Department of Botany, University of Rajasthan, Jaipur, India

*Corresponding author: Indu Singh Sankhla, Department of Botany, University of Rajasthan, Jaipur India. E-mail Id:induiitkgp@gmail.com

Copyright: © Yadav A, et al. 2025. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article Information: Submission: 06/06/2025; Accepted: 11/07/2025; Published: 16/07/2025

The present investigation deals with the phenotypic, biochemical and plant growth promoting activities of root nodule bacteria of Melilotus indicus. A total of 51 isolates were isolated from different sites in the Central Aravalli region. In this study, we found that bacterial colonies showed variation in their colonial characteristics, but most of the colonies were white, opaque, mucilaginous and raised. In salinity tolerance, most of the isolates showed high tolerance and grew up to 4-5% salt concentration, but growth was reduced. In the pH tolerance test, all isolates showed growth from pH 5 to 10. In our investigation, we found that root nodule bacterial strains of Melilotus indicus plants have diverse biochemical and plant growth promoting activities. Sugar utilization and antibiotic resistance varied among isolates; fructose was the most utilized sugar, while adonitol was the least. Most isolates were resistant to nitrofurantoin and carbenicillin. Catalase and oxidase activities were present in most isolates. Many isolates produced IAA (42), ammonia (43), and solubilized phosphate (14). Pectinase, cellulase and amylase activity were also noted in a few isolates. The findings highlight the functional diversity of Melilotus indicus root nodule bacteria and their potential applications in sustainable agriculture. Overall results indicated that Melilotus indicus is associated with a wide range of rhizobia. Such type of root nodule bacterial strains can be utilized as a part of biofertilizers to enhance legume crop productivity, sustainable agriculture and reduce the use of chemical fertilizers.

Keywords:Aravalli; Root Nodule Bacteria; Plant Growth Promoting Activity; Melilotus Indicus

Nitrogen is a critical essential element for all living organisms. It aids in plant growth, development and crop yield. However, its availability in the environment is approximately 78%, but not in a plant accessible form. In this era, with an increasing population, nutrient paucity in soil leads to a global food production chain crisis. To feed the people, it is vital to amplify the food production and yield of crops. For this, farmers use chemical nitrogen fertilizers which not only extortionate but also lead to an imbalance in microbial diversity, decrease soil fertility and cause environmental pollution, such as polluting water resources[1-3]. Globally, Nitrogen fertilizer consumption was 112.5 million tons in 2015 and is currently approximately 120 million tons, which is likely to increase with the world population[4,5]. So, it is crucial to and an alternative approach which not only ecofriendly but also economical and enhances the yield. Biological nitrogen fixation is the prime naturally governing alternative in which microsymbionts convert free nitrogen to plant accessible form either symbiotically or free-living. The primary source of nitrogen in the ecosystem is the symbiotic relationship. Legumes and microsymbionts, symbiotic relationships makeup over 60% of total BNF [6]. Fabaceae, more oen known as legumes, is the third largest family aer Asteraceae and Orchidaceae and it consists of around 770 genera and 19500 species[7,8]. Rhizobia have the ability to form root and stem nodules and establish symbiotic relationships with leguminous plants [9].Currently, rhizobia with 21 genera are classified into three classes: alpha proteobacteria, beta proteobacteria and gamma proteobacteria [10].

Melilotus is a forage legume of Fabaceae; it comprises approximately 25 species of annual or perennial herbs distributed throughout the world, native to Africa, Europe and Asia [11,12]. In India, three species (Melilotus albus, Melilotus offcinalis and Melilotus indicus) have been reported, while two Melilotus indicus and Melilotus albus have been reported in Rajasthan [13]. Melilotus indicus (Indian sweet clover) is a Eurasian species, is now found globally. It naturally grows in the Aravalli range, the oldest folded mountain system, known for its rich legume biodiversity. Although much research has focused on cultivated crops, the microsymbionts of wild legumes in the Aravalli region remain understudied [14].

Recently, wild legume microsymbionts have drawn interest due to their capability to bear stressful conditions like drought, salinity and temperature. So, there is a scope to characterize their microsymbionts and inoculate them in cultivated plants to increase productivity and soil fertility. Due to little attention to the diversity of its microsymbionts, Melilotus indicus from the Aravalli region was selected for its potential as a host plant to explore and utilize native root nodule bacteria in sustainable agriculture.

A survey was conducted during the winter season (December- March) to check the nodulation status of Melilotus indicus. Nodules were collected from different sites of Jaipur and Ajmer during February - March. The plants were excavated with a root system during collection, and nodules were excised. Subsequently, the morphological characteristics of collected nodules were recorded and anatomical studies were done through transverse and longitudinal sections of fresh root nodules.

