Effect of Biofertilizer on Agronomic Properties of Phaseolus Vulgaris

Effect of Biofertilizer on Agronomic Properties of Phaseolus Vulgaris

*Tolani, M. S., Aborisade, W. T. and Enu, K.B.

Department of Microbiology Unit, Kwara State University, Malete, Nigeria

*Corresponding Author

DOI: https://doi.org/10.51244/IJRSI.2025.12020060

Received: 07 February 2025; Revised: 13 February 2025; Accepted: 15 February 2025; Published: 15 March 2025

ABSTRACT

Biofertilizers have attracted considerable attention among agronomists and soil scientists due to their potential for promoting sustainable agricultural practices. This study investigates the effects of biofertilizers on some agronomic traits of Phaseolus vulgaris and rhizobacterial populations. A mesocosm experiment was conducted in a greenhouse over five weeks using a randomised block design with six replications. The treatments included microbial inoculants (Rhizobium sp., Azotobacter sp. and Pseudomonas sp.), a consortium of these microorganisms, NPK 315 fertilizer and positive control. Key parameters measured were seed germination, leaf, stem and shoot growth, as well as rhizosphere bacterial populations. Results showed significant variability in seed germination rates. Treatment F (NPK 315) achieved the highest germination rate (90% by Day 6), statistically similar to the positive control. Treatment C (Pseudomonas sp.) had the lowest germination rates (p < 0.05). Treatments A (Rhizobium sp.), B (Azotobacter sp.) and F (NPK 315) produced the greatest leaf lengths, while Treatment E (positive control) had the shortest (p < 0.05). Treatment B (Azotobacter sp.) outperformed others in leaf width and it also led to the highest shoot heights, although with some variability. Treatment C (Pseudomonas sp.) consistently showed the lowest shoot height. Stem diameter growth was most stable in Treatment D (Consortium), while Treatments E and F showed significant improvements after Week 2 (p < 0.05). Treatment B (Azotobacter sp.) consistently promoted the highest leaf growth, while Treatment E had the fewest leaves (p < 0.05). Treatment D (Consortium) had the highest bacterial populations, indicating a synergistic effect. Treatments A and B had moderate bacterial loads, while Treatment C (Pseudomonas sp.) exhibited the lowest bacterial growth. This research demonstrates the potential of biofertilizers, particularly Azotobacter sp., in enhancing agronomic traits and microbial activity in Phaseolus vulgaris.

Keywords: Agronomic properties, Azotobacter, Rhizobium, Pseudomonas, Phaseolus vulgaris

INTRODUCTION

The global demand for food continues to rise, driven by population growth and changing dietary preferences (Godfray et al., 2018). This increasing demand places immense pressure on agricultural systems to produce more food while minimising environmental impact. In Africa, where agriculture is a cornerstone of many economies, sustainable agricultural practices are crucial for ensuring food security and economic development (Raimi et al., 2017). However, several challenges identified to hinder crop productivity in Africa, include poor soil quality, extreme weather conditions such as high temperatures and drought, economic constraints, limited access to technology and inefficient farming practices (Gregory et al., 2005; Tully et al., 2015). Currently, among the methods employed in farm management for high crop yields and enhancing productivity are the use of organic and chemical fertilizers (Rahman et al., 2018).

Chemical fertilizers have transformed agriculture by increasing crop yields and enhancing global food production (Penuelas et al., 2023). However, their excessive use has resulted in significant environmental and health challenges. Nutrient runoff from agricultural fields pollutes water bodies, leading to eutrophication and damaging aquatic ecosystems (Albou et al., 2024). Prolonged use of chemical fertilizers degrades soil health, diminishing its fertility and nutrient retention capabilities (Howe et al., 2024). Furthermore, the processes involved in the production of chemical fertilizers contribute to greenhouse gas emissions, worsening climate change (Menegat et al., 2022). Additionally, exposure to specific chemicals in fertilizers can pose health risks to humans, including respiratory issues and a higher likelihood of cancer (Ahmad et al., 2024). To address these problems, it is essential to adopt sustainable practices such as the use of biofertilizers.

Biofertilizers are bio-based fertilizers that contain living microorganisms capable of promoting plant growth by enhancing nutrient availability in the soil (Daniel et al., 2022). These microorganisms include nitrogen-fixing bacteria, phosphate-solubilizing bacteria (PSB) and mycorrhizal fungi. Unlike chemical fertilizers that can lead to soil degradation and environmental pollution, biofertilizers offer an eco-friendly alternative that not only improves soil health but also enhances plant growth (Jana et al., 2024). As part of their benefits, biofertilizers can bolster plant resistance to diseases by suppressing soil-borne pathogens and fostering a healthier rhizosphere environment thereby reducing the need for chemical pesticides (Ghimirey et al., 2024).

The global biofertilizer market is expanding, driven by countries such as Argentina, Canada, China, Europe, India and the United States. These nations recognize the advantages of biofertilizers and are promoting their adoption, as evidenced by their developed biofertilizer markets (Aloo et al., 2022). These natural products are gaining recognition for their ability to improve agricultural productivity while minimizing reliance on chemical inputs (Ammar et al., 2023). In Africa, particularly in Nigeria, the adoption of biofertilizers is increasing due to their environmental benefits and potential to improve crop yields (Irewale et al., 2024). However, their widespread use is still limited compared to chemical fertilizers. Several factors contribute to this low adoption rate, including lack of awareness, limited access, doubts about effectiveness, high initial costs and insufficient technical support (Raimi et al., 2021).

