promot- by mixed inoculation in the presence of full dose of N-fertilizer ing substances. The growth of plants expressed as dry weights and the highest values of black cumin plants in this case, being was significantly affected by inoculation with azotobacter and 0.36 g at the harvest stage. Inoculation with the mixture of application of inorganic nitrogenous fertilizer. It was observed Azo. chroococcum significantly overcame the control. Bacterial that maximal dry weights of plants (being 63.52 g/plant), inoculation was significantly increased than uninoculated. respectively, were recorded in treatments mixed – inoculant Moreover, mixed inoculation highly significantly than other with a symbiotic bacteria in the presence of half dose of inor- bacterial treatment. However, the application of N2-fixer ganic N-fertilizer at the fruiting stage. It was also found that improved oil content of plants supplemented with inorganic inoculation with Azo. chroococcum gave slightly less fresh N-fertilizer. Inoculation with the mixture and application of and dry weights of black cumin plants, in comparison with the half dose of N-fertilizer induced the highest oil percentage, inoculation with the mixture. Uninoculated plants gave dry being 37.5% at the fruiting stage (harvesting), giving about 2 weights not more than 26.17 g/plant, respectively at the fruit- fold of oil yield at the same N-fertilizer dose over the uninoc- ing stage and supplemented with full dose N-fertilizer. It has ulated plants. It could be concluded that impact was recorded been stated that inoculation could be expected to improve between using either full or half doses of inorganic N-fertilizer, plant growth mainly if the bacteria were established and grown and also between single or mixed inoculation treatment. well in the rhizosphere. However, Gaskins et al. (1985) sug- The application of organic manure combined with biofertil- gested that N2-fixers increased the production of plant growth izers (Nitrobein plus Phosphorene) significantly increased lim- regulators, which stimulate plant growth. Kostove et al. (1991) onene and carvone content in Carum curvi El-Latif (2002). reported that organic manure and interaction between N2-fix- Moreover, inoculation with diazotrophs and phosphate dis- ers and phosphobacteria showed significant increase in tomato solving bacteria was significantly enhanced the productivity plant growth and fresh and dry weights comparing with each of Cuminum cyminum and Pimpinella anisum plants and its application. oil yield content by Abdel-Aziez (2006).

Branching rate, weight of 100 seeds and fixed oil content Oil constituents It is obvious from data in Table 6 that the highest rate of Data in Table 7 show that the percentage of the active material branching was obtained in black cumin plants inoculated with i.e. Linolenic acid in case of the bacterization with the mixture the mixture of Azo. chroococcum strains in the presence of half gave the maximum value 77.85% in descending order com- dose of inorganic N-fertilizer, being 63 branching rate / plant pared to the control treatment which gave 65.14%. However, at the fruiting stage. Plants inoculated with Azo. chroococcum the inoculation with the mixture in the presence of half dose only gave less figures of branching. However, uninoculated of N fertilizer increased the oil constituents of black cumin plants gave the lowest rate of branching, being 14, 21 and 25 two times comparing to the control. branching rate/plant in treatments supplemented with 1/2, 3/ The increment in some measurement found in this study 4 and full dose of inorganic N-fertilizer or without any N-addi- could be attributed to the inoculation of mixed culture of tion, respectively at the fruiting stage. No significant differ- Azo. Chroococcum which fix atmospheric nitrogen, produce ences were recorded between plants received full or 3/4 dose many enzymes included pectinases, cellulases, produces some of mineral N-fertilizer. The obtained results were in agreement siderophores, salicylic acid, plant growth promoting sub-

