Exploration and Biochemical Profiling of Native Azotobacter and Azospirillum Isolates from Chhattisgarh and Uttar Pradesh Soils

Introduction

Spinach (Spinacia oleracea), a major leafy vegetable of the Amaranthaceae family, is widely cultivated in India due to its high nutritional value and adaptability to diverse agro-climatic conditions (Hanif et al., 2006; Kisan et al., 2015; Tewani et al., 2016) [2,3,4] . In states like Chhattisgarh and Uttar Pradesh, favorable soil and water availability support its cultivation, making it an important crop for local farmers (Ministry of Agriculture and Farmers Welfare, India). Spinach leaves are rich in vitamins A, C, and K, minerals like iron and magnesium, and bioactive compounds such as flavonoids and phenolics, which contribute to human health. However, spinach productivity can be affected by nutrient deficiencies, poor soil health, and environmental stresses. Modern agriculture often relies heavily on chemical fertilizers, which may disrupt beneficial soil microorganisms, reduce soil fertility, and increase input costs for marginal farmers (Itelima et al., 2018) [9] . Biofertilizers containing PGPR offer an ecofriendly alternative by improving nutrient availability, enhancing plant growth, and maintaining soil microbial balance. Among PGPR, Azotobacter and Azospirillum are free-living nitrogen-fixing bacteria that support crop growth by fixing atmospheric nitrogen, producing phytohormones like indole-3acetic acid, and improving nutrient and water uptake. They also reduce soil-borne diseases and mitigate abiotic stresses such as salinity and drought (Bashan & De-Bashan, 2005; Dobbelaere et al., 2003; Rashid et al., 2016) [1,5,14] . The natural genetic and phenotypic diversity of native rhizobacterial populations provides opportunities to select superior strains for biofertilizer development (Raaijmakers & Weller, 2001; Hartmann & Amarger, 1991) [10,11] . Isolation and characterization of locally adapted strains are therefore crucial to develop effective, environmentally safe biofertilizers tailored to specific agro-climatic regions (Zhou et al., 2020) [6] . Such an approach enhances crop productivity while reducing dependency on chemical fertilizers, supporting sustainable agricultural practices. 12): 714 -719 [7] . Their use in crops like spinach can increase yield by 10-40% under controlled and field conditions (Kawalekar, 2013) [12] . Solid and carrier-based formulations using lignite, humus, peat, or wheat bran improve microbial shelf-life and ease of application (Bhattacharjee & Dey, 2014) [13] .

Scope of the Present Study

The present paper focuses exclusively on the isolation and biochemical characterization of Azotobacter and Azospirillum obtained from the spinach rhizosphere. The detailed evaluation of their inoculation effects on spinach growth parameters, including plant height, biomass, chlorophyll content, and NPK accumulation, will be reported in a subsequent research paper (hereafter referred to as "the second paper").

Experimental Site

The present study was conducted during the Master's research period (2024-2025) in the Microbiology Laboratory of the Department of Microbiology, College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya (IGKV), Raipur, Chhattisgarh. All experimental work from handling soil samples to isolating and characterizing bacterial cultures was carried out in a well-maintained laboratory environment, ensuring aseptic conditions and reliable observations throughout the research.

Materials and Methods

Sample Collection

Soil samples were collected from the rhizosphere of spinach plants across Chhattisgarh and Uttar Pradesh, including Dharsiwa, Surajpur, Balrampur, Nardaha (Raipur), Sarguja, Sakti, Achanakmar Biosphere Reserve, and research fields of Indira Gandhi Krishi Vishwavidyalaya, Raipur. Samples were taken at 0-15 cm depth using sterile soil augers to ensure collection of microorganisms closely associated with roots. Samples were immediately transferred to sterilized polythene bags, labeled, and stored at 4 °C to minimize microbial changes prior to analysis.

Culture Media

Jensen's medium was used for Azotobacter, and Okon's medium for Azospirillum. Media were prepared using standard protocols, sterilized at 121 °C for 15-20 minutes, and poured under aseptic conditions. Media were routinely checked for contamination.

Isolation of PGPR (Azotobacter & Azospirillum)

One gram of soil was suspended in 9 mL of sterile distilled water to prepare a stock solution, followed by serial dilutions up to 10⁻⁹. From suitable dilutions, 0.1 mL was spread on Jensen's or Okon's medium and incubated at 28-30°C for 24-48 hours. Well-separated colonies were selected and purified by repeated streaking to obtain single, uncontaminated cultures, which were maintained on slants at 4 °C for further analysis.

Colony Morphology

Colony size, shape, margin, elevation, surface texture, and pigmentation were observed for each isolate to provide preliminary identification cues.

Gram Staining

Smears were prepared on sterilized slides, air-dried, and heat-fixed. Sequential staining with crystal violet, Gram's iodine, 95% ethanol, and safranin was performed. Microscopic examination provided cell shape, arrangement, and Gram reaction.

Biochemical Characterization

Catalase Test: Fresh colonies were tested with 3% hydrogen peroxide. Rapid bubbling indicated positive catalase activity.

Starch Hydrolysis

Results and Discussion

Isolation of PGPR

Nine bacterial isolates (Azotobacter and Azospirillum) were successfully obtained from spinach rhizosphere soils and maintained as pure cultures.

Colony Morphology and Gram Staining

Azotobacter: Gram-negative, spherical or short rod-shaped cells; off-white, circular, convex colonies with smooth surfaces and entire margins.

Azospirillum: Gram-negative, slightly spiral cells; dark bluish, circular, convex colonies with smooth surfaces and entire margins. Representative plate images (Plate 4 -4.5) illustrate these distinctions.

Gram stain of Azotobacter

Gram stain of Azospirillum Plate 4: Gram staining of Azotobacter & Azospirillum isolates.

Azospirillum isolates in slants. Azotobacter isolates in slants

Biochemical Characteristics

Comparative Analysis and PGPR Potential

Morphological and biochemical differences were observed between the two genera, while functional similarities were noted within each genus. Biochemical tests confirmed oxidative stress tolerance (catalase) and ability to use citrate as carbon source. Minor differences in starch hydrolysis and uniformly negative methyl red reactions suggest stable metabolic profiles. These native strains demonstrated potential as plant growthpromoting rhizobacteria suitable for biofertilizer development.

Summary of Findings

All nine isolates were Gram-negative, showed genusspecific colony morphology, positive catalase and citrate activity, and minor variability in starch hydrolysis. Negative methyl red results confirmed the absence of strong acid production. The isolates possess characteristics associated with effective PGPR.

Conclusion

Nine native strains of Azotobacter and Azospirillum were successfully isolated and characterized from spinach rhizosphere soils of Chhattisgarh and Uttar Pradesh. Morphological and Gram staining traits differentiated the two genera, while biochemical profiling revealed positive catalase and citrate activity and minor variability in starch hydrolysis. Negative methyl red reactions confirmed metabolic stability. These isolates demonstrated key plant growth-promoting traits, indicating their suitability for biofertilizer development in sustainable crop production. Further studies on field inoculation and spinach growth performance will be addressed in the next phase.

References

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