Azotobacter: A Plant Growth Promoting Rhizobacteria a Biofertilizer for Wheat Crop

Vijay Kumar1*, Poonam2, Sapna Sharma3, Anjana4 and Minakshi Thakur5

1, 2, 3, 4 & 5 Department of Microbiology, Career Point University, Hamirpur (H.P.) 176041, INDIA

* Correspondence: E-mail: vijaybhatiadreams@gmail.com

DOI: http://dx.doi.org/10.33980/jbcc.2019.v05i02.006

(Received 22 Sep, 2019; Accepted 23 Nov, 2019; Published 14 Dec, 2019 )

ABSTRACT: In the present context, the best alternative of chemical fertilizer is necessary because of its adverse effects on the soil health. There are several alternatives available to enhance the soil fertility one of them is Azotobacter. It is a free-living N2- fixer diazotroph that has several beneficial effects on the crop growth and yield. It helps in synthesis of growth regulating substances like Auxins, Cytokinins and Giberellic Acid (GA). In addition, it stimulates rhizospheric microbes, protects the crops from major phytopathogens, improves nutrient uptake and ultimately boost up biological nitrogen fixation. The abundance of these bacteria in soil is related to many factors, mostly soil pH and fertility.

Keywords: Wheat; PGPR’s; Azotobacter; Biological nitrogen fixation and Soil fertility.

INTRODUCTION: Wheat is the major grain that sustains humanity. Wheat grows in temperate climate and it is staple food for 35% of world’s population. On other hand, it provides more calories and protein in the diet than any other crop. Wheat (Triticum spp., most commonly T. aestivum) is a cereal grain (botanically, a type of fruit called a caryopsis) originally from the Levant region but now cultivated worldwide 1. In 2016, world production of wheat was 749 million tonnes, making, it the second most-produced cereal after maize (1.03 billion tonnes), with more than rice (499 million tonnes) (United Nations, Food and Agriculture Organization. 2016) . Since 1960, world production of wheat and other grain crops has tripled and is expected to grow further through the middle of the 21 st Century.2.

This grain is grown on more land area than any other commercial food (220.4 million hectares, 2015-16) Food and Agriculture Organization Corporate Statistical Database (FAOSTAT, 2015). World trade in wheat is greater than for all other crops combined. Globally, wheat is the leading source of vegetal protein in human food, having a protein content of about 13%, which is relatively high as compared to other major cereals and staple foods.

When eaten as the whole grain, wheat is a source of multiple nutrients and dietary fiber, and is associated with lower risk of several diseases, including coronary heart disease, stroke, cancer and diabetes. 3

Nutrition: In 100 grams, wheat provides 327 calories and is a rich source (20% or more of the Daily Value, DV) of multiple essential nutrients, such as protein, dietary fibre, manganese, phosphorus and niacin. Several B vitamins and other dietary minerals are in significant content. Wheat seeds contain 13% water, 71% carbohydrates, and 1.5% fat. Its 13% protein content is comprised mostly of gluten as 75-80% of total wheat protein which upon digestion contributes amino acids for human nutrition. 4

Production and consumption: In 2016, global wheat production was 749 million tonnes, Food and Agriculture Organization (FAO 2016). Wheat is the primary food staple in North Africa and the Middle East, and is growing in uses in Asia. Unlike rice, wheat production is more widespread globally, though 47% of the world total in 2014 was produced by just four countries – China, India, Russia and the United States, Food and Agriculture Organization Corporate Statistical Database (FAOSTAT 2014).

During the past century, industrialization of agriculture has provoked a significant and essential productivity increase, which has led to a greater amount of food available to the general population. Along with this abundance the appearance of serious environmental and social problems came with the package: problems that must be faced and solved in the not too distant future. Nowadays, it is urgent to maintain that high productivity, but it is becoming urgent to alter as little as possible the environment. Clearly we must then head for a more environmentally sustainable agriculture while maintaining ecosystems and biodiversity. One potential way to decrease negative environmental impact resulting from continued use of chemical fertilizers, herbicides and pesticides is the use of plant growth-promoting rhizobacteria (PGPR). This term was first defined by Kloepper and Schorth (1978)5 to describe soil bacteria that colonize the rhizosphere of plants, growing in, on or around plant tissues that stimulate plant growth by several mechanisms.7

Table 1: Top Wheat producers in 2018-19.