Seed treatment methods involve the application of chemical agents to seeds before sowing. These agents can be synthetic or naturally derived and sometimes beneficial microorganisms are used in pre-sowing treatments. Seed treatments are highly effective, providing precise product delivery to the field and excellent pest control (Aborisade et al., 2021). Moreover, seed treatments reduce human and environmental exposure to agrochemicals, making them an environmentally friendly option for pest and pathogen control. Irfan et al. (2020) highlighted the effectiveness of seed treatments in protecting plants from pests and pathogens, particularly during critical developmental stages. Additionally, these treatments are user-friendly for growers, crop-responsive and safe for the environment.

Various methods for applying biofertilizers in seed treatment have been developed, including direct seed coating, soaking seeds in diluted solutions and using filter paper soaked in biofertilizer solutions (Mhada et al., 2021). These methods ensure that beneficial microbes are readily available during critical stages of seed germination and early plant development. As research continues to explore the multifaceted benefits and applications of biofertilizers, their role in modern agriculture is expected to expand significantly, offering promising solutions for enhancing crop productivity sustainably.

The experimental designs helps to minimize the impact of any spatial variations withing the greenhouse and increases the statistical power of the experiment, the replicates for each treatment enhances reliability of result.

Phaseolus vulgaris, commonly known as the common bean, is one of the most widely cultivated legumes in the world, valued for its nutritional benefits and economic importance (Uebersax et al., 2023). This annual crop is essential for food security, especially in developing countries, where it provides a staple source of protein, carbohydrates and vital micronutrients (Lisciani et al., 2024). To optimize bean production, effective management practices are crucial; these practices ensure that the plants receive the necessary nutrients, promote healthy growth and maximize yields. By implementing sustainable farming methods, farmers can enhance soil health, decrease dependence on excessive chemical fertilizers and improve long-term bean production. Nevertheless, supplemental fertilization is often required to meet the nutrient needs of the crop and achieve optimal yields (Du et al., 2021). Therefore, this study examines the effect of biofertilizer on the agronomic properties of Phaseolus vulgaris.

MATERIALS AND METHODS

The Study Area

The study was conducted at the greenhouse facility of Federal University Lokoja farm, Felele, Lokoja Nigeria.

The coordinate of the study area is within the Latitude 7° 47’ N and Longitude 6° 40’ E. The area is characterized by bimodal patterns of rainfall from April to October and a dry season that lasts from November to March (Olutimayin and Aribisala, 2020).

Figure 1: Location of the study area

Screening and Isolation of bacterial groups from soil samples

Selective media used to isolate nitrogen-fixing bacteria, phosphate-solubilizing bacteria and potassium-solubilizing bacteria were Ashby’s mannitol agar, Pikovskaya’s agar and Aleksandrov’s agar respectively.  Surface soil samples (0-10 mm depth) of Federal University Lokoja farm areas were collected with a clean soil auger and stored in a sterile container. The bulk soils were sieved through a 2 mm mesh to remove debris. Five grams from the soil samples were then suspended in a 250 ml conical flask. The suspension was incubated in an orbital shaker at room temperature (28 ^oC) for 5 days.

One millilitre (1 ml) from the broth culture was subjected to serial dilution up to 10^-6 concentrations plated on their respective media plates and incubated at room temperature for 72 hours. Morphologically distinct colonies were observed and subsequently, sub-cultured to obtain pure colonies of the isolates. The isolates were stored on agar slant for further studies.

Identification of Bacterial Isolates

Following the standard method, the bacterial isolates were identified based on their morphological (size, shape, colour and elevation), biochemical (Gram reactions, urease test, sugar utilization test, catalase test and oxidase test) and molecular characteristics (Chesbrough et al., 2003).

Effect of Biofertilizer Treatments on the Growth of Phaseolus vulgaris (L.)

The experimental field was laid out in a Complete Randomized Design based on 6 treatments and 6 replicates, in a unit plot size of 1.80 m x 1.00 m with a plant-to-plant spacing of 30cm and 20cm, respectively..  The 6 treatment groups used for this experiment were: “A”: 20g of Rhizobium biofertilizer to 1 kg of bean seed; “B”: 20g of Azotobacter  biofertilizer to 1 kg of bean seed; “C‟: 20g of Pseudomonas biofertilizer to 1 kg of bean seed; “D‟: 20g of Consortium (Rhizobium ₊ Azotobacter  ₊ Pseudomonas) biofertilizer to 1 kg of bean seed; “E”:(control), untreated seeds; “F”: 5g of  NPK fertilizer to 1 kg of bean seed.

Phaseolus vulgaris (L.) seed (1 kg) was purchased from a certified agro-products vendor (MANSON AGRO AND CHEMICALS), in Lokoja. The seeds were subjected to water-floating viability tests prior to sowing. The viable seeds were treated with biofertilizer through immersion into a suspension of bacterial culture of 0.5 McFarland’s standard for 20 minutes. The treated seeds of Phaseolus vulgaris (L.) were then planted and the germination rate was observed between 3^rd to 10 days after planting. Other growth parameters (Stem diameter(cm), shoot height(cm), leaf length(cm), leaf width(cm), leaf number) were observed at 6-day intervals over 35 days of the planting period.

