Harnessing Native Synergy: Screening And Evaluation Of Multi-Spectrum Indigenous Rhizobium Strains For Sustainable Agriculture

Modern agriculture faces the dual challenge of increasing food production for a rapidly growing global population while minimizing environmental degradation. Although synthetic chemical fertilizers substantially improved crop productivity during the Green Revolution, their excessive and prolonged use has resulted in serious ecological and health concerns, including soil degradation, groundwater contamination, nutrient imbalance, and greenhouse gas emissions (Tilman et al., 2002; Savci, 2012). Nitrous oxide (Nâ‚‚O), a major byproduct of nitrogen fertilizer application, possesses a global warming potential nearly 300 times greater than carbon dioxide (Galloway et al., 2008; Snyder et al., 2009). Furthermore, nitrate leaching into drinking water has been associated with methemoglobinemia (“blue baby syndrome”) and elevated cancer risks in adults (Camargo and Alonso, 2006; Ward et al., 2018). With global fertilizer consumption projected to exceed 205 million tonnes by 2025 (FAO, 2019), the development of sustainable and environmentally compatible agricultural alternatives has become imperative.

Among the promising alternatives, plant growth-promoting rhizobacteria (PGPR) have gained considerable attention due to their ability to enhance plant productivity while reducing dependency on chemical fertilizers (Vessey, 2003; Backer et al., 2018). Within this group, Rhizobium species represent one of the most extensively studied and agriculturally important symbiotic bacteria. These Gram-negative, motile microorganisms establish symbiotic associations with leguminous plants through the formation of specialized root nodules, where atmospheric nitrogen (N₂) is converted into plant-available ammonia (NH₃) via biological nitrogen fixation (BNF) mediated by the nod, nif, and fix gene clusters (Oldroyd et al., 2011; Masson-Boivin and Sachs, 2018). This natural process significantly reduces the requirement for synthetic nitrogen fertilizers while improving soil fertility and ecosystem sustainability.

Beyond nitrogen fixation, Rhizobium exhibits multiple plant growth-promoting traits that contribute to enhanced crop productivity and soil health. These bacteria facilitate nutrient mobilization through POâ‚„ and K solubilization, synthesize phytohormones such as indole-3-acetic acid (IAA), cytokinins, and gibberellins, and improve plant tolerance against abiotic stresses including drought and salinity (Richardson et al., 2009; Sharma et al., 2013; Egamberdieva et al., 2017). Additionally, the production of compounds such as lumichrome, riboflavin, siderophores, and extracellular enzymes contributes to improved nutrient acquisition, organic matter decomposition, and maintenance of a balanced rhizospheric microbial community (Dakora, 2003; Ahmad et al., 2008; Glick, 2012).

Commercial Rhizobium-based biofertilizers often show inconsistent performance in the field because non-native strains may not adapt well to local soil and environmental conditions (Malusá et al., 2012; Bashan et al., 2014). In comparison, indigenous Rhizobium strains are naturally suited to their native environment and usually perform better in terms of survival, root colonization, and nodulation efficiency. This makes them valuable candidates for developing effective region-specific biofertilizers (Thrall et al., 2007; Zhang et al., 2020).

This study focused on isolating and characterizing indigenous multi-spectrum Rhizobium strains from five leguminous crops. The isolates were examined for their biochemical traits, compatibility with beneficial soil microorganisms, and ability to promote plant growth under controlled pot conditions. The study highlights their potential as sustainable and locally adapted biofertilizers for improving crop growth and soil health.

3. MATERIALS AND METHODS

Sample Collection and Surface Sterilization:

Healthy root nodules were collected from five leguminous crops, namely Arachis hypogaea (groundnut), Vigna mungo (black gram), Vigna unguiculata (cowpea), Vigna radiata (green gram), and Cajanus cajan (pigeon pea), grown in agricultural fields at Bilda village, Tal. Phulambri, Dist. Chhatrapati Sambhajinagar, MS., India (20.0458562°N, 75.4015061°E). The nodules were separated aseptically, surface sterilized using standard procedures. (Vincent, 1970).

