Isolation and characterization of plant growth-promoting rhizobacteria from rhizospheric soil of hill paddy

1. Introduction

Rice (Oryza sativa L.) is an essential grain and often referred as one of the major staple foods for approximately half of the world’s population. It is primarily cultivated in Asia, accounting for about 90% of global rice production (Muthayya et al., 2014). It plays a significant economic, agricultural, and cultural role globally. In term of cultivation, rice cultivation can be divided into two types – lowland rice and upland rice. In Malaysia, both of the types of cultivation are widely practiced, with lowland rice cultivation majorly practiced in the Peninsular Malaysia, while in East Malaysia (Sabah and Sarawak) predominantly for upland rice (Che Omar et al., 2022). In contrast to lowland rice, which the paddy will be submerged; the upland rice cultivation refers to the cultivation of rice on hilly or mountainous regions. The rice varieties cultivated as upland rice commonly termed as hill paddy (padi huma/padi bukit) (Che Omar et al., 2022).

The productivity of hill paddy remains low, contributed by various factors, including poor soil fertility as reported by Department of Agriculture (2018). The current practice of conventional fertilizer application might be ineffective, given that upland rice cultivation is prone to nutrient leaching and continuous fertilizer application can be counter-productive (Wang et al., 2016). There has been a growing need and interest in identifying environmentally friendly approaches to improve productivity of hill paddy in Sarawak. Researchers have extensively explored the application of biofertilizer as a sustainable method to enhance soil health. It is known for its ability to increase nutrient availability by stimulating natural soil processes (Chatterjee et al., 2019). This involve the employment of products or mixtures of living microorganisms that will improve nutrient supply to plants, promote growth, restore soil health, and suppress plant pathogens; thereby reducing reliance on conventional fertilizers to support sustainable agriculture and environmental conservation (Amoo et al., 2019; Ferreira et al., 2019).

One of the common types of biofertilizers is plant growth-promoting rhizobacteria (PGPR). It helps to support the well-being of crops through various direct and indirect mechanisms, such as nitrogen fixation, solubilization of phosphate and potassium, producing vital phytohormones like auxin and gibberellins, and indirectly by suppressing plant pathogens (He et al., 2019). Also, numerous studies have reported significant successes in the application of PGPR on rice, including improved tillering and grain formation and grain yield, highlighting its impacts on the growth of paddy and soil health (Xiao et al., 2020; Ghaffari et al., 2018; Mitra et al., 2018). Given their ability to stimulate plant growth and enhance nutrient efficiency, PGPR offer an eco-friendly alternative to chemical inputs, making them a promising approach for improving hill paddy productivity while supporting sustainable agriculture.

The main objective of this study is to isolate PGPR from the rhizospheric soil of hill paddy. The isolates were screened and characterized through a series of in vitro assays to evaluate their plant growth-promoting traits before being partially identified using 16S rRNA sequencing. Also, mustard (Brassica juncea) was selected as a model plant to evaluate the effectiveness of the isolates, due to its rapid growth cycle and adaptability to diverse climates, making it suitable for experiments requiring timely results (South Carolina Department of Agriculture, 2002). The findings from this study provide a foundation for the development of a biofertilizer based on the selected PGPR, contributing to the more fruitful cultivation of hill paddy. Further research, including field trials and mechanistic studies, is recommended to validate and optimize the use of these PGPR strains.

2. Materials and Methods

2.1 Collection site

Rhizospheric soil samples were collected from a field cultivated with hill paddy (Fig. 1), located at Universiti Putra Malaysia Bintulu Campus, Sarawak (3.2048° N, 113.0708° E). The sampling was conducted during the hill paddy reproductive phase (approximately 80 days after sowing) using an auger at a soil depth of 0-15 cm. Five replicates of the sample were collected and pooled to form a composite sample. Intact hill paddy plants were gently uprooted, and the loosely adhering soil was removed by gentle shaking. The soil firmly attached to the roots, referred to as rhizospheric soil, was collected in sterile containers and immediately transferred to the Laboratory of Microbiology, Faculty of Agricultural and Forestry Sciences, Universiti Putra Malaysia, Bintulu Campus. The sampling and isolation were conducted on the same day to prevent contamination.

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Fig. 1.

Field cultivated with hill paddy as the sampling site.