Country

Millions of tonnes

China

126.2

India

95.8

Russia

59.7

United States

55.1

France

39.0

Canada

29.3

Germany

27.8

Pakistan

26.0

Australia

25.3

Ukraine

24.1

World

720

Source: UN Food and Agriculture Organization

Rhizosphere : The rhizosphere is coined more than hundred years ago by Hiltner (1904). 8 It is a nutrient-rich habitat for microorganisms, where severe, intense interactions take place between the plant, soil, and microfauna.9 They may have positive, negative or no visible effect on plant growth. Plant growth and productivity is highly affected by these interactions. Different type of microorganisms such as bacteria, fungi, protozoa, algae coexist among them. Bacteria contribute most to plant health by releasing many organic exudates, thus creating a selectively sensitive environment where diversity is less. Out of them plant growth promoting bacteria (PGPR) are most abundant among all others in the rhizosphere.10

Role of PGPR: PGPR are free living bacteria that resides in soil. They either directly or indirectly assist rooting11. They play different roles in the soil which proves beneficial for plant health and productivity. They colonize the rhizosphere and protect plant from its pathogens, produce secondary metabolites such as antibiotics that suppress harmful rhizobacteria, produce siderophores, and phytohormones, can fix atmospheric nitrogen, and help in providing nutrition uptake by solubilizing phosphate and produce biologically active substances which influence the plant growth and development.12

Occurrence and forms of PGPR: The mechanism by which PGPR exerts their beneficial effect on plants can be very diverse. They can establish themselves on root’s surface or inside the roots. PGPR can be classified into extracellular plant growth promoting rhizobacteria (ePGPR) that may exist in the rhizosphere, on the rhizoplane or in the spaces between the cells of root cortex. The bacterial genera such as Agrobacterium, Arthrobacter, Azotobacter , Azospirillum, Bacillus, Flavibacterium, Pseudomonas and Serratia belong to ePGPR. The other category is intracellular plant growth promoting rhizobacteria (iPGPR) that locates generally inside the specialized nodular structures of root cells Rhizobium spp.13

Azotobacter: Azotobacter spp. are Gram negative, free-living, aerobic soil dwelling14, oval or spherical bacteria that form thick-walled cysts (means of asexual reproduction under unfavorable condition), can grow well on N-free medium.15 There are around six species in the genus Azotobacter16 some of which are motile by means of peritrichous flagella, others are not. They are typically polymorphic and their size ranges from 2-10 μm long and 1-2 μm wide15. The Azotobacter genus was discovered in 1901 by Dutch microbiologist and botanist (founder of environmental microbiology).A.chroococcum is the first aerobic free-living nitrogen fixer. 17

These bacteria utilize atmospheric nitrogen gas for their cell protein synthesis. This cell protein is then mineralized in soil after the death of Azotobacter cells thereby contributing towards the nitrogen availability of the crop plants. Azotobacter spp. is sensitive to acidic pH, high salts, and temperature.18 Azotobacter has beneficial effects on crop growth and yield through, biosynthesis of biologically active substances, stimulation of rhizospheric microbes, producing phyopathogenic inhibitors.19-20

Besides being quite expensive and making high cost of production, chemical fertilizers have adverse effect on soil health and microbial population. In such situation, biofertilizers can be the best alternative for enhancing soil fertility. Being economic and environmental friendly, biofertilizers can be used in crop production for better yield. Similarly, microbial products are considered safer, self-replicating, target specific, which is regarded as major component of integrated nutrient management from soil sustainability perspective.21

Role of Azotobacter in soil:

Azotobacter in soil: The presence of Azotobacter sp. in soils has beneficial effects on plants, but the abundance of these bacteria is related to many factors, soil physico-chemical (e.g. organic matter, pH, temperature, soil moisture) and microbiological properties.22 Its abundance varies as per the depth of the soil profile.23 Azotobacteria are much more abundant in the rhizosphere of plants than in the surrounding soil and that this abundance depends on the crop species.24

Nitrogen fixation: Nitrogen is the component of protein and nucleic acids and chlorophyll. Thus, nitrogen supply to the plant will influence the amount of protein, amino acids, protoplasm and chlorophyll formed. Therefore, adequate supply of nitrogen is necessary to achieve high yield potential in crop.

