Isolation and Characterization of Plant Growth Promoting Rhizo Bacteria (PGPR) from Banana Rhizosphere

Jai Godheja^1,*, Divya Soni²

ITM University, India

¹jaig@itmuniversity.org

 *Corresponding Author: jaig@itmuniversity.org

Abstract

Rhizobacteria are predominantly found in soil and help enhance plant growth by forming colonies around plant roots. Agriculturists have identified ways to improve plant growth by applying this soil inoculum to different varieties of plants. This study is based on the “Isolation and characterization of plant growth-promoting rhizobacteria (PGPR) from the banana rhizosphere.” Soil samples were collected from the NBRI garden premises, Lucknow (U.P.). A total of 15 bacterial colonies were observed and identified as Staphylococcus aureus spp. based on microscopic and biochemical characterization. PGPR activity was analyzed on banana plants, and the results revealed that the bacterial isolates contributed to faster growth of the banana plants.

Keywords: Rhizobium, PGPR, Biofertilizer, Rhizobacteria, Rhizosphere, Actinomycetes.

Introduction

The need for this study arises from the growing demand for sustainable and eco-friendly agricultural practices. The excessive use of chemical fertilizers and growth enhancers in modern agriculture can lead to soil degradation, reduced microbial diversity, and increased toxicity in crops. Therefore, there is a pressing need to identify natural alternatives that can promote plant growth without harmful effects.

PGPR (plant growth-promoting rhizobacteria) are soil bacteria often found in association with plant roots and are widely studied for their plant growth-enhancing properties. Bananas are in high demand in tropical regions, and their demand in non-tropical regions is also increasing, making them an important fruit worldwide. Good-quality and fast-growing banana plants are therefore required, and thus studies on rhizobacteria are being conducted.

The rhizosphere layer is rich in nutrients due to microbial degradation activities in the soil. The rhizosphere of banana roots can be studied to increase the overall productivity of the plant. PGPR are also used as biocontrol agents, as certain chemicals secreted by rhizobacteria help banana plants fight infections, either through direct antagonistic interactions with pathogens or by inducing resistance mechanisms in the plant.

In biotechnological industries, bioremediation techniques are used in processes related to food, chemical, pharmaceutical, and mining sectors. PGPR are less expensive than chemical fertilizers (Gupta et al., 2015; Sharma et al., 2013). Bacterial biofertilizers enhance plant growth through several mechanisms, such as the production of phytohormones that can be absorbed by plants, mobilization of soil nutrients for uptake, protection of plants under stressful conditions by reducing the negative impacts of stress, and defense against plant pathogens, thereby reducing plant diseases (Garcia et al., 2015).

To protect plants from soil-borne pathogens, successful colonization of growth-promoting rhizobacteria in the rhizosphere is essential (Yuan et al., 2015; Arun et al., 2023). The host plant utilizes nitrogen fixed by rhizobacteria associated with banana roots, both externally and internally (Mia et al., 2010; Thamilmaraiselv et al., 2018). In banana “Prata Ana” roots, the genetic diversity of endophytic bacteria has been characterized (Souza et al., 2014).

Currently, the management of plant infections is very challenging and time-consuming, as at least 4,000 infectious agents have been identified across major biomes (Mhatre et al., 2019). For sustainable agriculture, crops need to be equipped with traits such as drought tolerance, salt tolerance, disease resistance, heavy metal stress tolerance, and improved nutritional value (Vejan et al., 2016).

Plant Growth-Promoting Rhizobacteria (PGPR) play a significant role in sustainable agriculture by enhancing plant growth through multiple direct and indirect mechanisms. According to Glick (2012), PGPR improve plant growth by producing phytohormones such as indole-3-acetic acid (IAA), facilitating nutrient uptake, and protecting plants against pathogens. Similarly, Vessey (2003) described PGPR as an effective alternative to chemical fertilizers due to their ability to improve soil fertility and plant productivity.

