Characterization of different Rhizobium isolates of lentil (Lens Culinaris) collected from different locations of Kolhapur district

Introduction

Lens culinaris L., commonly known as lentil or Masur, is an important cool-season legume of the family Leguminosae, widely valued for its protein-rich seeds, which serve as a staple dietary component, particularly as dal and as an affordable alternative to meat (Aykroyd et al., 1982) [1] . India ranks as the second-largest producer of lentil in the world after Canada, and leads the world in area under lentil cultivation. In India, the crop is grown extensively in Madhya Pradesh, Uttar Pradesh, Rajasthan, Maharashtra and Bihar. Nutritionally, lentils are rich in protein, complex carbohydrates, minerals and vitamins, along with bioactive compounds such as antioxidants and phytoestrogens (Dhull et al., 2022; Siva et al., 2017) [10,22] . As a legume, lentil is capable of fixing substantial amounts of atmospheric nitrogen through its symbiotic association with Rhizobium bacteria, with reported nitrogen fixation ranging from 10 to 129 kg N/ha depending on environmental conditions (ICARDA, 1989). Under favourable conditions, Rhizobium can fix about 57-100 kg N/ha, thereby enhancing soil nitrogen availability for plant uptake (Wagner, 2012) [25] . However, effective native Rhizobium strains are not present in all soils, which limits nodulation and nitrogen fixation efficiency. In such situations, isolation and inoculation of superior indigenous strains is essential to ensure efficient nodulation and improved productivity. Increasing concerns over the environmental and health hazards associated with excessive use of chemical fertilizers have intensified the search for eco-friendly alternatives such as biofertilizers. Since the efficiency of Rhizobium strains varies with soil type, host genotype and agro-climatic conditions, it becomes essential to isolate and characterize native strains from the region of interest before their use as inoculants. Keeping this in view, the present study was undertaken to isolate, purify and characterize different Rhizobium isolates from root nodules of lentil collected from different locations of Kolhapur district, based on their morphological, cultural and biochemical characteristics.

Materials and Methods

The present investigation was carried out at the Department of Plant Pathology and Agricultural Microbiology, Govt. College of Agriculture, Pune, during 2025-26.

Collection of root nodule samples

Eight field-grown lentil plants having root nodules were randomly sampled from eight different sites located in the Gadhinglaj, Chandgad, Ajara and Bhudargad tehsils of Kolhapur district, with two villages selected from each tehsil. Plants were carefully uprooted and the loosely attached soil was removed by gentle shaking. Roots bearing mature, healthy (pink) nodules were washed thoroughly under running tap water and the nodules were collected in sterile polythene bags, properly labelled, transported to the laboratory, and stored at 4 °C until further use.

Isolation, purification and maintenance of Rhizobium isolates

Isolation of Rhizobium was carried out following the methods described by Somasegaran and Hoben (1994) [23] . Healthy pink nodules were surface sterilized in 0.1% mercuric chloride (HgCl 2 ) for 3 minutes, followed by 75% ethanol for 3 minutes, and rinsed thrice with sterile distilled water. Sterilized nodules were aseptically crushed with a sterile glass rod in 1 ml sterile water, and serial dilutions were prepared. Aliquots (0.1 ml) from 10 -5 , 10 -6 and 10 -7 dilutions were streaked onto Congo Red Yeast Extract Mannitol Agar (CR-YEMA) medium and incubated at 28 ± 2 °C for 3 days. Translucent, white, elevated colonies typical of Rhizobium were selected and purified by repeated streaking on Yeast Extract Mannitol Agar (YEMA) medium. Pure cultures were maintained on YEMA slants at 4-5 °C. Isolates were designated using abbreviations derived from the host legume, tehsil and village of collection.

Morphological characterization

Gram staining was performed to determine cell shape, arrangement and Gram reaction, following standard procedure. Colony morphology (shape, size, elevation and colour) was recorded after 2-4 days of incubation (Nivaas et al., 2019) [16] . Motility was assessed using stab inoculation into motility medium and incubation at 28 °C for 24 hours. A 3% potassium hydroxide (KOH) test was performed to confirm the Gram reaction, wherein the formation of a viscous, thread-like strand upon mixing bacterial growth with KOH solution indicated a Gram-negative reaction (Suslow et al., 1982) [24] .

Cultural characterization

The cultural characteristics of the isolates were studied by growing them on four different solid media viz., Nutrient Agar (NA), Potato Dextrose Agar (PDA), Congo Red Yeast Extract Mannitol Agar (CR-YEMA) and Jensen's Agar medium. Sterilized media were aseptically dispensed into Petri plates, inoculated with the respective isolates, and incubated at room temperature (27 ± 2 °C) for eight days. Colony colour, shape, texture, margin and elevation were recorded on the seventh day of incubation, and colony diameter was measured and analysed statistically using a Completely Randomized Design (CRD).

