One of the nature's most dynamic environments is the rhizosphere, which is the microecological interface between plant roots and soil. It is created by complex interactions between plants, soil, and a broad range of microorganisms, including bacteria, fungi, protozoa, and archaea (Philippot et al. 2013; Vetterlein et al. 2020). These microbial communities play a crucial role in plant health by affecting growth regulation, nutrient uptake, stress tolerance, and disease defense. Consequently, the rhizospheric microbiota is preferentially shaped by the root exudates that plants release, which are made up of sugars, amino acids, organic acids, and secondary metabolites (Ali and Glick 2024; Chen and Liu 2024). As a result, the rhizosphere is a closely related plant-microbe system that is essential to the sustainability of agriculture and ecosystems. In recent decades, the possibility of using rhizospheric microbial populations to improve soil health, decrease the usage of chemical fertilizers, and increase plant growth and resistance has come to light (Wei et al. 2024). Studies on model plants and key crops like maize, wheat, and rice have demonstrated that the rhizosphere is home to beneficial microbes like phosphate solubilizers, nitrogen-fixing bacteria, and biocontrol agents (Kour et al. 2019; Imade and Babalola 2021; Di Benedetto et al. 2017; Ngalimat et al. 2021). Although many therapeutic plants are valued for their ability to produce secondary metabolites and are often cultivated in low-input systems, little is known about their rhizosphere microbiomes.

Long pepper, or Piper longum L., is a perennial climbing vine that is indigenous to South Asia and is a member of the Piperaceae family. Due to its medicinal qualities, which include antibacterial, anti-inflammatory, hepatoprotective, and anticancer actions, it is highly valued in ancient medical systems such as Ayurveda, Siddha, and Unani (Biswas et al. 2022). Its medical effectiveness is attributed to bioactive substances such piperine, piperdardine, and piperlongumine. There is a growing need for Piper longum to be grown using sustainable methods that preserve or improve its phytochemical content due to the increased interest in natural products and herbal remedies around the world (Prasad et al. 2025). However, little is understood about the microbial communities in the rhizosphere that could affect its metabolite production, growth, and health.

Historically, the variety of soil microbiomes has been difficult for culture-based methods to fully capture because a large number of soil microorganisms are not culturable in standard laboratory settings. Microbial ecology has undergone a radical transformation thanks to the direct study of DNA made possible by developments in next-generation sequencing (NGS) technology, particularly metagenomic sequencing (Dubey et al. 2022; Raghav et al. 2024). Metagenomics provides information about the functional potential of microbial communities in addition to their taxonomic composition by identifying genes linked to the nutrient cycle, stress adaptation, and plant–microbe interactions (Kumar and Yadav 2024). In this study, we used high-throughput Illumina sequencing to conduct a comprehensive metagenomic analysis of the P. longum rhizosphere. Through gene annotation across major functional databases, this study sought to clarify the functional potential of the microbial communities residing in the rhizosphere of P. longum while also characterizing their taxonomic diversity. Finding dominant microbial taxa that may contribute to improving soil fertility, bolstering plant health, and maintaining ecosystem stability received special attention. This work lays the groundwork for future functional studies centered on microbiome-assisted strategies for sustainable cultivation, increased plant productivity, and improved phytochemical quality in medicinal plants by offering an integrated overview of the P. longum rhizosphere microbiome.

The fertility status and suitability for microbial activity of the rhizospheric soil of Piper longum were evaluated by analyzing its physicochemical properties as shown in Table 1. With a pH of 7.52 ± 0.02 and a neutral to slightly alkaline reaction, the soil showed ideal conditions for microbial growth and nutrient availability. A moderate amount of soluble salts that is within the permissible range for healthy plant growth is indicated by the electrical conductivity (EC), which was measured at 639.21 ± 0.04 dS/m. While the levels of potassium (22.36 ± 0.007 kg/ha) and phosphorus (24.06 ± 0.10 kg/ha) demonstrated balanced nutrient availability necessary for plant metabolism and microbial functioning in the rhizosphere, the macronutrient composition showed a nitrogen content of 310.19 ± 0.03 kg/ha, indicating moderate fertility. Low to medium organic matter was indicated by the organic carbon content of 0.44 ± 0.008%. These soil parameters collectively show a biologically active, fairly fertile rhizosphere that supports the functional potential revealed by metagenomic profiling and is favorable for plant growth as well as the development of a metabolically diverse microbial community.

To explore the rhizospheric bacterial community of P. longum metagenomic analysis was performed which yielded high-quality paired-end sequencing genomic DNA on the Illumina platform which generated 19,942,187 read pairs, yielding approximately (~ 5.92 Gb) of data with Q30 values exceeding 90%, which ensured high-confidence base calls. The quality-filtered reads were assembled de novo using metaSPAdes, resulting in the 97,432 scaffolds with a total length of 52.26 Mb, an average scaffold size of 536 bp, an N50 of 489 bp, and a maximum scaffold size of 44,108 bp as provided in Table 2. The assembly statistics provided indicated a comprehensive as well as well-represented metagenomic dataset with a sufficient coverage for downstream analysis. Further, genes were predicted using Prodigal (v2.6.3) which identified 45,876 protein-coding genes, with an average gene length of 334 bp and a total gene length of 15.36 Mb, providing a robust catalog of coding sequences for taxonomic and functional annotation as shown in Table 3.