Evaluating the bacterial load in the rhizosphere region of growing Phaseolus vulgaris seedlings

A suspension of 1 g from the composite soil samples from seedlings of Phaseolus vulgaris in 9 ml distilled water was prepared and further diluted serially (ten-fold). About 0.1 ml of 10⁵ diluted concentrations were inoculated onto solid agar media using the pour plate method in three replicates. The inoculated plates were inverted and incubated at room temperature (25 ± 2 ^oC) for 48 hours before enumeration by counting the visible colonies formed. The media used for the estimation of rhizobia and phosphate solubilizing bacterial Congo-Red-Yeast-extract-Mannitol agar, Pseudomonas agar, Ashby agar and Nutrient agar respectively.

Soil samples were collected from the rhizosphere of Phaseolus vulgaris seedlings at different growth stages on the 35^th day after planting. The samples were placed in sterile plastic bags and transported to the laboratory under cold conditions (4°C) for immediate processing. To determine the bacterial load, 1 g of soil was suspended in 9 mL of distilled water and serially diluted to 10⁻⁷ fold. A 0.1 mL aliquot from each dilution was plated onto Nutrient Agar using the spread plate technique and incubated at 28 °C for 48 hours. After incubation, colony-forming units (CFUs) were counted and recorded as CFU/g of soil. Distinct bacterial colonies were selected based on morphological characteristics such as shape, colour, elevation, and margin. Gram staining was performed to differentiate bacterial types, while biochemical tests (catalase, oxidase, motility, and sugar fermentation) were conducted for further characterization.

Data analysis

The data collected were subjected to Analysis of Variance (ANOVA) and the differences among the mean values were established using the Least Significant Difference (LSD) at a 5% level of significance. Analyses were carried out on the IBM-SPSS version 23.

 RESULTS AND DISCUSSION

Impact of Biofertilizer on germination rate and other agronomic parameters of Phaseolus vulgaris

The results of the germination rate and other agronomic parameters of Phaseolus vulgaris were interpreted statistically. If two or more treatments share the same letter (a, b, c) within a column (day or week), it means their mean parameters are not significantly different from each other at the 5% significance level. If two treatments have different letters within a column, it means their mean parameters are significantly different from each other at the 5% significance level.

The treatment used for the experimental design as follows : A= Rhizobium sp.; B = Azotobacter sp. ; C= Pseudomonas sp. ; D= Rhizobium sp ₊ Azotobacter sp.₊ Pseudomonas sp., which shows the result as seen in the Figures below.

Figure 2: Graphical representation of germination rate over time.

Figure 3: Graphical representation of leaf length over weeks for different treatments.

Figure 4: Graphical representation of leaf width over weeks for different treatments.

Figure 5: Graphical representation of Shoot Height over weeks for different treatments.

Figure 6: Graphical representation of Stem diameter over weeks for different treatments

Figure 7: Graphical representation of Number of Leaves over weeks for different treatments.

Figure 8: Graphical representation of Average bacteria load over weeks for different treatments.

The germination rates for treated Phaseolus vulgaris (common bean) seeds are shown in Table 1. The first signs of growth rates were seen on the third day after planting across all treatments. On day 3, the germination rates were 23.33% for treatment A, 26.67% for B, 23.33% for C, 33.33% for D, 33.33% for E, and 46.67% for F. By day 4, these rates increased to 43.33% (A), 46.67% (B), 33.33% (C), 43.33% (D), 50.00% (E), and 50.00% (F). On day 5, the rates rose further to 70.00% (A), 60.00% (B), 43.33% (C), 63.33% (D), 70.00% (E), and 70.00% (F). By day 6, the germination rates reached 83.33% (A), 70.00% (B), 56.67% (C), 83.33% (D), 90.00% (E), and 90.00% (F).

There were noticeable differences in germination rates among the treatments over the days. Treatment F (NPK 315) consistently performed the best, reaching 90% germination by day 6, which was similar to the positive control (E). In contrast, treatment C (Pseudomonas) had the lowest germination rates, showing significant differences from the others starting from day 4 (p < 0.05). Treatments A, B, and D showed moderate germination rates, with some similarities on certain days, but treatment C remained significantly lower on most days (p < 0.05).

The strong performance of NPK 315 isn’t surprising, as chemical fertilizers like this provide a quick boost of essential nutrients such as nitrogen, phosphorus, and potassium, which support rapid germination and early growth. Studies, such as one by Singh et al. (2022), have shown that NPK fertilizers often outperform microbial treatments in the early stages due to their immediate nutrient availability. However, over time, chemical fertilizers may not be as sustainable, which is where microbial treatments can play a role in maintaining soil health in the long term.

Rhizobium has been seen to improve seed germination and plant growth, especially in legumes. Research by Zhang et al. (2020) highlights its effectiveness in nitrogen fixation and growth promotion, which explains why its germination rates improved on days 5 and 6. Azotobacter releases growth-promoting substances like gibberellins, but its effects on germination tend to be more noticeable in later growth stages. While Azotobacter supports plant growth, its immediate impact on germination isn’t as strong as that of chemical fertilizers or Rhizobium.

The lower germination rates with Pseudomonas align with its known role as a biocontrol agent rather than a germination enhancer. Research by Saeed et al. (2021) suggests that Pseudomonas is more effective in suppressing pathogens and colonizing roots, which may explain its slower impact on germination. When Rhizobium, Azotobacter, and Pseudomonas were combined (the Consortium), the treatment showed moderate to high germination rates. This aligns with studies indicating that microbial mixtures can work synergistically to support plant growth through nutrient mobilization, nitrogen fixation, and pathogen suppression, as noted by Timofeeva et al. (2023).