Biochemical characterization

Biochemical characterization of the isolates was carried out using standard assays, namely the catalase test (Blazevic and Ederer, 1975) [7] , methyl red test, starch hydrolysis test (Eckford, 1927) [11] , oxidase test (Collins and Lyne, 1970) [8] , and indole formation test (Gordon and Weber, 1951) [13] . Each test was performed test along with appropriate controls, and the reaction of each isolate was recorded as positive (+) or negative (-) based on the visible response.

Statistical analysis

The colony diameter data were analysed using a Completely Randomized Design (CRD), and treatment differences were tested through analysis of variance (ANOVA), following the standard statistical methods described by Panse and Sukhatme (1985) [18] .

Results and Discussion

Isolation and purification of Rhizobium isolates Root nodule samples were collected from lentil fields located across eight villages in the Chandgad, Gadhinglaj, Ajara and Bhudargad tehsils of Kolhapur district. Isolation of bacteria from the root nodules was carried out using CR-YEMA medium and the serial dilution technique. Out of the eight samples processed, four Rhizobium isolates were successfully obtained, originating from Bhadgaon, Halkarni, Uttur and Kadgaon, and were designated as RLGB1, RLCH 3 , RLAU 5 and RLBK 7 , respectively (Table 1).

Morphological characterization of Rhizobium isolates

All four isolates consistently exhibited a Gram-negative reaction, indicated by pink coloration of the cells after Gram staining. Microscopic examination revealed rod-shaped cells occurring singly or in pairs, reflecting typical bacillary and diplobacilli arrangements commonly seen in Rhizobium species. All isolates were motile and gave a positive 3% KOH test, confirming their Gram-negative nature (Table 2). This result is in agreement with Nushair et al., (2017) [17] , who reported that Rhizobia strains isolated from lentil root nodules formed circular, pink and convex colonies, and were Gram-negative, rod-shaped and motile. Similar morphological features were also reported by Wani and Khan (2013) [26] and Zafar et al., (2012) [27] , who observed that Rhizobium isolates from the lentil rhizosphere were consistently Gram-negative and rod-shaped, features typical of beneficial soil bacteria involved in nitrogen fixation.

Cultural characterization of Rhizobium isolates

The colony characteristics of the four isolates differed depending on the culture medium used (Table 3). On Nutrient Agar, RLGB 1 formed white, irregular, slimy colonies with lobate margins and raised elevation, while RLCH 3 , RLAU 5 and RLBK 7 exhibited variation in colour and margin. On CR-YEMA medium, RLGB 1 produced pink-creamy, circular, smooth colonies with entire margins and convex elevation, while the remaining isolates formed creamy to white-creamy, circular, smooth colonies. On Jensen's medium, colonies were largely creamy and mucoid with either raised or convex elevation, while on PDA medium, colonies were creamy, mucoid to slimy, with smooth margins. The mean colony diameter of the isolates also varied among the different media ( These findings are in agreement with Deka and Azad (2006) [9] , who characterized fifteen Rhizobium isolates on enriched CR-YEMA medium and reported considerable variation among isolates with respect to colony configuration, margin, elevation and colour. Similarly, Satyadev et al., (2018) [21] isolated nine Rhizobium meliloti strains from fenugreek root nodules on CR-YEMA medium and reported whitish, translucent, sticky, circular colonies measuring 3.2 to 5.2 mm in diameter, with variation observed mainly in colony size.

Biochemical characterization of Rhizobium isolates

Biochemical characterization, including catalase, oxidase, indole formation, starch hydrolysis, methyl red and urease tests, was carried out for all four isolates (Table 5). All the isolates tested positive for catalase, oxidase, starch hydrolysis, methyl red and urease tests, while all four

Conclusion

Out of eight root nodule samples collected from different locations of Kolhapur district, four Rhizobium isolates, namely RLGB 1 (Bhadgoun), RLCH 3 (Halkarni), RLAU 5 (Uttur) and RLBK 7 (Kadgaon), were successfully isolated and purified using CR-YEMA medium. All four isolates were Gram-negative, pink-staining, rod-shaped, motile and gave a positive KOH test. CR-YEMA medium supported the maximum colony growth among the four tested media, and RLGB 1 (13.73 MM) recorded the maximum mean colony diameter. All isolates were positive for catalase, oxidase, starch hydrolysis, methyl red and urease tests, and negative for indole formation, confirming their identity as Rhizobium species. Based on their morphological, cultural and biochemical characteristics, isolates RLGB 1 and RLBK 7 showed the most maximum growth and merit further evaluation for their nitrogen-fixing efficiency and plant growth-promoting potential in lentil.

Acknowledgement

AcknowledgementThe author is thankful to the Department of Plant Pathology and Agricultural Microbiology, Govt. College of Agriculture, Pune, and BNF Mahatma Phule Krishi Vidyapeeth, Rahuri, Maharashtra, for providing the necessary laboratory facilities to carry out this study.

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