Cultural, Microscopic, Growth, and Biochemical Characterization of Rhizobium Isolates:

Preliminary characterization of the isolates was performed on Yeast Extract Mannitol Agar (YEMA) supplemented with Congo red (CR) and Bromothymol blue (BTB) following incubation at 28±2°C for 48–72 h. Colony morphology, dye absorption, and colour changes were recorded for identification of typical Rhizobium colonies (Purwaningsih et al., 2021). Microscopic characterization was carried out using Gram staining (Gram, 1884).

Growth characteristics were evaluated in Yeast Extract Mannitol (YEM) broth by measuring optical density (OD₆₀₀ and OD₆₂₀) and pH changes. Fast-growing isolates were selected for further studies (Somasegaran et al., 1994; Vincent et al., 1970).

Biochemical characterization included carbohydrate utilization, substrate degradation, enzymatic assays, and plant growth–promoting traits. Tests such as TSI, sugar fermentation, starch hydrolysis, citrate utilization, catalase, oxidase, gelatinase, lipase, urease, lysine decarboxylase, phosphate solubilization, IAA production, ammonia production and IMViC were performed using standard protocols (Hajna, 1945; Pikovskaya, 1948; Cappuccino and Sherman, 2014, Møller 1955, Sierra 1957). Results were interpreted based on colour changes, clear zones, gas production, and other visible reactions.

DNA Isolation of Selected Rhizobium Isolates:

Genomic DNA was isolated from selected fast-growing Rhizobium isolates using standard extraction and purification procedures. DNA quality and quantity were assessed by agarose gel electrophoresis and spectrophotometric analysis. The purified DNA was further used for PCR amplification and molecular characterization (Sambrook and Russell, 2001; Ausubel et al., 1995).

Microbial Compatibility Assay:

The compatibility of selected Rhizobium isolates with common soil microorganisms was tested using the cross-streak method on nutrient agar plates. Microorganisms including Escherichia coli, Azotobacter, Pseudomonas, Aspergillus niger, and Aspergillus flavus were grown alongside the Rhizobium cultures to observe compatible or inhibitory interactions (Granada Agudelo et al., 2023).

Pot Assay for Evaluation of Plant Growth Promotion and Nodulation:

A pot assay was performed to evaluate the plant growth–promoting and nodulation ability of selected Rhizobium isolates using fenugreek (Trigonella foenum-graecum) seeds. Sterilized soil was filled in pots, and healthy seeds were sown under aseptic conditions. The experimental setup included an uninoculated control, individual treatments with isolates RHZ7 and RHZ20, and a consortium of RHZ2, RHZ7, RHZ12, RHZ20, and RHZ23 grown in Yeast Extract Mannitol (YEM) broth. The respective inocula were applied to the pots, which were maintained under regular watering conditions. Plant growth parameters and nodulation were periodically observed and recorded in all treatments (Vincent et al., 1970; Somasegaran et al., 1994).

Protein Profiling of Fenugreek Leaves using SDS-PAGE:

Protein profiling of fenugreek leaves from control and Rhizobium-treated plants was performed using SDS-PAGE following the method of Laemmli (1970). Leaf proteins were extracted, centrifuged, and equal quantities of protein samples were denatured and loaded onto SDS-PAGE gel. Following electrophoresis, gels were stained with Coomassie Brilliant Blue to visualize protein bands. Banding patterns of treated and control samples were compared using a protein marker to assess variations in protein expression.

4. RESULTS AND DISCUSSION

Cultural and Microscopic Characterization:

Distinct Rhizobium colonies on YEMA look as if circular, smooth, translucent, and mucoid after incubation. Poor Congo red absorption and the blue-to-yellow colour change on Bromothymol blue medium indicated typical Rhizobium characteristics and acid production. Gram staining showed Gram-negative, rod-shaped cells occurring singly or in small groups, confirming the purity and identity of the isolates.

CONCLUSION

This study demonstrates that indigenous Rhizobium isolates possess strong potential as sustainable biofertilizers. The selected strains enhanced plant growth, root development, and nodulation in Fenugreek, while also showing compatibility with native soil microflora. These conclusions support their possible use as eco-friendly alternatives to chemical fertilizers for improving soil fertility and crop productivity while maintain soil microbial peace.

Further research involving molecular identification, field trials, and formulation development will help establish efficient, region-specific biofertilizers for sustainable agricultural applications.

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