The positive control performed similarly to NPK 315, likely due to its nutrient-rich composition or growth-promoting additives. This matches findings from Khandakar (2023), which showed that nutrient-rich treatments can significantly improve early seed germination and plant health.

The effects of coating agents on the average leaf length of Phaseolus vulgaris seedling are presented in Table 2. Over 5 weeks period of observation, the average leaf length (cm) of experimental common bean seedlings ranged from 5.92- 6.45; 5.92– 7.00; 3.78 – 4.93; 4.08– 4.95 ,3.75-4.37 and 5.38– 6.27 for the treatment A, B, C, D, E and F respectively. Treatments A (Rhizobium sp.), B (Azotobacter  sp.), and F (NPK 315) consistently show the highest leaf lengths, with no significant difference among them by the 5th week (p > 0.05).Treatment C (Pseudomonas sp.) and D (Consortium) show intermediate leaf lengths in most weeks but are not as effective as A, B, or F. Treatment E (Positive control) consistently has the lowest leaf length throughout the study, showing a significant difference compared to other treatments (p < 0.05).

These findings are in line with what other research has said about using microbial inoculants and nutrient boosts in legume plants. Rhizobium and Azo           tobacter are known nitrogen-fixing bacteria that help improve growth by making more nitrogen available around the roots. Previous studies by Bhattacharyya and Jha (2012) and Bashan et al., (2014) have shown that plants treated with these bacteria grow better, especially when the soil is low in nitrogen. A review by Saad et al., (2020) supports this by pointing out how Rhizobium and Azotobacter increase nitrogen for legumes, leading to better overall growth, which matches the longest leaf lengths we see in Treatments A and B. Treatments C and D (Pseudomonas sp. and a blend of Rhizobium, Azotobacter, and Pseudomonas) performed okay, but not as well as A and B.

While such combinations can generally help plant growth by tackling multiple needs (like nitrogen fixing, phosphate solubilization, and fighting off pests), some research indicates that these mixed microbial interactions can also lead to competition and lower effectiveness (Saini et al ., 2021). Treatment F (NPK 315) showed equally good leaf lengths and was gradually consistent, although it didn’t outperform A and B by a lot. This highlights how balanced nutrients like nitrogen, phosphorus, and potassium are key for growth, much like what microbial inoculants do. The steady but moderate performance of the NPK fertilizer (Treatment F) is also backed by studies like Liy et al., (2024), which found that NPK contributes to plant growth by offering quick nutrient access, but the long-term effects often rely on how well organic amendments release nutrients over time.

The effects of biofertilizer on the average leaf width of Phaseolus vulgaris seedling are presented in Table 3. Over 5 weeks period of observation, the average leaf length (cm) of experimental common bean seedlings ranged from 3.42-3.72, 3.78-4.17, 2.40-3.10, 2.67-3.32, 2.33-2.75, 3.16-3.80 for the treatment A, B, C, D, E and F respectively. Treatment B (Azotobacter sp.) showed the best and most consistent performance in promoting leaf width, significantly outperforming Treatment A only in Week 3 (p < 0.05). Treatments A and F also performed well, though Treatment F had more variations. Treatments C and D showed moderate results, while Treatment E consistently had the lowest leaf width. Thus, Treatments B and A were the most effective in promoting leaf width over the five-week period, with Treatment B being the top performer. Bashan and de-Bashan (2010) pointed out that Azotobacter helps plants grow by making more nitrogen available, which leads to wider, healthier leaves. And it really shows with those high leaf widths. Past research, like what Saeed et al., (2021) found, showed that Rhizobium can really boost leaf area in leguminous plants, and that’s evident in Treatment A’s strong and steady leaf widths. NPK fertilizer works quite well for increasing leaf width, but it’s not as steady as the microbial inoculants which makes synthetic fertilizers, to show that while NPK supplies essential nutrients for growth, the plant reactions can differ based on things like soil quality and water supply (Liu et al., 2024). Different microbes, which could hold back its leaf width results when compared to the individual strains. The results from the microbial consortium in Treatment D (which includes Rhizobium, Azotobacter, and Pseudomonas) fit with what’s been found in past research about the complicated nature of using mixed microbial inoculants. While combining these microbes can have its benefits like fixing nitrogen and fighting off pathogens , some studies (like Saleem et al., 2019) suggest that the competition can lower overall effectiveness. That might explain why the consortium’s leaf width wasn’t as impressive as the single-species treatments. The low leaf width for Treatment E really highlights how crucial it is to have either microbial inoculation or some nutrient boosts for optimal growth. This highlighted what literature has said about Phaseolus vulgaris, emphasizing that its growth is heavily reliant on nitrogen and other nutrients (Kebede, 2021).

The effects of biofertilizers on the average shoot length of Phaseolus vulgaris seedling are presented in Table 4. Over 5 weeks period of observation, the average shoot length (cm) of experimental common bean seedlings ranged from 17.50-25.42,21.67-27.15,6.00-17.83,11.50-17.18,12.83-16.48,17.16-23.43 for the treatment A, B, C, D, E and F respectively. Treatment B (Azotobacter sp.) consistently produced the highest shoot height across the five weeks, though it also showed significant variability, particularly in later weeks. This suggests that while Treatment B promotes strong shoot growth, the results are less consistent (p > 0.05). Treatment C (Pseudomonas sp.) consistently had the lowest shoot heights but with low significant differences, indicating more uniform but lower growth (p < 0.05). Treatments D and E showed similarly low shoot heights but with less significant differences, particularly by Week 4 and Week 5. Overall, Treatment B was the most effective in promoting shoot height, though with significant differences in results, while Treatment E was the least effective.