Table 9 Effect of inoculation with Azotobacter chroococcum combined and/or inorganic N-fertilization on NPK. Treatment N0 1/4N N 1/2 N3/4 1N N Protein P K N Protein P K N Protein P K N Protein P K N Protein P K Uninoculated 1.03 6.44 0.22 0.38 1.45 9.06 0.24 0.39 2.13 13.31 0.24 0.51 3.25 20.31 0.27 0.53 3.55 22.19 0.28 0.60 Azo.4 2.60 16.25 0.34 0.63 3.63 22.69 0.36 0.71 3.64 22.75 0.36 0.92 4.29 26.81 0.39 0.94 4.75 29.69 0.42 0.95 Azo.5 2.42 15.13 0.31 0.62 2.64 16.50 0.33 0.70 3.66 22.88 0.34 0.80 3.67 22.94 0.35 0.90 4.69 29.31 0.39 0.92 Azo.9 3.20 20.00 0.29 0.62 3.49 21.81 0.30 0.70 3.86 24.13 0.32 0.78 4.73 29.56 0.33 0.89 5.76 36.00 0.40 0.90 Azo.23 3.65 22.81 0.29 0.60 3.70 23.12 0.30 0.69 4.75 29.69 0.31 0.78 4.79 29.94 0.34 0.90 5.82 36.37 0.39 0.90 Mixed inoculation 3.70 23.12 0.36 0.69 4.89 30.56 0.43 0.79 5.61 35.06 0.46 0.95 5.60 35.00 0.48 0.95 5.60 35.00 0.49 0.96 Total nitrogen % Total phosphorus % Total potassium % Factors: Biofertilizers = 0.007 Factors: Biofertilizers = 2.324 Factors:Biofertilizers = 0.007 N-Fertilizers = 0.006 N-Fertilizers = 2.122 N-Fertilizers = 0.006 Interaction Biofertilizers · N-Fertilizers = 0.126 Interaction Biofertilizers · N-Fertilizers = 5.197 Interaction Biofertilizers · N-Fertilizers = 0.126 106 S.M. Abdel-Aziez et al.

in the degradation of the insoluble nutrient minerals. This Table 10 Antimicrobial action of the essential oil of black ﬁnding is in harmony with that obtained by Girgis, 2006 and cumin plants against some pathogenic bacteria. Girgis et al., 2008). The use of plant growth promoting rhizo- Pathogenic bacteria Zone diameter of inhibition (mm) bacteria PGPR including N2-faxed Azotobacter as biofertiliz- Listeria monocytogenes 12 ers was suggested as a sustainable solution to improve plant Staphylococcus aures 10 nutrient and production (Vessey, 2003). Moreover, inoculation Salmonella sp. 12 by PGPR or with combined inoculation increase the bioavail- Shigella sp. 10 ability of P and K in the soils which may lead to increasing P E. coli 8 uptake and plant growth, was reported by many researchers (Lin et al., 2002; Sahin et al., 2004; Eweda et al., 2007.

The antibacterial effect of the essential oil of black cumin plant stances and solubilizing fixed phosphate, all these factors help- against pathogenic bacteria ing in increasing growth, productivity and oil content of black cumin crop cultivated in sandy soil. The crude fixed oil extracted from black cumin seeds has powerful antibacterial properties against diverse genera of some Chemical characteristics as affected by Azo. Chroococcum pathogenic bacteria. Significant growth inhibition was inoculants and/ or inorganic N-fertilization in black cumin recorded for, Listeria monocytogens followed by Salmonella sp., Staphylococcus aureus, Shigella sp. and Escherichia coli Nitrogen content in black cumin plant was considerably in descending order and the diameter of the inhibition zones increased by inoculation with N -fixer and supplementation 2 were 12, 12, 10, 10 and 8 ml, respectively Table 10. The former with inorganic nitrogen Table 8. Mixed inoculation with the results are in line with El-Sayed (2006) who studied the effect mixture of Azo. chroococcum slightly increased plant nitrogen of black cumin seed oil (N. sativa) for antimicrobial effective- content reaching maximum, being 4.25% in the case of using ness against different pathogenic microorganisms. These the half dose inorganic N-fertilization, in comparison with pathogens comprised three foodborne pathogens. Crude essen- 2.30% in the same inoculation treatment but without mineral tial oil of N. sativa showed a promising effect against some of N-fertilizer at the Flowering stage. Uninoculated treatments the tested organisms. Abou-Zied (2011) tested the effect of dif- gave the lowest N-contents of plants in the absence or presence ferent concentrations of different essential oils such as fennel, of half or full the dose of inorganic N-fertilizer. It is well black cumin and anise against different pathogenic microor- known that the biofertilizers are useful for recycling elements, ganism namely, (Streptococcus pneumonia, Yeast, Proteus reserving natural resources and for protection from increasing vulgaris, Shigella dysenteriae, Acinetobacter spp., Salmonella the pollution due to extensive use of mineral fertilizers (Girgis, typhi, Gemella spp., Klebsiella pneumonia, E. coli, Staphylococ- 2006). Also, they increase the amount of fixed nitrogen in the cus aureus, Pseudomonas aerugenosa and Staphylococcus plants. Bacterial inoculation was significantly increased than epidermis). It was found that the oil extracted from the seeds uninoculated. Mixture inoculation was highly significantly of plants have powerful antibacterial properties against these than other bacterial treatment. No difference was detected bac- diverse genus of bacteria. The influence was different and terial strains among isolates Azo.4, Azo.5 and Azo.9. Azo.23 depending on the bacterial strain. showed more effective than other strain. The inoculation mix- The inoculation with each Azo. chroococcum strain/or mixture of Azo. chroococcum enhanced plant phosphorus and ture of strains increased the effective material in seeds of black potassium percentage reaching its highest, (being 0.58%, cumin which has antibacterial effect on the pathogenic bacteria 0.68%) at the fruiting stage for plants received full dose of used in this study. N-fertilizer. Plants inoculation with Azo. chroococcum only or their mixture with half or full dose of N-fertilizer increased N, P and K% in plants over uninoculated plants (without References N-fertilizer). The difference in the nitrogen, phosphorus and potassium content in plant given half or full dose of inorganic A.O.A.C., 1990. Association of Official Agriculture Chemists, Pub- lished by Association of Official Agricultural Chemists, Washing- nitrogen are insignificant. These findings confirm in part with ton, DC, USA, 1008 p. those obtained by Kandeel and Sharaf (2003) who found that Abdel-Aziez, Samah M., 2006. Influence of biofertilizers on the inoculation of Majorana hortensis with Azo. chroococcum, productivity of active constituents of some medicinal plants. M.Sc. Bacillus. circulans and vesicular arbuscular mycorrhizae or Thesis Fac. Agric., Ain Shams Univ., Cairo, Egypt, 95 p. their mixture in the presence of full or half dose of recom- Abdel-Malek, Y., Ishac, Y.Z., 1968. Evaluation of methods used in mended rate of N, P and K fertilization gave higher content counting Azotobacter. J. Appl. Bact. 31, 269–275. of N, P, K in plant herb than the control. The chemical char- Abou-Zied, M.T., 2011. Microbial infection control in Zagazig acteristics or constituents of mineral contents and protein of university surgery hospitals. M.Sc. Thesis Botany Department black cumin seeds were significantly increased Table 9. Also ‘‘Microbiology’’ Faculty of Science, Zagazig University, Benha inoculated seeds with suspension of Azo. chroococcum either Branch, pp. 155–157. Abu-Al-Basal, Mariam A., 2009. In vitro and in vivo anti-microbial individually or with mixed of the seeds compared to uninocu- effects of Nigella sativa Linn. seed extracts against clinical isolates lated treatments. Rhizobacterial inoculating seeds to stimulate from skin wound infections. Am. J. Appl. Sci. 6 (8), 1440–1447. other rhizospheric microorganisms hence the enhancement of Allen, C., Gay, J., Simon-Buela, L., 1997. A regulatory locus, pehSR, plant growth could be attributed to the increased uptake of controls polygalacturonase production and other virulence func- nutrients from activated microorganisms. Liu et al. (2006), tions in Ralstonia solanacearum. Am. Phytopathol. Soc. 10 (9), reported that exopolysaccharides may play an important role 1045–1064. Improvement of productivity and quality of black cumin 107