Taxonomic classification was performed using Kaiju software, unraveling the microbial community associated with Piper longum which was found to be quite diverse; however, it was strongly dominated by the phylum Proteobacteria, that accounted for 14,015 hits (~ 42% of classified genes). Other major phyla that constituted the majority of the bacterial population in the P. longum rhizosphere with includes, Actinobacteria (4555 hits) which accounted for 13.9% of the total classified genes, Thaumarchaeota (13.16%), Chloroflexi (8.21%), Acidobacteria (7.28%), Candidatus rokubacteria (2.47%), and Nitrospirae (1.92%) whereas the least found phylum was that of Candidatus Fraserbacteria, Candidatus Roizmanbacteria and Thermodesulfobacteria (4 hits or 0.012% of relative abundance) as shown in Fig. 1. By digging into the finer taxonomic resolution, the most abundant class was found to be of Gammaproteobacteria, with relative abundance of 26.01%, followed by Betaproteobacteria with 17.09%, Actinobacteria with 12.02% and Nitrososphaeria 11.03%, respectively (Fig. 2). Similarly, the order level of classification revealed that Nitrososphaerales have a relative abundance of 15.82% was the dominant order, which was mirrored by the dominance of Nitrososphaeraceae (20.28%) at the family level (Figs. 3 & 4). At the genus level, Nitrososphaera was found to be the most abundant with relative abundance of 23.58% followed by Acinetobacter (13.11%), Rubrobacter (6.36%) and Nitrospira (5.06%), while the most abundant species detected was Candidatus Nitrososphaera gargensis with relative abundance of 11.21%, followed by Acidobacteria bacterium (9.74%) (Figs. 5 & 6). The preponderance of nitrifying archaea, such as Nitrososphaera, is indicative of the existence of an active ammonia-oxidizing microbial community. This community may be responsible for nitrogen cycling in the rhizosphere of Piper longum, indicating a community structure that is not only very diverse but also functionally relevant.

A thorough understanding of the metabolic capacity of the Piper longum microbiome was obtained by the functional analysis of the predicted genes using COGNIZER (v0.9b). The COG analysis identified 23,705 terms, with amino acid transport and metabolism accounting for the highest prevalence (2649 terms) which was followed by prediction of general function (2488 terms), carbohydrate transport and metabolism (2100 terms), and energy generation and conversion (1900 terms) (Fig. 7). This distribution emphasizes the microbial community's metabolic activity, especially in the areas of energy metabolism and nutrient cycling.

The metagenome sequencing from the current work has been submitted to the NCBI website under the accession number PRJNA600362. A variety of protein databases, including KEGG, Pfam, GO, COG, and FIG, were used to annotate the datasets with functional hierarchy information for the functional predictions (Fig. 8). The GO database produced the greatest number of hits, followed by the KEGG and Pfam databases. The COG functional analysis identified 23705 terms, and the KEGG annotation assigned 27,446 terms to 3594 KO classes, with the majority (54.6%) pertaining to metabolic pathways such as amino acid biosynthesis, carbohydrate metabolism, and energy production pathways. The next most prevalent functional category was environmental information processing (25.5%), followed by genetic information processing (12.7%), showing the existence of genes involved in two-component systems, ABC transporters, and transcriptional control as shown in (Fig. 9). The annotation of Pfam domains revealed 24,583 functional domains in 2930 distinct Pfam families, indicating a great variety of structural and catalytic proteins. Further clustering of 15,148 words into 4518 functionally homologous groupings using FIGfam analysis suggests conserved functionality across several taxa.

The Gene Ontology (GO) annotation generated 33,522 words over 952 functional categories, with the largest representation in biological processes such as metabolic activity, stimulus response, and cellular functions. A substantial number of genes were classified as molecular function categories such as catalytic activity and binding, as well as cellular component categories related to membrane proteins and intracellular activities. These findings collectively indicate that the microbiome associated with Piper longum is physiologically flexible, with a focus on nutrition mobilization, energy metabolism, and environmental adaptation. These findings collectively show that the microbiome of P. longum is rich and functionally diversified, dominated by ammonia-oxidizing archaea and Proteobacteria, and that metabolic pathways linked to nitrogen, carbohydrate, and amino acid cycling are strongly enriched. This functional potential highlights the ecological relevance of the P. longum microbiome in maintaining plant growth, improving the availability of nutrients, and possibly affecting the manufacture of secondary metabolites.