The atmosphere comprises of ~78% nitrogen as an inert, in unavailable form. Above every hectare of ground there are ~80000 tones of this unavailable nitrogen. In order to be con­verted to available form it needs to be fixed through either the industrial process or through Biological Nitrogen Fixation (BNF). Without these nitrogen-fixers, life on this planet may be difficult.

Nitrogen (N) deficiency is frequently a major limiting factor for crops production25. Nitrogen is an essential plant nutrient, widely applied as N-fertilizer to improve yield of agriculturally important crops. An interesting alternative to avoid or reduce the use of N-fertilizers could be the exploitation of Plant Grow­th-Promoting Bacteria (PGPB) capable of enhancing growth and yield of many plant species, several of agronomic and ecological significance. Azotobacter spp. are non-symbiotic heterotrophic bacteria ca­pable of fixing an average 20 kg N/ha/per year.22

Bacterization helps to improve plant growth and to increase soil nitrogen throu­gh nitrogen fixation by utilizing carbon for its metabolism. 26

Seed inoculation with Azotobacter and nutrient uptake: Seed Inoculated with Azotobacter helps in uptake of N, P along with micronutrients like Fe and Zn, in wheat, these strains can potentially be used to improve wheat nutrition27. Seed inoculation of Azotobacter profoundly contribute to increase yield by supplying nitrogen to the crops. Inoculation of seeds with Azotobacter chroococcum increased carbohydrate and protein content of two corn varieties (Inra210 and Inra260) in greenhouse experiment. 28

There is increment in Maize biomass with the application of manure and Azotobacter.29 In nitrogen-deficient sand, seed inoculation increased plant length, dry weight, and nitrogen content in addition to a significant increase in soil nitrogen.30

Seeds of wheat (Triticum aestivum) were inoculated with 11 bacterial strains of A. chroococcum, Research result showed that all A. chroococcum strains had positive effect on the yield and N concentrations of wheat.31

Role in growth substances production and promotion: Besides, nitrogen fixation, Azotobacter produces, Thiomin, Riboflavin, Nicotin, Indol Acetic Acid and Gibberellins. When Azotobacter is applied to seeds, seed germination is improved to a considerable extent. (Brake J and Hilger F 1965)32 showed that Azotobacter produced Indol-3-Acetic Acid (IAA) when tryptophan was added to the medium. (Hennequin JR, Blachere H 1996)33 found only small amounts of IAA in old cultures of Azotobacter to which no tryptophan was added.

Bacteria of the genus Azotobacter synthesize auxins, cytokinins, and GA-like substances, and these growth materials are the primary substance controlling the enhanced growth of wheat.34 These hormonal substances, which originate from the rhizosphere or root surface, affect the growth of the closely associated plants. (Eklund.1990)35 demonstrated that the presence of Azotobacter chroococcum in the rhizosphere of tomato and cucumber is correlated with increased germination and growth of seedlings. Phytohormones (auxin, gibberellin and cytokinins) can stimulate root development.

High Gibberillic acid production was detected in Azotobacter (71.42 %) isolates. Higher phosphate solubilization was detected in the isolates of Azotobacter (74.28 %) followed by Pseudomonas (63.00 %). Gibberellins applied in small quantities to the soil or rosettes on the leaves and shafts of certain plant produces an increase in height. In grains such as wheat and corn, they also cause an increase in length of the leaves. In some cases they also increase the fresh weight and dry weight. However it doesn’t produce any effect on the growth of the roots.31

These responses suggest that Azotobacter probably influences the development of plants by producing growth-regulating substances. Therefore, Azotobacter spp. is often regarded as a member of “Plant Growth Promoting Rhizobacteria (PGPR)”.36