In banana cultivation, several studies have demonstrated the beneficial effects of PGPR. Thiruvengadam et al. (2020) reported that PGPR inoculation enhances banana plant growth, root architecture, and nutrient absorption. Likewise, Khan et al. (2019) showed that PGPR significantly improve plant biomass and yield under field conditions. The mechanisms of PGPR action have been widely studied. Lugtenberg and Kamilova (2009) explained that PGPR promote plant growth directly through nitrogen fixation, phosphate solubilization, and phytohormone production, and indirectly by suppressing plant pathogens through antibiotic production and induced systemic resistance. Kloepper et al. (2004) further emphasized the role of PGPR in inducing systemic resistance in plants, thereby enhancing their defense against diseases.

PGPR also function as biocontrol agents. Studies by Compant et al. (2005) demonstrated that PGPR can inhibit phytopathogens through competitive exclusion and secretion of antimicrobial compounds. In banana, Pseudomonas fluorescens has been reported to suppress Fusarium oxysporum, the causative agent of Panama disease (Saravanan et al., 2004). Similarly, Bacillus subtilis has shown antagonistic activity against several soil-borne pathogens (Chen et al., 2006).

The impact of chemical fertilizers on soil microbial diversity has also been well documented. Jaizme-Vega et al. (2004) reported that excessive use of chemical fertilizers reduces beneficial microbial populations in the soil, negatively affecting long-term soil health. Recent studies have focused on exploring rhizospheric microbial diversity. Ekka et al. (2015) reported the presence of diverse PGPR strains in the rhizosphere capable of nitrogen fixation and phosphate solubilization. Although extensive research has been conducted on commonly known PGPR such as Bacillus, Pseudomonas, and Rhizobium, there is limited research on unconventional bacterial species. Studies by Artursson et al. (2006) suggest that exploring lesser-known microbial populations could reveal new PGPR candidates with significant agricultural potential.

Banana is an economically important crop with increasing global demand, requiring improved growth and productivity. Thus, there is a need to isolate and characterize effective PGPR strains from the banana rhizosphere and evaluate their potential in enhancing plant growth. This study aims to address this gap by identifying and testing rhizobacteria that can serve as sustainable biofertilizers for banana cultivation.

Materials and Method

Sample Collection of Soil from Banana Rhizosphere
Rhizospheric soil samples were collected from banana plants at the NBRI garden, Lucknow (U.P.). Soil samples were collected at a depth of 5–10 cm. The samples were collected in aseptic bags and immediately stored under cold conditions (4°C).

Isolation of PGPR Microbes from Soil Samples
One gram of rhizospheric soil from the banana plant was transferred into 10 ml of sterile water blank in a culture tube and vortexed. Serial dilutions of 10⁻³ and 10⁻⁴ were prepared. Nutrient agar plates were then inoculated and incubated at 37°C.

Identification of PGPR Microbes
Gram staining of all 15 isolates was performed using crystal violet (1% aqueous) as the primary stain and safranin (0.1%) as the counterstain.

Plant Growth-Promoting Rhizobacteria Characterization

IAA Test
Seventy-two-hour-old bacterial broth culture was centrifuged at 3000 rpm. Two ml of the supernatant was mixed with two drops of orthophosphoric acid and 4 ml of Salkowski reagent (50 ml of 35% perchloric acid and 1 ml of 0.5 M FeCl₃ solution) and incubated for 30 minutes.

Phosphate Solubilization Test
Bacterial isolates were cultured on selective Pikovskaya medium containing tricalcium phosphate for 24 hours. The formation of clear zones indicated a positive phosphate solubilization test.

Ammonia Production Test
Nessler’s reagent (0.5 ml) was added to bacterial cultures pre-incubated in peptone water for 48–72 hours. The tubes were kept for 15–20 minutes and observed for the development of a brown to yellow color, indicating a positive result for ammonia production.

Biochemical Characterization of PGPR Strains

Catalase Test
A drop of bacterial broth culture was placed on a slide, and bubble formation was observed after adding a drop of 3% H₂O₂.