Anderson, J.P.E., 1982. Soil respiration. In: Miller, A.L.R.H., Keeney, Hach, C.C., Brayton, S.V., Kapelove, A.B., 1985. Powerful kjeldahl D.R. (Eds.), Methods of Soil Analysis, part 2, second ed. ASA and nitrogen method using peroxy mono sulfuric acid. J. Agric. Food SSSA, Madison, Wisc, pp. 837–871. Chem. 33, 1117–1123. Bentley-Mowat, J.A., 1966. Activity of gibberellins A. to the Avena Hofte, M., 1993. Classes of microbial siderophores. In: Barton, L.L., first leaf. Bioassay and location of chromatography Ann-Bot. N.S., Hemming, B.C. (Eds.), Iron Chelation in Plants and Soil Micro- vol. 30, p. 165. organisms. Academic press, San Diego, Calif, pp. 3–26. Black, C.A., Evans, D.D., Ensminger, L.E., White, J.L., Clark, F.E., Holland, M.A., 1997b. Methylobacterium and plants. Recent Res. 1965. Methods of soil analysis part1.Chap. 18, water capacity. By Dev. Plant Physiol. 1, 207–213. Klute A., ASA Monograph series No. 9, Amer. Soc. Agron. Holt, J., Krieg, N., Sneath, P., Staley, J., Williams, S., 1994. Bergey’s Madison, Wisconson, USA. Manual of Determinative Bacteriology, ninth ed. Williams and British pharmacopoeia, 1968. Biological Assay of Active Constituent. Wilkins, Baltimore, pp. 77–135. The pharmaceutical press, London, pp. 484–486. Hosseinzadeh, H., Bazzaz, B.S.F., Haghi, M.M., 2007. Anti-bacterial Buntt, J.S., Rovira, A.O., 1955. Microbiological studies of some activity of total extracts and essential oil of Nigella sativa L. seeds subantarctic soil. J. Soil Sci. 6, 119–128. in Mice. Pharmacology 2, 429–435. Cordovilla, M.P., Berrido, S.I., Ligero, F., Lluch, C.L., 1999. Jackson, M.L., 1958. Soil Chemical Analysis. Prentice-Hall Inc., Rhizobium strain effects on the growth and nitrogen assimilation England, Cliffs, pp. 219–221. in Pisum sativum and Vicia faba L. plants. Plant. Sci. Limerick 140 Jacobs, M.B., Gerstein, M.J., 1960. Hand Book of Microbiology. In: (2), 127–136. De. Van (Ed.), Nostrand Co., Inc., New York, USA, pp. 139–202. Cottenie, A., Verloo, M., Kiekens, L., Velgh, G., Camerlynck, R., James, C.S., 1995. Analytical Chemistry of Foods. Blackie Academic and 1982. Chemical analysis of plant and soils. State Univ. Ghent professional publisher, an imprint of Chapman and Hall, 178 p. Belgium, 44–45. Jones, R.L., Varner, J.El., 1967. The Bioassay of Gibberellins Plant Dubey, R.C., Maheshwari, D.K., Kumar, Varun, Pandey, R.R., 2012. Berl., vol. 72, pp. 155–161. Growth enhancement of Sesamum indicum L. by rhizosphere- Kandeel, A.M., Sharaf, M.S., 2003. Productivity of Majorana hortensis competent Azotobacter chroococcum and its antagonistic activity L. plants as influenced by the interactions between mineral and against Macrophomina phaseolina. Arch. Phytopathol. Plant Pro- biological fertilization. J. Agric. Sci. Mansoura Univ. 28 (2), 1450– tect. 45 (4), 437–454. 1475. Duijff, B.J., Recorbet, G., Bakker, P.A.H.M., Loper, J.E., Lemanceau, Khoramdel, S., Kouchaki, Ali Reza, Nasiri Mahalati, Mahdi, Ghor- P., 1999. Microbial antagonism at the root level is involved in the bani Reza, Mahdi, 2008. Application effects of biofertilizers on the suppression of Fusarium wilt by the combination of nonpathogenic growth indices of black cumin (Nigella sativa) 8. Iran. J. Field Fusarium oxysporum Fo.47 and Pseudomonas putida WCS. 358. Crops Res. 6 (2), 285–294. Phytopathology 89, 1073–1079. Kostove, O., Rankov, V., Aranacova, G., Lynch, J.M., 1991. El-Latif, Abd, 2002. Effect of organic manure and biofertilizer on Decomposition of sawdust and bark treated with celluloses- Caraway plants. J. Agric. Sci. Mansoura Univ. 27 (5), 3463–3472. decomposing microorganisms. Biol. Fertil. Soil 11 (2), 105–110. El-Sayed, M.A.M., 2006. Effect of biofertilizers application on the Krieg, N.R., Holt, J.G., 