Research by Bashan et al., (2010) and Timofeeva and others in 2023 suggests that Azotobacter is capable of plant height and biomass improvement by augmenting nitrogen availability. But, the variability observed in this investigation aligns with previous findings that the effectiveness of Azotobacter is significantly dependent on environmental factors such as soil fertility and the efficiency of microbial colonization (Khan et al., 2020). Previous research by Saeed et al., (2021) align with this study’s findings, indicating that Rhizobium inoculation significantly enhances shoot height and biomass in Phaseolus vulgaris, showing reduced variability compared to non-symbiotic nitrogen-fixers like Azotobacter.  According to Liu et al., (2023), NPK fertilizers play a critical role by supplying essential nutrients for plant growth, although variability in nutrient absorption may result in inconsistent growth outcomes, as significant differences seen in in shoot heights within the NPK treatment observed in this study. Goswami et al., (2016) observed that Pseudomonas sp. is more capable as a plant health promoter than as a direct growth enhancer as noted in this study. Research conducted by Saleem et al., (2019) indicates that consortium treatments can provide multiple benefits, despite the potential for inner competition among species to reduce overall effectiveness in fostering shoot growth. The findings of this study support the idea that consortium approaches may yield balanced but reduced growth outcomes in contrast to single-species inoculants like Azotobacter. This phenomenon can be anticipated, as the control group lacks beneficial microbial inoculants or fertilizers that help to enhanced nutrient uptake and growth in plants.

The effects of coating agents on the average stem diameter of Phaseolus vulgaris seedling are presented in Table 5. Over 5 weeks period of observation, the average stem diameter (cm) of experimental common bean seedlings ranged 0.22-0.22,0.27-0.25,0.25-0.22,0.27-0.27,0.18-0.27,0.18-0.23 for the treatment A, B, C, D, E and F respectively. Treatment D, which comprised Rhizobium sp., Azotobacter sp., and Pseudomonas sp., exhibited the highest consistency and efficacy in enhancing stem diameter growth over the entire duration of the study, with no statistically significant variations noted (p > 0.05). Treatment B, featuring Azotobacter sp., experienced a temporary decrease in stem diameter during Week 2; however, it demonstrated significant recovery and approached the performance level of Treatment D by the conclusion of the experiment. Treatments E (the Positive control) and F (NPK 315) displayed marked improvements in stem diameter following Week 2 (p < 0.05), suggesting a delayed which didn’t change growth response. Treatment A, characterized by the presence of Rhizobium sp., maintained a stable but comparatively lower stem diameter throughout the study, with the exception of a decrease observed in Week 3 (p < 0.05). Treatment C, which included Pseudomonas sp., exhibited consistent growth without notable fluctuations, indicating its moderate effectiveness in promoting stem diameter expansion. Research conducted by Bashan et al., (2010) and Khan et al., (2020) asserts that Azotobacter significantly enhances plant biomass and stem development. The observed fluctuations in stem diameter throughout this study may reflect the process showing fluctuations of nitrogen fixation, subject to external environmental conditions and dynamics of root colonization. The progression observed in Treatment F characterized by an initial increase in stem diameter followed by little change is in accordance with findings presented by Liu et al., (2024), which indicate that chemical fertilizers can promote rapid initial growth that subsequently decreases as nutrient resources are exhausted.

Research conducted by Saleem et al., (2019) supports the research that use of  microbial consortia yields significant benefits for plant growth, as various microorganisms can complement one another’s roles. The increased and more consistent stem diameters recorded in Treatment D affirm that the integration of Rhizobium, Azotobacter, and Pseudomonas can result in superior overall growth compared to each microbial treatment administered individually.  Treatment A (Rhizobium sp.) and Treatment C (Pseudomonas sp.) reflect lower stem diameters when compared to the other treatments, Rhizobium sp. is popular for its symbiotic interactions with legumes that augment nitrogen fixation and booster root and overall plant growth. Although stem diameter may not represent the primary growth parameters influenced by Rhizobium, investigations such as those by Saeed et al., (2021) suggest that it does contribute positively to the overall strength and structural development of plants, these aligns with the moderate stem diameters recorded in this study.

The effects of coating agents on the average leaves number of Phaseolus vulgaris seedling are presented in Table 6. Over 5 weeks period of observation, the average leaves number of experimental common bean seedlings ranged 4.00-9.17,4.00-8.83-,2.67-7.50,2.50-7.00,2.16-6.17,3.50-7.17 for the treatment A, B, C, D, E and F respectively. Treatments A (Rhizobium sp.) and B (Azotobacter  sp.) were the most effective in promoting leaf growth, with the highest number of leaves by Week 5 and consistent increases each week (p > 0.05).Treatments C (Pseudomonas sp.), D (Consortium), and F (NPK 315) showed moderate growth, with significant improvements after Week 1, though they generally had fewer leaves than Treatments A and Treatment E (Positive control) consistently had the fewest leaves, showing significantly lower growth compared to the other treatments (p < 0.05), indicating that inoculants and fertilizers are crucial for promoting leaf development in Phaseolus vulgaris seedlings.