For isolation, the excavated root nodules were washed with tap water and sterilized according to standard methods [15]. Sterilized nodules were squeezed by sterile forceps and then streaked on Congo Red Yeast Extract Mannitol Agar plates (a selective media for root nodule bacteria) and incubated at 28°C for 48-72 hours. To attain the pure culture, single colonies were picked up and re-streaked. Once suffcient growth was observed, these plates were stored at 4°C for further study of phenotypic, biochemical and plant growth promoting activities of purified isolates.

Colony characteristics of root nodule bacterial strains, such as colony colour, mucilage production, gumminess, elevation, shape, margin, texture, opacity, and surface appearance, were recorded for each isolate. For this, all isolates were streaked on YEMA plates and incubated at 28°C for 48-72 hours and observed regularly to monitor the development and distinct features of the bacterial colonies.

All isolated root nodule bacterial strains were characterized for their phenotypic attributes such as acid or alkali production, NaCl tolerance, pH tolerance, sugar utilization ability and intrinsic antibiotic resistance pattern.

For determination of NaCl tolerance, all the bacterial isolates were streaked on the YEMA plates supplemented with dierent NaCl concentrations (0.5%, 1%, 2%, 3%, 4% and 5%) to assess their tolerance to salinity. Aer inoculation, plates were incubated at 28°C for 3-4 days and the growth of isolates at dierent concentrations was noted [16].

To assess tolerance to acidic or alkaline pH, bacterial cultures were streaked on to YEMA plates with a pH range of 5 to 10 and incubated for 48–72 hours at 28°C. The pH of the medium was adjusted using 1N NaOH and 1N HCl to achieve the target pH range. [16]. The visible growth at different pH values was recorded as positive results.

This test was performed to check whether bacterial isolates show an acidic or alkaline reaction. For this, YEM broth supplemented with bromothymol blue was used. Inoculated tubes were incubated at 28°C and 100 rpm for 3-4 days in a shaking incubator. Initially, broth colour was green at neutral pH; acid producing isolates changed it to yellow, while alkali producing isolates changed it too blue [15].

All bacterial strains were tested for their sugar utilization pattern with 21 different sugar discs. For this, 24 well plates containing Andrade’s peptone water were inoculated with freshly grown root nodule bacterial cultures. One sugar disc was placed in each well. The plates were incubated at 28°C for 48 hours. A positive sugar utilization result was indicated by the development of a pink colour in the medium.

The antibiotic susceptibility or resistance of root nodule bacterial strains was assessed using the antibiotic disc method [16]. For this, YEMA plates were used as medium. Fresh cultures of root nodule bacterial strains were evenly spread (swabbed) on the media and antibiotic discs were placed on the plates. Further, these plates were incubated at 28°C for 24-48 hours. The presence of clear zone (zone of inhibition) around each disc was recorded as susceptibility of isolates for the respective antibiotics.

All the isolates were characterized for their various biochemical and PGP activities including oxidase activity, amylase production, indole production, nitrate reductase, citrate utilization, gelatin hydrolysis, catalase activity, protease activity [16], cellulase activity [17], phosphate solubilization [18], Indole Acetic Acid production [19] and ammonia production [20].

The nodulation status of Melilotus indicus at seven sampling sites including the Rajasthan University Campus, and Niwaru (Jaipur), as well as Doomara, Govindgarh, Rampura Dabla, Akhepura and Pisangan (Ajmer), was successfully studied. [Figure 1A-1B] illustrates the overview and survey of various sampling sites. During the survey,

Melilotus indicus was consistently observed at all sampling sites [Figure 1C-1D] with nodulation present at each location across both Jaipur and Ajmer, indicating its widespread presence and symbiotic activity in these regions. Aer careful examination of the excavated root system, it was observed that nodules were mainly present on secondary and tertiary roots rather than primary and crown regions. Morphology of the root nodules showed variation during their developmental stages as shown in [Figure 1E]. In the initial stage, root nodules were globular to oblong, but as they matured, they became elongated and branched. The colour of nodules was dark brown to black when they matured but initially, they were light brown. Similar to the present study, the morphology of root nodules of 3 genera (Trifolium, Melilotus and Medicago) of the tribe Trifoliae was investigated by other researchers and it was found that in these genera initially the nodules were spherical, but matured ones were cylindrical or variously lobed [21]. A similar study was also conducted on 5 wild legumes (Alhagi murarum, Melilotus indicus, Medicago intertexta, Trifolium resupinatum and Trigonella hamosa) of the Nile Valley and it was observed that the nodules of Melilotus indicus, Medicago intertexta were elongated and sometimes branched, while nodules of Alhagi murarum, Trifolium resupinatum and Trigonella hamosa were globose to elongated [22]. For anatomical studies, we took transverse and longitudinal sections of root nodules and subsequently stained them with toluidine blue and then observed them under the microscope. The sections of root nodules clearly showed the presence of vascular supply and an infection zone (bacteroid) within the cortex region as shown in [Figure 2A]. Similar findings were reported by other researchers, who observed bacteroid-filled infection zones in nodules collected from the Thar Desert [23,24].

