The current agricultural system is heavily dependent on the intensive use of chemical fertilizer and pesticides. However, application of synthetic chemical inputs in agriculture has negative effects on water, soil, and human and animal health (Nicolopoulou-Stamati et al., 2016; Hirt, 2020). Rice is the staple food of Bangladesh and many Asian and African countries and high-yielding rice varieties require extensive application of synthetic chemical fertilizers such as nitrogen (N), phosphorus (P) potassium (K) for achieving higher yield for ensuring food security on these continents (Hazell, 2010). Bangladesh consumes approximately 6 million metric tons of chemical fertilizers annually for rice and other crop cultivation, of which about 80 percent are imported (Rahman et al., 2018b). The four major imported chemical fertilizers in Bangladesh are urea, triple super phosphate, diammonium phosphate, and muriate of potash (MOP; Rahman et al., 2018b; Singh et al., 2021). To maintain the substantial increase in rice yield to feed the ever increasing population of rice growing countries, farmers need to apply higher doses of these fertilizers to the field (Rahman et al., 2018b). A large proportion of the applied synthetic chemical fertilizers are lost in various ways and pollute the environment (Rahman et al., 2018a; Singh et al., 2021). Sadly, due to the deterioration of soil health by the use of synthetic fertilizers over a long time, the yields of rice crops have not increased as they have increased before (Singh, 2018). Furthermore, to offer fertilizers at an affordable price to farmers, the government of Bangladesh spends huge funds every year subsidizing them. The natural mineral sources of these chemical fertilizers are also finite. Therefore, demand remains for searching alternative ways that would help to promote growth and increase yield of rice. Plant probiotic bacteria have shown high promise in significantly reducing the dependency on synthetic chemical fertilizers for growth and yield of rice (Trân Van et al., 2000; Khan et al., 2016; dos Santos et al., 2020). Application of these renewable bioresources could also reduce production costs and improve soil health in a sustainable manner.

The microbial world is the largest unexplored reservoir of biodiversity, which may act as a major benefit for agriculture, industrial and medicinal applications (Maheshwari, 2011; Geisen et al., 2019). One of the potential ecologically and economically sustainable strategies for the reduction of the dependence on chemical fertilizers could be the discovery of plant probiotic bacteria from native plants and use them as biofertilizer and/or biostimulants (Islam and Hossain, 2012; Islam et al., 2016). Application of an appropriate plant probiotic bacterium or a consortium of elite probiotic bacteria has been found to significantly enhance growth and yield of crop plants with significant reduction of the requirements for chemical fertilizers (Islam et al., 2016; Menéndez and Paço, 2020). A large number of plant probiotic bacterial genera exist, such as Bacillus, Pseudomonas, Paraburkholderia, Klebsiella, Rhizobium, Rahnella, Enterobacter, Delftia, Azospirillum, Streptomyces, and they have been used in commercial formulation as biofertilizer in many countries, which are now a multibillion-dollar business worldwide (Zambrano-Mendoza et al., 2021). In addition to growth promotion through diverse direct and indirect mechanisms, they also protect plants from pathogens by producing antimicrobial substances and enhance stress resistance to plants by inducing the expression of stress-responsive genes in the host plant (Harman et al., 2001; Zambrano-Mendoza et al., 2021). Plant probiotic bacteria that express aminocyclopropane-1-carboxylic acid (ACC) deaminase activity protects plants from growth inhibition by flooding and anoxia, drought, high salt, the presence of fungal and bacterial pathogens, nematodes, and the presence of metals and organic contaminants (Gamalero and Glick, 2011; Kandel and Kandel, 2015). To promote plant-growth, probiotic bacterial inoculants must either establish themselves in the soil or become associated with the host plant. The effects of microbial inoculation could be positive or negative depending on the microflora of root and rhizosphere (Ambrosini et al., 2016). Since only about 5% of these microorganisms are cultivable, the effects of the application of probiotic bacteria on the microbial diversity and population remained vastly unknown. Better understanding of the effect of applied plant growth promoting probiotic bacteria (PGPB) on the change in diversity and populations of the native bacteria in roots and rhizosphere are important for better understanding of the tripartite interactions which is critical for their sustainable application in crop production.