The high leaf counts observed in Treatments A and B are consistent with findings from Zhang et al., (2020),  reported earlier that Rhizobium and Azotobacter, allows  increase in  leaf production. Nitrogen is critical for chlorophyll synthesis and vegetative growth, which explains the enhanced leaf production in these treatments. The rapid leaf growth observed in Treatment F during the early weeks aligns with the well-established effects of NPK fertilizers, which provide essential macronutrients for plant growth. However, the high variability in leaf number in Week 3 is consistent with studies by Verma et al., (2019), which suggest that chemical fertilizers can lead to inconsistent plant responses if not applied in a balanced manner or if nutrient uptake is limited by other factors. The moderate leaf counts observed in Treatment C are consistent with research by Timofeeva et al., (2023), which found that Pseudomonas can enhance plant growth, though its effects may be more pronounced in root development and stress tolerance rather than in leaf production.  However, in this study, the results were similar to those of individual inoculants, suggesting that the benefits of microbial consortia may depend on the specific plant-microbe interactions and environmental conditions. Prasad et al., (2019) have highlighted that while consortia can improve plant growth, their effectiveness may vary depending on factors such as soil type and plant species. This aligns with the baseline growth observed in untreated plants, which lack the enhanced nutrient availability provided by microbial inoculants or chemical fertilizers.

The effects of coating agents on the average Bacterial load of Phaseolus vulgaris seedling are presented in Table 7. Over 5 weeks period of observation, the average Bacterial load (Cfu / g x 10⁷) range from of experimental common bean seedlings 33-83,35-73,20-54,94-129,25-81,30-62 for the treatment A, B, C, D, E and F respectively. Treatment D (Consortium of Rhizobium sp., Azotobacter sp., and Pseudomonas sp.) had the highest and most consistent bacterial load throughout the experiment (p < 0.05), indicating the synergistic effect of using multiple bacterial species to enhance the rhizosphere’s bacterial population. Treatments A (Rhizobium sp.) and B (Azotobacter sp.) performed similarly, with moderate bacterial growth, consistently lower than Treatment D but significantly higher than Treatment C (p < 0.05).  Treatment C (Pseudomonas sp.) had the lowest bacterial loads, showing significantly lower values compared to other treatments (p < 0.05). Treatments E (Untreated seeds – Positive control) and F (NPK 315) showed intermediate bacterial loads, with Treatment F slightly outperforming Treatment E, but both were still significantly lower than Treatment D (p < 0.05).

The findings from this study align with previous research highlighting the effectiveness of microbial consortia in promoting bacterial colonization in the rhizosphere. For instance, Wei et al., (2024) demonstrated that the combined inoculation of multiple beneficial microbes (such as Rhizobium, Azotobacter, and Pseudomonas) synergistically enhances bacterial populations and improves plant growth parameters, which supports the high bacterial loads observed in Treatment D in this study. Similarly, Hungria et al., (2015) showed that combine inoculation of Rhizobium and Azotobacter increases bacterial counts in the rhizosphere, which is consistent with the results from Treatments A and B.

The relatively lower bacterial loads in the single-inoculum treatments (Pseudomonas sp. in Treatment C) aligns the findings of Sarma and Saikia (2014), who reported that while Pseudomonas alone can benefit plant growth, its effect on bacterial populations is more limited compared to when it is applied in combination with other microbes.

Control Treatments (E and F) also demonstrated lower bacterial loads than the consortium, Additionally, the control treatments (E and F) having lower bacterial loads compared to biological inoculants aligns with research by Timofeeva et al., (2023), which indicates that while chemical fertilizers like NPK provide essential nutrients, they do not foster the same level of microbial proliferation in the rhizosphere as biofertilizers.

CONCLUSION

The research illustrates that the synergistic application of microbial inoculants, particularly the consortium comprising Rhizobium sp., Azotobacter sp. and Pseudomonas sp., significantly improves growth and bacterial populations in Phaseolus vulgaris. Treatment F (NPK 315) outperformed others in facilitating early seed germination and leaf development. In contrast, Treatment B (Azotobacter sp.) consistently exhibited superior overall growth performance across multiple criteria. Conversely, Treatment C (Pseudomonas sp.) demonstrated suboptimal performance in most metrics, indicating its limited effectiveness when utilized independently.

RECOMMENDATION

Farmers and agricultural practitioners should be trained on the benefits of using biofertilizers and microbial inoculants over conventional chemical fertilizers. Workshops and field demonstrations could help in disseminating this knowledge. Additional studies should explore the long-term impacts of microbial inoculants on soil health and crop yields. Investigating the interactions between different microbial species in consortia could further enhance understanding and efficacy in agricultural applications.

Contribution to knowledge

This study makes a valuable contribution to the field of sustainable agriculture by demonstrating the efficacy of biofertilizers in improving the growth of Phaseolus vulgaris and enhancing soil microbial activity. It provides evidence-based insights that can guide farmers, policymakers, and researchers in adopting more sustainable farming practices, ultimately contributing to food security an environmental conservation.

Acknowledgement:The authors acknowledge Suraj Tolani Muhammed  for sponsoring the research and the staff of Eden research laboratory and Federal university Lokoja.