A total of 51 root nodule bacterial strains were isolated and purified from root nodules of Melilotus indicus. All isolates showed immense growth on CR-YEMA media. Colony characteristics of isolates showed variations [Table 1], but most of the colonies were white, opaque, mucilaginous, with smooth margins and raised [Figure 2B]. Similar to our results, variations in colony characteristics of the root nodule bacterial strains were also reported by other researchers[23,24]. In the NaCl tolerance test, 30 isolates showed growth up to 4%, while 11 isolates grew up to 5% concentration, which means they show high tolerance towards salinity. Results for NaCl tolerance are shown in [Table 2] and [Figure 2D]. However, the growth of isolates was decreased as compared to the control with the increasing salt concentration. Salt tolerant isolates are a good opportunity to fix nitrogen in highly saline and alkaline soil to enhance the yield. Salt tolerant rhizobial inoculants mitigate the effect of salinity and enhance yield in soybeans [25]. Similarly, high salinity tolerance (up to 6%) of root nodule bacteria associated with Medicago littoralis and Melilotus indicus was observed in the Algerian Sahara [26]. Various phenotypic and biochemical characteristics of each isolate are shown in [Table 2] . In the pH tolerance test, all isolates

showed visible growth on culture media having a pH of 5 to 10. Under low pH (acidic) conditions, the growth of isolates was significantly reduced, whereas at high pH (alkaline) conditions, growth remained relatively stable compared to the control (Table 2) (Figure 2E). These findings suggest a strong adaptive capability of the isolates to alkaline conditions, reflecting their ecological suitability for the naturally alkaline soils of the Aravalli region. All isolates tested for sugar utilization and intrinsic antibiotic resistance test showed significant variation in their carbon utilization (Table 3)(Figure 2C) and intrinsic antibiotic resistance pattern (Table 4) (Figure 2G). In the present study, we found that out of 21 sugars, fructose was the most and adonitol was the least utilized sugar by the isolates. Different utilization patterns of sugar can be used to identify the taxon [27]. Dierent sugar utilization patterns from the current investigation showed resemblance with the study conducted on the Rhizobium strain of Vicia faba root nodules in Ethiopia [28]. In Intrinsic antibiotic resistance pattern, an investigation conducted on Rhizobium strains of chickpea root nodules in Turkey, they observed that their majority of isolates show resistance to chloramphenicol, kanamycin and streptomycin [29]. In our investigation of antibiotic resistance, most of the isolates showed resistance towards nitrofurantoin and carbenicillin antibiotics. In the arid region of Morocco, endophytes associated with chickpea (Cicer arietinum), faba bean (Vicia faba), lentil (Lens culinaris) and common bean (Phaseolus vulgaris) were tested for antibiotic resistance and found that isolates were resistant to erythromycin, ciprooxacin, ampicillin and tetracycline [30]. In our acid-alkali production test, 5 isolates showed alkaline production, while 32 isolates showed acid production and the remaining 14 isolates showed neutral reaction [Table 2] [Figure 2F]. A study conducted on characterization of root nodules microsymbionts of Trigonella foeniculum in Western Rajasthan found that most of their isolates are acid producing, which is a characteristic of fast-growing bacteria [31]. The results of acid-alkali production from the present investigation

showed resemblances with the study conducted on root nodules microsymbionts of Mung bean in Gypsiferous soil. They found that 9 out of 10 isolates are acid producing (fast growing) [32] A similar study was conducted on microsymbionts of Zornia gibbosa in the Aravalli range and found that out of 41 isolates 8 isolates showed acid production while 8 isolates showed alkali production [33].