1984. In Bergey’s Manual of Systemic productivity of Nigella sativa cultivated in desert sandy soils and Bacteriology Genus Azotobacter sp., vol. (II). Williams, Wilkins, efficiency of produced seeds against some pathogenic microorgan- Baltimore, USA, pp. 225–229. isms. Ph.D thesis, Fac. Agric., Benha. University, Egypt, 115 p. Leeman, M., Den Ouden, F.M., Van Pelt, J.A., Dirkx, F.P.M., Steijl, El-Shazly, M.M., 2003. Studies on some factors affecting productivity H., Bakker, P.A.H.M., Schippers, B., Den-Oden, F.M., Van-Pelt, of growth regulators by Azotobacter in desert soil. M.Sc. Thesis, J.A., 1996. Iron availability affects induction of systemic resistance Fac. Sci., Al-Azhar Univ., 35 p. to Fusarium wilt of radish by Pseudomonas fluorescens. Phytopa- Enyedi, A.J., Yalpani, N., Silverman, P., Raskin, I., 1992. Localiza- thology 86 (2), 149–155. tion, conjugation, and function of salicylic acid in tobacco during Lethman, D.S., 1968. A new cytokinin bioassay and naturally the hypersensitive reaction to tobacco mosaic virus. Proc. Natl. occurring cytokinin complex I. In: Wightman, F., Setterfield, G. Acad. Sci. USA 89, 2480–2484. (Eds.), Biochemistry and Physiology of Plant Growth Substances. Eweda, Wedad E., Selim, Sh.M., Mostafa, M.I., El-Fattah, Dalia A. Runge Press, Ottawa, pp. 19–35. Abd, 2007. Use of Bacillus circulans as bio- accelerator enriching Lin, Q.M., Rao, Z.H., Sun, Y.X., Yao, J., Xing, L.J., 2002. composted agricultural wastes E-identification and utilization of Identification and practical application of silicate-dissolving bacte- the microorganism for compost production. In: Proceedings of the ria. Agric. Sci. China 1, 81–85. 12.th Conference of the Microbiology. Organized by the Egyptian Liu, W., Xu, X., Wu, X., Yang, Q., Luo, Christie, P., 2006. Society of Applied Microbiology (ESAM), 18–20 March, Giza, Decomposition of silicate minerals by Bacillus mucilaginosus in Egypt, pp. 43–65. liquid culture. Environ. Geochem. Health 28, 133–140. Gaskins, M.H., Albrecht, S.L., Hhubbel, D.H., 1985. Rhizosphere Mali, G.V., Bodhankar, M.G., 2009. Antifungal and Phytohormone bacteria and their use to increase productivity; a review. Agric. Production Potential of Azotobacter chroococcum Isolates from Ecosyst. Environ. 12, 99–116. Groundnut (Arachis hypogea L.) Rhizosphere. India. Asian J. Exp. Girgis, M.G.Z., 2006. Response of wheat to inoculation with phos- Sci. 23 (1), 293–297. phate and potassium mobilizers and organic amendment. Annals Murphy, J., Riley, J.H., 1962. A modified single solution for the Agric. Sci., Ain Shams Univ. Cairo 51 (1), 85–100. determination of phosphate in natural waters. Annal Chem. Acta Girgis, M.G.Z., Khalil, Heba M.A., Sharaf, M.S., 2008. Evaluation of 27, 31–36. the rock phosphate and potassium solubilizing potential of Bacillus Odeniyi, O.A., Onilude, A.A., Ayodele, M.A., 2009. Production strains in vitro. Aust. J. Basic Appl. Sci. 2 (1), 68–81. characteristics and properties of cellulose/polygalacturonase by a Giti-Emtizi, Zahra Ethemadifar., Habibi, Mohammad H., 2004. Bacillus coagulans strain from a fermenting palm-fruit industrial Production of extra-cellular polymer in Azotobacter and biosorp- residue. Afr. J. Microbiol. Res. 3 (8), 407–417. tion of metal by exopolymer. Afr. J. Biotechnol. 3 (6), 330–333. Pedersn, W.L., Chakrabarty, K., Klucas, R.V., Vidaver, A.K., 1978. Goel, A.K., Laura, R.D.S., Pathak, G., Anuradha, G., Goel, A., 1999. Nitrogen fixation (acetylene reduction) associated with roots of Use of biofertilizers: potential, constraints and future strategies winter wheat and sorghum in Nebraska. Appl. Environ. Mirobiol. review. Int. J. Trop. Agric. 17, 1–18. 35 (1), 129–135. Goreja, W.G., 2003. Black Seed: Nature’s Miracle Remedy. Amazon Piper, C.S., 1950. Soil and Plant Analysis, first ed. Int. Publishers, New Herbs Press, New York, ISBN: 09742962-1-X, 54 p. York, pp. 47–79. 108 S.M. Abdel-Aziez et al.