Conflict of interest: No conflict of interest

Author contributions: Tolani M.S conceived the idea and wrote the manuscript, Tolani M.S carried out the research , Enu K.B collected the data and analyzed it, Aborishade T.W supervised the research work and edited the manuscript.

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Table 1: Germination rate of Phaseolus vulgaris L. seed as observed on the 3^rd, 4^th ,5^th and 6^th days after planting

Treatments (g/kg)	Germination rate (%/day)
	3rd	4th	5th	6th
A	23.33 ± 0.08c	43.33 ± 0.08a	70.00 ± 0.11a	83.33 ± 0.02ab
B	26.67 ± 0.10c	46.67 ± 0.10a	60.00 ± 0.18b	70.00  ± 0.17b
C	23.33 ± 0.10c	33.33 ± 0.10b	43.33 ± 0.15c	56.67 ± 0.15c
D	33.33 ± 0.10b	43.33 ± 0.08a	63.33 ± 0.08ab	83.33 ± 0.08ab
E	33.33 ± 0.10b	50.00 ± 0.11a	70.00 ± 0.11a	90.00 ±0.11a
F	46.67 ± 0.10a	50.00 ± 0.11a	70.00 ± 0.11a	90.00 ± 0.11a

Key A= Rhizobium sp.; B = Azotobacter  sp. ; C= Pseudomonas sp. ; D= Rhizobium sp ₊ Azotobacter  sp.₊ Pseudomonas sp. ;E=Positive control; F= NPK 315; Values were presented as the x σ (i.e. Mean plus / minus standard deviation of mean); Mean value with the same letter in the column are not significantly different from one another at 5% level of significant

Table 2: Average length of leaves of Phaseolus vulgaris L.  seedling per week  after planting

Treatments(g/kg)	Length of leaves (cm/week)
	1st	2nd	3rd	4th	5th
A	5.92 ± 0.49a	5.95 ± 0.50a	6.17 ± 0.21a	6.30 ± 0.42a	6.45 ± 0.34a
B	5.92 ± 2.11a	6.45 ± 2.24a	6.75 ± 2.08a	6.77 ± 1.04a	7.00 ± 1.15a
C	3.78 ± 0.34b	4.27 ± 0.50bc	4.32 ± 0.42b	4.73 ± 0.87b	4.93 ± 0.83b
D	4.08 ± 1.11a	4.62 ± 0.20bc	4.63 ± 0.82b	4.90 ± 1.07b	4.95 ± 1.09b
E	3.75 ± 0.69b	3.83 ± 0.79c	3.88 ± 0.66c	3.96 ± 0.66c	4.37 ± 0.72b
F	5.38 ± 2.05a	5.58 ± 1.93ab	5.78 ± 1.95ab	6.03 ± 1.76a	6.27 ± 1.59a

Key A= Rhizobium sp.; B = Azotobacter  sp. ; C= Pseudomonas sp. ; D= Rhizobium sp ₊ Azotobacter  sp.₊ Pseudomonas sp. ;E=Positive control; F= NPK 315; Values were presented as the x σ (i.e. Mean plus / minus standard deviation of mean); Mean value with the same letter in the column are not significantly different from one another at 5% level of significant

Table 3: Average Width of leaves of Phaseolus vulgaris L. seedling per week   after planting

Treatment (g/kg	Width of leaves (cm/week)
	1st	2nd	3rd	4th	5th
A	3.42 ± 0.58a	3.63 ± 0.38a	3.67 ± 0.17ab	3.70 ± 0.42a	3.72 ± 0.32a
B	3.78 ± 0.34a	3.70 ± 1.25a	3.82 ± 0.80a	3.95 ± 0.22a	4.17 ± 0.52a
C	2.40 ± 0.24b	2.65 ± 0.50b	2.78 ± 0.43c	2.85 ± 0.52b	3.10 ± 0.41a
D	2.67 ± 0.38b	3.13 ± 0.37a	3.18 ± 0.32b	3.28 ± 0.37a	3.32 ± 0.50a
E	2.33 ± 0.57b	2.43 ± 0.45b	2.58 ± 0.39c	2.65 ± 0.36b	2.75 ± 0.45b
F	3.16 ± 0.79a	3.32 ± 0.80a	3.42 ± 0.98ab	3.67 ± 1.09a	3.80 ± 1.08a

Key A= Rhizobium sp.; B = Azotobacter  sp. ; C= Pseudomonas sp. ; D= Rhizobium sp ₊ Azotobacter  sp.₊ Pseudomonas sp. ;E=Positive control; F= NPK 315; Values were presented as the x σ (i.e. Mean plus / minus standard deviation of mean); Mean value with the same letter in the column are not significantly different from one another at 5% level of significant

Table 4: Average Shoot height of Phaseolus vulgaris L. seedling per week  after planting

Treatments(g/kg)	Shoot height  (cm/week)
	1st	2nd	3rd	4th	5th
A	17.50 ± 1.52a	18.00 ± 2.21a	19.67 ± 2.55a	22.00 ± 3.41a	25.42 ± 5.04a
B	21.67  ± 4.76a	22.92 ± 3.74a	24.03 ± 4.45a	27.00 ± 5.55a	27.15 ±5.32a
C	6.00 ± 0.24c	13.00 ± 2.76b	14.55 ± 2.32b	17.50 ± 5.43b	17.83 ± 5.23b
D	11.50 ± 1.05b	13.58 ± 1.28b	14.67 ± 0.32b	15.92 ± 1.69b	17.18 ± 1.50b
E	12.83 ± 1.94b	14.42 ± 3.32b	14.50 ± 1.05b	15.33 ± 1.33b	16.48 ± 1.20b
F	17.16 ± 5.31a	19.67 ± 3.06a	21.33 ± 3.13a	22.67 ± 4.46a	23.43 ± 4.87a