Methyl Red (MR) and Voges–Proskauer (VP) Test
For the MR test, bacterial broth cultures were observed for color change after adding 5 drops of methyl red reagent.
For the VP test, bacterial broth cultures were observed for color change after adding 6 drops of 5% alpha-naphthol. The solution was aerated, followed by the addition of 2 drops of 40% potassium hydroxide, and observed for the development of a pink-red color at the surface within 30 minutes.

Starch Hydrolysis Test
Starch agar was prepared and autoclaved at 121°C and 15 psi for 15 minutes. The medium was poured into plates and allowed to solidify. The microbes were streaked using a loopful of a 24-hour-old pure culture. Plates were incubated at 37°C for 24 hours. After incubation, iodine solution was added to observe zones of clearance.

Urease Test
The broth medium was inoculated with the test organism and incubated at 35°C with the cap loosely closed in ambient air for 18–24 hours.

Motility Test
Slant cultures of nutrient agar medium were inoculated using a single-line streaking method and observed for 24 hours at 37°C.

Growth Observation Test
Three samples of baby banana plants were used to observe growth after inoculation with bacterial isolates of different concentrations. The first plant served as the control, with no bacterial inoculum added. The second plant (Plant A) was treated with 5% bacterial inoculum. The third plant (Plant B) was treated with 10% bacterial inoculum.

Result

Isolation and Staining
Fifteen colonies from the 10⁻³ dilution plate were selected and streaked onto freshly prepared nutrient agar medium, followed by incubation at 37°C. After Gram staining, all the bacterial isolates were found to be Gram-positive cocci.

Plant Growth-Promoting Rhizobacteria Characterization

In the IAA test, the 18th isolate showed a positive result, whereas the 3rd, 4th, 6th, 8th, 10th, 12th, and 14th isolates showed weakly positive results. The 2nd, 15th, 16th, 17th, and 20th isolates showed negative results.

In the ammonia production test, isolates 6, 8, 10, 18, and 20 showed positive results. Isolates 3rd, 4th, 14th, and 17th showed weakly positive results, while the 2nd and 12th isolates showed negative results.

The phosphate solubilization results indicated that PGPR isolates 6th and 10th showed higher zones of clearance compared to other isolates (3rd, 4th, and 18th) on Pikovskaya medium.

 Table 1- PGPR test result

Biochemical characterization of PGPR strains

Isolates 3, 4, 6, 10, and 18 showed positive results in the catalase test. In the MR and VP tests, all the isolates showed positive results. In the starch hydrolysis test, a clear transparent zone was observed around all the cultured isolates, indicating that the bacteria were able to hydrolyze starch and the test was positive.

The urease broth was inoculated with bacterial isolates and incubated at 37°C for 24 hours. A color change from orange-yellow to pink was observed, indicating that the isolates were capable of producing the urease enzyme, and thus tested positive.

In the motility test, after inoculation, isolate 6 showed a positive result, whereas isolates 10 and 18 showed negative results.

Table 2- Biochemical test result

As per the biochemical tests performed the unknown bacterial species were identified as S. aureus (Table 3.)

Table 3- Biochemical characterization

Growth observation

Fig-1 (a) Growth of Banana plant (Control) (b) Growth of Banana plant after 7 days (Control) (c) Growth of Banana plant after 14 days (Control)

Fig-2 (a) Banana plant growth Plant A (PGPR, 0 Days) (b) Banana plant growth Plant A (PGPR, 7 Days) (c) Banana plant growth Plant A (PGPR, 14 Days)

Fig-3 (a) Banana plant growth Plant B (PGPR, 0 Days) (b) Banana plant growth Plant B (PGPR, 7 Days) (c) Banana plant growth Plant B (PGPR, 14 Days)

Table 4- Growth result of the banana plant after 7 and 14 days

Graph for growth of the stem and leaves in 7 days:

The graph shows the increase in stem length and the increase in leaves length and no. of leaves after 7 days:

1.                The plant taken as Control increased in 0.5 cm in length and the no. of leave increased by 1 extra leaf.