Metagenomics analysis opens a new window to explore the microbial diversity of various ecosystems without direct culturing of the microorganisms (Hoque et al., 2021; Rafiqul Islam et al., 2022). This approach is becoming increasingly popular in large-scale genomics applications as a way to study the taxonomic and functional composition of microbial communities from environmental, agricultural, and clinical settings (French et al., 2021; Rahman et al., 2021; Hoque et al., 2022). Metagenomics can improve our understanding of the importance of microbes to plants and the associations that exist among them (Melcher et al., 2014) that can be used for better understanding the complex interactions between crop plants and soil microbiome critical for crop production (Melcher et al., 2014; Trivedi et al., 2017). Novel antibiotics and enzymes are among the early discoveries from metagenomics. This state-of-the-art technology is also now being applied to identify unknown pathogen(s) in outbreaks of emerging and re-emerging diseases (Hoque et al., 2019, 2021; Rafiqul Islam et al., 2022). Targeted 16S/18S/ITS amplicon sequencing is a powerful and affordable tool for clinical microbiota analysis (Islam et al., 2021). It can also be used to determine gut microbial species and their abundance, and allows to the monitoring of human health and well-being. Moreover, metagenomics can support the development of probiotics (Xu et al., 2019).

Our previous studies showed that the application of PGPB such as Paraburkholderia spp. strain BRRh-4 and Delftia sp. strain BTL-M2 significantly improves plant growth and increases grain yield of rice under reduced application of N, P, and K fertilizers (Khan et al., 2016). So far, the known mechanisms of these two bacteria in plant growth promotion are the production of indole-3-acetic acid (IAA), fixation of atmospheric nitrogen and solubilization of essential mineral nutrients such as phosphorus and potassium (Khan et al., 2016). However, detailed information is not available on whether the application of plant probiotic Paraburkholderia sp. and Delftia sp. modulate diversity and population of bacteria in roots and rhizosphere soils of the treated rice plants. Therefore, we hypothesized that the application of probiotic bacterial strains, BRRh-4 and BTL-M2 improves growth and yield of rice under nutrient deficient soils by improving the diversity of the bacteriome in roots and rhizosphere soils of rice. To test this hypothesis, we evaluated the effects of P. fungorum strain BRRh-4, and Delftia sp. strain BTL-M2 on growth, yield of rice under varying fertilizer doses and analysed the diversity and population of the bacterial community in rice roots and rhizosphere soils using the targeted 16S rRNA gene metagenomics sequencing.

The use of plant probiotic bacteria (PPB) with minimum or no chemical fertilizers is considered as an eco-friendly means to carry out intensified rice production without degrading soil health and environment. Over recent decades, attention in the application of PPB in rice production has increased dramatically due to their ability to act as plant-growth regulators (Harman and Uphoff, 2019). Crop plants harbour and rely on rhizosphere-associated microbiomes for nutrition, which is crucial to their productivity. The excessive use of nitrogenous, phosphorous and potash fertilization is currently one of the main problems affecting soil health, eutrophication and high input cost of rice production in intensive agriculture system (Ha et al., 2015; Dong et al., 2021). In this study, we demonstrated that application of two rice probiotic bacteria, P. fungorum strain BRRh-4 and Delftia strain BTL-M2 produce statistically equivalent or higher grain yield of rice with 50% reduced doses of recommended N, P, and K fertilizers compared to 100% doses of these fertilizers without the application of any probiotic bacteria. Furthermore, using metagenomics analysis, we demonstrated that application of probiotic bacteria significantly modulates diversity and population of bacteriome in roots and rhizosphere of the treated rice plants. Interestingly, we found the recruitment or abundance of the specific genera of bacteria only in the probiotic treated rice roots and rhizosphere soils (Table 5). Although some plant growth promoting traits of Delftia sp. and Paraburkholderia sp. have been reported (Chaudhary et al., 2012; Ha et al., 2015), this study for the first time demonstrated the application of these rice probiotic bacteria to improve growth and yield of rice likely through modulating and/or improving the diversity of the native bacteriome of rice and rhizosphere soils.

