Assessment of Azotobacter chroococcum ’s Ability to Produce Hydrolytic Enzymes and Enhance Growth of Vicia faba

: A. chroococcum isolate was examined for its ability to produce the hydrolytic enzymes chitinase, pectinase, protease, and lipase, in an effort to enhance the growth of fava bean ( Vicia faba ). Biological experiment was conducted in pots with complete random design (CRD). The experiment includes three treatments: control (plant without treatment) (P), NPK fertilizer (plant + fertilizer) (PF), and A. chroococcum inoculum (plant + A. chroococcum ) (PA). These treatments were performed with sterile and non-sterile soil, which were planted with fava beans. At the end of experiment (seven weeks from planting), length and weight of plant shoot and plant root were calculated. The results show that the isolate was able to produce chitinase, pectinase, protease, and lipase enzymes. Plant growth increased with fertilizer and A. chroococcum inoculum addition. Fertilizer treatments (PF) were the best growth indicators, with the averages of shoot length and weight, and root length and weight of 19.52 cm, 15.55 gm, 19.93 cm, and 10.52 gm respectively. The averages of these indicators in A. chroococcum treatment (PA) were 16.4 cm, 8.85 gm, 17.52 cm, and 6.88 gm respectively. The percentages of increase caused by A. chroococcum inoculum addition (PA) in contrast with control treatment (P) were 12.83% and 34.4% in plant shoot length and weight respectively, and 60.98% and 38.99% in plant root length and weight. In general, growth indicators were higher in non-sterile than sterile soil, but these increases were not significant.


Introduction:
Chemical fertilizers are used to achieve maximum crop production in agricultural fields.The frequent usage of this fertilizers is associated with health hazards, environmental pollution, and loss of soil fertility.Therefore biofertilizers, substances which contain living microorganisms, are gaining importance as they are non-hazardous, ecofriendly and enhance soil fertility.Several bacteria have been used as biofertilizers such as Azotobacter, Azospirillum, Pseudomonas and Rhizobium (1).Azotobacter are aerobic, free-living soil microbes which play an important role in the nitrogen fixation.The isolates of Azotobacter show positive nitrogen fixing capacity ranging from 3.16 to 12.66 mg N per gram of sucrose consumed (2).Azotobacter have a full range of enzymes needed to perform the nitrogen fixation such as ferredoxin, hydrogenase and an important enzyme nitrogenase (3).Being free living N 2 -fixer, Azotobacteria synthesize growth materials such as auxins, cytokinins, and gibberellin, which are the primary cultures (the optical density = 0.84 at 600 nm) and incubated at 30 ○ C. Formation of a clear zone after incubation time (3-7 days for Chitin, and Pectin, and 24h for milk agar) indicated the positive result for production of chitinase, pectinase and protease enzymes respectively (8,9).
Lipase production was investigated on mineral salt olive oil agar, which was inoculated with 24 h age of bacterial culture and incubated for 3-7 days at 30 ○ C. Bacterial growth, appearance and reduction of medium oil reflected a positive result (10).

Azotobacter inoculum
The A. chroococcum isolate used in this study was provided and identified by The Agriculture Research Center of Abu Ghraib (ARC).This bacterium was isolated from the rhizosphere of wheat.Morphologically, it was oval to spherical shape, gram-negative, motile and produced smooth white colonies on nutrient agar.Biochemically, it was positive for oxidase, catalase, urease and indole tests (11) (12).Inoculum preparation was achieved by growing of A. chroococcum isolate in 100 ml of the broth culture for six days at 30 ○ C, where the optical density of bacterial culture was 0.84 at 600 nm.

Plant seeds preparation
Fava bean (Vicia faba) seeds, which were obtained from ARC, were prepared by surface sterilization using 2% HgCl 2 and 95% Ethanol for 2 min, and then washed well with distilled water (13).

Soil preparation
Soil was obtained from Baghdad University fields from the depth of (10-15) cm, and Table (1) showed some of soil properties.Soil was divided into two groups: sterile and non-sterile soil, where the sterile soil was autoclaved three times at 121 ○ C for 1 hour each time (14).

Biological experiment
Sterile and non-sterile soil was distributed in 18 sterile, 5 Kg pots, 9 for each set of trials.Seeds, treated with inoculum, were soaked with 100ml of A. chroococcum fresh culture (the optical density = 0.84 at 600 nm) and mixed with sterilized Arabic gum.NPK fertilizer supplied by ARC.
Biological experiment performed (CRD) with three replicates and two factors; sterile and non-sterile soil and three treatments: control, include plant without treatment (P), NPK fertilizer (100 Kg. hec. - ) (PF), and addition of A. chroococcum inoculum (PA).Thus there were 18 experimental units (2*3*3=18).Seeds were cultured, pots were arranged randomly inside a plastic house and irrigated with tap water.

Plant growth promoting effect estimation
Seven weeks after germination, an assessment of length and weight of each plant shoot and plant root were conducted.

Statistical Analysis
The experiment was statistically analyzed using ANOVA, and the averages was compared with least significant difference (LSD) values at the level of 0.05 (15).