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3. Results

3.4 Plant test- Evaluation of plant growth-promoting ability in plants

An assessment of the overall performance of the plant growth-promoting activity by the rhizobacterial isolates was made based on the vigor index that incorporated the growth performance of the mustard plant, including germination percentage, root length, and shoot height of the seedlings. The bar charts (Fig. 2) illustrate the germination rate, shoot height, root length, and vigor index across nine different rhizobacterial inoculation treatments, including the control. In general, most of the rhizobacteria isolates have the ability to improve seed germination percentage and the subsequent growth of seedlings within 7 days after sowing. Although none of the treatments exhibited statistically significant overall improvements in the plant test, strain RRZ034 (T3) consistently recorded the highest mean values across all evaluated parameters. Notably, the vigor index of T3 increased by at least 79% compared to the control, highlighting its potential as an effective PGPR.

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Fig. 2.

Effects of different PGPR treatments (T1–T8) on germination percentage, shoot height, root length, and vigor index of hill paddy under laboratory conditions. The control (C) represents non-inoculated seeds. Each bar represents the mean ± standard error of triplicate measurements. Different letters (a, b, ab) above the bars indicate statistically significant differences among treatments according to Tukey’s HSD test at p < 0.05. Bars sharing the same letter are not significantly different from each other, whereas bars with different letters are significantly different.

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3.5 Molecular identification of the most promising rhizobacteria strain

Based on the screening results, rhizobacterial strain RRZ034 was selected for further identification. The 16S rRNA gene was sequenced, and homologous gene segments were compared using pairwise alignment. The partial 16S rRNA nucleotide sequence of RRZ034 showed a high level of identity (99-100%) with Pseudomonas nicosulfuronedens. Phylogenetic analysis (Fig. 3) revealed that RRZ034 clustered closely with Pseudomonas nicosulfuronedens strain LAM1902, with <99% sequence similarity to this previously reported strain. A phylogenetic tree was constructed using the Neighbor-Joining method with 1000 bootstrap replicates to assess branch reliability, performed in MEGA11 software. The analysis also indicated a close evolutionary relationship between RRZ034 and related species such as Pseudomonas nitroreducens, Pseudomonas multiresinivorans, and Pseudomonas nitritireducens.

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Fig. 3.

Phylogenetic tree based on 16S rRNA gene sequences shows the relationship between isolate RRZ034 and closely related Pseudomonas species.

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4. Discussion

In this study, 44 rhizobacterial isolates were successfully obtained from the rhizospheric soil of hill paddy. The presence of diverse PGPR in the environment associated with rice has been well-reported in previous studies. For instance, Basik et al. (2020) reported the isolation of over 300 bacterial strains from the rhizosphere of hill paddy in Kuching, highlighting a great diversity of genera and a significant proportion of novel isolates. Similarly, Othman et al. (2022) discovered rhizospheric bacteria from rice roots with zinc-solubilizing and biocontrol properties, consistent with the present finding of isolate RRZ034 that demonstrated antagonistic activity against pathogenic fungi. Furthermore, Syaziana et al. (2024) explained the potential of PGPR strains from rice irrigation systems, where a selected isolate from MR269 rice displayed multiple plant growth-promoting traits, including nitrogen fixation, phosphate and potassium solubilization, as well as production of siderophore and IAA, which promoted the overall growth performance of chili plants. These studies support the current findings and emphasize the rhizosphere as a potential reservoir for beneficial microbial strains.

The study revealed variability among treatments, with none showing statistically significant superiority. However, T3, which incorporated the PGPR strain RRZ034, consistently outperformed other treatments across all measured parameters. This performance is likely attributed to the strong plant growth-promoting traits of RRZ034 as demonstrated in the in vitro assays. The strain exhibited key functions such as nitrogen fixation and phosphate and potassium solubilization, which are essential for improving nutrient availability and uptake. These attributes are likely to include potassium solubilization, which is essential for improving nutrient availability and uptake. These attributes likely contributed to the enhanced seedling growth and vigor observed during the trials.

While the single-strain application of RRZ034 yielded promising results, it is important to consider the broader possibility of interactions among different PGPR strains. Its promising trend may become more pronounced with larger sample sizes or under real-world field conditions. The broader potential of co-inoculation strategies also deserves consideration. Previous studies have highlighted the synergistic effects of combining Pseudomonas spp. with other PGPR strains, particularly Bacillus spp. (Singh et al., 2022). Such combinations have been reported to improve nitrogen availability, enhance plant growth, and provide disease resistance (Poveda et al., 2022). Exploring similar co-inoculation approaches involving RRZ034 could enhance its efficacy and adaptability under diverse environmental conditions. However, further research is needed to confirm these findings across diverse agricultural settings and to better understand the mechanisms in which the PGPR strain RRZ034 promotes plant growth.

