Diversity and functional traits based indigenous rhizosphere associated phosphate solubilizing bacteria for sustainable production of rice

Abstract

Introduction: Rice, particularly Basmati rice, holds significant global importance as a staple food. The indiscriminate use of phosphate-based fertilizers during rice production has led to high residual levels of these chemicals in soil, impacting soil health and fertility. This study aimed to address this challenge by investigating the potential of phosphate solubilizing bacteria (PSB) in improving soil fertility and boosting the growth of Basmati rice.

Methods: Using amplicon-based 16S rDNA sequencing, bacterial isolation and cultivation, conducting greenhouse and field experiments, and PSB localization, we optimized the search for PSB inoculants to enhance Basmati rice growth.

Results and discussion: Rice rhizosphere prokaryote communities showed significant differences in microbial diversity and composition between between basmati and non-basmati rice cultivated areas. Dominant bacterial phyla included Proteobacteria, Acidobacteria, Actinobacteria, and Firmicutes, with Actinobacteria and Proteobacteria playing a crucial role in nutrient recycling. Isolation and optimization of PSB strains, including Acinetobacter sp. MR5 and Pseudomonas sp. R7, were carried out and soil microcosm studies confirmed their efficacy in increasing soil available phosphorus concentration. Response surface methodology revealed the relative importance of factors such as pH, inoculum density and incubation temperature in maximising phosphate solubilization. Microplot experiments demonstrated the effectiveness of optimized PSB inoculants in promoting Basmati rice growth, with significant increases in plant height, tiller number, biomass, and grain yield compared to uninoculated controls. A consortium of PSB proved superior to single-strain inoculants, even with reduced chemical fertilizer application. Field trials at several rice growing sites confirmed the positive impact of the PSB consortium on grain yield, soil phosphorus availability, and plant phosphorus uptake. The competence and persistence of the inoculated strains in the rhizosphere was confirmed by FISH and BOX Polymerase Chain Reaction (BOX-PCR). This work highlights the potential of PSB-based biofertilizers to improve soil fertility, promote sustainable rice production and reduce the negative environmental impacts of chemical fertilizers. Future research would focus on scaling up these findings for widespread adoption in agriculture and exploring their applicability to other crops and agroecosystems.

Keywords: Basmati rice; biofertilizers; microbial diversity; next-generation sequencing; nutrient recycling; sustainable agriculture.

Figure 1

The map shows the positions of the study sites within the chosen region. Each marker indicates where samples were collected for microbiological analysis, covering a range of environmental conditions to ensure thorough data collection (A). Evenness, diversity, and richness of rhizosphere soil sample of Basmati rice (B). Venn diagram showing the bacterial genera shared among the rhizosphere soil samples of Basmati rice collected from different areas of the rice belt and Faisalabad (C). Relative abundance of taxonomic groups (D) and functional groups (E) detected by Illumina Sequencing of 16S rRNA gene in the rhizosphere soil sample of Basmati rice collected from different areas of rice belt and Faisalabad. Site 1 = Sheikhupura, Site 2 = Gujranwala, and Site 3 = Faisalabad.

Figure 2

Soil microcosm to study the effect of Acinetobacter sp. MR5 and Pseudomonas sp. MR7 at 15 DPI, 30 DPI, and 60 DPI on (A) soil available P (B) bacterial phosphatase activity and (C) total bacterial population. Means are an average of three biological replicates and means followed by the same letter differ non-significantly at p = 0.01 according to LSD using 3-way ANOVA.

Figure 3

The interaction of different variables in response to inoculation of Acinetobacter sp. MR5 and Pseudomonas sp. MR7 on solubilized phosphate. 3D surface curves were plotted against two independent variables and keeping other variable at their central (0) level. The 3D curves and contour plots from the interactions between variables of the calculated response are (A,D): relative effect of pH and temperature on phosphate solubilization while keeping inoculum concentration at its central level. (B,E) relative effect of temperature and inoculum size on phosphate solubilization while keeping temperature at its central level. (C,F) relative effect of pH and inoculum size on phosphate solubilization while keeping pH at its central level.

Figure 4

Biokinetics to study the phosphate solubilization activity of two Acinetobacter sp. MR5 and Pseudomonas sp. MR7. Effect of inoculum on (A) Specific growth rate. (B) P solubilization (C) effect of inoculum on pH. Acinetobacter sp. MR5 (0.022 and 0.038% inoculum w/v wet cells) and Pseudomonas sp. MR7 (0.008 and 0.014% inoculum w/v wet cells) were used for inoculation in Pikovskaya broth and kept at 36 ± 2°C. The supernatant was collected at different time intervals (4, 12, 18, 22, and 34 h) and plotted against the time for activity. Means are an average of three biological replicates, ± represents standard deviation.

Figure 5

Principal component analysis (PCA) showing the response of bacterial inoculation treatments at three different soils: (A) Loaded as soil type (upper panel) and (B) treatments (lower panel) (total variance explained: 82%). T1 = 80% uninoculated control, T2 = 80% PSB inoculated treatments, and T3 = 100% uninoculated control. S1 = Sheikhupura, S2 = Gujranwala, and S3 = Faisalabad.

Figure 6

Re-isolation of inoculated PSB colonies (A). Plate showing re-isolated colonies of MR5 and MR7 (B, C). Confocal Laser Scanning Microscopy of rice roots at 35 days post inoculation of P solubilizing bacteria in the microplot experiment under net house conditions. Control treatments without inoculation (D) and from the PSB inoculation of MR5 (E) and MR7 (F). Plant growth-promoting attributes of re-isolated PSB strains for confirmation of inoculated PSB: P solubilization and IAA production were detected quantitatively by high-performance liquid chromatography (HPLC), gluconic acid production and siderophores production (G). Values are an average of six biological replicates. MR5: Acinetobacter sp.’ MR7 and Pseudomonas sp. The photograph of the gel indicates re-isolated colonies of PSB that are similar morphologically to the inoculated consortium (H).