Key A= Rhizobium sp.; B = Azotobacter  sp. ; C=[i] Pseudomonas sp. ; D= Rhizobium sp ₊ Azotobacter  sp.₊ Pseudomonas sp. ;E=Positive control; F= NPK 315

Table 5: Average stem diameter of Phaseolus vulgaris L. seedling per week   after planting

Treatments(g/kg)	Stem diameter (cm/week)
	1st	2nd	3rd	4th	5th
A	0.22 ± 0.05a	0.22 ± 0.05a	0.18 ± 0.03b	0.22 ± 0.05a	0.22 ± 0.05a
B	0.27  ± 0.05a	0.18 ± 0.03b	0.27  ± 0.05a	0.27  ± 0.05a	0.25  ± 0.05a
C	0.25  ± 0.05a	0.25  ± 0.05a	0.27 ± 0.05a	0.22 ± 0.05a	0.22  ± 0.05a
D	0.27  ± 0.05a	0.27  ± 0.05a	0.28  ± 0.04a	0.27  ± 0.05a	0.27  ± 0.05a
E	0.18 ± 0.03b	0.18 ± 0.03b	0.27  ± 0.05a	0.27  ± 0.05a	0.27  ± 0.05a
F	0.18 ± 0.03b	0.18 ± 0.03b	0.27  ± 0.05a	0.23  ± 0.05a	0.23 ± 0.05a

Key A= Rhizobium sp.; B = Azotobacter  sp. ; C= Pseudomonas sp. ; D= Rhizobium sp ₊ Azotobacter  sp.₊ Pseudomonas sp. ;E=Positive control; F= NPK 315; Values were presented as the x σ (i.e. Mean plus / minus standard deviation of mean); Mean value with the same letter in the column are not significantly different from one another at 5% level of significant

Table 6: Average number of leaves of Phaseolus vulgaris L. seedling per week  after planting

Treatments(g/kg)	Numbers of leaves /week
	1st	2nd	3rd	4th	5th
A	4.00 ± 0.89a	4.67 ± 0.52a	5.50 ± 1.22a	6.67 ± 1.03a	9.17 ± 0.75a
B	4.00 ± 1.22a	4.83 ± 0.33a	5.50 ± 0.55a	6.68 ± 1.29a	8.83 ± 1.60a
C	2.67 ± 0.38b	4.50 ± 0.55a	4.83 ± 0.33a	5.83 ± 1.17a	7.50 ± 0.77a
D	2.50 ± 0.54b	4.33 ± 0.52ab	4.50 ± 0.55ab	5.33 ± 1.51ab	7.00 ± 0.63ab
E	2.16 ± 0.64b	3.83 ± 0.88b	4.17 ± 0.75b	4.33 ± 0.52b	6.17 ± 0.75b
F	3.50 ± 1.64ab	4.83 ± 0.33a	6.00 ± 2.68a	6.33 ± 1.51a	7.17 ± 1.33ab

Key A= Rhizobium sp.; B = Azotobacter  sp. ; C= Pseudomonas sp. ; D= Rhizobium sp ₊ Azotobacter  sp.₊ Pseudomonas sp. ;E=Positive control; F= NPK 315; Values were presented as the x σ (i.e. Mean plus / minus standard deviation of mean); Mean value with the same letter in the column are not significantly different from one another at 5% level of significant

Table 7: Average bacterial loads in the in the rhizosphere region of Phaseolus vulgaris L. seedlings

Treatments(g/kg)	Average Bacterial loads (×105  )/cfu/g
	1st	2nd	3rd	4th	5th
A	33.00± 6.23b	45.00± 8.03b	59.00 ± 5.89b	70.00 ± 7.47b	83.00 ± 7.19b
B	35.00 ±4.08b	45.00± 4.08b	59.00 ± 5.93b	66.00  ±3.44b	73.00 ± 2.38b
C	20.00± 4.42c	27.00± 6.48c	36.00 ± 7.80c	45.00 ± 0.50c	54.00 ± 5.96c
D	94.00± 5.76a	103.00±8.50a	115.00±13.19a	123.00±15.06a	129.00±14.84a
E	25.00±5.48c	37.00 ±5.18bc	50.00 ± 7.75bc	65.00 ± 5.48b	81.00 ± 6.57b
F	30.00±1.79bc	38.00 ±5.83bc	46.00 ± 8.11bc	54.00 ± 7.72bc	62.00± 5.58bc

Key A= Rhizobium counts on CREYMA agar; B = Azotobacter  counts on Ashby agar  ; C= Pseudomonas counts on Pseudomonas agar. ; D= Rhizobium sp ₊ Azotobacter  sp.₊ Pseudomonas sp. = Consortium(Total bacterial counts) on Nutrient agar;E= Untreated seeds(Total bacterial counts) on Nutrient agar; F = NPK 315 (Total bacterial counts) on Nutrient agar; Values were presented as the x σ (i.e. Mean plus / minus standard deviation of mean); Mean value with the same letter in the column are not significantly different from one another at 5% level of significant