A. chroococcum ability to produce hydrolytic enzymes
A. chroococcum isolate was able to form hydrolytic zones on chitin, pectin and milk agar (Table 2), so it was able to produce chitinase, pectinase and protease enzymes (8 and 9).The isolate also was able to grow and reduced the oil in mineral salt olive oil agar, verifying the ability to produce a functional lipase enzyme (16).Production of hydrolytic enzymes including cellulose, pectinase, protease, chitinase and lipase by some rhizospheric bacterial community indicate its benefit and plant growth promoting activity (17).Rodrigues and his team (18)

Azotobacter chroococcum inoculum effect on plant shoot -Shoot length
The average shoot length of fava bean in non-sterile soil treatments (17.02 cm) was more than in sterile soil treatments (16.61 cm), but this difference is not significant.Azotobacter inoculum increased shoot length in sterile and non-sterile soil (Figs. 1 and 2).The percentage of increase caused by Azotobacter inoculum addition was 12.83 %.Fertilizer treatments show significant increase in shoot length from control treatments.There were no significant differences between fertilizer treatment (18.97 cm) and Azotobacter treatment (16.8 cm) in sterile soil.While these two treatments (20.07 and 16 cm respectively) exhibited significant differences in non-sterile soil, this may have related to the computation of indigenous microorganisms.Azotobacter inoculum increased the growth by supplying the crop with nitrogen through asymbiotically fixation and phytohormone synthesis.Kalay and his team (19) mention that applying A. chroococcum on choy sum (Brassica rapa L.) did not increase the yield.Felipe and his team (5) observed a beneficial effect on greenhouse planted cotton inoculated with A. chroococcum, and suggested that co-inoculation allow to reduce nitrogen fertilization doses up to 50%.

Shoot weight
There were no significant differences between the averages of plants shoot weight in sterile soil treatments (10.09 gm) and non-sterile soil treatments (10.56 gm).Fertilizer treatments (15.03 and 16.07 gm respectively) showed significant increase in shoot weight from the control (6.92 and 6.25 gm respectively) and Azotobacter treatments (8.33 and 9.37 gm respectively) (Fig. 3).Shoot weight gained increase by 34.4% in Azotobacter treatment compared with control treatment, which may have been related to production of indol-3-acetic acid and siderophores, which can increase nutrient availability and improve plant growth (20).Felipe and his team (5) evidenced an increase ranging between 8 and 53% in all parameters of plant growth, suggesting that A. chroococcum inoculation has a higher potential to promote cotton plant growth.

Azotobacter chroococcum inoculum effect on plant root -Root length
The results in figs.(4 and 5) show that fertilizer treatments exhibited highest length of bean plant root in non-sterile and sterile soil (19.6 and 20.26 cm respectively) followed by Azotobacter treatments (16.6 and 18.43 cm respectively).Azotobacter inoculum increased the length of plant roots in contrast with control treatments, with an increase of 60.98%.Root lengths in non-sterile soil (16.85 cm) are improved better than in sterile soil (15.37 cm).Wani Sartaj and his team (21) mentioned that Azotobacteria affect the growth of the closely associated higher plants.They can benefit plant development through multiple mechanisms of action, directly through the production of substances which promote growth and increase nutrient availability in soil and their uptake, or indirectly through the suppression of plant pathogens (20).

Root weight
The treatments of non-sterile soil presented no significant increase in the average of fava bean plants root weight (8.05 gm) from sterile soil treatments (6.84 gm).Fertilizer treatments obtained maximum root weight in sterile soil and non-sterile soil (10 and 11.03 gm respectively), which differ significantly other treatments (fig.6).Azotobacter inoculum enhanced the roots weight of bean plant in sterile soil and non-sterile soil by 6.13 and 7.63 gm respectively.The percentage of increase in inoculum treatments from control treatments was 38.99%.This increase may have been related to synthesized hormonal substances

Conclusion:
The concept of this study is to use natural alternatives that are effective and not harmful to the environment.Therefore, A. chroococcum isolate, which is known for its capability to fix atmospheric nitrogen, was used.Moreover, Azotobacter strains have been well known for their siderophores, indol-3-acetic acid and exopolysaccharides (EPS) production, which may benefit plant growth/nutrient availability and soil water retention properties.This isolate was also able to produce chitinease, pectinase, protease and lipase enzymes.A. chroococcum isolate improved length and weight of fava bean plants shoot and root.Therefore, such addition should be recommended to replace chemical fertilizers, albeit partially, in order to reduce the risk of chemical toxicity and preserve soil fertility and health.inoculants in sustainable agricultural productivity.Springer, India.2016; 229-244.17

Treatments sterile soil non-sterile soil which
(1))inate from the rhizosphere or root surface(21).Tiwari and his team(1)reported that Azotobacter sp.inoculum enhanced plant shoot length, root length, leaves number, shoot and root fresh and dry biomass, but mixed inoculations of Rhizobium sp. and Azotobacter sp.revealed further enhancement.