Dry matter accumulation: There is increment in dry matter accumulation in Azotobacter inoculated plants; it stimulates development of foliage, roots, branching, flowering and fruiting which is triggered by fixed nitrogen and plant growth regulator like substance produced. It also increases plant tolerance to lack of water under adverse condition.37 Similar result put forwarded by (Sandeep et al. 2011) which revealed that there is better growth response of Azotobacter inoculated plants as compared to non-inoculated control plants. Better crop growth response ultimately results in better dry matter accumulation.38

Biochemical effects: Several strains of Azotobacter are capable of producing amino acids when grown in culture media amended with different car­bon and nitrogen sources.39 Substance like amino acid produ­ced by these rhizobacteria are involved in many processes that explain plant-growth promotion. Biochemical analysis of chloro­phyll, nitrogen, phosphorous, potassium and protein content was higher in Azotobacter inoculated plants as compared to non-ino­culated control plants.40

Anti-pathogenic response : Azotobacter spp. are capable to produce siderophore, they bind to the available form of iron Fe3+ in the rhizosphere, thus making it unavailable to the phytopathogens and protecting the plant health; similarly Hydrogen Cyanide (HCN) production was higher in traits of Azotobacter (77.00 %). Azotobacter secretes an antibiotic with a structure similar to anisomycin, which is a documented fungicidal antibiotic. Azotobacter, in sufficient numbers, will out-compete pathogens for food. Some of the pathogens that have been controlled by Azotobacter in the soil and on the leaf include: Alternaria, Fusarium, Collectotrichum, Rhizoctonia, Microfomina, Diplodia, Batryiodiplodia, Cephalosporium, Curvularia, Helminthosporium and Aspergillus etc.36

Effects of chemical fertilizer inoculation with Azotobacter: Combined application of bio-fertilizer with 50% of chemical fertilizers (N and P) has significant effect in plant growth, plant height, number of branches, fresh and dry weight of saf flower in comparison with chemical fertilizers alone. Similarly, application of Azotobacter biophosphate and organic fertilizers, with half dose of chemical fertilizers increase the economic yield of wheat.41 Efficiency of Azotobacter found decreased with increased N level42 . The best combination was recorded with NH4Cl at 0.1 g/L whereas, action of copper in Azotobacter found toxic even in very low concentration. The population of Azotobacter may suffer due to high amount of nitrates and the acidic environment created because of chemical fertilizer.43

Effect of pesticides in Azotobacter: Balajee & Mahadevan, (1990)44 reported that, the effect of herbicide 2, 4-D and its products; p-chlorophenoxy-acetic acid and p-chlorophenol were utilized by A. chroococcum as carbon source, which ultimately stimulate nitrogenase enzyme. Similar result found by (Kanungo et al. 1995)45 which revealed insecticide carbofuran also stimulates nitrogenase enzyme activity. Martinez et al. 1991)46 found that, herbicide simazine have no effect on A. chroococcum growth either on standard medium or on dialysed soil and sterilized soil medium. The presence of 50-300 mg of simazine per ml of culture or in one gram of soil did have a stimulating effect on Azotobacter. When Azotobacter is grown in presence of simazine, the cells have a higher ATP content than the control. Whereas organophosphorous insecticides profenofos and chloropyrifos had adverse effect on the number of aerobic nitrogen fixers and decreased nitrogen fixation.47

Azotobacter in nutrient cycling: Azotobacter makes availability of certain nutrients like Carbon, Nitrogen, Phosphorus and Sulphur through accelerating the mineralization of organic residues in soil and avoid uptake of heavy metals. Azotobacter can be an important alternative of chemical fertilizer because it provides nitrogen in the form of ammonia, nitrate and amino acids without situation of over dosage, which might be one of the possible alternatives of inorganic nitrogen source (eg. Urea). It also helps to sustain the plant growth and yield even in case of low phosphate content soil, as well as helps in uptake of macro and certain micro nutrients which facilitates better utilization of plant root exudates itself.48