2.                The plant taken as Plant A did not increase in stem length and the no. of leaves increased by 2 extra leaf.

3.                The plant taken as Plant B increased in 0.5 cm in stem length and the no. of leaves increased by 1 extra leaf.

Graph for growth of the stem and leaves in 14 days:

The graph shows the increase in stem length and the increase in leaves length and no. of leaves after 14 days:

1.       The plant taken as Control increased in 1.5 cm in length and the no. of leave increased by 1 extra leaf.                       

2.       The plant taken as Plant A increased in 2 cm in length and the no. of leaves increased by 2 extra leaf.             

3.       The plant taken as Plant B increased in 1.5 cm in length and the no. of leaves increased by 1 extra leaf.

Fig 4- Growth of Banana plant (Control, Plant A, Plant B) after 7 and 14 days

DISCUSSION

PGPR bacteria promote plant growth in a natural and safe manner. Chemical fertilizers and synthetic growth hormones can be harmful in certain ways. This study demonstrates the isolation of Staphylococcus aureus, which exhibited plant growth-promoting properties. Other genera such as Agrobacterium, Bacillus, Enterobacter, Klebsiella, Lysinibacillus, Micrococcus, and Rhizobium are also known to enhance plant growth through mechanisms such as nitrogen fixation, phosphate solubilization, and biocontrol (Ekka et al., 2015; Thiruvengadam et al., 2020; Khan et al., 2019; Chen et al., 2006).

In this study, significant plant growth was observed, indicating the effectiveness of PGPR as growth-promoting agents. Nowadays, many agricultural practices rely on chemicals to accelerate plant growth within a short period; however, this may increase toxicity in plants and reduce soil fertility. In contrast, PGPR are naturally occurring microorganisms in the rhizosphere and are not harmful to plant growth.

Staphylococcus aureus was isolated from the banana rhizosphere, and tests for phosphate solubilization and ammonia production were performed. The bacterium showed positive results in all PGPR characterization tests. It was then applied to the soil of baby banana plants, which were observed for about one week and showed significant growth.

Previous studies have reported that the application of chemical fertilizers can reduce the number of beneficial soil microorganisms that play a crucial role in plant growth (Jaizme et al., 2004). The use of chemical growth enhancers for rapid crop production can also decrease soil fertility over time. Similarly, prolonged use of inorganic fertilizers reduces the population of PGPR microorganisms in the soil.

PGPR can be used as biofertilizers to promote plant growth and protect plants against various diseases. In our study, banana plants showed improved growth following inoculation with isolated PGPR. It was observed that inoculated plants exhibited better growth, improved seedling health, and enhanced survival rates (Apastambh et al., 2016).

This study highlights the isolation and characterization of Staphylococcus aureus based on morphological and biochemical properties, along with its plant growth-promoting activities such as IAA production and phosphate solubilization. Similar studies have reported the role of Pseudomonas and Bacillus species in promoting plant growth through such mechanisms (Arturson et al., 2006).

Overall, this study suggests that inoculation with PGPR can serve as a sustainable alternative to reduce the use of pesticides and chemical fertilizers in agriculture. The use of beneficial microorganisms such as PGPR offers an eco-friendly approach for improving plant productivity (Glick et al., 2012).

Novelty of the Study

The study reports the isolation and characterization of Staphylococcus aureus from the banana rhizosphere, which is relatively uncommon as PGPR studies typically focus on genera like Bacillus, Pseudomonas, or Rhizobium.

It demonstrates the plant growth-promoting potential of Staphylococcus aureus, suggesting a new or less-explored PGPR candidate.

The research provides experimental evidence of enhanced growth in banana plants using these isolates, highlighting a specific application in banana cultivation.

The work contributes to the exploration of rhizospheric microbial diversity from the NBRI garden, Lucknow, adding location-specific microbial insights.

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