Reeves, M., Pine, L., Neilands, J.B., Bullows, A., 1983. Absence of Tans-Kersten, J., Guan, Y., Allen, C., 1998. Ralstonia solanacearum siderophore activity in Legionella sp. Grown in iron deﬁcient pectin methylesterase is required for growth on methylated pectin media. J. Bacteriol. 154, 324–329. but not for bacterial wilt virulence. Appl. Environ. Microbiol. 64 Richards, L.A., 1954. Diagnoses and improvement of saline and (12), 4918–4923. alkaline soils, USA. Dept. Agric. Handbook No. 60, pp. 17–133. Thompson, J.P., Skerman, V.B.D., 1979. Systematic examination of Rizzolo, A.C., Baldo, Polesello, A., 1991. Application of high the bacterial strains. In: Azotobacteriaceae. The Taxonomy and performance liquid chromatography to the analysis of niacin and Ecology of the Aerobic Nitrogen-Fixing Bacteria. Academic press biotin in Italian almond cultivars. J. Chromatogr. 553, 1–2. inc., London, pp. 143–255. Sahin, F., Cakmakci, R., Kantar, F., 2004. Sugar beet and barley Van Berkum, P., Neyra, C.A., 1979. Nitrogenase activity in isolated

yields in relation to inoculation with N2-ﬁxing and phosphate sorghum roots. The effect of biocarbonate and inorganic nitrogen. solubilizing bacteria. Plant Soil 265, 123–129. Plant Physiol. 57, 533. Salman, M.T., Khan, R.A., Shukla, I., 2008. Anti-microbial activity of Vessey, J.K., 2003. Plant growth-promoting rhizobacteria as biofertil- Nigella sativa linn. Seed oil against multi-drug resistant bacteria izers. Plant Soil 255, 571–586. from clinical isolates. Nat. Prod. Rad. 7, 10–14. Waller, R.A., Duncan, B.D., 1969. A way for the symonetric multiple Sayed, A.V., Hossein, A.F., 2011. Investigation of biofertilizers comparison problem. Amer. Stat. Assoc. J. 3, 1485–1503. inﬂuence on quantity and quality characteristics in Nigella sativa Watanabe, F.S., Olsen, S.R., 1965. Test of an ascorbic acid method for

L.. J. Horticulture For. 3 (3), 88–92. determining phosphorus in water and NaHCO3 extracts from soil. Schleicher, P., Saleh, M., 2000. Black Cumin: The Magical Egyptian Soil Sci. Soc. Am. Proc. 29, 677–678. Herb for Allergies, Asthma, Skin Conditions and Immune Disor- Yao, T., New, P.B., 1984. Key to genera of family Azotobacteriaceae. ders. Healing Arts Press, Rochester, Vermont, pp. 31–85. In: Bergeys Manual of Systematic Bacteriology, vol. 1. Williams & Schollhorn, R., Burris, R.H., 1967. Acetylene as a competitive Wilkins, Baltimore, USA, pp. 225–229.

inhibitor of N2 ﬁxation. Proc. Natl. Acad. Sci. USA 58, 213–216. Zhang, W., Xue, Y., Ma, Y., Zhou, P., Ventosa, A., Grant, W.D., Silva, E.G., Borges, M.F., Medina, C., Piccoli, R.H., Schwan, R.F., 2007. Salinicoccus alkaliphilius sp. Nov., a novel alkaliphile and 2005. Pectinolytic enzymes secreted by yeasts from tropical fruits. moderate halophile from Bear soda lake in inner Mangolia. Femes Yeast Res. 5, 859–865. Autonomus Region, China. Int. J. Syst. Evol. Microbiol. 52, Snedecor, G.W., Cochran, W.G., 1982. Statistical Methods, 8th ed. 789–793. The Iowa State Univ., Press, Ames, Iowa, USA, 507 p..