In this study, we found that seed germination rate was higher in both Delftia strain BTL-M2 (89%) and P. fungorum strain BRRh-4 (88%) treated rice compared to untreated controls (77%). Previous studies have reported a number of endophytic bacteria with high nitrogen fixing enzyme activity and probiotic potential that were isolated from rice (Chaudhary et al., 2012; Khan et al., 2017). Enhancement of the rice seed germination rates, seedling growth and yield of rice in nutrient deficient soils by probiotic bacteria have also been reported in a recent report (Khan et al., 2017). A previous study has reported that both BTL-M2 (89%) and BRRh-4 strains produced phytohormone IAA and solubilize phosphorus in vitro (Khan et al., 2017). The enhanced root and shoot growth in rice seedlings might be linked with the production of IAA and phosphorus solubilization ability of the bacterial strains. An association of phytohormone production with root and shoot growth promotion by probiotic bacteria have been reported (Khan et al., 2017; Méndez-Gómez et al., 2021). Several studies have reported that plant probiotic bacteria influence plant biomass production by the production of phytohormone, solubilization of phosphorus and some other mechanisms (Hu et al., 2021). The increased shoot weight of rice seedlings could be linked with the production of IAA, fixation of atmospheric nitrogen and solubilization of essential nutrient elements in soils by the bacterial strain BRRh-4 and BTL-M2 (Sharma and Shukla, 2021).

Several lines of evidence suggest that plant-associated P. fungorum and Delftia sp. promote growth and dry matter production by various mode of actions (Singh et al., 2021). Supplementing synthetic fertilizers partially with the plant-growth-promoting probiotic bacteria is an alternate option to reduce chemical fertilizer (CF) usage in crop production (Singh et al., 2021). One of the interesting findings of our current study is that application of both Delftia strain BTL-M2 and P. fungorum strain BRRh-4 with 50% recommended doses of N, P, and K fertilizers produced almost equivalent or higher yield of rice compared to the application of 100% N, P, and K fertilizers only. Our results suggest that application of these probiotic bacteria as biofertilizer could reduce 50% requirement of chemical fertilizers (CF) in rice. In earlier studies, we found that both Paraburkholderia and Delftia solubilize minerals, phosphates and potassium, fix atmospheric nitrogen and produce IAA in vitro. The presence of the applied bacterial population and modulated population and diversity of the native bacteriome shown in this might be involved in growth and yield promotion of rice (Khan et al., 2017; Rahman et al., 2018a). Various plant growth promoting traits including fixation of atmospheric nitrogen by both Paraburkholderia and Delftia spp. separately have been reported (Trân Van et al., 2000; Han et al., 2005; Rahman et al., 2018a). Kobua et al. (2021) previously demonstrated that the combined application of CF + plant growth promoting rhizobacteria (PGPR) improved the growth and yield of rice. Conversely, Nguyen et al. (2019) and Akram et al. (2013) reported that the tiller number of monocot plants such as rice was less (significantly) affected by CF + probiotic bacteria treatments than the crop height and other vegetative parameters. In fact, plants treated with some elite strains of probiotic bacteria form resilient cell structures and have better metabolic processes along the culm area (Parra-Cota et al., 2014; Kobua et al., 2021). However, the results obtained in the current study in pot culture condition are needed to be confirmed by field experiment. A large body of the literature indicate that the application of probiotic bacteria enhance the growth and yield of crop by both direct and indirect mechanisms including phytohormone production, nutrient acquisition, inhibiting the growth of pathogenic fungi and also inducing systemic resistance in the host plants (Khan et al., 2016; Hu et al., 2021; Méndez-Gómez et al., 2021). Our study demonstrated that two rice probiotic bacteria with various plant growth promoting traits are potential candidates for the development of biofertilizers to reduce the cost of rice production and improve the soil health. A further large field evaluation of these bacteria alone or in combination is needed before recommending them as biofertilizers for rice.

Interestingly, we found that our applied bacterial population significantly low in the rhizosphere soils and roots but a remarkable change of the diversity and population structure occurred in other bacteriome in both rhizosphere and roots due to their application. We assume that the modulation in bacteriome could be due to the interactions and/or cross-talk of the applied bacteria with the native bacteria in rice rhizosphere that ultimately resulted in the higher yield of rice. A further study is warranted to clarify the functional aspects of the changed bacteriome that impacted to the growth and yield of rice (Khan et al., 2017). Microbial communities in the rhizosphere play a key role in solubilisation and availability of essential nutrient elements to the plants and thus influence plant health, growth and yield (Friesen et al., 2011). In turn, rhizosphere soil microorganisms heavily rely on root exudates, such as carbon metabolites and other nutrients, as growth substrates. Plant species directly influence soil microbial communities through these root exudates which change as the plant matures (Berg and Smalla, 2009). A number of studies have shown that microbial taxa identified through the metagenomic analysis are all widely present in soil, including species from the Actinobacteria, Chloroflexi, Planctomycetes, Proteobacteria, and Verrucomicrobia phyla (Berg and Smalla, 2009; Zhang and Lv, 2020). Our study also reported the rice associated bacteria cultivated in the soils of Bangladesh under the treatment of probiotic bacteria with or without the application of major chemical fertilizers used in rice cultivation. The beneficial effects of the application of probiotic bacteria on rice and many other crops have been reported (Yanni et al., 2001; Yadav et al., 2020; Ngalimat et al., 2021). For example, soil bacteria such as Pseudomonas, Bacillus and Rhizobium increase the concentration of plant available phosphorus (P) in soil by releasing phosphatase enzymes or organic chelates (Lazcano et al., 2021). Additionally, certain strains of Bacillus, Pseudomonas, Paraburkholderia and Acinetobacter are thought to increase the availability and uptake of micronutrients such as Zn and Fe for mung bean, rice and corn (Goteti et al., 2013; Lazcano et al., 2021).