The variations in biochemical enzymatic activity of root nodule bacterial isolates were reported by various authors[26-28, 33]. In the present study, all isolates were characterized for their biochemical properties and plant growth-promoting activities. The results are presented in (Table 2) and (Table 5) respectively, and illustrated in (Figure 2A-2M). In addtion, percentage of root nodule bacterial strains showing sugar utilization, antibiotic resistance, various biochemical and PGP activities are shown in (Table 6) for more clarity and understanding. In our study, 34 isolates showed negative results for gelatin hydrolysis. Similar results were observed in root nodules bacterial strains isolated in Pisum sativum, who stated that the majority of isolates showed a negative result of gelatin hydrolysis [34]. Only one isolate (MI46) was found positive for indole production. A study conducted on root nodule bacteria of Zornia gibbosa in the central Aravalli range had similar results, in which they found only one isolate (Z22) positive for indole production[33]. Indole production results were also observed in rhizobial strains of lablab, cowpea and elephant plants [35].

Out of 51 isolates, 50 and 45 isolates showed a positive result for catalase and oxidase activity, respectively. With high catalase and oxidase activity, root nodule bacterial isolates enhance nitrogen fixation. Similarly, all 8 rhizobial isolates associated with Cajanus cajan in Telangana showed positive for catalase activity, while 7 isolates showed oxidase activity [36]. Rhizobium strains isolated from Cicer arietinum and lentis also showed positive for catalase and oxidase activity [37,38]. Citrate utilization activity showed whether root nodule bacterial isolates utilize citrate as a carbon source or not. In our findings, 10 isolates showed a positive result for citrate utilization, while 14 isolates showed nitrate reduction. Root nodule bacterial strains of Lablab purpureus and Vigna sinens is resulted in

negative for citrate utilization [39], while another study found positive for all isolates in root nodule bacterial strains (Mesorhizobium) in chickpea [40]. Cellulase and amylase activity are significant in the field of biotechnology and various industries. In our study, 10 isolates showed cellulase activity. Similar results were observed in the central Aravalli region, 27 % isolates associated with root nodules of Zornia gibbosa exhibited cellulase activity[33]. Similary in Ethiopian region 48% root nodule bacterial strains asociated with groundnut exhibited cellulase activity [41]. In addition, only two isolates (MI26 and MI34) from the present study showed starch hydrolysis. The ability of the isolates to hydrolyze starch indicates their potential role in decomposing complex carbohydrates, which may contribute to improved nutrient availability and support plant growth.

Root nodule bacterial isolates were investigated for multiple plant growth promoting activities (Table 5). Aer nitrogen, phosphorus is the most critical element that limits plant growth. However, it is found in high concentrations in some soils, but the plants utilizable form of phosphorus is finite in soil because either, it forms insoluble precipitates with metals (reaction with highly reactive Al3+ and Fe3+ in acidic, and Ca2+ in calcareous or normal soils) or is found in organic form which is directly not assimilated by plants [42,43]. In the present study 14 isolates showed a positive result for phosphate solubilization. Phytohormones are low molecular weight chemical messengers that coordinate cellular activities and stimulate plant growth, and development such as Indole acetic acid [44]. Out of 51 isolates, 42 isolates showed positive results for indole acetic acid production. Similarly, the positive result for IAA and phosphate solubilization was observed by other researchers [33,45]. In addition, the majority of isolates (43) gave positive results for ammonia production, while 7 isolates showed protease activity. A similar study, in the context of ammonia production, was reported from isolates of root nodules of Sulla flexuosa [46]. During legume rhizobia symbiosis, rhizobia faced penetration problems due to pectin. So the pectinase activity may enhance penetration and nodule organogenesis. Out of 51, only 5 isolates showed a positive result for pectinase activity. Similar findings were reported for root nodule bacterial strain of Glycine max. In their study, they found 5 isolates (out of 17) were positive for pectinase activity [47]. In the present investigation, we found the diversity between rhizobial isolates in terms of biochemical and PGP activities. Applying beneficial microsymbionts that possess plant growth-promoting features would help to remediate the crop soil by reducing the need for chemical fertilizers and enhancing adaptability for sustainable agriculture.

Melilotus indicus is a good fodder plant and is widely distributed in the Aravalli region. In the present study, it was found that Melilotus indicus had a well nodulated root system in all sampling sites in the Central Aravalli region. All the 51 root nodule bacterial isolates from the current investigation showed high tolerance towards salinity and had significant variation in their sugar utilization and intrinsic antibiotic resistance patterns. These isolates were highly diverse in their physiological and biochemical characteristics and also had plant growth-promoting activities. Therefore, these isolates can be utilised as part of biofertilizers that can reduce the use of chemical nitrogen fertilizers and favour sustainable agriculture.