CONCLUSION: Azotobacter spp. are free living, non-symbiotic, heterotrophic bacteria capable of fixing an average of 20 kg N/ha per year. These bacteria are regarded as Plant Growth Promoting Rhizobacteria (PGPR) which synthesize growth substance that enhances plant growth and development and inhibit major phytopathogenic growth by secreting inhibitors. It also helps in nutrient uptake and produces some biochemical substances such as protein, amino acids etc. Azotobacter improves seed germination and has beneficiary response on Crop Growth Rate (CGR). It helps to increase nutrient availability and to restore soil fertility for better crop response. It is an important component of integrated nutrient management system due to its significant role in soil sustainability. More research is necessary in future to explore the potentiality of Azotobacter in soil fertility.

REFERENCES:

1. Shewry, Peter R. “wheat,” Journal of Experimental Botany. 2009, 60(6), 1537-1553.

2. Godfrey, D.; Hawkesford, M. J.; Powers, S. J.; Miller, S.; Shewry, P. R. Effects of crop nutrition on wheat grain composition and end use quality.Journal of Agriculture and Food Chemistry. 2010, 58, 3012- 3021.

3. Shewry, P. R.; Hey, S. J. The contribution of wheat to human diet and health. Food Energy Security. 2015, 4(3), 178- 202.

4. Shewry, P. R. Wheat gluten proteins. In:wheat Gluten Protein Analysis (P. R. Shewry and G. Lookhart eds) 2002, pp. 1-17, American Association of Cereal Chemists, St. Paul, Minnesota, USA.

5. Kloepper, J. W.; and Schroth, M. N. Plant growth-promoting rhizobacteria on radishes. In: Proceeding of the 4th International Conference on Plant Pathogenic Bacteria . 1978, Vol. 2, (pp. 879-882).

6. Filippi, M. M. C.; da Silva, G. B.; Silva- Lobo, V. L.; Cortes, M. V. C. B.; Moraes, A. J. G.; Prabhu, A. S. Leaf blast (Magnaporthe oryzae ) suppression and growth promotion by rhizobacteria on aerobic rice in Brazil. Biol Control. 2011, 58, pp. 160- 166.

7. Yu, X. M.; Ai, C. X.; Xin, L.; Zhou, G. F. The siderophore- producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper. Eur J Soil Biol. 2011, 47, pp. 138-145.

8. Hiltner, L. About recent experiences and problems the field of soil bacteriology with special Consideration of green manure and fal­low.Arbeiten der Deutschen Land wirtschaftlichen Gesellschaft. 1904, 98, 59-78.

9. Antoun, H.; Prevost, D. Ecology of plant growth promoting rhi­zobacteria. In: Siddiqui ZA (Ed.), PGPR: biocontrol and biofertilization, Springer, Dordrecht. 2005, pp. 1-65.

10. Sivasakthi, S.; Usharani, G.; Saranra, J. N. Biocontrol potentiality of plant growth promoting bacteria (PGPR) Pseudomonas fluorescens and Bacillus subtilis: A review. Afri Jourl of Agri Rersch. 2014, 9, 1265-1277.

11. Mayak, S.; Tirosh, T.; Glick, B. R. Effect of wild-type and mutant plant growth promoting rhizobacteria on the rooting of mung bean cuttings. Journal of plant growth regulation. 1999, 18(2), 49-53.

12. Arshad, M.; Frankenberger, W. T. Jr. Plant growth regulating substances in the rhizosphere: microbial production and functions. Adv Agron. 1998, 62, 146-151.

13. Figueiredo, M. V. B.; Seldin, L.; Araujo, F. F.; Mariano, RLR. Plant growth promoting rhizobacteria: fundamentals and applications. Maheshwari, DK (Ed.), Plant Growth and Health Promoting Bacteria. Springer- Verlag, Berlin, Heidelberg. 2011, pp. 21-42.

14. Gandora, V.; Gupta, R. D.; Bhardwaj, K. K. R. Abundance ofAzotobacter in great soil groups of North–West Himalayas.J Indian Soc Soil Sci. 1998, 46(3), 379-383.