A strong relationship between soil fertility parameters and the functional competence of the microbial community in the Piper longum rhizosphere is highlighted by the combination of metagenomic insights and soil nutrient profiling. Plant-microbe symbiosis and microbially mediated nutrient cycling are supported by the moderate organic carbon content and balanced nutrient composition. The ecological and biotechnological significance of the P. longum microbiome is highlighted by these interactions, which not only maintain soil health but may also have an impact on the biosynthesis of pharmacologically significant compounds like piperine. The present research offers novel insights into the taxonomic constitution, functional potential, and ecological importance of the related microbial community by conducting a thorough metagenomic analysis of the Piper longum microbiome. The number of scaffolds and a N50 value of 489 bp demonstrate the high-quality and outstanding coverage of the dataset produced by high-throughput Illumina sequencing in tandem with de novo assembly. The assembly and gene prediction outcomes were similar to those reported for other medicinal plants, including Tribulus terrestris, Piper nigrum, Calatropis procera, where plant-associated microbial communities have been investigated using comparable sequencing depths (Rahimlou et al. 2025; Vaz et al. 2025; Li et al. 2016; Ramadan et al. 2021).

As demonstrated by the taxonomic profiling, Proteobacteria clearly dominated the rhizosphere and endophytic communities in medicinal plants. Proteobacteria are often the most abundant phylum because of their diverse metabolic capacities and capacity to colonize plant tissues (Vincze et al. 2024). The high prevalence of betaproteobacteria and gammaproteobacteria among the Proteobacteria indicates a metabolically active community that can react quickly to changes in nutrient levels in the rhizosphere (Manapure et al. 2022). Given that these ammonia-oxidizing archaea are important participants in the nitrification process and aid in the conversion of ammonia to nitrite, a crucial stage in the nitrogen cycle, the identification of Nitrososphaerales and the dominance of Candidatus Nitrososphaera gargensis seems particularly significant (Xiang et al. 2023; Clark et al. 2021). Their presence strengthens the possibility that the rhizospheric zone of P. longum is a nitrogen cycling hotspot, which could improve the intake and growth of nitrogen by plant. The robust vegetative development and the generation of bioactive metabolites under natural conditions may be partially explained by these microbial interactions. The metabolic adaptability of the P. longum microbiome was further highlighted by functional annotation. A microbiome that is actively involved in nutrient mobilization is indicated by the enrichment of COG categories linked to energy production, carbohydrate metabolism, and amino acid transport and metabolism (Sefrji et al. 2025; Xu et al. 2021). Through the synthesis of precursors for secondary metabolites, such as alkaloids like piperine, amino acid metabolism affects plant health in addition to being essential for microbial proliferation (Narayanan et al. 2022).

With notable presence in pathways related to nitrogen, sulfur, and carbon cycle, the KEGG pathway analysis showed that over half of the annotated genes fell under the metabolic category. These results are consistent with recent research on various microbiomes of medicinal plants, which emphasize the role of environmental information processing and nutrient cycling in promoting plant development and metabolite biosynthesis (Semenzato and Fani 2024; Singh et al. 2022). Remarkably, genes linked to stress response and xenobiotic degradation were also found, indicating that the Piper longum microbiome is capable of assisting the plant in overcoming abiotic stress and possibly enhancing resistance in harsh environmental circumstances (Pandey et al. 2023; Chaudhary et al. 2022).

Additionally, our findings offer a useful resource for comprehending interactions between microbes and plants in relation to the generation of secondary metabolites. Since the availability of nitrogen is a known regulator of alkaloid production, the presence of microbial genes involved in nitrogen cycling, phenylpropanoid metabolism, and amino acid biosynthesis may have an indirect effect on piperine biosynthesis (Gao et al. 2022). It may be possible to determine whether particular microbial species actively control the piperine biosynthesis pathway by future functional investigations, such as transcriptomics and metabolomics. Targeted enrichment of these species may increase the efficiency of nitrogen utilization in Piper longum culture, and the discovery of nitrogen-transforming archaea like Nitrososphaera opens up possibilities for the development of biofertilizer (Li et al. 2023). Overall, this study creates a baseline microbiome profile for Piper longum, exposing a metabolically active and taxonomically varied microbial community that may play roles in secondary metabolism, stress adaption, and nutrient cycling. These discoveries broaden our knowledge of the ecological functions of microorganisms linked to plants and point to potential uses in precision farming, where piperine productivity could be increased through microbiome engineering.

Plant health and soil nutrient dynamics depend on the taxonomically diverse and functionally rich microbial community found in the rhizosphere of Piper longum, according to metagenomic research. The abundance of Proteobacteria and ammonia-oxidizing archaea such as Nitrososphaera and Candidatus Nitrososphaera gargensis indicates that the rhizosphere is likely to be an active site of nitrogen transformation. Functional gene annotation demonstrated a significant representation of metabolic pathways associated with energy synthesis, amino acid transport, and environmental adaptation, as well as a microbiome that is precisely regulated to support plant development in a variety of soil conditions. Together, the data suggest that the rhizospheric microbiota of P. longum is important for the cycling of nutrients and may indirectly affect the production of compounds that are relevant to pharmacology, such as piperine. This study highlights the importance of using beneficial microbial consortia for sustainable cultivation and increased phytochemical production, while also offering a basic understanding of the P. longum microbiome. Future multi-omics methods that combine transcriptome and metabolomic studies will be essential to identify possible taxa for the development of bioinoculants in medicinal plant agriculture as well as to pinpoint the exact microbiological processes underlying the production of secondary metabolites.