The 44 rhizobacterial isolates obtained in this study did not exhibit high functional diversity; many demonstrated consistent PGP traits, including phosphate and potassium solubilization, IAA production, and antifungal activity. The selected strain, RRZ034, was molecularly identified as Pseudomonas nicosulfuronedens, consistent with the strain LAM1902 previously described by Li et al. (2021). LAM1902 was isolated from a microbial consortium capable of degrading nicosulfuron, a herbicide, and used it as the sole nitrogen and energy source. Similarly, RRZ034 was isolated from a hill paddy farm established through conventional land-clearing practices involving herbicide application. This suggests a potential adaptation of RRZ034 to chemically disturbed environments. In line with this, Riddech et al. (2024) reported that their Pseudomonas nicosulfuronedens strain AP01 exhibited high phosphate solubilization, strong pathogen suppression, and significantly enhanced the growth of roselle plants. RRZ034 showed comparable characteristics, with notable solubilization indices for both phosphate and potassium, further highlighting its potential as an effective PGPR.

Given that the soil properties can influence the microbial diversity and activity, it is possible that these properties may have supported the natural presence and functionality of Pseudomonas nicosulfuronedens RRZ034 in the hill paddy rhizosphere. The moderately acidic pH of the rhizospheric (pH 5.2 in water) aligned well with the growth preferences of Pseudomonas species, which are known to thrive in such conditions (Wu et al., 2021). Although the soil had low total nitrogen (0.07%) and a limited cation exchange capacity (7.99 mEq/100g), the Pseudomonas nicosulfuronedens was able to persist may be due to its ability to adapt and contribute to nutrient availability. Notably, the low available phosphorus level (4.00 mg/kg) in the soil may stimulate the phosphate-solubilizing activity of this bacterium, as Pseudomonas strains typically demonstrated efficient phosphate uptake (Li et al., 2024). These soil characteristics can collectively trigger the natural occurrence and functional role of Pseudomonas nicosulfuronedens in promoting plant growth under nutrient-limited environments.

Although P. nicosulfuronedens has been known for several years, studies on its role as a PGPR remain limited. However, the genus Pseudomonas is widely recognized for its plant growth-promoting capabilities. Numerous studies, including those by Pandey et al. (2020) and Issifu et al. (2022), have reported that Pseudomonas spp. frequently exhibit traits such as phosphate solubilization, IAA production, biocontrol activity, and stress alleviation. Among them, P. aeruginosa is often cited as an effective PGPR. For instance, Ghamdamgahi et al. (2022) and Chandra et al. (2020) reported its ability to promote plant growth through phosphate solubilization, siderophore and IAA production, and effective suppression of pathogens such as Alternaria alternata, Aspergillus flavus, and Fusarium oxysporum.

The identification of RRZ034 as a Pseudomonas sp. connects this study to a broader body of research emphasizing the importance of this genus in sustainable agriculture. Pseudomonas spp. are well-regarded for their contributions to biofertilization and biocontrol. According to Okur et al. (2018), microbial biofertilizers like Pseudomonas spp. enhance soil fertility, stimulate nutrient uptake, and improve crop yield by colonizing the rhizosphere or plant interior. These microbial inoculants, when applied to seeds, plant surfaces, or soil, not only support plant development but also protect against pests and diseases, making them a sustainable and cost-effective solution for modern agriculture (Raghuwanshi, 2012; Glick et al., 2020).

5. Conclusions

This study identified several rhizobacterial isolates from the hill paddy rhizosphere, with eight strains demonstrating multiple plant growth-promoting traits in vitro. Among these, strain RRZ034 (Pseudomonas nicosulfuronedens) emerged as the most promising candidate. Although none of the treatments resulted in statistically significant differences in plant growth parameters, T3, which involved strain RRZ034, consistently recorded the highest mean values for germination percentage, root length, shoot height, and vigor index. The consistent performance across all parameters suggested potential biological relevance and highlighted RRZ034 as a promising strain for further investigations and trials. In the future, strain RRZ034 can be explored further for optimized application in crop production. Also, although Pseudomonas nicosulfuronedens is not a novel species, its plant growth-promoting traits remain largely underexplored. Future studies should focus on optimizing its formulation using compatible and effective carriers such as biochar or alginate beads to enhance its viability, stability, and shelf life. Additionally, field trials are essential to investigate its effectiveness in improving plant growth, yield, and soil health under natural environmental conditions. These efforts are critical to assess its potential as an effective and sustainable biofertilizer.