15. Salhia, B. The Effect of Azotobacter chrococcumas Nitrogen biofertilizer on the growth and yield of Cucumis sativus . Deanery of Higher Education Faculty of Science, Master of Biological Sciences, Botany: The Islamic University Gaza. 2013.

16. Martyniuk, S.; Martyniuk, M. Occurrence of Azotobacter spp. in some polish soils. Polish J Environ Stud. 2003, 12(3), 371–374.

17. Beijerinck M. W. Ueber Oligonitophile Mikroben, Zentralblattfiir Bakteriologie, Parasitenkunde, Infektionskrankheiten and Hygiene. Abteilung II. 1901, 7, 561–582.

18. Tchan, Y. T. New P. B. Azotobacteraceae. In: Holt JG, Williams, et al. editors. Bergey’s Manual of Systematic Bacteriology Volume 1. Baltimore, USA. 1989, p, 220–229.

19. Chen, J. The combined use of chemical and organic fertilizers and/ or biofertilizer for crop growth and soil fertility . International workshop on Sustained Management of the Soil–Rhizosphere System for Efficient Crop Production and Fertilizer Use, Thailand. 2006, p, 1–10.

20. Lenart, A. Occurance Characteristics and Genetic Diversity ofAzotobacter chroococcum in Various Soils of Southern Poland.Pol J Environ Stud. 2012, 21(2), 415–424.

21. Nagananda, G. S.; Das, A.; Bhattachrya, S. et al. In vitro studies on the Effects of Biofertilizers (Azotobacter and Rhizobium) on Seed Germination and Development of Trigonella foenum–graecum L. using a Novel Glass Marble containing Liquid Medium. International Journal of Botany. 2010, 6(4), 394–403.

22. Kizilkaya, R. Nitrogen fixation capacity of Azotobacter spp. Strains isolated from soils in different ecosystems and relationship between them and the microbiological properties of soils.J Environ Biol. 2009, 30(1), 73–82.

23. Vojinoviv, Z. Microbiological properties of main types soil in Serbia for nitrogen cycling. Journal for Scientific Agricultural Research. 1961, 43, 3– 25.

24. Kalaigandhi, V.; Kannapiran, E.; Harimuraleedharan. Azotobacter population in Rhizosphere and Non–Rhizosphere sediments of Tondi Coast.International Journal of Biological Technology. 2010, 1(1), 63–65.

25. Dong, H.; Li, W.; Eneji, A. E. Nitrogen rate and plant density effects on yield and late–season leaf senescence of cotton raised on a saline field.Field Crops Research. 2012, 126, 137–144.

26. Monib, M.; Hosny, I.; Fayez, M. Effect of Azotobacter inoculation on plant growth and soil nitrogen. Zentralbl Bakteriol Naturwiss. 1979, 134(2), 140– 148.

27. Rajaee, S.; Alikhani, H. A.; Raiesi, F. Effect of Plant Growth Promoting Potentials of Azotobacter chroococcum Native Strains on Growth, Yield and Uptake of Nutrients in Wheat [2007–10]. Agris records. 2007, 11(41), 285–297.

28. Kizilog, I. U.; Bilen, F. T.; Ataplu, N. Effect of Inoculation Eight Azotobacter chroococcum and Nitrogen Fertilizer on Plant Growth of Corn (Zea mays) Carbohydrate and Protein Contents.Ziraat Fakultesi Dergisi Ataturk Universitasi. 2001, 32, 215–221.

29. Meshram, S. U.; Shende, S. T. Response of Maize toAzotobacter chroococcum. Plant and Soil. 1982, 69, 265–273.

30. Monib, M.; Malek, A. E. Y.; Hosny, I et al. Seed inoculation with Azotobacter chroococcum in sand cultures and its effect on nitrogen balance. Zentralbl Bakteriol Naturwiss. 1979, 134(3), 243–248.

31. Kizilkaya, R. Yield response and nitrogen concentrations of spring wheat (Triticum aestivum) inoculated withAzotobacter chroococcum strains. Ecological Engineering. 2008, 33(2), 150.156.