The plant microbiome has potential for improving crop productivity and sustainability (Kim and Lee, 2020). In this study, we characterized the bacteria communities associated with rice roots and rice rhizosphere soils after the application of rice probiotic bacteria, BRRh-4 and BTL-M2 with varying doses of chemical fertilizers. We detected 133 and 161 genera in CF and CF + probiotic bacteria treated rice groups, respectively (Supplementary Figure S3A). Some of these genera such as Prevotella were abundant in rice root samples and other genera were more abundant with rhizosphere soils. Prevotella species have been isolated from human and animal gut samples, and also from root and soil samples of the irrigated rice (Ueki et al., 2007; Tett et al., 2021). This genus is also detected in the rumen of the ruminant as high abundance after feeding rice straw supplemented with nitrogenous substances (Latham et al., 2018). Prevotella species are found to play a significant role in breakdown of carbohydrate in the gut of animals (Precup and Vodnar, 2019). Furthermore, P. paldividence was isolated as hemicellulose decomposing bacteria from plant residue and rice roots in irrigated rice-field soils (Ueki et al., 2007). Therefore, the high abundance of Prevotella in our irrigated rice root and rhizosphere might be partly linked with the cow dung application in the pot soils. The genus Prevotella plays major roles in carbon, carbohydrate and nitrogen metabolism (Kim et al., 2017). In term of ‘One Health Concept’, interlinking of different ecosystems by Prevotella is one of the best examples of soil-water-plant–animal-human continuum.

In the present study, we recovered several unique bacterial taxa of rice roots belonging to the genera of Chryseobacterium, Exiguobacterium, Novosphingobium, Massilia, Nocardioides, Methylosinus, Streptomyces, Sphingomonas, and Paenibacillus. Many of these genera have shown in vitro and/or in planta beneficial effects on promoting the growth, productivity and/or improve disease resistance of plants (Zhou et al., 2020). For instance, the beneficial probiotic bacteria of Chryseobacterium genus can inhibit pathogens, and modulate other beneficial microbes (Jinendiran et al., 2019), and many strains of Exiguobacterium are known as potential sources for acquisition and fixation of nutrients (Pandey, 2020). Novosphingobium, a novel rhizosphere-associated bacterium with plant beneficial properties isolated from saline-tolerant pokkali rice (Krishnan et al., 2017). The probiotic bacteria Sphingomonas increases plant growth rate and alters the rhizosphere microbial community structure of Arabidopsis thaliana under drought stress (Luo et al., 2019). The Gram-positive rhizosphere bacterium, Paenibacillus promotes plant growth and produces various antibiotics (Jeong et al., 2019). Discussing the functional characters of an endophytic community colonizing rice roots, del Barrio-Duque et al. (2020) speculated that the endophytic community of rice play a vital role in plant growth promotion, improvement of stress tolerance, biocontrol pathogenic microorganisms, and bioremediation (del Barrio-Duque et al., 2020).

Our study catalogued the bacteriome in rice roots and rhizosphere, and demonstrated how the application of rice probiotic Paraburkholderia and Delftia with or without application of chemical fertilizers modulate the bacterial community in both roots and rhizosphere soils. Our results indicate that bacterial populations are highly influenced by the application of CF alone and/or in combination with probiotic bacteria. Interestingly, combined application CF and probiotic bacteria had the highest bacterial diversity. Furthermore, this higher diversity of bacteria in root and rhizosphere of rice was correlated with the higher growth and grain yield. These results suggest that manipulation of bacterial community associated with rice by the application of probiotic bacteria could be an alternative and sustainable approach for reduction of the dependency of synthetic chemical fertilizers in rice production. Obviously, the enhancement of microbial diversity by the application of probiotic bacteria Paraburkholderia and Delftia sp. might improve the soil health.