32. Brakel, J.; Hilger, F. Etude qualitative et quantitative de la synthese de substances de nature auxinique parAzotobacter chroococcum in vitro. Bull Inst Agron Stns Rech Gembloux. 1965, 33, 469–487.

33. Hennequin, J. R.; Blachere, H. Recherches sur la synthese de phytohormones et de composes phenoliques par Azotobacter et des bacteries de la rhizosphere. Ann Inst Pasteur. 1966, 3, 89–102.

34. Azcorn, R.; Barea, J. M. Synthesis of auxins, gibberellins and cytokinins by Azotobacter vinelandi and Azotobacter beijerinckii related to effects produced on tomato plants. Plant Soil. 1975, 43, 609–619.

35. Eklund, E. Secondary effects of some Pseudomonads in the rhizosphere of peat grown cucumber plant. In: Pharis RP, et al. editors. Hormonal Regulation of Development. 1970, 3, p, 613.

36. Althaf, H. S.; Srinivas, P. Evaluation of plant growth promoting traits by Pseudomonas and Azotobacter Isolated From Rhizotic soils of two selected agro forestry tree species of godavari belt region, India.Asian J Exp Biol Sci. 2013, 4(3), 431–436.

37. Yasari, E.; Patwardhan, A. M. Effect of (Azotobacter and Azosprillium) Inoculants and Chemical Fertilizers on Growth and Productivity of Canola (Brassica napus L.). Asian Journal of Plant Sciences. 2007, 6(1), 77–82.

38. Sandeep, C.; Rashmi, S. N.; Sharmila V. Growth Response of Amaranthus gangeticusto Azotobacter chroococcum Isolated from Different Agroclimatic Zones of Karnataka. Journal of Phytology. 2011, 3(7), 29–34.

39. Lopez, G. J.; Pozo, R. B.; Lopez, S. Liberation of amino acids by heterotrophic nitrogen fixing bacteria. Amino Acid. 2005, 28(4), 363–367.

40. Naseri, R.; Azadi, S.; Rahimi, M. J. et al. Effects of Inoculation with Azotobacter chroococcum and Pseudomonas putida on yield and some of the important agronomic traits in Barley (Hordeum vulgar L).International Journal of Agronomy and Plant Production. 2013, 4(7), 1602–1610.

41. Ojaghloo, F.; Farahvash, F.; Hassanzadeh, A. et al. Effect of inoculation with Azotobacter and barvar phosphate bio–fertilizers on yield of safflower (Carthamus tinctorius L.). Journal of Agricultural Sciences. Tabriz Branch: Islamic Azad University. 2007, p, 25–30.

42. Soleimanzadeh, H.; Gooshchi, F. Effects of Azotobacter and nitrogen chemical fertilizer on yield and yield components of wheat (Triticum aestivum L.). World Applied Sciences Journal. 2013, 21(8), 1176–1180.

43. Gul, FS. Growth and nitrogen fixation dynamics of Azotobacter chroococcum in nitrogen–free and OMW containing medium. The middle east technical university. 2003, p, 1–12.

44. Balajee, S.; Mahadevan, A. Influence of chloroaromatic substances on the biological activity of Azotobacter chroococcum.Chemosphere. 1990, 21(1– 2), 51–56.

45. Kanungo, P. K.; Adhya, T. K.; Rao, V. R. Influence of repeated applications of carbofuran on nitrogenase activity and nitrogen – fixing bacteria associated with rhizosphere of tropical rice. Chemosphere. 1995, 31(5), 3249–3257.

46. Martinez, T.; Salmeron, M. V.; Gonzalez, L. Effect of simazine on the biological activity of Azotobacter chroococcum.Soil Science. 1991, 151(6), 459–467.

47. Martinez, T.; Salmeron, M. V.; Gonzalez, L. Effects of an organophosphorus insecticide, profenofos on agricultural soil microflora.Chemosphere. 1992, 24(1), 71–80.

48. Levai, L.; Szilvia, V.; Nora, B et al. Can wood ash and biofertilizer play a role in organic agriculture. Agronomski Glasnic.2008, 3, 263–271.