A hallmark of our research findings is that we found the abundance or recruitment of specific genera of known plant growth promoting bacteria in rice by the application of rice probiotic bacteria, Paraburkholderia and Delftia. The abundance/presence of Sphingomonas, Novosphingomonas and Massilia only with the treatment of rice with a strain of Paraburkholderia (BRRh-4) was highly interesting and merit further investigation for understanding the mechanism involved. Our results indicate that the growth promotion and yield increment of rice were not only achieved by the effects of the applied probiotic bacteria but also largely by the effects of the modulated bacteriome and other unknown microbiome.

In the current study, probiotic bacteria application significantly modulates the structures, diversity and population of the rhizosphere bacteria of rice. These modulations might be linked with the significant growth-promotion and grain yield increase in rice by the application of BRRh-4 and BTL-M2. Production of phytohormones, solubilisation of essential nutrient elements (e.g., P and K) and fixation of atmospheric nitrogen in soils by the Paraburkholderia and Delftia spp. have been reported (Trân Van et al., 2000; Han et al., 2005; Rahman et al., 2018a). A further shotgun metagenomic study is needed to elucidate the mechanisms of these (BRRh-4 and BTL-M2) elite strains of probiotic bacteria on growth promotion, yield increase and microbial diversity in rice cultivated in the field conditions. Although the microbiome of rice roots and rhizosphere has become documented and discussed in several reports (Kim and Lee, 2020; Santos-Medellín et al., 2021), our study demonstrated that the application of two rice probiotic bacteria significantly improve the growth and yield of rice and modulate the bacterial diversity and population in roots and rhizosphere soils of treated rice plants. The performances of these bacteria on rice root and soil microbiome in different edaphic conditions should be evaluated for understanding the precise role in sustainable rice production with reduced chemical fertilizers.

The microbiome inhabiting in rice roots and rhizosphere soils are taxonomically and phylogenetically diverse. Although they play a vital role in growth, health and productivity of rice, little is known about the interactions of rice plants and these microbial communities at a molecular level. In the current study, we demonstrated that two elite strains of plant probiotic bacteria, P. fungorum strain BRRh-4 and Delftia sp. strain BTL-M2 significantly improve seed germination and growth of rice plants both in vitro and in planta. In the pot experiments, both BRRh-4 and BTL-M2 with 50% reduced doses of recommended N, P, and K fertilizers produced almost equivalent yield of rice treated with 100% recommended doses of chemical fertilizers without application of bacteria. The amplicon sequencing analysis revealed that a combination of chemical fertilizers and P. fungorum strain BRRh-4 or Delftia sp. strain BTL-M2 significantly increased the diversity, structure and compositions of associated bacteriome in both in rice roots and rhizosphere soils. These probiotic bacteria may likely increase the availability and use efficiency of the N, P and K fertilizers by modulating rice root-associated microbiome. Our results indicate that application of BRRh-4 and BTL-M2 could reduce 50% N, P, and K fertilizers requirements for rice production without compromising the yield. Interestingly, we found the recruitment and/or abundance of specific bacterial genera in the rice treated with the specific probiotic bacteria which merits further investing to understand the molecular cross-talks in the rhizobiocomplex in rice. The novel findings of our study indicate that elite species of plant probiotic bacteria have tremendous potential for use as biofertilizers as an alternative to the costly and hazardous synthetic fertilizers, which are commonly used in conventional rice production. However, a large-scale field experiment and further metagenomics analyses are required for a better understanding of the effects of modulated microbiome on growth and yield of rice by the application of probiotic bacteria alone or in combination under reduced level of chemical fertilizers. The role of applied probiotic bacteria on health of rice cultivated soils also needs to be investigated.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

TI conceived, designed and supervised the study. F, DRG and NUM carried out field and laboratory experiment, interpreted the results and drafted the manuscript. MNH and TIS executed the bioinformatics analysis, interpreted the results and drafted the manuscript. TI and AGS contributed intellectually to the interpretation and presentation of the results. All authors contributed to the article and approved the submitted version.

This study was supported by the grant from Bangladesh Academy of Science (BAS)-USDA-PAL project CR-11 to TI.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.